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florentgbelidji

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pubmed-running

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PMC4153017

male 8-week - old wistar rats ( 200 250 g ; specific pathogen - free ( spf ) ; japan shizuoka laboratory center ) were individually housed in cages ( 33 23 23 cm ) under controlled conditions of temperature ( 245 1c ) , humidity ( 55 5 % ) and light ( 12 h light12 h dark cycle ) . rats were provided with a stock diet ( charles river formula ( crf-1 ) ; oriental yeats co. ltd ) and water ad libitum . they were accustomed to treadmill running at a 0 % gradient and a speed of 25 m / min for the first 10 min and 85 m / min for the last 10 min ( kn-73 , natsume seisakusho co. ltd ) two times during 1 week of the acclimatisation period . all procedures were performed in accordance with the animal experimentation guidelines of sugiyama jogakuen university . milk ( meiji co. ltd ) , sports drink ( pocari sweat ; otsuka co. ltd ) and dextrin ( cyclic cluster dextrin ; esaki glico co. ltd ) were used . mps was obtained by overnight dialysis of milk in visking membrane tubes ( medical science co. ltd ) . mes was prepared by mixing kcl , nacl and kh2po4 ( wako pure chemical co. ltd ) . the composition and osmolality of the experimental solutions are shown in table 1 . in experiment 2 , [ 2-c]acetic acid was added to experimental drinks ( cambridge isotope laboratories inc . ) at a concentration of 01 l / ml . table 1.components of experimental drinksenergy density ( kj / l)lipid ( g / l)osmolality ( mosm / l)na ( mmol / l)k ( mmol / l)water0019060milk285639255186383sports drink1130034019550mps6310321633mes28560141217383mps , milk protein solution ; mes , milk electrolyte solution . . a total of eighty - three rats were starved for 20 h from 17.00 hours and were deprived of fluids from 11.00 hours on the day of the experiment . they ran on a treadmill for 60 min ( 11.00 to 12.00 hours ) at a 0 % gradient and a speed of 25 m / min for the first 10 min and 85 m / min for the last 50 min . rats were housed in custom - made metabolism cages for urine collection from 12.00 to 17.00 hours . average body weight reduction from the baseline values was 31 g and body fluid loss was 12 % of pre - exercise body weight . the experimental drinks water , milk , sports drink , mps and mes were orally administered at the dose of 24 % of baseline body weight ( average 60 ml ) using a stomach sonde under diethyl ether anaesthesia at 13.00 hours . after administration , urine was collected every 1 h for 4 h and was stored below 20c . urine was collected and weighed , and osmolality and na and k concentrations were measured . a total of fifteen rats were treated as in experiment 1 except that they were housed in a respiratory metabolic chamber to collect respiratory gas . the experimental drinks water , milk , mps and sports drink contained [ 2-c]acetic acid at a dose of 01 l / ml . respiratory co2 analysis was performed using an open - circuit metabolic gas analysis system connected directly to a mass spectrometer ( model arco2000 ; arcosystem ) . room air was pumped through the chambers at a rate of 05 litres / min . expired air was dried in a thin cotton column and then directed to a ms o2/co2 analyser . the peak time of co2 emission was used as the index of gastric emptying rate for each experimental drink . a total of sixty rats were treated as in experiment 1 and administered the experimental drinks water , milk , mps and sports drink . blood was collected from the common iliac vein under ether anaesthesia at 30 min , 1 h and 2 h after the administration of drinks . osmolality of plasma and urine was measured cryoscopically using an osmometer ( osmomat 030 ; gonotec ) . the concentrations of na and k in plasma and urine were analysed using hplc . briefly , na , k , mg and ca ions were separated in a mobile phase buffer ( 075 mm - sulfuric acid , 025 mm - copper sulfate ( ii ) ; ph 24 ) on a super ic - cation column ( 46 mm inner diameter 150 mm ; tosoh co. ) , and ions were detected using an indirect uv method ( 230 nm ) . plasma aldosterone levels were measured using an elisa kit ( enzo biochem inc . ) . the between - group comparisons of single - point measurements were made using one - way anova followed by tukey 's post hoc test . the time course studies of urine osmolality and electrolyte excretion were compared using two - way anova followed by bonferroni post hoc tests . significance was set at p < 005 . all statistical analyses were performed using prism software ( windows version 4.03 ; graphpad software ) . the mean body mass loss during the dehydration protocol was 12 % of the initial body weight . the volumes of urine excreted each hour over the course of the experiment ( 4 h ) are presented in fig . drinks ingested following exercise significantly altered cumulative volumes of urine excreted over the course of the 4-h recovery period ( p < 00001 ) . urine excretion was significantly less in the milk group compared with that in the water group ( p < 005 ) . the volume of urine excreted by rats in sports drink , mps and mes groups was more than that by rats in the water group and less than that by rats in the milk group . additionally , the volume of urine collected by the mes group drastically increased 3 h after rehydration , although there were no significant changes in urine volume between mes- and milk - administered groups for the initial 2 h of rehydration . 1.cumulative urine excretion over time after consumption of the five experimental drinks in expt 1 : water ( ---- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein solution ( mps ; ---- ) ; milk electrolyte solution ( mes ; ---- ) . the right y - axis indicates body fluid retention expressed as a percentage relative to the water group . values are means , with standard errors represented by vertical bars ( n 13 , 14 , 18 , 19 and 19 for water , milk , sports drink , mps and mes , respectively ) . mean values with unlike letters were significantly different ( p<005 ) . cumulative urine excretion over time after consumption of the five experimental drinks in expt 1 : water ( ---- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein solution ( mps ; ---- ) ; milk electrolyte solution ( mes ; ---- ) . the right y - axis indicates body fluid retention expressed as a percentage relative to the water group . values are means , with standard errors represented by vertical bars ( n 13 , 14 , 18 , 19 and 19 for water , milk , sports drink , mps and mes , respectively ) . mean values with unlike letters were significantly different ( p<005 ) . the cumulative volume of urine collected from milk , mps and mes groups was 55 , 31 , and 23 % less than that from the water group 4 h after rehydration , respectively . these percentages reflect the retention of body fluid during the 4-h recovery period ( fig . furthermore , the sum of body fluid retentions in the mps ( 31 % ) and mes ( 23 % ) groups equalled that in the milk group ( 55 % ) . thus , milk protein could contribute to 56 % ( i.e. 31/55 % ) of the body fluid retention of milk . in preliminary studies comparing whole milk to fat - free milk ( data not shown ) and mes ( 2866 kj / l ) to mes without dextrin ( 0 kj / l ; data not shown ) , the energetic content of the drinks showed no significant effect on the volume of urine excreted . there was a significant difference in the urinary electrolyte concentrations after consumption of rehydration solution ( table 2 ) . rats in milk and mes groups , whose drinks contain more electrolyte than the other experimental drinks , showed significantly larger urinary na and k excretion compared with that in the water and mps groups at 2 h after rehydration ( p < 005 ) . however , urinary na excretion tended to be lower in rats administered milk compared with those administered mes 1 h after rehydration , whereas urinary k excretion was not significantly different between these groups . table 2.urinary electrolyte excretion and osmolality following rehydration ( expt 1)*(mean values with their standard errors)time after rehydration ( h)1234anova ( p)mean sem mean sem mean sem mean sem drinktimedrink timena ( mmol / l)water20051504390615034000010000105968milk7512204672052929035sports drink450669161132820131mps815058211183419744mes17241203351732222834k ( mmol / l)water20437131502908831430000010000100001milk335686831077566244263sports drink29452379673916524836mps28630284305058541568mes43635591734113326030osmolality ( mosm / kg)water26445088737623028388059793040150952709671milk621914338750158191728846610961sports drink31144923543864429410993571593mps406182842001014539185672421237mes59565927258121737634232679379mps , milk protein solution ; mes , milk electrolyte solution . mean values with unlike superscript letters were significantly different ( p < 005).*n 13 , 14 , 18 , 19 and 19 for water , milk , sports drink , mps and mes , respectively . urinary electrolyte excretion and osmolality following rehydration ( expt 1 ) * ( mean values with their standard errors ) mps , milk protein solution ; mes , milk electrolyte solution . mean values with unlike superscript letters were significantly different ( p < 005 ) . n 13 , 14 , 18 , 19 and 19 for water , milk , sports drink , mps and mes , respectively . urine osmolality differed significantly depending on the drink administered ( p < 005 ; fig . urine osmolality was significantly higher than that in the water group at 2 and 3 h after administration ( p < 005 ) . in the mes group , urine osmolality was also significantly higher than that in the water group at 2 h ( p < 005 ) , but the difference disappeared at 3 h after rehydration . at 3 h of rehydration , urine osmolality in mps - administered rats was less than that of the milk group and more than that of the water group . 2.plasma aldosterone levels after consumption of the four experimental drinks in expt 3 : water ( ) ; milk ( ) ; sports drink ( ) ; milk protein solution ( ) . values are means , with standard errors represented by vertical bars ( n 15 ) . mean values with unlike letters were significantly different ( p < 005 ) . plasma aldosterone levels after consumption of the four experimental drinks in expt 3 : water ( ) ; milk ( ) ; sports drink ( ) ; milk protein solution ( ) . values are means , with standard errors represented by vertical bars ( n 15 ) . mean values with unlike letters were significantly different ( p < 005 ) . gastric emptying of milk was slightly slower than that for other experimental drinks , although this difference was ns . there were also no differences between gastric emptying times of mps , water and the sports drink ( table 3 ) . table 3.gastric emptying rate of experimental drinks ( expt 2)*(mean values with their standard errors)gastric emptying rate ( min)mean sem water545milk621sports drink507mps529mps , milk protein solution.*n 4 , 3 , 4 , and 4 for water , milk , sports drink and mps , respectively. no significant differences were observed . gastric emptying rate of experimental drinks ( expt 2 ) * ( mean values with their standard errors ) mps , milk protein solution . n 4 , 3 , 4 , and 4 for water , milk , sports drink and mps , respectively . no significant differences were observed . in comparing the two electrolyte - free drinks ( mps and water ) , plasma aldosterone levels were significantly higher in the mps group than in the water group . despite the na content of milk , plasma aldosterone levels in rats administered milk were higher than that those in rats administered water . conversely , plasma aldosterone levels were slightly lower after rehydration with the sports drink than with water because na lowers plasma aldosterone , although this difference was ns ( fig . 2 ) . plasma na concentration levels showed a significant decrease after administration of all the drinks . however , plasma na level increased again after administration of milk or mps , but not after the sports drink or water ( fig . plasma k levels increased immediately after administration of all the drinks , but decreased to basal levels 3 h after the sports drink or water . in contrast , plasma k concentrations remained high in both the milk and mps groups ( fig . 3.plasma sodium ( a ) and potassium ( b ) levels after consumption of the four experimental drinks in expt 3 : water ( ---- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein solution ( -- ) . values are means , with standard errors represented by vertical bars ( n 15 ) . mean values with unlike letters were significantly different ( p < 005 ) . plasma sodium ( a ) and potassium ( b ) levels after consumption of the four experimental drinks in expt 3 : water ( ---- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein solution ( -- ) . values are means , with standard errors represented by vertical bars ( n 15 ) . mean values with unlike letters were significantly different ( p < 005 ) . from 30 min to 2 h after administration , plasma osmolality in rats administered water and the sports drink decreased significantly compared with that in rats administered milk and mps ( p < 005 ) . at 4 h after administration , plasma osmolality in the mps group was maintained at the initial level , while a decrease in plasma osmolality was observed in the other three groups , but was much slower in the milk group than that in the sports drink or water groups ( fig . 4.plasma osmolality after consumption of the four experimental drinks in expt 3 : water ( -- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein solution ( -- ) . values are means , with standard errors represented by vertical bars ( n 5 ) . mean values with unlike letters were significantly different ( p < 005 ) . plasma osmolality after consumption of the four experimental drinks in expt 3 : water ( -- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein solution ( -- ) . values are means , with standard errors represented by vertical bars ( n 5 ) . mean values with unlike letters were significantly different ( p < 005 ) . rehydration after exercise is associated with smooth water absorption and total body fluid retention . in several previous studies , total body fluid retention has been shown to be superior in milk compared with that in isotonic electrolyte drinks . while milk is considered an effective post - exercise rehydration drink , the effect of milk protein itself on total body fluid retention has not been estimated . here , we showed that the cumulative volume of urine excreted following rehydration in rats with milk , mps and mes was 55 , 31 and 23 % less , respectively , than that with water 4 h after rehydration ( fig . 1 ) . the 32 % difference ( 5523 % ) between milk and mes may reflect the effects of milk protein on total body fluid retention , as both of these experimental drinks have equal electrolyte content and energy density . this difference of 32 % was nearly equal to the 31 % relative retention of urine in the mps group . given that mps contains milk proteins but not electrolytes , milk protein itself appears to contribute 56 % ( 31/55 % ) of the body fluid retention effect of milk . interestingly , urine volume from mes - administered rats rapidly increased 3 h after rehydration , while it was not different from that from the milk group for the first 2 h of rehydration ( fig . this discrepancy between mes and milk may be related to differences in the composition of these two experimental drinks , with the most typical candidate substance being milk protein . . also showed differences in urine volume after 3 h of rehydration with energy- and electolyte - matched carbohydrate and carbohydrate milk protein solutions . together , these findings suggest that milk protein is more effective in fluid retention than carbohydrate , although carbohydrate also exerts a mild influence on body fluid retention . although previous studies have indicated the effectiveness of milk protein on body fluid retention , the mechanisms behind this have not been determined . these reports indicate that protein clotting in the stomach delays the gastric emptying of milk . the half - emptying time of milk was estimated to be 14 % slower than that of the sports drink . we showed that the gastric emptying rate of mps is not different from that of water or the sports drink , while the gastric emptying rate of milk is 14 % slower as described by a previous report earlier ( table 3 ) . to estimate the gastric emptying rate , we used c - labelled acetic acid as a marker in a respiratory gas analysis system . absorption and oxidation of acetic acid are rapid , and can be utilised as a marker of gastric emptying rate . the gas analysis system used in this study was optimised for the small volume of respiratory gas emitted by rats , and was sufficiently accurate for the purpose of the study . the results show that milk protein itself does not lower the gastric emptying rate , and also indicates that this is slowed by interactions between protein and electrolyte fractions . in a previous human study , urine osmolality 2 h after milk intake was about three to nine times higher than that following intake of water and a sports drink . the same applied to the observation in the present study . urine osmolality after administration of milk was maintained at a higher level than that after administration of the sports drink or water ( table 2 ) . given that the elevation of urine osmolality was greater than that of na , k or cl , another plasma component derived from milk may contribute to this elevated osmolality . urinary na excretion in the mes group was higher than that in the milk group during the first 1 h of rehydration . on the other hand , differences in urinary k excretion between rats administered mes and milk were not observed during the first hour of rehydration , but were significantly different at 3 and 4 h of rehydration ( table 2 ) . we have demonstrated that raised plasma aldosterone contributes to the total body fluid retention 30 min and 1 h after administration of milk ( fig . 2 ) . however , as na ingestion suppresses plasma aldosterone levels , the difference between the milk and water groups was ns . the renin angiotensin aldosterone system is , from an evolutionary viewpoint , a well - preserved regulatory system with many morphological and physiological similarities in all vertebrate groups from fish to mammals . elevation of plasma aldosterone level causes the reduction of renal na excretion and the increasing of renal na retention . aldosterone secretion is known to be regulated by various factors such as the renin 2 ) in the mps group compared with the water group 30 min after administration . although the mechanism that induces aldosterone secretion is unclear , a 5-d high - protein diet ( 2 g / kg body weight / d ) caused increased plasma renin , aldosterone and vasopressin levels in human subjects . similar changes in vasoactive hormones may have occurred in the present study , as animals were orally administered approximately 1 g / kg body weight at a time after exercise . elevation of plasma aldosterone positively affects body fluid retention by conserving plasma na . we showed that plasma aldosterone and na levels in the mps group were higher than in rats administered water or the sports drink 13 h after administration of the drink ( fig . in contrast to the mps group , plasma na levels in the water group decreased linearly for 3 h after administration . at this time point plasma na levels were highest in the milk group , followed by the mps group , the sports drink group and the water group . , plasma osmolality in the mps group was maintained at a higher level than that in the water and sports drink groups for 2 h after rehydration ( fig . 4 ) . as mps contained no electrolytes , this elevated plasma osmolality and na retention can not be explained by the electrolyte content of the drink . therefore , higher plasma osmolality in the mps group can be partially explained by the higher na retention ( fig . 3(a ) ) and by other plasma components derived from milk , such as amino acids or their metabolites . most previous studies on body fluid retention have been conducted in human subjects by measuring the changes in body weight . although animal studies have several limitations in predicting the effect of milk protein solution on body fluid retention for human subjects , in this study , total body fluid retention was assessed using small laboratory animals . of interest is whether there is a specific component in milk that affects body fluid retention . given that it is difficult to obtain a large amount of effective fractions , experimental models using small laboratory animals are useful in evaluating the effects of small amounts of effective fractions on fluid retention . as precise urine collection is difficult in the small laboratory animal , we developed a protocol to measure fluid retention in rats , which requires precise measurement of urine volumes to avoid the confounding effect of body weight on volumes of faeces and urine . in conventional urine collection cages ( metabolism cage ) , the separation of urine and faeces achieves a urine recovery rate of 64 % . to improve this the authors consider that the effects of anaesthesia on the experimental results were negligible . inhalational anaesthetics influence renal function by reducing cardiac output and systemic blood pressure , and affecting the concentration of aldosterone , angiotensin ii , atrial natriuretic peptide and vasopressin levels . by means of anaesthesia by ether for 30 min , urine volume was reported to decrease by half in a human study . on the other hand , in the present study , as anaesthesia was performed for rapid oral administration , the aspiration of ether was within 2 min . therefore the influence of ether anaesthesia on the observation in the present study was negligible . in summary , milk protein was responsible for 56 % of the body fluid retention after milk consumption . milk protein caused elevated plasma aldosterone levels and suppressed the reduction of plasma osmolality without delaying gastric emptying time . these data show that milk proteins maintain plasma osmolality and suppress body fluid loss .

milk is an effective post - exercise rehydration drink that maintains the net positive fluid balance . however , it is unclear which components are responsible for this effect . we assessed the effect of milk protein solution ( mps ) obtained by dialysis on body fluid retention . milk , mps , milk electrolyte solution ( mes ) , sports drink and water were administered to male wistar rats at a dose of 6 ml / rat after treadmill exercise . total body fluid retention was assessed by urine volume 4 h after administration of hydrating liquids . the rate of gastric emptying was evaluated by a tracer method using 13c - labelled acetate . plasma osmolality , na and k levels , and urinary na and k were measured by hplc and osmometry , respectively . the gastric emptying rate was not delayed by mps . during 4 h of rehydration , cumulative urine volumes differed significantly between treatment groups ( p < 005 ) with 49 , 22 and 34 ml from water- , milk- and mps - fed rats , respectively . thus , mps elicited 50 % of the total body fluid retention of milk . plasma aldosterone levels were significantly higher in mps- and milk - fed rats compared with water - fed rats . plasma osmolality was maintained at higher levels in mps - fed rats than in water- and mes - fed rats ( p < 005 ) . cumulative urine na excretion was also suppressed in the milk- and mps - fed groups compared with the mes - fed group . our results demonstrate that mps obtained by dialysis clearly affects net body water balance without affecting gastric emptying after exercise . this effect was attributed to retention of na and water , and maintenance of plasma osmolality .

Experimental methods Results Discussion

PMC2635056

protein production production and purification of recombinant human tpp1 from a chinese hamster ovary ( cho ) expression system were conducted using modifications of methods described previously ( 9 ) . cells were cultured in 1050-cm roller bottles for 9 days with serum - free dulbecco 's modified eagle's / f-12 medium containing 1 g / ml kifunensin . conditioned media were harvested and adjusted to contain 2.5 m nacl as well as 20 mm tris , ph 8.0 , and 20 m cacl2 ( tc buffer ) . the solution was clarified by centrifugation and filtration and then applied to a butyl - sepharose 4 fast - flow column ( ge healthcare ) . after washing with 2.5 m nacl in tc buffer , the column was eluted with a 7.5-column volume linear gradient of 2.5 to 0 m nacl in tc buffer . fractions containing most of the pro - tpp1 were concentrated and equilibrated with 20 mm nacl / tc buffer by diafiltration . the solution was filtered , loaded onto a mono q12 column ( bio - rad ) , and washed with 4 column volumes of 20 mm nacl in tc buffer . the column was eluted with 10 column volumes of a linear gradient of 20 to 515 mm nacl in tc buffer . peak fractions containing pro - tpp1 were adjusted to contain 100 mm sodium citrate , ph 5.5 . protein was deglycosylated by incubation with endo hf ( new england biolabs , 26,500 units per mg of pro - tpp1 ) at 25 c for 15 h followed by 1 h at 37 c . the solution was buffer exchanged and repurified by mono q12 chromatography as described above . fractions containing pro - tpp1 were concentrated and applied to a superdex 75 column equilibrated in 150 mm nacl in tc buffer to separate pro - tpp1 monomer from a minor amount of aggregated material . a similar procedure was used to isolate glycosylated pro - tpp1 , omitting kifunensin from the cell culture media and the endo h digestion and second mono q12 chromatography steps . hexahistidine - tagged human pro - tpp1 was produced using an insect cell system and purified as described previously ( 14 ) except that all column buffers contained 20 m cacl2 , and a final superdex 75 gel filtration step was performed as described above . tpp1 activation kinetics proenzyme was diluted to the desired concentration using 0.1% triton x-100 , 150 mm nacl , 20 mm tris , ph 8.0 . for experiments using hexahistidine tpp1 , the protein was preincubated with 1 mm e-64 to inhibit an insect cell thiol protease contaminant that was present in trace amounts in some preparations . proenzyme ( 20 l ) was mixed with 180 l of 0.1% triton x-100 , 150 mm nacl , and 100 mm sodium formate adjusted with hcl to the indicated ph . the reaction was incubated at 25 c for the indicated time , and the activation process was terminated by diluting 1 part sample into 9 parts ice - cold 0.1% triton x-100 , 150 mm nacl , 500 mm sodium acetate , ph 5.0 , frozen on dry ice , and stored at -80 c . the amount of enzymatically active tpp1 ( total final tpp1 concentration typically 1.5 nm ) was measured using the previously described kinetic fluorescence assay ( 35 ) using 200 m ala - ala - phe - amc substrate in 0.1% triton x-100 , 150 mm nacl , 500 mm sodium acetate , ph 5.0 . data for activation conducted at ph 4.0 were fit to a one - phase exponential association model using built in functions in prism version 5.01 ( graphpad software ) . the activation reactions at ph 2.5 to 3.5 were fit to an irreversible consecutive reaction model where the inactive tpp1 zymogen ( z ) is converted to an intermediate ( i ) with partial activity , which is then converted into a fully active mature form ( m ) as shown in reaction 1 , using this model , one can calculate the activity at any given time from equation 1 , ( eq.1)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}\;\;v_{t}\hspace{1em}=\hspace{1em}v_{max}({\alpha}_{1}[{\mathrm{z}}]_{t}\hspace{1em}+\hspace{1em}{\alpha}_{2}[{\mathrm{i}}]_{t}\hspace{1em}+\hspace{1em}{\alpha}_{3}[{\mathrm{m}}]_{t})\;\;\end{equation*}\end{document } here vt denotes the observed tpp1 enzymatic activity at any given time ; vmax denotes the enzymatic activity of the preparation when existing as the fully activated form ; denotes the relative enzymatic activity of the different forms , and for any given time [ z]t + [ i]t + [ m]t is a constant that corresponds to the total added tpp1 , with all existing as z at time = 0 . 36 using prism version 5.01 , with the following constraints : no activity for the zymogen ( 1 = 0 ) , partial activity for the intermediate form ( 0 2 1 ) , and maximal activity for the mature form ( 3 = 1 ) . pro - tpp1 was activated as described above . for matrix - assisted laser desorption ionization time - of - flight mass spectrometry ( maldi - tof ms ) , reactions were terminated by mixing with an equal volume of ice - cold solution containing 0.5% trifluoroacetic acid , 50% acetonitrile , mixed with sinapinic acid matrix , and analyzed using an applied biosystems / mds - sciex 4800 mass spectrometer operated in the linear mode . for sds - page analysis , reactions were terminated using reducing sample buffer and fractionated on 10% nupage bistris gels using mops running buffer ( invitrogen ) . gels were either stained using sypro ruby ( molecular probes ) or transferred to membranes , stained with coomassie blue , and relevant regions of the membrane excised and sequenced as described previously ( 9 , 14 ) . crystallization of pro - tpp1purified endo h - deglycosylated pro - tpp1 was dialyzed into tc buffer containing 100 mm nacl , concentrated to 7 mg / ml using a 30,000 centrifugal device ( millipore ) , and filtered through 0.22-m pore size cellulose acetate filters ( corning costar ) . initial crystallization conditions were identified by high throughput screening at the hauptman woodward medical research institute , buffalo , ny . in the optimized condition , crystals of pro - tpp1 ranging in size between 50 100 100 m and 50 100 300 m appeared in 3 days when set up and grown at 4 c using hanging drop diffusion by mixing 1 l of pro - tpp1 solution at 5 mg / ml and 1 l of reservoir solution containing 0.1 m citrate , 4 - 7% polyethylene glycol 6000 , ph 5.0 . data for two native , one mercury - derivatized , and one lead - derivatized crystals were collected to 1.85 at x4c at the national synchrotron light source , brookhaven national laboratory , upton , ny . the lead and mercury derivatives were obtained by soaking the crystals overnight at 4 c in reservoir solutions containing heavy atom salts at concentrations of 2.5 mm pb(no3)2 and 0.5 mm c2h5hgcl . cryoprotection was achieved with passage of the crystals held in cryoloops through 1-l drops of 50% ethylene glycol for 1 s and flash - cooling in liquid n2 stream . for all crystals , all four data sets were processed and scaled in the primitive orthorhombic p212121 space group using denzo and scalepack ( 37 ) ( table 1 and supplemental table s1 ) . initial attempts for structure solution with molecular replacement using a homology model of tpp1 ( 33 ) or homologous structures ( 24 , 27 , 28 ) did not yield reliable solutions . furthermore , attempts for solving the structure by multiple isomorphous replacement using multiple heavy atom derivatives were unsuccessful because of lack of isomorphism between the datasets ( supplemental table s1 ) . subsequently , the crystal structure was solved by single wavelength anomalous dispersion ( sad ) technique using the mercury dataset . four mercury sites were obtained from the anomalous differences using shelx programs ( 38 ) with the hkl2map software ( 39 ) . the sites were used for sad phasing at 2.5 resolution through solve / resolve ( 40 , 41 ) as implemented in the software package phenix ( 42 ) . taking advantage of the anisomorphism among the data sets , the sad phases were improved and extended to 1.9 resolution through multiple crystal form averaging four data sets ( two native , mercury - soaked and lead - soaked ) using dmmulti ( 43 ) integrated into ccp4 version 6.0 package ( 44 ) . the initial model consisting of 511 of the 544 residues was obtained by automated chain tracing with the arp / warp routine ( 45 ) using the averaged phases . the remaining residues were modeled following iterative cycles of maximum likelihood and isotropic temperature refinement with refmac 5.1.2.4 ( 46 ) , and manual model building using coot ( 47 ) . the translation / libration / screw ( tls ) motion determination server ( 48 - 50 ) was used to determine the optimal number of tls groups ( five groups used ) and to generate parameters that were used in the final stages of refinement in refmac 5.1.2.4 ( 46 ) . initial models for solvent molecules were obtained from the hic - up server ( 51 ) . the final model , refined to 1.85 with rwork and rfree of 0.179 and 0.205 , respectively , includes all 544 residues , 1 bound ca ion , 4 n - acetyl - d - glucosamine ( glcnac ) groups , 4 ethylene glycol molecules , and 237 water oxygen atoms ( table 1 ) . the structure was validated using the molprobity server ( 52 ) , which reported 97.05% of the residues in the ramachandran favored regions . loop regions thr - leu and glu - thr were modeled using a combination of composite omit and difference fourier maps calculated with map algorithms within cns version 1.2 ( 53 ) , and side chain conformations for residues in the aforesaid loops as well as for residues lys , glu , gln , arg , and arg were modeled as the most common rotamer , because of disordered electron density corresponding to these regions . table 1data collection and refinement statisticsvalues corresponding to highest resolution shells are shown in parentheses . data collection and refinement statistics values corresponding to highest resolution shells are shown in parentheses . structural analyses superposition of structural models was accomplished using lsqkab ( 54 ) from the ccp4 program suite version 6.0 ( 44 ) . structural comparisons were made using the secondary structure matching server ( 55 ) , and overall root mean square deviation ( r.m.s.d . ) the protein - protein interaction server ( 56 , 57 ) , the imoltalk server ( 58 ) , and the whatif web interface ( 59 ) were used for analyses of interdomain and lattice contacts . buried surface areas were calculated using cns version 1.2 ( 53 ) , and all structural figures were generated using pymol version 1.0 ( 60 ) . electrostatic potential calculations analysis of ph dependence on interdomain binding energy in pro - tpp1 was accomplished using the adaptive poisson - boltzmann solver version 0.5.1 ( apbs ) ( 61 ) . the pdb2pqr server ( 62 , 63 ) implementing the parse forcefield and the propka program ( 64 ) to assign specific protonation states at different ph conditions ( ph 2.5 - 8.0 ) was used to convert the pro - tpp1 protein data bank ( pdb ) coordinates to three sets of pqr files , one for the intact zymogen ( residues 20 - 563 ) ( zph ) , one for the propiece ( residues 20 - 195 ) ( pph ) , and the third for the mature domain ( residues 196 - 563 ) ( mph ) for a given ph condition . these ph - specific pqr files were used as input for electrostatic binding energy calculations at that ph using a linearized poisson - boltzmann equation ( pb ) using cubic b - spline discretization of the charge distributions . pb calculations were performed at 298 k with a dielectric constant of 78.0 for water and 2.0 for the protein interior . the ion concentrations were set to 0.15 m with ionic radius of 2.0 . care was taken to remove self - interaction energies by using exactly the same grid spacing , length and center , determined by the psize.py script provided in the apbs distribution . the electrostatic binding energy at a given ph ( beph ) is calculated by apbs as shown in equation 2 , ( eq.2)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}\;\;be_{{\mathrm{ph}}}\hspace{1em}=\hspace{1em}z_{{\mathrm{ph}}}\hspace{1em}-\hspace{1em}(p_{{\mathrm{ph}}}\hspace{1em}+\hspace{1em}m_{{\mathrm{ph}}})\;\;\end{equation*}\end{document } production of deglycosylated tpp1initial attempts to obtain suitable crystals from previously characterized glycosylated recombinant human pro - tpp1 produced in insect cells ( 14 ) and in cho cells ( 9 ) were unsuccessful ( data not shown).5 given that heterogeneity in glycosylation may have been a complicating factor , we tried different methods to deglycosylate the protein produced from cho cells . in our hands , peptide : n - glycosidase f was not completely effective in removing oligosaccharides unless the protein was denatured , suggesting that steric hindrance prevented cleavage of the glcnac - asn bond . endo h , which cleaves between the first two asn - linked glc - nac residues , was partially effective in deglycosylating the native molecule , but some n - linked glycans were retained , reflecting the presence of oligosaccharides that had been processed to endo h - resistant forms ( 15 , 65 ) . we therefore cultured the cho cells in the presence of the endoplasmic reticulum mannosidase i inhibitor kifunensin , which prevents further processing of n - linked glycans to endo h - resistant forms ( 66 ) . when treated with endo h , pro - tpp1 produced in this manner yielded a single species by sds - page analysis , corresponding to a deglycosylated form in which four single asn - linked glc - nac monosaccharides remain , as confirmed by the crystal structure ( supplemental fig . properties of deglycosylated tpp1glycosylation has been reported to be critical for the folding , processing , and/or stability of tpp1 in cultured cells ( 17 , 67 ) . in this study , it was therefore important to compare the functional properties of deglycosylated tpp1 prepared for structural studies and the previously well characterized glycosylated native protein . purified pro - tpp1 is a zymogen that undergoes low ph - triggered autoactivation and processing ( 9 , 13 , 14 , 16 , 68 ) . the kinetics for autoactivation of deglycosylated pro - tpp1 were essentially identical to those for glycosylated protein produced from untreated cho cells or a hexahistidine - tagged protein produced from insect cells ( fig . rates were greatly accelerated at lower ph values and showed biphasic kinetics at ph 3.5 and below ( fig . 1 ) . we also measured the activity of preactivated glycosylated and deglycosylated tpp1 using two different fluorogenic substrates , ala - ala - phe - amc and arg - nle - nle - amc . the kcat values ( vmax / protein concentration ) for the deglycosylated and glycosylated proteins were similar ( fig . although it appeared that the deglycosylated protein had slightly higher activity , the difference fell within the variation that we typically observe with independent preparations of glycosylated tpp1 . 2 , a and b ) and substrate specificity constant ( kcat / km ) ( fig . our results do not rule out a role for glycosylation in folding or trafficking of pro - tpp1 , nor do they address the role of the terminal asn - linked glcnac that remains after endo h - deglycosylation . however , they do demonstrate that endo h - deglycosylated tpp1 is equivalent in terms of autocatalytic and enzymatic function to fully glycosylated tpp1 . highly purified human recombinant tpp1 preparations ( filled circles , hexahistidine - tagged pro - tpp1 from an insect cell expression system ; open circles , fully glycosylated pro - tpp1 from cho cells ; and open squares , endo h - deglycosylated pro - tpp1 from kifunensin - treated cho cells ) were incubated at the indicated ph for the specified time before stopping the reaction and measuring tpp1 activity as described under experimental procedures . data for each experiment were normalized to plateau values , all of which were comparable . the curves shown are fit to the reaction curves for the hexahistidine - tagged pro - tpp1 preparation as described under experimental procedures . highly purified human recombinant tpp1 preparations ( filled circles , hexahistidine - tagged pro - tpp1 from an insect cell expression system ; open circles , fully glycosylated pro - tpp1 from cho cells ; and open squares , endo h - deglycosylated pro - tpp1 from kifunensin - treated cho cells ) were incubated at the indicated ph for the specified time before stopping the reaction and measuring tpp1 activity as described under experimental procedures . data for each experiment were normalized to plateau values , all of which were comparable . the curves shown are fit to the reaction curves for the hexahistidine - tagged pro - tpp1 preparation as described under experimental procedures . figure 2.enzymatic properties of glycosylated and endo h - deglycosylated tpp1 . cho cell - produced enzyme preparations described in fig . enzyme activity measurements were conducted using eight different concentrations of either ala - ala - phe - amc ( 800 to 6.25 m ) or arg - nle - nle - amc ( 400 to 3.12 m ) substrate . final reactions contained 150 mm nacl , 0.1% triton x-100 , and either 100 mm sodium formate , ph 2.5 , or sodium acetate ( all other ph values ) adjusted to the indicated ph using hcl . kinetic constants were calculated using the built - in michaelis - menten curve fitting functions in prism version 5.01 . c , circles , ala - ala - phe - amc substrate ; squares , arg - nle - nle - amc substrate . enzyme activity measurements were conducted using eight different concentrations of either ala - ala - phe - amc ( 800 to 6.25 m ) or arg - nle - nle - amc ( 400 to 3.12 m ) substrate . final reactions contained 150 mm nacl , 0.1% triton x-100 , and either 100 mm sodium formate , ph 2.5 , or sodium acetate ( all other ph values ) adjusted to the indicated ph using hcl . kinetic constants were calculated using the built - in michaelis - menten curve fitting functions in prism version 5.01 . c , circles , ala - ala - phe - amc substrate ; squares , arg - nle - nle - amc substrate . crystal structure of pro - tpp1 reveals a conserved subtilisinlike fold we have determined the crystal structure of the inactive precursor of endo h - deglycosylated form of tpp1 at 1.85 resolution ( supplemental fig . s1 and fig . the data processed in the p212121 space group with one molecule in the asymmetric unit with a mathhews coefficient and solvent content of 2.37 / da and 48.0% , respectively . the structure is markedly similar to the glycosylated form of pro - tpp1 determined at 2.35 resolution in the p21212 space group with two molecules in the asymmetric unit ( pdb i d 3ee6 ) . superposition of c coordinates of deglycosylated pro - tpp1 with those of the two chains of glycosylated pro - tpp1 yield r.m.s.d . values of 0.61 and 0.66 , respectively , with differences in positioning of a few solvent exposed loops and conformations of a couple of active site residues ( as discussed below ) . pro - tpp1 exhibits a characteristic bi - domain precursor structure with a c - shaped prodomain ( residues ser - phe ) tightly packed against a more globular catalytic domain ( residues leu - pro ) connected by a 24-residue linker ( gly - gly ) . the propiece has an antiparallel-/antiparallel- fold and the catalytic domain has a typical subtilisin - like fold . the n - terminal portion ( residues 172 - 179 ) of the propiece linker is well ordered and threads through the substrate - binding groove making strong short distance contacts ( one hydrogen bond , one salt bridge , and three hydrophobic interactions ) with the catalytic domain . in contrast , the remaining portion of the linker ( residues 180 - 195 ) loops out of the interface making only minimal long range van der waal contacts . the conformation of this portion of the linker is stabilized by a few strong lattice contacts involving residues 180 - 190 as discussed below . interestingly , the well structured nature of the n - terminal portion of the linker is supported by identical conformations and its involvement in interdomain contacts in both glycosylated and deglycosylated structures . the flexible nature of the c - terminal portion of the linker is emphasized by its involvement in lattice contacts in the deglycosylated structure and by its absence in the glycosylated structure , possibly because of disorder . furthermore , a tls motion analysis ( 48 - 50 ) for evidence of thermal motion detects significant flexibility in the linker region as compared with the rest of the protein ( supplemental fig . such flexibility may be important in positioning the linker for autocatalytic cleavage during zymogen activation ( see below ) . stereo view of pro - tpp1 backbone depicted in coil representation with the propiece shown in cyan and blue and the catalytic domain in gold and red , for helices and strands , respectively , and the linker loop shown in magenta . side chains of active site catalytic triad ( ser , glu , and asp ) and oxyanion hole ( asp ) residues are shown in cpk mode . glycosylation sites with residual glcnac moieties ( circles , asn , 1 ; asn , 2 ; asn , 3 ; and asn , 4 ) and disulfide bridges ( squares , cys - cys , 1 ; cys - cys , 2 ; and cys - cys , 3 ) are depicted in stick mode . stereo view of pro - tpp1 backbone depicted in coil representation with the propiece shown in cyan and blue and the catalytic domain in gold and red , for helices and strands , respectively , and the linker loop shown in magenta . side chains of active site catalytic triad ( ser , glu , and asp ) and oxyanion hole ( asp ) residues are shown in cpk mode . glycosylation sites with residual glcnac moieties ( circles , asn , 1 ; asn , 2 ; asn , 3 ; and asn , 4 ) and disulfide bridges ( squares , cys - cys , 1 ; cys - cys , 2 ; and cys - cys , 3 ) are depicted in stick mode . the structure has marked similarities with those of other s53 family members ( 24 , 27 , 28 , 30 ) in terms of the overall fold , barring minor differences in the presence or absence of a few secondary structural elements in both the propiece and mature domain and orientation of the linker ( supplemental figs . superpositions of c coordinates of corresponding regions of pro - tpp1 with propiece of prokumamolisin ( pdb i d 1t1e ) ( 28 ) , and mature enzymes of sedolisin ( pdb i d 1ga4 ) ( 24 ) , kumamolisin ( pdb i d 1gtg ) ( 27 ) , and kumamolisin - as ( pdb i d 1sn7 ) ( 30 ) yield r.m.s.d . values of 2.17 , 2.07 , 1.81 , and 1.92 , respectively . compared with prokumamolisin , the propiece and mature domain in pro - tpp1 are more compact with 550 more buried surface area than the former , whereas the respective linkers are oriented at an angle of 30 ( supplemental fig . the total buried surface area between the propiece and mature domain in pro - tpp1 is 2200 a with 22 direct and 10 water - mediated hydrogen bonds , 3 salt bridges , and 6 hydrophobic interactions ( supplemental fig . s5 and supplemental table s2 ) . key features of the s53 family members , including c positions of catalytic residues forming the charge transfer complex ( viz . ser , glu , asp , and asp ) , two cis - peptide bonds , and the calcium - binding site are conserved . three cis - pro residues are found in pro - tpp1 ( pro , pro , and pro ) , of which the former two are completely conserved in the bacterial homologs ( supplemental fig . s3 ) . the structural role and binding partners of the octahedrally coordinated calcium ion , including the side chain carboxyl groups of asp and asp , backbone carbonyl groups of val , gly , and gly , and a bound water molecule , are conserved within the s53 family . in addition , three disulfide linkages unique to pro - tpp1 ( viz . cys - cys , cys - cys , and cys - cys ) stabilize the fold . the lone cys is completely buried within the core of the mature domain and is within hydrogen bonding distance of the side chain carboxyl of asp ( see below ) . endo h treatment leaves one glcnac moiety at each n - linked glycosylation site . asn , asn , asn , asn , and asn ) are utilized ( 67 ) . we find clear evidence for single glcnac moieties at all sites except for asn , a surface residue , which is clearly not modified in our structure . the structure of the glycosylated form of pro - tpp1 also shows that asn is not utilized ( pdb i d 3ee6 ) . it is formally possible that this site is heterogeneously glycosylated and that crystallization conditions have selected out the four - sugar glycosylated form from a population of four- and five - sugar forms . in our structure , the glcnac moiety linked to asn participates in lattice contacts with a symmetry - related molecule ( supplemental fig . s6 and supplemental table s3 ) . active site of pro - tpp1 and maintenance of the zymogen in the inactive state the active site in pro - tpp1 contains an sed ( ser - glu - asp ) catalytic triad , similar to all other members of the s53 family of serine peptidases and contrasting with the shd catalytic triad of canonical serine peptidases . the replacement of the his residue with the acidic residue glu in the s53 family allows the active site serine to function in nucleophilic attack of substrate at acidic ph ( 14 ) , with the glu abstracting the hydroxyl proton from the serine ( 14 , 23 - 26 , 34 ) . in addition , there is a second conserved asp residue ( asp ) , designated here as d , in place of a conserved asn in the subtilisin family . the role of the latter is to bind and stabilize the oxyanion of the tetrahedral intermediate of the peptidase reaction by hydrogen bond interactions , as described by nmr analyses and high resolution crystal structures of members of the subtilisin family bound to inhibitors acting as transition - state analogs that mimic the tetrahedral intermediate ( reviewed in ref . the catalytic efficiency in proteolytic enzymes is significantly increased by transition - state stabilization of the tetrahedral intermediate in the reaction . computational studies on kumamolisin - as suggest that the d residue has multiple roles in catalyzing peptide bond hydrolysis , serving as a general acid to protonate the substrate during formation of the tetrahedral intermediate and subsequently functioning as a general base , extracting a proton from the tetrahedral intermediate during conversion to the acyl enzyme ( 73 , 74 ) . in tpp1 , these roles are likely to be performed by asp . the d residue is conserved in the s53 family , suggesting a common proton transfer mechanism that distinguishes these enzymes from canonical serine proteases . figure 4.comparison of the active site of pro - tpp1 zymogen with that of mature kumamolisin . backbones are shown as ribbon representations with featured side chains in ball - and - stick mode , bound water molecules depicted as red spheres , and hydrogen bonds shown as dashed lines with distances in . a , stereo view of tpp1 showing catalytic residues in a conformation that is recalcitrant to activity . in tpp1 , glu ( e ) , asp ( d ) , and ser ( s ) form the catalytic triad , and asp ( d ) forms part of the stabilizing oxyanion hole . side chain oxygen atoms are shown in red and backbone carbonyl oxygens in yellow . a bound water molecule ( 1 ) occupies the position expected for one of the carboxyl oxygens of glu , if the active site were poised for activity . one of the carboxyl oxygens of asp forms a hydrogen bond to a water molecule ( 2 ) , and the other is within weak hydrogen bond distance to the sulfhydryl of unpaired cys . the side chain hydroxyls of ser and ser participate in a strong hydrogen bond . a third water molecule ( 3 ) makes a strong hydrogen bond with the ser hydroxyl and is also within hydrogen bond distance of the asp carboxyl constituting the oxyanion hole , close to where the carbonyl oxygen of the scissile peptide bond is expected to be positioned . b , comparison of catalytic machinery of pro - tpp1 with that in mature kumamolisin . least square superpositions of c coordinates of the pro - tpp1 catalytic domain with those of all available structures of s53 family members were performed and yielded similar results . superposition of tpp1 with mature kumamolisin ( pdb i d 1gtl ) ( 27 ) is shown with ribbon depictions in gray and green , respectively . although the corresponding oxyanion d residues in tpp1 and kumamolisin are in similar conformations , the corresponding catalytic glu and asp residues are both in different conformations . furthermore , the s residue in tpp1 ( ser ) is in a different rotameric conformation , and its c position is displaced by 1.5 from the corresponding s ( ser ) in kumamolisin , resulting in different hydrogen bond schemes with the active site ser . comparison of the active site of pro - tpp1 zymogen with that of mature kumamolisin . backbones are shown as ribbon representations with featured side chains in ball - and - stick mode , bound water molecules depicted as red spheres , and hydrogen bonds shown as dashed lines with distances in . a , stereo view of tpp1 showing catalytic residues in a conformation that is recalcitrant to activity . in tpp1 , glu ( e ) , asp ( d ) , and ser ( s ) form the catalytic triad , and asp ( d ) forms part of the stabilizing oxyanion hole . ser ( s ) is completely conserved in the s53 family . side chain oxygen atoms are shown in red and backbone carbonyl oxygens in yellow . a bound water molecule ( 1 ) occupies the position expected for one of the carboxyl oxygens of glu , if the active site were poised for activity . one of the carboxyl oxygens of asp forms a hydrogen bond to a water molecule ( 2 ) , and the other is within weak hydrogen bond distance to the sulfhydryl of unpaired cys . the side chain hydroxyls of ser and ser participate in a strong hydrogen bond . a third water molecule ( 3 ) makes a strong hydrogen bond with the ser hydroxyl and is also within hydrogen bond distance of the asp carboxyl constituting the oxyanion hole , close to where the carbonyl oxygen of the scissile peptide bond is expected to be positioned . b , comparison of catalytic machinery of pro - tpp1 with that in mature kumamolisin . least square superpositions of c coordinates of the pro - tpp1 catalytic domain with those of all available structures of s53 family members were performed and yielded similar results . superposition of tpp1 with mature kumamolisin ( pdb i d 1gtl ) ( 27 ) is shown with ribbon depictions in gray and green , respectively . although the corresponding oxyanion d residues in tpp1 and kumamolisin are in similar conformations , the corresponding catalytic glu and asp residues are both in different conformations . furthermore , the s residue in tpp1 ( ser ) is in a different rotameric conformation , and its c position is displaced by 1.5 from the corresponding s ( ser ) in kumamolisin , resulting in different hydrogen bond schemes with the active site ser . the crystal structure of pro - tpp1 reveals a number of features that help maintain the zymogen in an inactive state , with the active site assuming a geometry that is suboptimal for catalysis . low ph - mediated pro - tpp1 zymogen maturation is likely to be associated with different positioning of key elements at the active site as well as different protonation states of active site carboxylates that might favor conformations poised for catalysis . although the c positions of key catalytic residues , including sedd are superimposable with those of corresponding residues in all previously determined structures of bacterial homologs ( 24 , 27 , 28 , 30 ) , the side chain conformations and hydrogen bond network at the active site in pro - tpp1 are different ( fig . as revealed by several high resolution crystal structures of free and inhibitor - bound mature enzymes of sedolisin , kumamolisin , and kumamolisin - as , the geometry of an active site poised for catalysis is characterized by a charge transfer complex involving the key sed residues with hydrogen bonds between one of the side chain carboxyl oxygens of the glu with the hydroxyl of the ser and the other with one of the carboxyl oxygens of the asp . in contrast , in pro - tpp1 , side chains of both glu and asp are directed away from each other and from the active site ser . such alternate rotameric conformations of these catalytic residues allow hydrogen bonds distinct from those characteristic of a charge transfer complex ( fig . propka predictions ( 64 ) yield pka values of 3.6 and 6.4 for glu and asp in the mature enzyme , respectively ( supplemental table s4 ) . from this it follows that such rotameric conformations for the glu and the asp , as observed in pro - tpp1 , might arise from electrostatic charge repulsions and could likely be favored at neutral ph , at which both of these residues are predicted to be deprotonated and negatively charged . however , at the crystallization ph condition , ph 5.0 , only glu and not asp is predicted to be deprotonated . the active site glu is also found in dual conformations in some inhibitor complexes in structures of homologs crystallized at higher ph ( 25 ) . in addition , the conformations of glu and asp in the glycosylated form of pro - tpp1 are similar to those found in mature enzymes of bacterial homologs . these results suggest that the acidic residues at the active site most likely exist in equilibrium between alternate rotameric states and that different conditions favoring different ph - driven protonation states increase the probability of one state over another . the model was constructed by docking a hexapeptide substrate , aaffaa , with a fixed position of the carbonyl oxygen of the p1 residue ( phe ) relative to the catalytic domain of tpp1 , based on the prokumamolisin structure ( pdb i d 1t1e ) ( 28 ) , onto the catalytic domain of tpp1 in which the side chains of active site residues glu and asp were reoriented in the presumed active conformation as found in mature bacterial homologs . a ribbon diagram and a transparent surface rendition of the catalytic domain are shown in gray , a ball - and - stick depiction of the modeled hexapeptide substrate in yellow , and the superimposed linker with side chains of featured residues shown in ball - and - stick mode in cyan . this model is an approximation because the positions of the m3 helix with glu and asp as well as m4 with ser together with the m4-m5 loop with asp are likely to differ in the mature enzyme ( fig . the n terminus of the tripeptide substrate is likely to interact with the asp side chain carboxyl groups . the predicted protonation state of asp ( see text ) is consistent with a role in binding to an unsubstituted positively charged n terminus of the p3 residue of a putative substrate , thus providing a possible structural basis for the tripeptidyl peptidase activity . in addition , in the absence of crystal lattice contacts , positively charged arg of the linker is likely to participate in electrostatic interactions with negatively charged asp of the oxyanion hole , potentially restricting its role in general acid and base catalysis . the model was constructed by docking a hexapeptide substrate , aaffaa , with a fixed position of the carbonyl oxygen of the p1 residue ( phe ) relative to the catalytic domain of tpp1 , based on the prokumamolisin structure ( pdb i d 1t1e ) ( 28 ) , onto the catalytic domain of tpp1 in which the side chains of active site residues glu and asp were reoriented in the presumed active conformation as found in mature bacterial homologs . a ribbon diagram and a transparent surface rendition of the catalytic domain are shown in gray , a ball - and - stick depiction of the modeled hexapeptide substrate in yellow , and the superimposed linker with side chains of featured residues shown in ball - and - stick mode in cyan . this model is an approximation because the positions of the m3 helix with glu and asp as well as m4 with ser together with the m4-m5 loop with asp are likely to differ in the mature enzyme ( fig . the n terminus of the tripeptide substrate is likely to interact with the asp side chain carboxyl groups . the predicted protonation state of asp ( see text ) is consistent with a role in binding to an unsubstituted positively charged n terminus of the p3 residue of a putative substrate , thus providing a possible structural basis for the tripeptidyl peptidase activity . in addition , in the absence of crystal lattice contacts , positively charged arg of the linker is likely to participate in electrostatic interactions with negatively charged asp of the oxyanion hole , potentially restricting its role in general acid and base catalysis . furthermore , in both structures of the glycosylated and endo h - deglycosylated forms of pro - tpp1 , the side chain hydroxyl group of the active site ser is also involved in a strong hydrogen bond with the side chain hydroxyl of ser at 2.6 . interestingly , this ser ( designated here as s ) is completely conserved within the s53 family ( supplemental fig . it is noteworthy that in the crystal structures of mature enzymes of the bacterial homologs , the region of the protein containing the conserved s residue exhibits a different conformation and consequently a distinct hydrogen bond geometry with the active site ser ( fig . , the s residue ser is in a different rotameric conformation , and its c position is displaced by 1.5 away from that of the pro - tpp1 s ser . this geometry supports a hydrogen bond between the backbone carbonyl of the active site ser and the side chain hydroxyl of ser at 2.8 ( backbone - side chain bond ) and another between the side chain hydroxyl of the active site ser and the backbone carbonyl of ser at 3.5 ( side chain - backbone bond ) , in place of a strong side chain - side chain hydrogen bond in pro - tpp1 . with lack of mutational data on this residue , its contribution to the catalytic machinery is not certain but is worth consideration because of its complete conservation . in pro - tpp1 the differences in positioning of structural elements of this region compared with those in mature enzymes are likely propagated as a result of interdomain contacts involving residues c - terminal to ser , viz . within the m4-m5 loop and the m5 strand ( region iii in supplemental fig . the side chain conformation of the d residue asp constituting the oxyanion hole is indistinguishable from the corresponding residue in homologous structures except in prokumamolisin where the corresponding asp is in a different rotameric conformation . in addition , a bound water molecule ( water 3 ) occupies a similar position as the backbone carbonyl of the scissile peptide bond of a substrate as observed upon superposition of the pro - tpp1 catalytic domain with an inhibitor bound homologous structure ( 27 ) . this water makes strong hydrogen bonds with the side chain oxygens of active site ser and asp ( fig . zymogens are commonly kept in their inactive states by occlusion of their active sites by propeptides that function as competitive inhibitors , occupying the substrate - binding site in a nonhydrolyzable configuration ( reviewed in ref . 75 ) . to test whether the pro - tpp1 linker can function in this role , we docked a model hexapeptide substrate to the catalytic domain in which the side chain conformations of glu and asp were reoriented to assumed active conformations , based on the structures of mature enzymes of bacterial homologs . however , when the linker was superimposed on the docked model , the p3 and p3 residues overlap with linker residues pro and arg , respectively , suggesting steric occlusion . the docking study further suggests that the linker residue arg would hinder approach of the substrate to the s2 and s3 subsites ( fig . 5 ) . in addition , a putative salt bridge between the positively charged arg and negatively charged asp is also likely to render a nonfunctional oxyanion hole . this is consistent with the pka of 4.7 predicted for asp in the zymogen form ( supplemental table s4 ) . a corresponding salt bridge between arg and the oxyanion hole asp observed in prokumamolisin has been implicated in substrate - mediated inactivation ( 28 ) . however , in pro - tpp1 this salt bridge is absent ( > 5.0 ) , presumably because of crystal lattice interactions involving the linker region c - terminal to arg ( supplemental fig . s6 ) . structural basis for tripeptidyl peptidase activity the substrate - binding subsite and the active site pocket are likely to undergo subtle changes in the mature enzyme with different rotameric conformations of catalytic triad and neighboring residues ( such as ser , see above ) . nevertheless , a docked model of a hexapeptide substrate aaffaa revealed that the asp side chain is a likely candidate for binding to the positively charged unsubstituted n termini of p3 residues of substrates , thus facilitating release of tripeptides after catalysis ( fig . 5 ) . asp is completely conserved in all mammalian tpp1 proteins but not among the bacterial homologs ( supplemental fig . s3 ) . interestingly , in the latter , a neighboring trp side chain blocks this region . in pro - tpp1 , the asp carboxyl is 5.3 away from the n terminus of the modeled hexapeptide , although this is likely to change in the mature domain . in addition , pka predictions using the propka program ( 64 ) indicate that the pka of asp is 6.2 when it is buried as part of pro - tpp1 , whereas it is 3.9 when exposed in the mature enzyme . from this it follows that at ph < 3.9 it is likely to be protonated when its binding to the positively charged n terminus of a substrate is suboptimal . this correlates with the km profile of the enzyme , which reveals a substantial increase in km below ph 4.0 ( fig . such a role for asp was also suggested from the previously predicted homology model for tpp1 ( 33 ) , supported by site - directed mutagenesis ( 34 ) . figure 6.model for autoproteolytic endopeptidase cleavage during ph - dependent pro - tpp1 zymogen maturation . a , ms analysis of autoproteolytic cleavage products . endo h - deglycosylated pro - tpp1 was incubated at either 25 c for 0.5 min ( ph 2.5 and 3.0 ) , 6.3 min ( ph 3.5 ) , 13 min ( ph 4.0 ) , or at 37 c for 18 h ( ph 4.5 ) . indicated m / z are those of the predicted tpp1 fragments spanning residues 20 - 168 ( 16640 ) , 20 - 181 ( 18024 ) , and 20 - 189 ( 19029 ) . b , endopeptidase cleavage sites and positions of ionizable residues at the interdomain interface . the pro - tpp1 backbone is shown with the catalytic domain in orange , the prodomain in cyan , and the propiece linker in magenta . the endopeptidase cleavage sites ( 1 , ser - leu ; 2 , gln - val ; and 3 , asp - phe ) and active site residues ( ser , glu , asp , and asp ) are depicted in stick mode . the c positions of ionizable interface residues with pka values predicted to be within the relevant physiological ph range for tpp1 ( supplemental table s4 ) are shown as spheres : 1 , glu ; 2 , glu ; 3 , glu ; 4 , glu ; 5 , asp ; 6 , asp ; 7 ; asp ; 8 , glu ; and 9 , asp . details of these interactions are shown in supplemental fig . s4 and supplemental table s2 . c , titration curve of binding energies of the propiece and mature domain of pro - tpp1 . a schematic depiction of pro - tpp1 is shown with colors , and endopeptidase cleavage sites and interdomain contacts involving ionizable residues are indicated as in b. the active site cleft is depicted as a triangle within the catalytic domain . the geometry of one side of the cleft ( top half of triangle ) defined by the acidic residues is subject to ph - driven protonation states , whereas the structure of the other side ( lower half of triangle ) defined by ser and ser is subject to restructuring by propagation of long range prodomain interactions . at higher ph , the prodomain and mature domains are tightly bound , and the active site catalytic machinery is preformed , and the active site exists in equilibrium between two states , one suboptimal for activity ( hatched triangle with mouth closed ) and one poised for activity ( white triangle with open mouth ) . as the ph is lowered , changes in protonation states progressively disrupt interdomain interactions and affect the rotameric conformations of the active site residues favoring an active site geometry poised for catalysis . as the ph is lowered to ph 4.5 , electrostatic interactions holding the 3-strand leaf of the prodomain -sandwich weaken and peel away from the interface . this is likely to result in repositioning of the linker loop bringing cleavage site 1 close to the active site . at progressively lower ph , interactions involving the 4-strand leaf of the propiece this allows sufficient kinking in the linker conformation so that site 2 can be brought close to the active site and targeted for endopeptidase action . at ph < 3.0 , the interdomain binding interface is completely disrupted , allowing access to site 3 . model for autoproteolytic endopeptidase cleavage during ph - dependent pro - tpp1 zymogen maturation . a , ms analysis of autoproteolytic cleavage products . endo h - deglycosylated pro - tpp1 was incubated at either 25 c for 0.5 min ( ph 2.5 and 3.0 ) , 6.3 min ( ph 3.5 ) , 13 min ( ph 4.0 ) , or at 37 c for 18 h ( ph 4.5 ) . indicated m / z are those of the predicted tpp1 fragments spanning residues 20 - 168 ( 16640 ) , 20 - 181 ( 18024 ) , and 20 - 189 ( 19029 ) . b , endopeptidase cleavage sites and positions of ionizable residues at the interdomain interface . the pro - tpp1 backbone is shown with the catalytic domain in orange , the prodomain in cyan , and the propiece linker in magenta . the endopeptidase cleavage sites ( 1 , ser - leu ; 2 , gln - val ; and 3 , asp - phe ) and active site residues ( ser , glu , asp , and asp ) are depicted in stick mode . the c positions of ionizable interface residues with pka values predicted to be within the relevant physiological ph range for tpp1 ( supplemental table s4 ) are shown as spheres : 1 , glu ; 2 , glu ; 3 , glu ; 4 , glu ; 5 , asp ; 6 , asp ; 7 ; asp ; 8 , glu ; and 9 , asp . details of these interactions are shown in supplemental fig . s4 and supplemental table s2 . c , titration curve of binding energies of the propiece and mature domain of pro - tpp1 . a schematic depiction of pro - tpp1 is shown with colors , and endopeptidase cleavage sites and interdomain contacts involving ionizable residues are indicated as in b. the active site cleft is depicted as a triangle within the catalytic domain . the geometry of one side of the cleft ( top half of triangle ) defined by the acidic residues is subject to ph - driven protonation states , whereas the structure of the other side ( lower half of triangle ) defined by ser and ser is subject to restructuring by propagation of long range prodomain interactions . at higher ph , the prodomain and mature domains are tightly bound , and the active site catalytic machinery is preformed , and the active site exists in equilibrium between two states , one suboptimal for activity ( hatched triangle with mouth closed ) and one poised for activity ( white triangle with open mouth ) . as the ph is lowered , changes in protonation states progressively disrupt interdomain interactions and affect the rotameric conformations of the active site residues favoring an active site geometry poised for catalysis . as the ph is lowered to ph 4.5 , electrostatic interactions holding the 3-strand leaf of the prodomain -sandwich weaken and peel away from the interface . this is likely to result in repositioning of the linker loop bringing cleavage site 1 close to the active site . at progressively lower ph , interactions involving the 4-strand leaf of the propiece this allows sufficient kinking in the linker conformation so that site 2 can be brought close to the active site and targeted for endopeptidase action . at ph < 3.0 , the interdomain binding interface is completely disrupted , allowing access to site 3 . model for autoproteolytic endopeptidase cleavage of pro - tpp1maldi - tof - mass spectrometric ( ms ) analysis revealed differences in the size of the liberated propeptide at different ph values ( fig . this indicates that the zymogen can utilize three different endopeptidase cleavage sites ( fig . 6b ) as follows : 1 ) ser - leu site within ph range 4.0 - 4.5 ; 2 ) gln - val site within ph range 3.0 - 4.0 ; and 3 ) asp - phe site within ph range 2.5 - 4.0 . cleavages at sites 1 and 2 have been reported previously ( 13 ) . calculations using apbs electrostatic analysis software ( 61 ) with pro - tpp1 domains revealed that the binding energy between the propiece and mature domain is more positive ( i.e. interactions are less stable ) with lower ph ( fig . a similar profile is also observed for calculations done using prokumamolisin coordinates ( data not shown ) . key salt bridge interactions as well as hydrogen bonds contributed by ionizable residues may be disrupted at low ph where acidic residues are likely to become protonated . this conclusion is supported by a recent report that the extent of inhibition of tpp1 activity by the prodomain decreases with lower ph and is negligible at ph 3.5 ( 68 ) . in addition , complex formation between kumamolisin - as e78h / d164n mutant mature domain and wild - type propiece was observed to be less at lower ph ( 29 ) . these results are consistent with a ph - induced change in binding between the propiece and the mature domain . in pro - tpp1 , the binding interface has many electrostatic bonds and ionizable residues with pka values within the physiological ph range ( fig . predictions of pka values of ionizable residues at the interdomain interface reveal that these residues cluster into groups that map to specific regions of the binding interface ( supplemental fig . these regions are associated with distinct ranges of pka values , suggesting ph - specific disruption of domain contacts . we propose a model of activation that takes into the account the ph - induced change in binding together with the ph - specific cleavage site preference ( fig . the cleavage site his - phe is positioned close to the active site ser ( ser ) ( supplemental fig . s4 ) . this corresponds to cleavage site 1 ( ser - leu ) in pro - tpp1 , which is relatively close to the active site ser , with the carbonyl carbon of the scissile peptide being 12.0 away . in the absence of the lattice interactions that alter the position of the linker in our crystals grown at ph 5.0 however , under the conditions that we use in our in vitro activation reactions , autocatalytic cleavage at ph 4.5 and above is extremely slow , with the intact pro - tpp1 zymogen persisting for days ( data not shown ) . it is likely that tight binding between the prodomain and catalytic domain at this ph holds the zymogen in a conformation that favors the nonoptimal active site geometry , and that the small amount of cleavage observed might arise from local breathing motions that allow acquisition of a conformationally active catalytic site . however , given that the prodomain retains high affinity for the catalytic domain and remains associated even after cleavage ( 68 ) , the equilibrium would favor the inactive conformation . at lower ph , the prodomain - catalytic domain interface could become destabilized , allowing assumption of a more conformationally optimal active site in the catalytic domain , as well as reorientation of the prodomain and linker . after endoproteolytic cleavage , the liberated propeptide as well as portions of the linker region still attached to the catalytic domain could then be further digested by the free catalytic domain ( see below ) . consistent with this model , rates of pro - tpp1 activation are greatly accelerated in the presence of denaturants such as urea ( supplemental fig . in addition , the environment of late endosomes and lysosomes may be conducive to destabilization of the prodomain - catalytic domain complex , allowing rapid activation at the physiological ph of these organelles ( ph 6 to 4.5 ) ( 76 , 77 ) . for instance , glycosaminoglycans have been shown to accelerate autoactivation of pro - tpp1 at mildly acidic ph ( 16 ) , as do liposomes containing bis(monoacylglycero)phosphate ( also called lysobisphosphatidic acid),6 an anionic lipid enriched in late endosomes ( 78 ) . nonetheless , decreasing the ph to optimize the protonation states of residues in the catalytic center , combined with reorientation of the catalytic serine and glutamic acid that would be favored by destabilization of the prodomain - catalytic domain interaction , are likely to be key elements in acquisition of activity . analysis of hexahistidine - tagged glycosylated recombinant tpp1 using sds - page ( fig . 7c ) , and maldi - tof ms ( data not shown ) corroborates the ms analysis of the endo h - deglycosylated untagged tpp1 ( fig . 6a ) , and it also reveals that autocatalytic processing involves a series of ph - dependent endoproteolytic cleavages and tripeptidyl aminopeptidase cleavages . at ph 4.0 , the predominant initial cleavage products correspond to an intermediate starting at val ( fig . 7a , band marked f2 and maldi - tof ms ; data not shown ) . over time , the intermediate undergoes two tripeptidyl - peptidase cleavages , yielding an intermediate starting at thr and the mature protein starting at leu ( fig . , the predominant initial cleavage products correspond to an intermediate starting at phe ( fig . 7 , a , band marked , and c ) and residues 20 - 168 of the propeptide ( fig . 7a , band marked f3 and maldi - tof ms ; data not shown ) . with increasing time , the intermediate undergoes a second endoproteolytic cleavage to liberate the intermediate form starting at val with subsequent tpp1 cleavage as above ( fig . initial endoproteolytic cleavages of peptide bonds asp - phe and gln - val occur both at ph 3.0 and 2.5 ( fig . in addition to the tripeptidylpeptidase cleavages of the form with an n - terminal val observed at ph 4.0 , there is also processing of the form starting at phe , yielding intermediates with n - terminal gly and arg . this is entirely consistent with the known exopeptidase specificity of tpp1 , where peptides with prolines at either p1 or p1 are extremely poor substrates ( 20 ) , thus preventing further exopeptidyl digestion past arg . 6a ) ( 13 ) could not be processed by progressive tripeptidyl peptidase cleavages to yield mature tpp1 because of both frame and specificity considerations , and would be predicted to undergo a second endoproteolytic cleavage at gln - val to yield the form with an n - terminal leu . the liberated propeptide has an n terminus sysp that is unlikely to be efficiently digested by tpp1 exopeptidase activity . thus , in the absence of any additional proteases , its hydrolysis probably involves tpp1 endoproteolytic cleavage . activation at ph 4.0 can be accelerated by other proteases ( data not shown ) , and given the protease - rich environment of the lysosome , it is likely that downstream hydrolysis of the initial propeptide cleavage product is not rate - limiting in vivo . it has been recently reported that the propeptide can inhibit the activity of mature tpp1 at neutral and mildly acidic ph , with an ic50 in the micromolar range at ph 4.0 ( 68 ) . our results on comparing zymogen cleavage and acquisition of tpp1 activity are consistent with this . there was a marked lag between the two events at ph 4.0 ( fig . in contrast , the appearance and disappearance of the free propeptide could be fit with a two - step consecutive irreversible reaction model ( 36 ) , with the second phase having a t of 7 h , paralleling that of acquisition of tpp1 activity . thus , the lag is likely to reflect the time required for proteolysis of the inhibitory prodomain . the lag at lower ph values was much less pronounced , with the difference in the half - times between the first phase of the activation reaction and zymogen cleavage differing by at most a factor of 2 and both processes occurring rapidly ( fig . 7b , ph 2.5 , 3.0 , and 3.5 ; t cleavage 3 , 6 , and 20 s ; and t activation 5 , 12 , and 20 s , respectively ) . this is consistent with rapid dissociation of the cleaved propeptide at more acidic ph values . activation reactions were conducted using 1 - 1.5 m pro - tpp1 , and aliquots ( typically 0.5 - 1 g ) were analyzed by sds - page as described under experimental procedures . z , position of 66-kda glycosylated pro - tpp1 zymogen ; asterisk , 49-kda intermediate ; m , 46-kda glycosylated mature tpp1 ; f1 , f2 , and f3 , fragments cleaved at sites 1 , 2 , and 3 , respectively , with assignment of f1 being based on maldi - tof analysis of deglycosylated tpp1 ( fig . b , comparison of proteolytic processing and acquisition of tpp1 activity . two to four gels for each ph were used for the analysis and background - corrected intensity data normalized for each gel . the green upward pointing triangles indicate disappearance of zymogen at each time point , normalized to the intensity at t = 0 ( e.g. 1 - zt / zo ) . the red downward pointing triangles indicate levels of liberated propeptide ( f1 + f2 + f3 ) , normalized to intensity of the highest value obtained for the reaction time course . open circles indicate tpp1 activity measurements on activation reactions initiated using 1.5 m zymogen . for comparison , data from fig . although the time course was similar at both concentrations for ph 4 and 3.5 , there were noticeable differences at ph 2.5 and 3.0 . for the latter reactions , the initial phase for acquisition of activity was similar , but the second phase was noticeably slower for the high concentration reactions . the reason for this is not entirely obvious , but it may reflect experimental considerations such as some reassociation of the prodomain after the activation reactions were arrested by dilution into ph 5.0 buffer . similarly , the faster time course and coincidence of the acquisition of enzymatic activity and endoproteolysis at ph 4.0 that we observed previously ( 14 ) may reflect differences in experimental design , where the reactions were terminated by dilution into ph 4.0 buffer and then assayed immediately for tpp1 activity . under these conditions , the propeptide would be expected to be largely dissociated from the mature protein . protein ( 17 pmol / time point ) was resolved by sds - page and transferred to membranes . bands of molecular mass ranging between 46 and 49 kda were analyzed by chemical sequencing . the table lists the amount of each form in picomoles as estimated from the sequencing data ( see experimental procedures for details ) . activation reactions were conducted using 1 - 1.5 m pro - tpp1 , and aliquots ( typically 0.5 - 1 g ) were analyzed by sds - page as described under experimental procedures . z , position of 66-kda glycosylated pro - tpp1 zymogen ; asterisk , 49-kda intermediate ; m , 46-kda glycosylated mature tpp1 ; f1 , f2 , and f3 , fragments cleaved at sites 1 , 2 , and 3 , respectively , with assignment of f1 being based on maldi - tof analysis of deglycosylated tpp1 ( fig . b , comparison of proteolytic processing and acquisition of tpp1 activity . two to four gels for each ph were used for the analysis and background - corrected intensity data normalized for each gel . the green upward pointing triangles indicate disappearance of zymogen at each time point , normalized to the intensity at t = 0 ( e.g. 1 - zt / zo ) . the red downward pointing triangles indicate levels of liberated propeptide ( f1 + f2 + f3 ) , normalized to intensity of the highest value obtained for the reaction time course . open circles indicate tpp1 activity measurements on activation reactions initiated using 1.5 m zymogen . for comparison , data from fig . although the time course was similar at both concentrations for ph 4 and 3.5 , there were noticeable differences at ph 2.5 and 3.0 . for the latter reactions , the initial phase for acquisition of activity was similar , but the second phase was noticeably slower for the high concentration reactions . the reason for this is not entirely obvious , but it may reflect experimental considerations such as some reassociation of the prodomain after the activation reactions were arrested by dilution into ph 5.0 buffer . similarly , the faster time course and coincidence of the acquisition of enzymatic activity and endoproteolysis at ph 4.0 that we observed previously ( 14 ) may reflect differences in experimental design , where the reactions were terminated by dilution into ph 4.0 buffer and then assayed immediately for tpp1 activity . under these conditions , the propeptide would be expected to be largely dissociated from the mature protein . protein ( 17 pmol / time point ) was resolved by sds - page and transferred to membranes . bands of molecular mass ranging between 46 and 49 kda were analyzed by chemical sequencing . the table lists the amount of each form in picomoles as estimated from the sequencing data ( see experimental procedures for details ) . the structure of pro - tpp1 provides some insights into disease - causing mutations in lincl ( discussed in the accompanying paper by pal et al . s8 ) . there are currently 57 mutations listed in the ncl mutation data base . roughly half ( 29 mutations ) are missense mutations , some of which could respond to small molecule chaperone therapy directed at stabilizing the aberrant protein and thus rescuing function . for these , the structure of pro - tpp1 may help in identifying such small molecule therapeutics . however , most individuals with lincl would not benefit from this approach as two mutations that encode totally nonfunctional proteins represent the majority of disease alleles ( 79 ) . one is an invariant 3 splice acceptor mutation in intron five that introduces a frameshift upstream of the phe codon ; the other is a nonsense mutation at codon arg that does not appear to efficiently respond to potential therapeutics that promote translation readthrough ( 80 ) . based on these and other considerations , any widely applicable therapy will need to target sufficient amounts of tpp1 to the brain to digest both newly arriving substrates and pre - existing lysosomal storage material . for enzyme replacement therapy using peripherally administered protein , it will be necessary to modify pro - tpp1 so that it can cross the blood - brain barrier ( e.g. creation of a chimeric protein containing elements that allow transcytosis ) . in addition , for both enzyme and gene therapy approaches , it would be extremely useful to engineer functional tpp1 derivatives that have a longer half - life in the lysosome , thus increasing the effectiveness of properly targeted protein .

late infantile neuronal ceroid lipofuscinosis is a fatal childhood neurological disorder caused by a deficiency in the lysosomal protease tripeptidyl - peptidase 1 ( tpp1 ) . tpp1 represents the only known mammalian member of the s53 family of serine proteases , a group characterized by a subtilisin - like fold , a ser - glu - asp catalytic triad , and an acidic ph optimum . tpp1 is synthesized as an inactive proenzyme ( pro - tpp1 ) that is proteolytically processed into the active enzyme after exposure to low ph in vitro or targeting to the lysosome in vivo . in this study , we describe an endoglycosidase h - deglycosylated form of tpp1 containing four asn - linked n - acetylglucosamines that is indistinguishable from fully glycosylated tpp1 in terms of autocatalytic processing of the proform and enzymatic properties of the mature protease . the crystal structure of deglycosylated pro - tpp1 was determined at 1.85 resolution . a large 151-residue c - shaped prodomain makes extensive contacts as it wraps around the surface of the catalytic domain with the two domains connected by a 24-residue flexible linker that passes through the substrate - binding groove . the proenzyme structure reveals suboptimal catalytic triad geometry with its propiece linker partially blocking the substrate - binding site , which together serve to prevent premature activation of the protease . finally , we have identified numerous processing intermediates and propose a structural model that explains the pathway for tpp1 activation in vitro . these data provide new insights into tpp1 function and represent a valuable resource for constructing improved tpp1 variants for treatment of late infantile neuronal ceroid lipofuscinosis .

EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION Supplementary Material

PMC3308029

techniques for directly assessing and quantifying rna by high - throughput sequencing , collectively known as rna - seq , have revealed unexpected complexity and diversity in human transcriptomes . many previously unknown transcripts that are being detected are low - abundance long intergenic non - coding rnas ( lncrnas ) , which seem to be crucial for function and in disease . a key advantage of rna - seq over previous microarray - based methods for assessing transcription is the ability to query all transcripts on a genome - wide scale without prior knowledge about the locations and structures of genes . however , this advantage of rna - seq has also been its achilles heel : transcriptomes are dominated by few highly abundant transcripts , and the frequent sampling of such transcripts in proportion to their abundances and lengths reduces the power to detect transcripts that are rare or short . an apt analogy for a genome biologist attempting to measure transcription of a short , low - abundance gene from genome - wide rna - seq data is the difficulty encountered by an astronomer attempting to detect a low magnitude star from images collected in a low resolution sky survey . the tradeoff between breadth and depth is one that astronomers have grappled with for a long time and have ultimately resolved with the development of telescopes that can limit the scope of a detector to areas of interest , along with guiding technology enabling deep sampling of a region over long exposures . this approach was the design principle for the hubble deep field , which focused a powerful detector on the darkest portions of the sky . with the bright ' foreground ' of nearby objects removed , an immense number of galaxies were discovered in what was previously thought to be empty space . have designed an analogous focused experiment for probing the transcriptome , rna captureseq , and describe a similar outcome : regions with only scattered coverage in genome - wide experiments are revealed to be loci with transcription of low - abundance rnas . a key aspect of captureseq is that the integrity of the transcriptome in the ' deep field ' is preserved : the relative proportions of transcripts sampled with captureseq are shown to be equivalent to the relative proportions in conventional rna - seq . mercer et al . first performed conventional rna - seq focusing on a primary human foot fibroblast cell line . de novo assembled transcripts from conventional rna - seq were combined with ' dark ' intergenic regions that seemed not to be transcribed to design capture arrays . provide numerous controls to show that the approach maintains library diversity without introducing pcr amplification bias or other biases . the enrichment provided by captureseq is estimated to be 380-fold more than conventional rna - seq in the targeted regions . therefore , the resolution achievable with captureseq ( in the targeted regions ) is approximately equivalent to what could be obtained with 10 billion conventional rna - seq reads . as mercer et al . point out , such depth is necessary for finding very - low - abundance transcripts and for accurately quantifying abundances . we reinforce the latter observation in figure 1 , which gives an example showing that extreme depth is necessary for accurate isoform abundance estimation for the dystrophin gene , a complex multi - isoform gene that can harbor mutations causing muscular dystrophy . the need for deep sequencing in the example arises from the overlap between the multiple isoforms of the gene and the ambiguity that this causes in read mapping . it has been estimated that only 60% of transcripts can be accurately quantified with 10 billion conventional rna - seq reads . to demonstrate the difficulty of accurate isoform - level abundance estimation on low - abundance genes , we simulated an rna captureseq experiment on the 18 isoforms of the dystrophin gene as annotated in refseq ( hg19 ) , shown in ( a ) . for each number of 76 bp paired - end fragments that aligned to the gene , we estimated abundances of each isoform using the online em algorithm ( for details of the model used see and for details of the implementation see ) . ( b ) the accuracy of isoform abundance estimation measured as the pearson correlation coefficient ( r ) of the logged relative abundance estimates compared with the true abundance used to generate the simulated data . because of the similarity of the isoforms , only 2.5% of fragments aligned uniquely to a single isoform on average , making the deconvolution particularly difficult . the bottom x - axis shows how many alignable paired - end fragments would be required to achieve the same r in a genome - wide experiment as in the captureseq simulation . here we assume 3.17 fragments per kilobase per million mapped reads ( fpkm ) for the gene , which is what we estimated from a sample encode dataset [ accession srr065495 ] . another striking result of mercer et al . is the number of previously unknown transcripts discovered and the corollary that current sequencing experiments are very far from saturation . the message is clearly ' seq and find ' and this is exactly what is happening in rna - seq . the experiments surveyed in are an order of magnitude smaller than the norm today , and it is reasonable to extrapolate that as the costs of sequencing drop precipitously , the average depth of sequencing in rna - seq experiments will increase by another order of magnitude in the next 3 years . given the observations above , it is natural to speculate that conventional rna - seq with 10 billion reads will be commonplace in the near future and to ask whether technologies such as captureseq are truly necessary . it certainly seems plausible that exome sequencing , which is to genome sequencing as captureseq is to conventional rna - seq , will eventually be replaced by routine whole genome sequencing . however , in addition to technological challenges that must be overcome to allow routine sequencing of 10 billion reads , there are also bioinformatics problems that must be solved if such data are to be useful . in particular , increased numbers of reads in rna - seq lead to the ' curse of deep sequencing ' , in which extra sequence actually reduces performance and accuracy in current processing pipelines . firstly , most existing algorithms for rna - seq quantification require loading a substantial fraction of the total number of sequenced reads into memory . algorithms whose running times are not linear in the number of reads are also likely to fail with large amounts of sequence data . secondly , reads have errors and are prone to various biases , which , while appearing at a fixed frequency , occur at greater numbers with increased reads . for example , if a sufficiently large amount of sequence is obtained , a recurring error in a highly abundant gene may appear to be a ( false ) novel isoform . it therefore becomes imperative with large amounts of sequence to correct for sequencing artifacts , and this can be computationally prohibitive . captureseq breaks the curse of deep sequencing by providing increased transcript resolution at fixed sequencing depth . this means that existing methods can be readily applied to the analysis of captureseq data , and indeed the authors show that the cufflinks suite of tools can be used for both assembly and quantification of captureseq data . mercer et al . emphasize the discovery of novel lncrnas in the deep field . they found 163 novel neighboring and antisense lncrnas around protein coding genes . in general , they found that captured lncrnas have very low expression of only 0.011 fpkm ( fragments per kilobase per million mapped reads ) . these findings follow on the heels of a recent comprehensive annotation of human lncrnas and together suggest that very rare transcripts may bestow individual transcriptional ' fingerprints ' on cells . estimate that the newly discovered lncrnas are present at an average copy number of 0.0006 transcripts per cell . this precise quantification together with evidence from captureseq that the sequenced fragments are samples from complete transcripts ( and not just ' noise ' ) points towards the presence of very rare transcripts , possibly even unique to individual cells . captureseq therefore motivates the development of other approaches to the enrichment of low - abundance transcripts . one complementary possibility is the further development of depletion approaches , which could selectively filter the highest - abundance transcripts before sequencing ; an example is removal of ribosomal rna already performed in many experiments . although the depletion approach may inadvertently remove lower - abundance rnas because of cross - hybridization , it offers a genome - wide approach to enrichment . furthermore , captureseq may have a similar bias in the opposite direction : repetitive sequence in the transcriptome might lead to captured rna from outside a targeted genomic region . regardless of how low - abundance transcripts are detected , mercer et al . have demonstrated the extent of discovery possible in the deep field . the functional relevance of ultra low - abundance transcripts is currently debated , and the question of whether rare transcripts regulate biologically important processes or are artifacts of stochastic transcription is a key open problem . however , there is increasing recognition that antisense lncrnas are present at many protein coding genes , including numerous proto - oncogenes , and that they regulate their associated genes via epigenetic modifications . the ability to see farther into the rna deep field with captureseq is therefore likely to lead to many exciting developments in genomic medicine thanks to better understanding of the aberrant transcription underlying human disease . fpkm : fragments per kilobase per million mapped reads ; lncrna : long intergenic non - coding rna ; rna - seq : rna sequencing .

initial high - throughput rna sequencing ( rna - seq ) experiments have revealed a complex and dynamic transcriptome , but because it samples transcripts in proportion to their abundances , assessing the extent and nature of low - level transcription using this technique has been difficult . a new assay , rna captureseq , addresses this limitation of rna - seq by enriching for low - level transcripts with cdna tiling arrays prior to high - throughput sequencing . this approach reveals a plethora of transcripts that have been previously dismissed as ' noise ' , and hints at single - cell transcription fingerprints that may be crucial in defining cellular function in normal and disease states .

The deep field Seq and find Breaking the curse of deep sequencing The single cell transcriptome RNA CaptureSeq and beyond Abbreviations Competing interests Acknowledgements

PMC3913666

during investigation of physical and genetic interactome of heat shock protein 90 ( hsp90 ) in saccharomyces cerevisiae several putative interacting proteins were identified , among them 2 protein pih1 and tah1 was named after their characteristic . protein interacting with hsp90 was termed as pih1 ( also known as nop17 ) while tpr containing protein associated with hsp90 was termed as tah1 . pih1 is well known to interact with rvb1 , rvb2 , and tah1 and form r2tp complex . later , human r2tp complex was purified from human cells containing similar component like ruvbl1/ruvbl2 , pih1d1 , and rpap3 . thus it seems that both yeast and human r2tp complexes are conserved while other mammalian r2tp complex also contains some components of the perfoldin complex . tah1 of r2tp complex is known to interact with hsp90 and form r2tp - hsp90 complex . the components of r2tp multiprotein complex of yeast and human have been studied by different groups and their importance has been explored in many cellular processes ( fig . 1 ) . ruvb1 and ruvb2 are the 2 proteins , which have been previously identified as chromatin remodeling factors later these proteins were found to be involved in the multiple cellular process like transcription , telomerase core complex assembly , cell cycle progression , apoptosis , snornps biogenesis , pikk mediated signaling , rna polymerase ii assembly , as well as in the mitosis . recently human ruvb1 and ruvb2 are in the focus because of their involvement in many human cancers like hepatocellular carcinomas , renal and gastric carcinomas . the biochemical characterization reports on ruvb like proteins established that these are active atpase and many reports have also shown their helicase activity . due to the presence of atpase activity and involvement in multiple cellular processes these proteins have been categorized as aaa+ proteins ( atpases associated with multiple cellular processes ) . the schematic of r2tp complex involvement in various activities was prepared on the basis of reported role in yeast and human . pih1 interacts with snornps factor nop1/fibrillarin , nop58 , tel2 , monad and play crucial role in snornps biogenesis and pikk signaling . tah1 is characterized as the cofactor of hsp90 and enhances the activity of hsp90 protein . human counterpart of tah1 is known as rpap3 which is almost 6 times bigger in terms of number of amino acid as well as in number of tpr repeat . complex has been found to play an important role in many processes like rna polymerase - ii assembly , snornps assembly , pikk signaling , and in the apoptosis . r2tp - hsp90 complex together with perfoldin like complex interacts with rna polymerase - ii and was found to be crucial for the assembly of the rna polymerase - ii . in yeast pih1 deletion led disruption of r2tp complex resulted into decreased snornas of box c / d . human ruvb1 and ruvb2 interact with almost all core box c / d snornps factor and pih1d1 directly interacts with nop1 , nop58 , and nop56 . ruvbs have been crucial for the box c / d and box h / aca snorna . the depletion of human ruvbl1 and ruvbl2 showed a decreased level of mature box c / d snorna , thus providing the evidence of r2tp complex involvement in snornp assembly and biogenesis . recently it has been discovered that r2tp complexes are also involved in the pikk ( phosphatidylinositol-3 kinase related protein kinase ) signaling pathway . ruvbl1 and ruvbl2 interact with all the 6 pikks ( atm , atr , dna - pkcs , mtor , smg-1 , and trrap ) in mammals . ruvbl1/ruvbl2 regulates the downstream signaling via phosphorylation led activation of atm , atr , mtor , smg-1 : chk2 , chk1 , p70s6k , and upf1 respectively . smg-1 is critical component of the mrna surveillance complex known to be involved in the non - sense mediated mrna decay . furthermore , r2tp - hsp90 complex is essential player in the stability and assembly of pikks . human r2tp complex component like ruvbl2 , rpap3 , and pih1d1 have been identified as the interacting partners of monad ( a subunit of perfoldin like complex ) that is involved in the apoptosis . in this article , we have extensively investigated the leishmania donovani and plasmodium falciparum genome for the putative components of the r2tp complex using bioinformatics approaches and further characteristic domain identification and modeling of the proteins was also done . the phylogenetic analysis was also performed for every component of the putative r2tp complex of l. donovani and p. falciparum . the genome wide screening of the l. donovani and p. falciparum led to the identification of all the components of the complex such as ruvb like1 , ruvb like 2 , pih1 , and tah1 . during investigation of physical and genetic interactome of heat shock protein 90 ( hsp90 ) in saccharomyces cerevisiae several putative interacting proteins were identified , among them 2 protein pih1 and tah1 was named after their characteristic . protein interacting with hsp90 was termed as pih1 ( also known as nop17 ) while tpr containing protein associated with hsp90 was termed as tah1 . pih1 is well known to interact with rvb1 , rvb2 , and tah1 and form r2tp complex . later , human r2tp complex was purified from human cells containing similar component like ruvbl1/ruvbl2 , pih1d1 , and rpap3 . thus it seems that both yeast and human r2tp complexes are conserved while other mammalian r2tp complex also contains some components of the perfoldin complex . tah1 of r2tp complex is known to interact with hsp90 and form r2tp - hsp90 complex . the components of r2tp multiprotein complex of yeast and human have been studied by different groups and their importance has been explored in many cellular processes ( fig . 1 ) . ruvb1 and ruvb2 are the 2 proteins , which have been previously identified as chromatin remodeling factors later these proteins were found to be involved in the multiple cellular process like transcription , telomerase core complex assembly , cell cycle progression , apoptosis , snornps biogenesis , pikk mediated signaling , rna polymerase ii assembly , as well as in the mitosis . recently human ruvb1 and ruvb2 are in the focus because of their involvement in many human cancers like hepatocellular carcinomas , renal and gastric carcinomas . the biochemical characterization reports on ruvb like proteins established that these are active atpase and many reports have also shown their helicase activity . due to the presence of atpase activity and involvement in multiple cellular processes these proteins have been categorized as aaa+ proteins ( atpases associated with multiple cellular processes ) . the schematic of r2tp complex involvement in various activities was prepared on the basis of reported role in yeast and human . pih1 interacts with snornps factor nop1/fibrillarin , nop58 , tel2 , monad and play crucial role in snornps biogenesis and pikk signaling . tah1 is characterized as the cofactor of hsp90 and enhances the activity of hsp90 protein . human counterpart of tah1 is known as rpap3 which is almost 6 times bigger in terms of number of amino acid as well as in number of tpr repeat . complex has been found to play an important role in many processes like rna polymerase - ii assembly , snornps assembly , pikk signaling , and in the apoptosis . r2tp - hsp90 complex together with perfoldin like complex interacts with rna polymerase - ii and was found to be crucial for the assembly of the rna polymerase - ii . in yeast pih1 deletion led disruption of r2tp complex resulted into decreased snornas of box c / d . human ruvb1 and ruvb2 interact with almost all core box c / d snornps factor and pih1d1 directly interacts with nop1 , nop58 , and nop56 . ruvbs have been crucial for the box c / d and box h / aca snorna . the depletion of human ruvbl1 and ruvbl2 showed a decreased level of mature box c / d snorna , thus providing the evidence of r2tp complex involvement in snornp assembly and biogenesis . recently it has been discovered that r2tp complexes are also involved in the pikk ( phosphatidylinositol-3 kinase related protein kinase ) signaling pathway . ruvbl1 and ruvbl2 interact with all the 6 pikks ( atm , atr , dna - pkcs , mtor , smg-1 , and trrap ) in mammals . ruvbl1/ruvbl2 regulates the downstream signaling via phosphorylation led activation of atm , atr , mtor , smg-1 : chk2 , chk1 , p70s6k , and upf1 respectively . smg-1 is critical component of the mrna surveillance complex known to be involved in the non - sense mediated mrna decay . furthermore , r2tp - hsp90 complex is essential player in the stability and assembly of pikks . human r2tp complex component like ruvbl2 , rpap3 , and pih1d1 have been identified as the interacting partners of monad ( a subunit of perfoldin like complex ) that is involved in the apoptosis . in this article , we have extensively investigated the leishmania donovani and plasmodium falciparum genome for the putative components of the r2tp complex using bioinformatics approaches and further characteristic domain identification and modeling of the proteins was also done . the phylogenetic analysis was also performed for every component of the putative r2tp complex of l. donovani and p. falciparum . the genome wide screening of the l. donovani and p. falciparum led to the identification of all the components of the complex such as ruvb like1 , ruvb like 2 , pih1 , and tah1 . in order to explore the components of r2tp complex in leishmania donovani , we used amino acid sequence of each component of the yeast and human r2tp complex as a basic tool to study . blast - p analysis of yeast ruvb like protein sequences in the leishmania donovani genome database showed many hit and their further screening on the basis of high score revealed that 2 ruvb like proteins are present in the leishmania donovani genome and their genedb numbers are ldbpk_343280.1 and ldbpk_342440.1 . amino acid sequence of both ruvb like proteins was retrieved and protein blast analysis with yeast as well as human genome shows that both have considerable homology with human pontin ( hs - ruvbl1 ) and reptin ( hs - ruvbl2 ) as well as with yeast rvb1 and rvb2 . further analysis of these sequences with interproscan showed that these proteins contain aaa+ domain / ruvb like domain ( fig . figure 2 . in - silico prediction of conserved motifs of ruvb from l. donovani using interproscan . ( a ) predicted motifs model of l. donovani ruvb1 was prepared , which shows that ldruvb1 contains characteristic aaa+ domain and belongs to pontin ( ruvb like1 helicase ) . ( b ) conserved motifs model of l. donovani ruvb2 was prepared , which shows that ld ruvb2 contains characteristic aaa+ domain and belongs to reptin ( ruvb like2 helicase ) . ruvb1 from l. donovani is 458 amino acids long protein and its theoretical pi is 5.76 and molecular weight is ~50 kda . while ruvb2 from l. donovani is 483 amino acids long protein and its calculated pi is 5.29 and molecular weight is ~54 kda . we further analyzed the ldruvbs sequences with the ruvb like proteins of plasmodium falciparum , plasmodium vivax , yeast , and human using multiple sequence alignment and result shows that both the ruvb like proteins contain walker motif a and walker motif b ( fig . 3 and 4 ) . both these motifs are well known for the atp binding and for the atpase activity . through mutational study of both these motifs it has been discovered that these are essential for the characteristic atpase activity of ruvb like protein in different organisms including yeast and human . multiple sequence alignment of ldruvb1 with yeast and human ruvb1 using clustalw at default parameters . the amino acid sequence of various conserved motifs like walker a , walker b , sensor i , arginine finger and sensor - ii motifs are boxed in green color . figure 4 . conserved motifs of l. donovani ruvb2 and comparison with yeast and human ruvb2 characteristic motifs . multiple sequence alignment of ldruvb2 with yeast and human ruvb2 using clustalw at default parameters . the amino acid sequence of various conserved motifs like walker a , walker b , sensor i , arginine finger , and sensor - ii motifs are boxed in green color . ldruvb1 was aligned with human pontin and yeast rvb1 and all the characteristic motifs of ruvb like protein are boxed in the green color ( fig . thus , from this study it is clear that ldruvb1 protein contains walker motif a , walker motif b , sensor - i , arginine finger motif , and sensor - ii motif . similarly ldruvb2 was aligned with reptin of human and yeast rvb2 , and the result shows that ldruvb2 also contains all the characteristic motifs shown in figure 4 . interestingly , both the proteins also contain characteristic dna binding domain ( fig . 2 ) . the bioinformatics based analysis was performed to obtain the structural insight of all these ruvb like proteins of r2tp complex . for structural modeling the sequences of both the l. donovani ruvbs were used for the prediction of 3-dimensional structure of these proteins using swissmodel homology - modeling server ( http://swissmodel.expasy.org/ ) . ldruvb1 primary sequence residues 12 to 451 showed ~64% identities to human ruvb - like 1 helicase . structural modeling of the l. donovani ruvb1 ( http://swissmodel.expasy.org ) provided a basic tool to compare with human ruvb1 structure . the ribbon diagram of the template is shown in figure 5a ( i ) and the predicted structure of ldruvb1 is shown in figure 5a ( ii ) . the ribbon diagram of the template bound with adp ( known crystal structure of this homolog , pdb number 2c9oa at http://www.rcsb.org ) and the predicted structures of ldruvb1 were superimposed , it is clear that these structures superimpose considerably and seems that both the proteins have similar adp binding site [ fig . ldruvb2 primary sequence residues 21454 showed ~51% identity to a different human ruvb - like helicase from h. sapiens . therefore the structural modeling of the ldruvb2 was done using the known crystal structure of this homolog as the template ( pdb number : 2xszd at http://www.rcsb.org ) . 5b ( iii ) ] and molecular graphic images were produced using the ucsf chimera package ( http://www.cgl.ucsf.edu/chimera ) from the resource for biocomputing , visualization , and informatics at the university of california , san francisco ( supported by nih p41 rr-01081 ) . these results show that the structure of ldruvb1 is almost similar to the human ruvb1 while ldruvb2 has entirely different structure and shares similarity with reptin ( human ruvb2 ) . thus , it is highly expected that ldruvb1 and ldruvb2 enzymatic activities are similar to the yeast ruvb1 and ruvb2 like proteins , which are essential for the viability and are involved in multiple cellular processes . the modeling of ldruvb1 and ldruvb2 was done using amino acid sequences at swissmodel server . the molecular graphic images were produced using the ucsf chimera package from the resource for biocomputing , visualization , and informatics ( http://www.cgl.ucsf.edu/chimera ) at the university of california , san francisco ( supported by nih p41 rr-01081 ) . ( a ) ( i ) template for ldruvb1 ; ( ii ) ldruvb1 ; ( iii ) superimposed image of template and ldruvb1 . ( b ) ( i ) template for ldruvb2 ; ( ii ) ldruvb2 ; ( iii ) superimposed image of template and ldruvb2 . the phylogenetic analysis of ldruvb like proteins with e. coli , yeast and human ruvbs was carried by using phylogeny.fr . with default parameters . further phylogenetic analysis was performed for ruvb like proteins of l. donovani with human , yeast , trypanosoma , xenopus , p. falciparum , plasmodium vivax , and plasmodium knowlesi . phylogenetic analysis further shows that ldbpk_343280.1 is closer to the pontin / rvb1 , while ldbpk_342440.1 is closer to the reptin / rvb2 ( fig . thus , these results further confirm the identification of ruvb like1 and ruvb like 2 proteins in the l. donovani and we named these proteins ldruvb1 and ldruvb2 , accordingly . in r2tp complex , ruvb1/ ruvb 2 has been found to tightly interact with another component of the same complex that is pih1 originally discovered in yeast and later in humans . for the identification of pih1 in l. donovani , we applied the same strategy as for the identification of ldruvb1 and ldruvb2 . pih1 sequence of yeast was used for blast - p search in the l. donovani genome and the results showed that it contains one protein quite homologous to the query sequence with high score . the genedb number of ldpih1 is ldbpk_354400.1 , which is 635 amino acids long and its calculated pi is 5.55 and molecular weight is ~70 kda . to further confirm this identification , we retrieved the ldpih1 sequence and blast - p was performed in the human genome , which shows that ldpih1 is similar to the human pih1 and shares 31% identity . we further analyzed the sequence of ldpih1 and pfpih1with interproscan and the results show that pih domain is present in both the sequences ( fig . 6a and b ) . the phylogenetic analysis of pih1 amino acid sequences of human , yeast and l. donovani , in human and yeast , pih1 acts as an adopter protein , which interacts with ruvb1/ruvb2 through n - terminal , while through c - terminal it interacts tightly with tah1 , another protein of the same complex . tah1 contains characteristic tpr motifs , which are known to mediate the interaction with hsp90 to form r2tp - hsp90 complex . in l. donovani , blast - p analysis of yeast / human tah1 amino acid sequence led to the identification of tah1 like protein , which is comparable to the human rpap3 ( tah1 homolog ) , thus we named this protein as ldrpap3 and its genedb number is ldbpk_081020.1 . yeast tah1 protein has only 111 amino acid and its homolog in human ( rpap3 ) has 665 amino acid while ldrpap3 contains 546 amino acid . further amino acid sequence of ldtah1 like protein was studied in silico with interproscan and the results show the characteristic feature like tricopeptide repeats motif ( tpr motifs ) , which seems quite similar to the human rpap3 ( fig . these results clearly show that l. donovani tah1 and pftah1 homologs are close to the human rpap3 , especially in terms of the number of amino acid and the arrangement of tpr repeat ( fig . this analysis was further supported by the phylogenetic analysis , which shows that l. donovani tah1 is closer to the human rpap3 as compared with the yeast tah1 ( fig . ( a ) predicted motifs model of ldpih1 was prepared with interproscan which shows that ldpih1 contains characteristic pih domain . ( b ) predicted motifs model of pfpih1 was prepared with interproscan , which shows characteristic pih domain . ( c ) phylogenetic analysis of ldpih1 and pfpih1 proteins with other pih1 proteins was carried by using phylogeny.fr . ( a ) predicted motifs model of ldtah1 was prepared with interproscan which shows that ldtah1 contain characteristic tpr domain . ( b ) predicted motifs model of pftah1 was prepared with interproscan which shows characteristic tpr domain . ( c ) phylogenetic analysis of ldtah1 and pftah1 proteins with other tah1 including yeast and human tah1 proteins was carried by using phylogeny.fr . two main components of the r2tp complex , such as ruvb1/ruvb2 , have been already identified and characterized recently . it is well known that 2 ruvb like proteins ( ruvbl1/ruvbl2 or rvb1/rvb2 or pontin / reptin ) are present in yeast to human while the identification of 3 ruvb like protein in p. falciparum opened the new avenue to explore the role of these proteins in malaria parasite . it is interesting that 3 ruvb like protein have also been identified in many parasites like p. vivax , p. knowlesi , trypanosoma cruzi , and schistosoma mansoni . hence , it seems that mode of regulation of ruvbs in these human parasites shares similar mechanism to control the ruvb like proteins led cellular processes . the phylogenetic analysis has revealed that pfruvb1 and pfruvb2 are closer to human / yeast ruvb like1 ( pontin / rvb1 ) , while pfruvb3 seems closer to the human / yeast ruvb like2 protein ( reptin / rvb2 ) . recently the detailed characterization of pfruvb1 confirmed its biochemical role as it contains both atpase as well as dna helicase activity . interestingly , pfruvb1 also showed some dna - independent atpase activity , thus it was speculated that in vivo it can serve as dna independent atpase . however , the presence of ss - circular / linear dna showed enhancement in the atpase activity of pfruvb1 . furthermore , the enhancement of atpase coupled helicase activity in pfruvb1 indicates its involvement in the nuclear functions like chromatin remodeling and dna damage repair etc . pfruvb3 , which is phylogenetically closer to human / yeast ruvb2 , shows that it contains dna dependent atpase activity , but lacked dna helicase activity from recombinant protein , but endogenous protein showed some helicase activity and walker motif a is essential for the atpase activity . for the identification of pih1 homolog in malaria parasite , initially we retrieved the yeast and human pih1 sequences and blast - p analysis was performed in the p. falciparum genome and results show that it contains one protein with high score and its plasmodb number is pf3d7_1235000 . this gene was further analyzed by the blast - p in the human genome , which shows that pfpih1 is similar to the human pih1d1 and shares ~27% identity and contains characteristic pih1 domain . we further analyzed the amino acid sequence of pfpih1 with human pih1d and yeast pih1 , which showed that pfpih1 aligned considerably with human and yeast pih1 and it contains characteristic domain of pih1 ( fig . 6b ) . the phylogenetic analysis of pih1 amino acid sequences of human , yeast , p. falciparum and l. donovani etc . showed that pfpih1 is also closer to the human pih1 similar to the ldpih1 ( fig . 6c ) . similarly , we also performed the bioinformatics based analysis to identify tah1 protein in p. falciparum genome . using the approach described above , yeast tah1 sequence was used as query and the results show that plasmodb contains at least 2 proteins ( pf3d71434300 and pf3d7_0213500 ) with tpr repeats . further , these protein sequences were extensively analyzed in silico with the pfam , scanprosite , smart , panther , etc . , to identify the characteristic motifs and the results showed that pf-3d71434300 possess similar pattern of the tpr motifs as in the yeast and human tah1 . all these results showed that pftah1 protein seems similar to the human rpap3 ( tah1 ) , thus we named this protein as pfrpap3 . yeast tah1 protein has only 111 amino acids , rpap3 has 665 amino acids , while pfrpap3 has 564 amino acids . the sequence alignment showed that pfrpap3 is close to the human rpap3 especially in terms of the number of amino acid and number of tpr repeat ( fig . 7b ) similar to the l. donovani rpap3 homolog . for structural modeling the sequences of ldtah1 and pftah1 a number of models were obtained for each sequence , but the models that were built using yeast co - chaperone sti1/hop as template were studied in detail . ldtah1 primary sequence residues 226 to 467 showed ~38% identity to the template yeast co - chaperone sti1/hop . the modeling of the template is shown in figure 8a and of ldtah1 using this template is shown in figure 8b . when the modeled structure of ldtah1 and the template were superimposed , it is clear that these structures superimpose partially ( fig . pftah1 primary sequence residues 242 to 491 showed ~37% identity to the template yeast co - chaperone sti1/hop . when the modeled structure of pftah1 and the template were superimposed , it is clear that these structures superimpose partially ( fig . molecular graphic images were produced using the ucsf chimera package ( http://www.cgl.ucsf.edu/chimera ) from the resource for biocomputing , visualization , and informatics at the university of california , san francisco ( supported by nih p41 rr-01081 ) . the modeling of ldtah1 and pftah1 was done using amino acid sequences at swissmodel server . ( a ) template , ( b ) ldtah1 , ( c ) superimposed image of template and ldtah1 , ( d ) pftah1 , ( e ) superimposed image of template and pftah1 . the molecular graphic images were produced using the ucsf chimera package from the resource for biocomputing , visualization , and informatics ( http://www.cgl.ucsf.edu/chimera ) at the university of california , san francisco ( supported by nih p41 rr-01081 ) . considering all the results of these investigations it seems that both parasites have similar kind of r2tp complex , which is composed of ruvb1/ ruvb2 , pih1 , and rpap3 proteins as has been discovered in the yeast and in human . in order to explore the components of r2tp complex in leishmania donovani , we used amino acid sequence of each component of the yeast and human r2tp complex as a basic tool to study . blast - p analysis of yeast ruvb like protein sequences in the leishmania donovani genome database showed many hit and their further screening on the basis of high score revealed that 2 ruvb like proteins are present in the leishmania donovani genome and their genedb numbers are ldbpk_343280.1 and ldbpk_342440.1 . amino acid sequence of both ruvb like proteins was retrieved and protein blast analysis with yeast as well as human genome shows that both have considerable homology with human pontin ( hs - ruvbl1 ) and reptin ( hs - ruvbl2 ) as well as with yeast rvb1 and rvb2 . further analysis of these sequences with interproscan showed that these proteins contain aaa+ domain / ruvb like domain ( fig . figure 2 . in - silico prediction of conserved motifs of ruvb from l. donovani using interproscan . ( a ) predicted motifs model of l. donovani ruvb1 was prepared , which shows that ldruvb1 contains characteristic aaa+ domain and belongs to pontin ( ruvb like1 helicase ) . ( b ) conserved motifs model of l. donovani ruvb2 was prepared , which shows that ld ruvb2 contains characteristic aaa+ domain and belongs to reptin ( ruvb like2 helicase ) . ruvb1 from l. donovani is 458 amino acids long protein and its theoretical pi is 5.76 and molecular weight is ~50 kda . while ruvb2 from l. donovani is 483 amino acids long protein and its calculated pi is 5.29 and molecular weight is ~54 kda . we further analyzed the ldruvbs sequences with the ruvb like proteins of plasmodium falciparum , plasmodium vivax , yeast , and human using multiple sequence alignment and result shows that both the ruvb like proteins contain walker motif a and walker motif b ( fig . 3 and 4 ) . both these motifs are well known for the atp binding and for the atpase activity . through mutational study of both these motifs it has been discovered that these are essential for the characteristic atpase activity of ruvb like protein in different organisms including yeast and human . multiple sequence alignment of ldruvb1 with yeast and human ruvb1 using clustalw at default parameters . the amino acid sequence of various conserved motifs like walker a , walker b , sensor i , arginine finger and sensor - ii motifs are boxed in green color . figure 4 . conserved motifs of l. donovani ruvb2 and comparison with yeast and human ruvb2 characteristic motifs . multiple sequence alignment of ldruvb2 with yeast and human ruvb2 using clustalw at default parameters . the amino acid sequence of various conserved motifs like walker a , walker b , sensor i , arginine finger , and sensor - ii motifs are boxed in green color . ldruvb1 was aligned with human pontin and yeast rvb1 and all the characteristic motifs of ruvb like protein are boxed in the green color ( fig . thus , from this study it is clear that ldruvb1 protein contains walker motif a , walker motif b , sensor - i , arginine finger motif , and sensor - ii motif . similarly ldruvb2 was aligned with reptin of human and yeast rvb2 , and the result shows that ldruvb2 also contains all the characteristic motifs shown in figure 4 . interestingly , both the proteins also contain characteristic dna binding domain ( fig . 2 ) . the bioinformatics based analysis was performed to obtain the structural insight of all these ruvb like proteins of r2tp complex . for structural modeling the sequences of both the l. donovani ruvbs were used for the prediction of 3-dimensional structure of these proteins using swissmodel homology - modeling server ( http://swissmodel.expasy.org/ ) . ldruvb1 primary sequence residues 12 to 451 showed ~64% identities to human ruvb - like 1 helicase . structural modeling of the l. donovani ruvb1 ( http://swissmodel.expasy.org ) provided a basic tool to compare with human ruvb1 structure . the ribbon diagram of the template is shown in figure 5a ( i ) and the predicted structure of ldruvb1 is shown in figure 5a ( ii ) . the ribbon diagram of the template bound with adp ( known crystal structure of this homolog , pdb number 2c9oa at http://www.rcsb.org ) and the predicted structures of ldruvb1 were superimposed , it is clear that these structures superimpose considerably and seems that both the proteins have similar adp binding site [ fig . ldruvb2 primary sequence residues 21454 showed ~51% identity to a different human ruvb - like helicase from h. sapiens . therefore the structural modeling of the ldruvb2 was done using the known crystal structure of this homolog as the template ( pdb number : 2xszd at http://www.rcsb.org ) . 5b ( iii ) ] and molecular graphic images were produced using the ucsf chimera package ( http://www.cgl.ucsf.edu/chimera ) from the resource for biocomputing , visualization , and informatics at the university of california , san francisco ( supported by nih p41 rr-01081 ) . these results show that the structure of ldruvb1 is almost similar to the human ruvb1 while ldruvb2 has entirely different structure and shares similarity with reptin ( human ruvb2 ) . thus , it is highly expected that ldruvb1 and ldruvb2 enzymatic activities are similar to the yeast ruvb1 and ruvb2 like proteins , which are essential for the viability and are involved in multiple cellular processes . the modeling of ldruvb1 and ldruvb2 was done using amino acid sequences at swissmodel server . the molecular graphic images were produced using the ucsf chimera package from the resource for biocomputing , visualization , and informatics ( http://www.cgl.ucsf.edu/chimera ) at the university of california , san francisco ( supported by nih p41 rr-01081 ) . ( a ) ( i ) template for ldruvb1 ; ( ii ) ldruvb1 ; ( iii ) superimposed image of template and ldruvb1 . ( b ) ( i ) template for ldruvb2 ; ( ii ) ldruvb2 ; ( iii ) superimposed image of template and ldruvb2 . the phylogenetic analysis of ldruvb like proteins with e. coli , yeast and human ruvbs was carried by using phylogeny.fr . with default parameters . further phylogenetic analysis was performed for ruvb like proteins of l. donovani with human , yeast , trypanosoma , xenopus , p. falciparum , plasmodium vivax , and plasmodium knowlesi . phylogenetic analysis further shows that ldbpk_343280.1 is closer to the pontin / rvb1 , while ldbpk_342440.1 is closer to the reptin / rvb2 ( fig . thus , these results further confirm the identification of ruvb like1 and ruvb like 2 proteins in the l. donovani and we named these proteins ldruvb1 and ldruvb2 , accordingly . in r2tp complex , ruvb1/ ruvb 2 has been found to tightly interact with another component of the same complex that is pih1 originally discovered in yeast and later in humans . for the identification of pih1 in l. donovani , we applied the same strategy as for the identification of ldruvb1 and ldruvb2 . pih1 sequence of yeast was used for blast - p search in the l. donovani genome and the results showed that it contains one protein quite homologous to the query sequence with high score . the genedb number of ldpih1 is ldbpk_354400.1 , which is 635 amino acids long and its calculated pi is 5.55 and molecular weight is ~70 kda . to further confirm this identification , we retrieved the ldpih1 sequence and blast - p was performed in the human genome , which shows that ldpih1 is similar to the human pih1 and shares 31% identity . we further analyzed the sequence of ldpih1 and pfpih1with interproscan and the results show that pih domain is present in both the sequences ( fig . 6a and b ) . the phylogenetic analysis of pih1 amino acid sequences of human , yeast and l. donovani , in human and yeast , pih1 acts as an adopter protein , which interacts with ruvb1/ruvb2 through n - terminal , while through c - terminal it interacts tightly with tah1 , another protein of the same complex . tah1 contains characteristic tpr motifs , which are known to mediate the interaction with hsp90 to form r2tp - hsp90 complex . in l. donovani , blast - p analysis of yeast / human tah1 amino acid sequence led to the identification of tah1 like protein , which is comparable to the human rpap3 ( tah1 homolog ) , thus we named this protein as ldrpap3 and its genedb number is ldbpk_081020.1 . yeast tah1 protein has only 111 amino acid and its homolog in human ( rpap3 ) has 665 amino acid while ldrpap3 contains 546 amino acid . further amino acid sequence of ldtah1 like protein was studied in silico with interproscan and the results show the characteristic feature like tricopeptide repeats motif ( tpr motifs ) , which seems quite similar to the human rpap3 ( fig . these results clearly show that l. donovani tah1 and pftah1 homologs are close to the human rpap3 , especially in terms of the number of amino acid and the arrangement of tpr repeat ( fig . this analysis was further supported by the phylogenetic analysis , which shows that l. donovani tah1 is closer to the human rpap3 as compared with the yeast tah1 ( fig . ( a ) predicted motifs model of ldpih1 was prepared with interproscan which shows that ldpih1 contains characteristic pih domain . ( b ) predicted motifs model of pfpih1 was prepared with interproscan , which shows characteristic pih domain . ( c ) phylogenetic analysis of ldpih1 and pfpih1 proteins with other pih1 proteins was carried by using phylogeny.fr . ( a ) predicted motifs model of ldtah1 was prepared with interproscan which shows that ldtah1 contain characteristic tpr domain . ( b ) predicted motifs model of pftah1 was prepared with interproscan which shows characteristic tpr domain . ( c ) phylogenetic analysis of ldtah1 and pftah1 proteins with other tah1 including yeast and human tah1 proteins was carried by using phylogeny.fr . two main components of the r2tp complex , such as ruvb1/ruvb2 , have been already identified and characterized recently . it is well known that 2 ruvb like proteins ( ruvbl1/ruvbl2 or rvb1/rvb2 or pontin / reptin ) are present in yeast to human while the identification of 3 ruvb like protein in p. falciparum opened the new avenue to explore the role of these proteins in malaria parasite . it is interesting that 3 ruvb like protein have also been identified in many parasites like p. vivax , p. knowlesi , trypanosoma cruzi , and schistosoma mansoni . hence , it seems that mode of regulation of ruvbs in these human parasites shares similar mechanism to control the ruvb like proteins led cellular processes . the phylogenetic analysis has revealed that pfruvb1 and pfruvb2 are closer to human / yeast ruvb like1 ( pontin / rvb1 ) , while pfruvb3 seems closer to the human / yeast ruvb like2 protein ( reptin / rvb2 ) . recently the detailed characterization of pfruvb1 confirmed its biochemical role as it contains both atpase as well as dna helicase activity . interestingly , pfruvb1 also showed some dna - independent atpase activity , thus it was speculated that in vivo it can serve as dna independent atpase . however , the presence of ss - circular / linear dna showed enhancement in the atpase activity of pfruvb1 . furthermore , the enhancement of atpase coupled helicase activity in pfruvb1 indicates its involvement in the nuclear functions like chromatin remodeling and dna damage repair etc . pfruvb3 , which is phylogenetically closer to human / yeast ruvb2 , shows that it contains dna dependent atpase activity , but lacked dna helicase activity from recombinant protein , but endogenous protein showed some helicase activity and walker motif a is essential for the atpase activity . for the identification of pih1 homolog in malaria parasite , initially we retrieved the yeast and human pih1 sequences and blast - p analysis was performed in the p. falciparum genome and results show that it contains one protein with high score and its plasmodb number is pf3d7_1235000 . this gene was further analyzed by the blast - p in the human genome , which shows that pfpih1 is similar to the human pih1d1 and shares ~27% identity and contains characteristic pih1 domain . we further analyzed the amino acid sequence of pfpih1 with human pih1d and yeast pih1 , which showed that pfpih1 aligned considerably with human and yeast pih1 and it contains characteristic domain of pih1 ( fig . 6b ) . the phylogenetic analysis of pih1 amino acid sequences of human , yeast , p. falciparum and l. donovani etc . showed that pfpih1 is also closer to the human pih1 similar to the ldpih1 ( fig . similarly , we also performed the bioinformatics based analysis to identify tah1 protein in p. falciparum genome . using the approach described above , yeast tah1 sequence was used as query and the results show that plasmodb contains at least 2 proteins ( pf3d71434300 and pf3d7_0213500 ) with tpr repeats . further , these protein sequences were extensively analyzed in silico with the pfam , scanprosite , smart , panther , etc . , to identify the characteristic motifs and the results showed that pf-3d71434300 possess similar pattern of the tpr motifs as in the yeast and human tah1 . all these results showed that pftah1 protein seems similar to the human rpap3 ( tah1 ) , thus we named this protein as pfrpap3 . yeast tah1 protein has only 111 amino acids , rpap3 has 665 amino acids , while pfrpap3 has 564 amino acids . the sequence alignment showed that pfrpap3 is close to the human rpap3 especially in terms of the number of amino acid and number of tpr repeat ( fig . 7b ) similar to the l. donovani rpap3 homolog . for structural modeling the sequences of ldtah1 and pftah1 a number of models were obtained for each sequence , but the models that were built using yeast co - chaperone sti1/hop as template were studied in detail . ldtah1 primary sequence residues 226 to 467 showed ~38% identity to the template yeast co - chaperone sti1/hop . the modeling of the template is shown in figure 8a and of ldtah1 using this template is shown in figure 8b . when the modeled structure of ldtah1 and the template were superimposed , it is clear that these structures superimpose partially ( fig . pftah1 primary sequence residues 242 to 491 showed ~37% identity to the template yeast co - chaperone sti1/hop . when the modeled structure of pftah1 and the template were superimposed , it is clear that these structures superimpose partially ( fig . molecular graphic images were produced using the ucsf chimera package ( http://www.cgl.ucsf.edu/chimera ) from the resource for biocomputing , visualization , and informatics at the university of california , san francisco ( supported by nih p41 rr-01081 ) . the modeling of ldtah1 and pftah1 was done using amino acid sequences at swissmodel server . ( a ) template , ( b ) ldtah1 , ( c ) superimposed image of template and ldtah1 , ( d ) pftah1 , ( e ) superimposed image of template and pftah1 . the molecular graphic images were produced using the ucsf chimera package from the resource for biocomputing , visualization , and informatics ( http://www.cgl.ucsf.edu/chimera ) at the university of california , san francisco ( supported by nih p41 rr-01081 ) . considering all the results of these investigations it seems that both parasites have similar kind of r2tp complex , which is composed of ruvb1/ ruvb2 , pih1 , and rpap3 proteins as has been discovered in the yeast and in human . r2tp complex is a recently discovered important multiprotein complex involved in diverse cellular process like snornp biogenesis , pikk signaling , rna polymeraseii assembly and in the apoptosis . pih1 act as adopter protein which tightly interacts with rvb1-rvb2 and with the tah1 to form r2tp macromolecular complex . tah1 of the r2tp complex further interacts with the hsp90 to form r2tp - hsp90 complex that has been found playing role in many cellular process . during this study , the genome wide in - silico screening of the l. donovani and p. falciparum led to the identification of ruvb like1 , ruvb like2 , pih1 , and tah1 genes . the in - silico characterization of conserved motifs helped further to speculate that this complex is also important for these parasites as in the yeast . detailed analysis of crucial components of r2tp complex , ldruvb like 1 and ld - ruvb like 2 , revealed the characteristic motifs like dna binding motif and atpase motifs . the phylogenetic analysis shows that pfruvb1 , pfruvb2 , and ldruvb1 are closer to the yeast / human ruvb1 , while pfruvb3 and ldruvb2 are closer to the yeast / human ruvb2 ( reptin ) . recently , in many studies it has been reported that ruvb like proteins are overexpressed in many cancers . later , it was found that the atpase activity is crucial for the cancer cell proliferation and has been proposed as suitable drug target . similarly , one of the p. falciparum ruvb like protein ( pfruvb3 ) has been found specific to the stage where rapid nuclear division led multiplication of parasite occurs . considering all these studies it seems that exploring the role of the parasite r2tp complex may reveal some critical roles in rapid multiplication of the parasite in human host . all the sequences of the components of the r2tp complex from human and yeast were retrieved from their genome database . the downloaded sequences were used for blast - p analysis in human genome and yeast genome . all the corresponding sequences of leishmania donovani and plasmodium used in this analysis were retrieved from genedb ( www.genedb.org ) and plasmodb ( plasmodb.org ) , respectively . the retrieved corresponding sequences were in - silico studied and various domains were manually assigned and confirmed by using pfam , prosite , smart , panther , etc . , integrated software , interproscan . multiple - sequence alignment was done by using mac vector , as well as clustalw , while phylogenetic tree analysis was done by using phylogeny.fr . the ribbon diagram of the template and the predicted structure of ldruvb1 and ldruvb2 were superimposed . molecular graphic images were produced using the ucsf chimera package ( http://www.cgl.ucsf.edu/chimera ) from the resource for biocomputing , visualization , and informatics ( supported by nih p41 rr-01081 ) .

recently discovered r2tp complex is an important multiprotein complex involved in multiple cellular process like snornp biogenesis , pikk signaling , rna polymerase ii assembly and apoptosis . within r2tp complex , pih1 tightly interacts with rvb1/rvb2 and with tah1 to form r2tp macromolecular complex . r2tp complex further interacts with hsp90 to form r2tp - hsp90 complex , which has been found critical in many cellular process . the genome wide screening of leishmania donovani and plasmodium falciparum led to the identification of ruvb like1 , ruvb like 2 , pih1 , and tah1 . therefore , we speculate that this complex is also important for these parasites as in the yeast . the detailed analysis of crucial components of r2tp complex , ld - ruvb like 1 , and ld - ruvb like 2 , revealed the presence of characteristic motifs like dna binding motif and atpase motifs . hsp90 is also reported from leishmania donovani and plasmodium falciparum suggesting that the r2tp complex further interacts with hsp90 to form r2tp - hsp90 complex . recently it has been discovered that ruvb like proteins are overexpressed in many cancers and their atpase activity is crucial for cancer cell proliferation and the human ruvbs have been proposed as suitable drug target for cancer . similarly one of the plasmodium falciparum ruvb like protein ( pfruvb3 ) has been found to be specific to the stage where nuclear division led multiplication of parasite take place . considering all these it seems that the r2tp complex may be playing some critical role both in the cancer cell proliferation in human and rapid multiplication of the parasites leishmania donovani and plasmodium falciparum .

Introduction R2TP complex and its involvement in cellular processes Results and Discussion Identification of the R2TP complex component of Identification of the R2TP complex in human malaria parasite Concluding Remarks Materials and Methods

PMC4544475

due to the availability of efficient fluorescent probes and labeling recipes , fluorescence microscopy allows the direct observation of cellular processes in fixed and living cells as well as in complete organisms with molecular specificity and high temporal resolution in three dimensions ( giepmans et al . 2006 ; haugland 2005 ; lippincott - schwartz and patterson 2009 ; pawley 2006 ) . due to the wave nature of light , however , the spatial resolution is limited to about half of the wavelength of the light in the imaging plane ( abbe 1873 ; rayleigh 1903 ) , that is , conventional confocal and wide - field fluorescence microscopy does not provide insight into the structural and dynamic organization of biological samples such as vital protein assemblies and machineries with a size of a few tens of nanometers . within recent years , different far - field microscopic approaches have been introduced that have found a way to bypass the so - called diffraction barrier exploiting concepts to distinguish fluorescence emission of fluorophores in an additional dimension , e.g. , absorption , fluorescence emission , and fluorescence lifetime ( betzig 1995 ; heilemann et al . 1998 ) , and thus localize their positions individually . however , the number of distinguishable spectroscopic characteristics of fluorophores is too low to distinguish thousands of fluorophores per diffraction - limited area that compose a typical biological structure . on the other hand , fluorescence emission of fluorophores is distinguishable also in time allowing the temporal separated fluorescence detection of fluorophores . thus , the key to super - resolution imaging is confinement of the number of simultaneously fluorescing fluorophores . this is achieved in a deterministic way by generating a light pattern that prevents the emission of fluorophores in a defined area , as used by stimulated emission depletion ( sted ) ( dyba and hell 2002 ; hell and wichmann 1994 ; willig et al . 2006 ) , and structured illumination microscopy ( sim ) ( gustafsson 2000 , 2005 ; heintzmann and cremer 1999 ; kner et al . 2009 ) or in a stochastic way by temporally modulating the emission of individual fluorescent molecules by photoactivation , photoconversion , or photoswitching , as used in photoactivated localization microscopy ( palm ) ( betzig et al . 2006 ; manley et al . 2008 ; shroff et al . 2007 ) , fluorescence photoactivation localization microscopy ( fpalm ) ( hess et al . 2006 , 2007 ) , stochastic optical reconstruction microscopy ( storm ) ( bates et al . 2007 ; huang et al . 2008b ; rust et al . 2006 ) , direct storm ( dstorm ) , ( heilemann et al . 2008 , 2009 ; van de linde et al . 2009 ; bock et al . 2007 ; flling et al . 2008 ; geisler et al . 2007 ; gunkel et al . 2009 ; lemmer et al . 2008 ; steinhauer et al . 2009 ) ( table 1 ) . historically , localization microscopy started with the efforts of lidke et al . ( 2005 ) to use the fluorescence intermittencies of semiconductor quantum dots for single - molecule localization . the authors envisioned that a high - resolution image consisting of individually localized points may be reconstructed and termed it pointillism however , because blinking characteristics of quantum dots can be best described by a power law ( kuno et al . 2000 ) , densely labeled structures are difficult to resolve . alternatively , transient binding of fluorescent probes to the target structure ( giannone et al . 2010 ; sharonov and hochstrasser 2006 ) or the conversion of fluorogenic probes by catalysts ( roeffaers et al . 2009 ) can be exploited to bypass the diffraction limit by the reconstruction of a super - resolved image.table 1single - molecule localization microscopy techniquestechniquebasic principleexperimental detailsreferencespointillismfluorescence intermittencies of emitters such as triplet - state blinking can be used to temporally separate fluorescence emission , localize emitters , and reconstruct a structure of interest ( e.g. , the cytoskeleton ) by essentially painting them in a fashionblinking of randomly distributed quantum dots on a coverslip ( qd655 ) ; ~1.000 photons per on - event ; off - state lifetime too short to demonstrate usability to resolve cellular structurelidke et al . ( 2005)palm photoactivated localization microscopy uses photoactivation or photoconversion of fluorescent proteins to generate a sparse subset of fluorescent molecules per framephotoactivatable and photoconvertible fluorescent proteins ( e.g. , pagfp , meos , and pamcherry ) ; no special buffer additions required ; typically , a few hundred photons per on - event ; ( betzig et al . ( 2007)storm stochastic optical reconstruction microscopy uses pairs of fluorophores ( e.g. , cy3cy5 ) to photoswitch the reporter ( cy5 ) in the presence of thiols between a bright on- and a reduced off - state with long lifetime ( > 1 s ) ; the activator ( cy3 ) is used for efficient reactivation of the reporterstandard fluorophore dye pairs ( cy5/cy3 and alexa 647/cy3 ) ; addition of 10100 mm of thiols such as mercaptoethylamine ( mea ) ; oxygen depletion ; off - state lifetimes of seconds to minutes ; ~3.000 photons per on - event of cy5/alexa 647bates et al . ( 2006)fpalm fluorescence photoactivation localization microscopy uses photoactivation or photoconversion of fluorescent proteins to generate a sparse subset of fluorescent molecules per framephotoactivatable fluorescent proteins ( e.g. , pagfp ) ; no special buffer additions required ; typically , a few hundred photons per on - statehess et al . ( 2006 , 2007)palmira palm with independently running acquisition uses an asynchronous acquisition protocol and a fast camera that allows matching the camera frame time to the average on time of fluorophoresfluorescent protein ( rsfastlime ) and standard fluorophore ( cy5 ) ; embedding in poly ( vinyl - alcohol ) ( pva ) to prolong triplet - state lifetime ; ~200 photons per on - state of rsfastlime , ~700 photons per on - state of cy5bock et al . ( 2007 ) dstorm direct storm photoswitches standard fluorescent probes in the presence of thiols between a bright on- and a reduced off - state with long lifetime ( > 1 s)standard fluorophores ( cyanine , rhodamine , and oxazine dyes , e.g. , atto- and alexa dyes ) ; addition of 10100 mm of thiols such as mercaptoethylamine ( mea ) ; off - state lifetimes of seconds to minutes ; 5005.000 photons per on - event dependent on dyeheilemann et al . / spectral position determination microscopy uses reversible photobleaching of fluorophores and fluorescent proteins under high irradiation intensitiesstandard fluorophores and fluorescent proteins ( e.g. , yfp ) mounted in an antifade reagent ; irradiation intensities of 10 kw cm1 mw cm ; > 1.000 photons detected per on - event of yfp moleculesbaddeley et al . ( 2009 ) , gunkel et al . ( 2008)gsdim ground state depletion microscopy followed by individual molecule return uses triplet and other metastable off - states of fluorophores for temporal separation of fluorescence emission of individual fluorophoresstandard fluorophores ( atto 532 , alexa 488 , texas red ) and fluorescent proteins ( e.g. , egfp and eyfp ) ; oxygen depletion or embedding in poly(vinyl - alcohol ) ( pva ) to prolong off - state lifetime to 10100 ms ; 8002.600 photons per on - eventflling et al . ( 2008 ) , hell ( 2009)blink microscopyengineering on- and off - states by controlling the photophysics of the fluorophores by electron transfer reactionsstandard fluorophores ( cy5 , atto 655 , atto 680 , atto 700 ) ; oxygen removal and addition of 100 m ascorbic acid to quench the triplet state and generate reduced fluorophores with off - state lifetimes > 20 ms ; 70700 photons per on - eventsteinhauer et al . ( 2008 ) , vogelsang et al . ( 2009)most techniques work optionally with a second laser ( 405 nm ) to increase the reactivation efficiency of the on - state ( the on - switching probability ) . in many cases , the reactivation efficiency of the readout laser is high enough to ensure a constant number of fluorophores residing in the on - state at any time of the experiment . with the exception of spdm , all other localization microscopy techniques apply excitation intensities of a few kw cm ( typically 13 kw cm ) for fluorescence readout and off - switching or photobleaching , respectively . the recovery or reactivation intensities are two to three orders of magnitude smaller single - molecule localization microscopy techniques most techniques work optionally with a second laser ( 405 nm ) to increase the reactivation efficiency of the on - state ( the on - switching probability ) . in many cases , the reactivation efficiency of the readout laser is high enough to ensure a constant number of fluorophores residing in the on - state at any time of the experiment . with the exception of spdm , all other localization microscopy techniques apply excitation intensities of a few kw cm ( typically 13 kw cm ) for fluorescence readout and off - switching or photobleaching , respectively . the recovery or reactivation intensities are two to three orders of magnitude smaller all stochastic single - molecule localization microscopy techniques ( table 1 ) share the same concept that only a sparse subset of fluorophores is allowed to stay in its fluorescent on - state at any time of the experiment this is achieved by stochastic activation of individual fluorophores , single - molecule detection using a wide - field fluorescence microscope equipped with a sensitive charge - coupled device ( ccd ) camera , and precise position determination ( localization ) , i.e. , fitting of ideal point - spread functions ( psfs ) to the measured photon distributions . as long as the distance between individual fluorophores residing simultaneously in their on - state ( i.e. , per frame ) is larger than the distance resolvable by the microscope ( > /2 on the ccd camera ) , individual fluorophores can be clearly localized . since the localization precision ( i.e. , the accuracy of position determination of a single fluorophore ) depends mainly on the number of collected photons , n , and on the standard deviation , , of the psf , single fluorophores can be localized with an accuracy approximated by /n for negligible background ( mortensen et al . while all single - molecule localization microscopy methods share the same concept of temporal separation of fluorescence emission , they use different concepts to accomplish this experimental requirement ( table 1 ) . to ensure that only a sparse subset of fluorophores resides in the on - state at any time of the experiment and that each fluorophore defining the structure of interest is detected as individual emitter and thus precisely localized , the generation of stable off - states with lifetimes exceeding seconds is of outstanding importance . while this task is comparatively easy to accomplish using photoactivatable fluorescent proteins , which are non - fluorescent at the beginning of the experiment , the use of standard organic fluorophores requires the transfer of the majority of bright fluorophores to a long - lived , stable off - state . this succeeds for most alexa and atto dyes belonging to the cyanine , rhodamine , and oxazine dye classes upon irradiation of an aqueous solution in the presence of millimolar concentrations of reducing thiols . the dyes are cycled between the singlet ground and first excited singlet state until the dye enters the triplet state . the triplet state can then be quenched to repopulate the singlet ground state either by oxygen or by the thiolat ( the actual reducing species ) to form a semi - reduced fluorophore radical anion , the fully reduced leuco form of the fluorophore , or a thiol adduct ( dempsey et al . finally , the singlet ground state is recovered by the oxidation of the reduced off - state , e.g. , with molecular oxygen . because the off - state of most organic fluorophores absorbs at 400 nm , photoinduced recovery can be applied to control the number of fluorophores residing in the on - state ( van de linde et al . since dstorm is easy to perform , i.e. , it works with commercial available fluorescent standard probes in aqueous solvents and standard protocols for immunostaining ( heilemann et al . 2008 , 2009 , 2011b , 2012 , 2013 ) , it is currently the most applied single - molecule localization microscopy concept using organic fluorophores . the simple thiol - assisted photoswitching mechanism ( millimolar concentrations of thiols such as -mercaptoethylamine , cysteine , or glutathione have to be added to the imaging buffer ) and the availability of efficient open - source software for two - dimensional ( 2d ) and three - dimensional ( 3d ) multicolor localization microscopy ( e.g. , rapidstorm ) ( wolter et al . 2010 , 2012 ) facilitated the acceptance and broad applicability of dstorm for different super - resolution imaging studies ( dempsey et al . , the feasibility and reliability of different localization microscopy techniques has to be judged by test samples with well - defined molecular structure and composition . among the various natural biological test samples , the nuclear pore complex ( npc ) with its well - defined molecular composition , subdiffraction dimension , and eightfold symmetry holds a special position ( adams and wente 2013 ) . because the npc is composed of a defined number of proteins and exhibits a central channel with a diameter of ~40 nm , it is suited for testing of the achievable resolution of different super - resolution imaging methods and counting the number of molecules in a biological structure . the combination of raw dstorm localization data and particle averaging techniques as first demonstrated by lschberger et al . ( 2012 ) can also be used to reconstruct average protein positions in multiprotein complexes such as the npc with nanometer resolution ( szymborska et al . it neglects , however , structural heterogeneity of the sample by averaging individual signals the strength of all single - molecule methods and does not enhance the optical resolution . whereas super - resolution imaging or localization of fluorophore - labeled target molecules does not rely on the number of fluorophores attached to the molecule per se , the labeling density determines the achievable resolution of complex biological structures . according to the information theory , the required density of fluorescent probes must be high enough to satisfy the nyquist in essence , this theorem states that the average distance between neighboring fluorophores ( the sampling interval ) must be at least two times smaller than the desired spatial resolution . for example , to resolve structural features with a spatial resolution of 20 nm , fluorophores must be placed and localized every 10 nm . for a 2d structure , a labeling density of approximately 10 fluorophores m or approximately 600 fluorophores within a circular diffraction - limited region is required ( sauer 2013 ) . thus , independent of the technique used , efficient and specific labeling with fluorescent probes is a decisive aspect of super - resolution imaging . however , they also exhibit a non - negligible size ( a cylinder with a length of 4.2 nm and diameter of 2.4 nm ) ( tsien 1998 ) . organic fluorophores such as rhodamine or carbocyanine dyes are substantially smaller ( ~1 nm ) but require a chemical labeling procedure such as standard immunofluorescence using primary and secondary igg antibodies with a size of ~10 nm ( weber et al . 1978 ) . alternatively , small labeled camelid antibodies ( nanobodies ) directed against green fluorescent protein ( gfp ) ( ries et al . 2012 ) with a smaller size ( 1.5 nm 2.5 nm ) can be used for efficient labeling . one of the most promising future labeling methods for high - density labeling is the 1,2,3-triazole linkage ( click chemistry ) between a fluorophore modified as an azide and a reaction partner ( e.g. , protein ) modified as an alkyne , or vice versa ( laughlin et al . this method , however , requires further improvements concerning specific labeling of the protein of interest in living and fixed cells . photoswitchable or photoactivatable fluorescent proteins ( pa - fps ) were the first choice for super - resolution imaging in living cells , as they enable stoichiometric labeling and non - pertubative imaging of the sample ( lippincott - schwartz and patterson 2003 ) . several studies have been conducted using pa - fps to unravel sub - diffractive morphologies and dynamics of cellular components ( table 2).table 2fluorophores used in live - cell single - molecule localization microscopy studiesfluorophoreabs/em ( nm)photons / on - eventtagadditivescamera frame rate ( hz)temporal resolution ( s)spatial resolutionreferencesoregon greenrhodamine490/514900snapnone100150303d : 50 nm xy ; 90 nm z jones et al . ( 2012)tmrrhodamine554/5801,100snapnone10015030 3d : 45 nm xy ; 82 nm z jones et al . ( 2008)tdeos fp569/5811,200fusion proteinnone100150 3d : 30 3d : 40 nm xy ; 80 nm z jones et al . ( 2011)eosfp fp571/581n / afusion proteinnone20 500 25 nmmanley et al . ( 2008)meos2 fp573/5841,200fusion proteinnone100150 3d : 303d : 40 nm xy ; 80 nm z jones et al . ( 2011)alexa 568rhodamine572/6001,700directly labeled-mercaptoethanol , oxygen scavenger500 3d : 303d : 40 nm xy ; 70 nm z jones et al . ( 2012)mitotracker redcationic rosamine581/644790noneoxygen scavenger500900 210 3040 nmshim et al . ( 2012)er - tracker redbodipy587/615820noneoxygen scavenger500 10 35 nmshim et al . ( 2012)alexa 647cyanine649/6703,500directly labeled , snap-mercaptoethanol , oxygen scavenger500 2d : 0.5 3d : 12 2d : 25 nm3d : 30 nm xy ; 50 nm z jones et al . ( 2010)atto 655oxazine663/6841,200snapnone100150 30 3d : 40 nm xy ; 80 nm z jones et al . ( 2012 ) fp fluorescent protein converted / activated form fluorophores used in live - cell single - molecule localization microscopy studies fp fluorescent protein converted / activated form one of the first realizations of live - cell super - resolution imaging investigated the structure of the actin protein mreb labeled with eyfp by palm ( biteen et al . furthermore , they introduced time - lapse palm to increase the effective labeling concentration and achieve a sub-40 nm spatial resolution . images were obtained with cycles of reactivation at 407 nm and imaging of eyfp at 514 nm with a frame rate of 10 hz and 12 min temporal resolution . palm was also successfully implemented to investigate the nanoscale dynamics of adhesion complexes by imaging paxillin fused to tdeosfp ( shroff et al . since the dynamics of these complexes occurs at a timescale of minutes , a series of palm images , acquired over a time > 20 min , with a temporal resolution of 2560 s was sufficient to study morphological changes . palm imaging revealed sub - diffractive structures and different dynamics within an adhesion complex and between complexes at a spatial resolution of about 60 nm . assembly and disassembly of adhesions in different cell lines were also studied . with fpalm imaging of pa - gfp , the motion of hemagglutinin ( ha ) clusters in the cell membrane of fibroblasts with a localization precision of ~40 nm was investigated ( hess et al . 2007 ) by calculating the distance distribution of ha molecules in consecutive frames with a time resolution of 100300 s. thus , an effective diffusion coefficient could be determined . a combination of single - particle tracking and palm imaging ( sptpalm ) was introduced to study the mobility and distribution of two membrane proteins ( vsvg and gag ) by manley et al . the experiments gave single - molecule trajectories at a high density ( 50 m ) and the mapping of single - molecule diffusion coefficients of vsvg and gag evidenced that vsvg proteins exhibit a larger mobile fraction than gag proteins . due to the comparatively low fluorescence quantum yield and photostability of fps , ( f)palm in living cells is restricted to slower cellular processes . imaging faster dynamic processes in cells requires higher temporal resolution , ideally without decreasing the spatial resolution . in this therefore , they are well suited for improving the spatiotemporal resolution in live - cell localization microscopy experiments 2008 ; miller et al . 2005 ) offer an elegant method for specific labeling of proteins with organic dyes in living cells ( table 2 ) . here , a polypeptide tag , which is genetically fused to the target protein , binds to a fluorescently labeled substrate . on the other hand , live - cell labeling with chemical tags is also challenging because the fluorescently labeled substrates have to exhibit high membrane permeability and negligible non - specific binding in living cells . thus , the labeling procedure has to be optimized for each protein and fluorescent substrate separately concerning substrate concentration and incubation time . furthermore , the amount of non - specific binding of fluorescently substrates to cellular organelles has to be carefully checked . nonetheless , some fluorescently labeled substrates are very well suited for specific labeling of proteins in living cells . the first demonstration of live - cell single - molecule localization microscopy with organic dyes was performed by dstorm employing the trimethoprim ( tmp ) tag ( wombacher et al . the cell - permeable dye atto 655 was used to label the edhfr tagged core histone protein h2b . since the cell provides its own reductive environment , mostly due to the presence of millimolar concentration of the thiol glutathione ( van de linde et al . 2012 ) , atto 655 molecules switch between a bright on- and a non - fluorescent off - state upon irradiation under physiological conditions . applying a sliding window analysis on the localization data , the dynamic movement of histone proteins could be observed at a temporal resolution of 10 s. slightly later , the snap - tag technology was also successfully applied for live - cell dstorm in various cell lines using the two rhodamine - based substrates snap - cell tmr - star and snap - cell 505 ( fig . 1 ) ( klein et al . the results demonstrated the formation of long - lived fluorophore off - states under physiological conditions , which can be effectively and repeatedly reactivated upon irradiation at 405 nm ( klein et al . because rhodamine dyes tend to bind non - specifically on glass surfaces , coverslips were coated with 2 m glycine.fig . 1live - cell dstorm with snap tags . a fluorescence image of histone h2b proteins in a cos-7 cell stained with snap - cell tmr - star ( 1 m ) . b fluorescence image of the same cell but with 532 nm excitation of ~1 kw cm , which induced photoswitching . c dstorm image reconstructed from 10,000 images ( acquired at 50 hz ) . adapted from ( klein et al . 2011 ) , with permission live - cell dstorm with snap tags . a fluorescence image of histone h2b proteins in a cos-7 cell stained with snap - cell tmr - star ( 1 m ) . b fluorescence image of the same cell but with 532 nm excitation of ~1 kw cm , which induced photoswitching . c dstorm image reconstructed from 10,000 images ( acquired at 50 hz ) . adapted from ( klein et al . 2011 ) , with permission the photoswitchability of some organic fluorophores in living cells was also used to study the organization of intracellular microcompartments ( appelhans et al . , mitochondrial proteins of the outer and inner membrane were labeled with tetramethylrhodamine ( tmr ) via the halotag . single - molecule tracking and localization of individual protein complexes showed protein - specific diffusion behavior within both membranes and their substructures like cristae , indicating mitochondrial compartmentalization . as the different chemical tags can be used orthogonally , multi - color super - resolution imaging is likewise possible . for single - molecule tracking of two different plasma membrane receptor proteins , a multi - color approach employing snap- and halotag has been demonstrated ( benke et al . the fluorophores used ( dy-547 for snap and alexa 488 for halo ) are spectrally well separated and were imaged sequentially . in addition , the study demonstrated that tracking is not restricted to membrane proteins , but is also applicable to intracellular proteins like h2b . using a snap- and clip tag in combination with the two dyes alexa 647 and tmr , dual - color live - cell dstorm of the beta-2-adrenergic receptor and h2b histone proteins has been demonstrated ( klein et al . the membrane receptor was labeled with the cell - impermeable dye alexa 647 and the nucleus with tmr . to induce photoswitching of alexa 647 , the medium was supplemented with 100 mm glutathione and an oxygen scavenger system . however , in all live - cell experiments using organic fluorophores , the generation of reactive oxygen species ( ros ) has to be considered ( van de linde et al . ros may already perturb the intracellular environment during the experiment and thus can be expected to be a serious source of cell damage . the possibility to combine palm and dstorm for two - color super - resolution imaging for live - cell imaging , the combination of pa - fps and chemical tags enabled also three - color palm / dstorm of the spatiotemporal organization of membrane receptors ( wilmes et al . fusion proteins of the type i interferon receptors ( ifnar1 and ifnar2 ) with pa - gfp and patagrfp were coexpressed with the actin - binding lifeact peptide ( riedl et al . three - color super - resolution imaging was performed through cycles of photoactivation at 405 nm and sequential excitation at 488 nm for pa - gfp , 568 nm for patagrfp , and 647 nm for atto 655 . the reconstructed images showed a cytoskeletal association of the two receptors , indicating an interaction with actin or actin - bound adaptor proteins ( wilmes et al . they bind directly to the membrane of mitochondria , endoplasmic reticulum , lysosomes , or the plasma membrane . these commercially available probes proved to be photoswitchable in living cells upon irradiation with the respective wavelength and reactivation at 405 nm ( shim et al . due to the high labeling density , the ultrastructural dynamics of organelles could be resolved with a spatial resolution of ~40 nm and temporal resolution of 115 s using multi - emitter fitting algorithms . time - lapse imaging of mitochondria with mitotracker red , for example , revealed thin tubular intermediates between mitochondria at fission and fusion events , which could not be resolved in conventional diffraction - limited imaging ( shim et al . 2012 ) . three - dimensional super - resolution imaging in live cells has also been demonstrated ( jones et al . clathrin - coated pits and their cargo transferrin were labeled with different fluorophores using snap - tag fusion proteins or direct labeling . due to high laser intensities used of up to 15 kw cm and the resulting fast switching kinetics of alexa 647 , a time resolution of 0.5 s and 25 nm spatial resolution was achieved in 2d and 2 s and 50 nm ( axial ) in 3d , respectively . only with alexa 647 , which was delivered into the cell by electroporation , the cup - like morphology of ccps could be resolved . very recently , a new near - infrared fluorophore has been introduced for live - cell imaging ( lukinavicius et al . 2013 ) . this new silicon rhodamine ( sir ) is highly cell - permeable and shows a fivefold increase in fluorescence upon binding to its target protein . the fluorogenic character results from the formation of the non - fluorescent spirolactone if the dye aggregates or binds unspecifically to hydrophobic structures , whereas it becomes fluorescent upon reaction with the respective protein tag . single - molecule localization microscopy is also feasible , as sir exhibits switching behavior under physiological conditions , which was shown for cells expressing h2b snap ( lukinavicius et al . in order to enable three - dimensional ( 3d ) super - resolution imaging , the axial symmetry of the psf has to be broken , that is , in conventional microscopy ( in the case of zero aberrations ) , the psf of a fluorophore slightly above and below the image plane appears equally blurred preventing the extraction of the accurate axial fluorophore position . to overcome the obstacle methods have been introduced that tweak the signal in a way that the psf above and below the image plane looks different . some of the ideas have already been successfully used in single - particle tracking , and with the ability to image only a few fluorophores defining a densely labeled structure at a time due to temporal separation of fluorophore emission , it was straightforward to use these established methods also for localization microscopy . methods used to infer information about the axial origin of a signal can roughly be split into four groups : ( 1 ) astigmatism , ( 2 ) biplane , ( 3 ) psf splitting into lobes , and ( 4 ) interferometric approaches ( table 3).table 3three - dimensional single - molecule localization microscopy techniquestechniquelateral / axial resolution ( fwhm ) ( nm)axial capture rangereferencesastigmatism ( polynomial)2030/60700.6 m ; with stacking up to 3 mhuang et al . al . ( 2009)prilm3847/822 mbaddeley et al . ( 2011)ipalm22.8/9.80.225 mshtengel et al . ( 2009 ) z - position determination via sigma difference lookup table direct z - position determination by comparison with experimental psf three - dimensional single - molecule localization microscopy techniques z - position determination via sigma difference lookup table direct z - position determination by comparison with experimental psf the astigmatism approach introduces a cylindrical lens into the detection path of the microscope system . this leads to a stretching of the psf because only one spatial direction is tightly focused while the other is defocused ( fig . , there is one point of equal psf widths ( which can be set to be z = 0 nm ) , and psfs originating from above or below are spread to a horizontal or vertical line , respectively . the rough axial position can be extracted from the orientation of the psf and the widths in x and y can be calibrated to yield exact z - coordinates . after the initial use in single - particle tracking ( holtzer et al . 2007 ; kao and verkman 1994 ) , the method has been broadly applied to single - molecule localization microscopy ( dani et al . 2012 ) . for calibration of the defocusing behavior , usually single fluorophores , quantum dots or small fluorescent beads the obtained psf widths in x and y are fitted with a polynomial of second order , which represents a physically derived model ( holtzer et al . 2007 ) or a fourth - order polynomial to account for imperfections in the optical system ( huang et al . 2008a ) . to avoid fitting of a more or less physically derived function to the calibration data , a lookup table can be created for the extraction of the actual axial position . in the open - source quickpalm plugin for imagej ( henriques et al . 2010 ) , the standard deviations of the calibration psf in x and y are determined and the known z - position is plotted against x y . the obtained straight line serves to look up the z - coordinates during the course of a measurement.fig . 2 a nucleus of a xenopus laevis a6 cell stained against the nuclear pore complex protein gp210 with pale white bar indicating the area where the x z - cross section b is taken ; c and d show the respective x y- and y z - views of the distal appendage protein cep152 of centrioles from a u2os cell ; e represents another pair of centrioles in a cos-7 cell . scale bar a , b 1 m ; c , d 200 nm ; e 500 nm ; color - code ( blue to red ) a , b 04.6 m ; c , d 0400 nm a nucleus of a xenopus laevis a6 cell stained against the nuclear pore complex protein gp210 with pale white bar indicating the area where the x z - cross section b is taken ; c and d show the respective x y- and y z - views of the distal appendage protein cep152 of centrioles from a u2os cell ; e represents another pair of centrioles in a cos-7 cell . scale bar a , b 1 m ; c , d 200 nm ; e 500 nm ; color - code ( blue to red ) a , b 04.6 m ; c , d 0400 nm to use biplane imaging , the detected signal has to be split into two parts with an optical delay added to one of the two signals . both signal paths can be either imaged on the same camera ( juette et al . 2008 ) , or if a second camera is at hand , the two signals can be detected separately . in the second scheme , a relative offset in the axial position between both image planes has to be realized . the psf widths detected on detector a and b encode the z - coordinate just as the ratio between the x- and y - psf widths using the astigmatism approach . the third family of 3d localization microscopy techniques uses additional optics to split the psf of a fluorophore into two lobes that rotate around each other with respect to the axial position of the emitter . it was first proposed to engineer the psf to a double - helix by the introduction of a phase mask , which is created by a polarization - sensitive spatial light modulator ( slm ) ( pavani et al . the two resulting lobes rotate around each other in a double - helix ( dh - psf ) manner , which baptized the technique . it was later proposed to use a simplified approach adding a phase ramp in the objective aperture to half of the signal ( baddeley et al . termed phase ramp imaging localization microscopy ( prilm ) , in contrast to dh - psf , one lobe remains fixed while the other rotates around it . both techniques have in common that the available axial range is significantly larger than in biplane- or astigmatic imaging ( baddeley et al . , the sample is placed between two objectives , and thus , the signal of a single fluorophore travels along two different light paths . exploiting the wave particle duality , upon superposition of the respective signals with a three - way beam splitter , every single photon will interfere with itself . depending on the axial position , i.e. , whether it is closer to objective a or b , the fluorophore will show distinct interference that gives information about the z - coordinate ( kanchanawong et al . interferometry so far yields the best axial localization precision but is limited in axial range to about half the emitted wavelength ( table 3 ) . most 3d localization microscopy approaches use high na oil - immersion objectives for surface confined total internal reflection fluorescence ( tirf ) irradiation . another very popular irradiation mode for intracellular imaging only available with oil immersion is highly inclined and laminated optical sheet ( hilo ) microscopy ( tokunaga et al . 2008 ) . in a sense , hilo is similar to tirf , where illuminating light is approaching the sample at a critical angle , so that only molecules very close to the coverslip are illuminated by the evanescent wave . however , instead of illuminating only the molecules in close proximity to the coverslip as in tirf , hilo illuminates by a highly inclined and thin beam under a particular angle allowing high irradiation intensities combined with high signal - to - noise ratio even inside of cells . unfortunately , using an oil - immersion lens for imaging while measuring in aqueous buffer induces aberrations due to the refractive - index mismatch at the coverslip buffer interface . thus , the volume image of a specimen will appear stretched along the optical axis , an effect well described for both confocal ( hell et al . furthermore , the refractive - index mismatch leads to spherical aberration along the z - axis that is difficult to correct ( deng and shaevitz 2009 ) . by localizing a single fluorescent particle placed in various but well - controlled depths by an optical tweezer , it was shown that the determined z - position could be up to 30 % erroneous . this issue is especially tricky , as the amount of false localization is not uniform along the z - axis , that is , if single fluorophores adsorbed on a bare glass surface were used for calibration and the subsequent measurement is taken slightly above the surface , the localization determination between coverslip and focal plane will perform precisely , while a systematic false coordinate determination will occur for fluorophores above the focal plane . both sorts of aberrations can be avoided by either adjusting the refractive index of the imaging buffer ( huang et al . 2008a ) or by using objectives that use immersion media with matching refractive index ( glycerol immersion for tissue imaging or water immersion for adherent cells ) . however , the resulting epifluorescence illumination geometry results in lower excitation intensities , higher background , and increased photodamage rendering live - cell super - resolution imaging experiments more difficult . in addition , when working with glycerol and water objectives , it is crucially important to ensure that the sample is aligned perfectly horizontally . it was shown that even small tilt angles of less than 1 deteriorate the concentric pattern of the psf ( arimoto and murray 2004 ) . the center of a psf above or below the focal plane will thus appear to be shifted to the same side , which is a nice indication for coverslip tilt if encountered . thus , it has to be affirmed that the stage is perfectly leveled before the immersion medium is changed . finally , it has to be considered that the time needed to fully image a large 3d volume is significantly higher compared to one single 2d snapshot because several layers have to be imaged . unfortunately , with longer imaging times , setups are more prone to drift , which causes severe difficulties for later image processing . drift compensation is usually performed by fluorescence tracking of non - blinking fiducials . using a hydrogel with a refractive index close to water , 100 nm fiducials can be stably immobilized and homogeneously distributed in 3d even in close vicinity of cells ( zessin et al . in order to enable super - resolution imaging of whole living organisms , novel long - term continuous imaging methods with high spatial resolution and minimal photodamage such as selective plane illumination microscopy ( spim ) ( huisken et al . 2004 ) or bessel beam plane illumination ( gao et al . 2012 ; planchon et al . 2011 ) are urgently needed . ideally , these methods are combined with the enhanced penetration depth and axial sectioning capabilities of two - photon microscopy . first , realizations of super - resolution imaging with confined two - photon activation ( york et al . 2011 ) ( fig . 3 ) and spim ( cella zanacchi et al . 2011 , 2013 ) have recently been demonstrated . besides the use of photoactivatable fluorescent proteins , so - called caged , non - fluorescent organic dyes that can be activated upon irradiation with uv or ir light , such as rhodamine nn dyes ( belov et al . a xy ( top ) and xz ( bottom ) maximum - intensity projections of pa - mcherry1vimentin . about 1 million unlinked localizations insets show further magnification of white rectangles in xy ( lines in xz ) maximum - intensity projection , highlighting individual vimentin fibrils in 60-nm - thick z - slices ( localizations are linked ) . arrow region of fibril with apparent width < 100 nm . b axial extent of vimentin network with z - location indicated as a color map . for clarity , localizations corresponding to 01.5 m ( top ) and 1.53 m ( bottom ) are shown separately . arrowheads in a and b indicate a fibril that persists over > 2 m axially . scale bars 3 m ( a ) , 600 nm ( insets ) , 3 m ( b ) . reproduced from york et al . ( 2011 ) , with permission three - dimensional palm imaging of a vimentin network . a xy ( top ) and xz ( bottom ) maximum - intensity projections of pa - mcherry1vimentin . about 1 million unlinked localizations was rendered in each view . insets show further magnification of white rectangles in xy ( lines in xz ) maximum - intensity projection , highlighting individual vimentin fibrils in 60-nm - thick z - slices ( localizations are linked ) . arrow region of fibril with apparent width < 100 nm . b axial extent of vimentin network with z - location indicated as a color map . for clarity , localizations corresponding to 01.5 m ( top ) and 1.53 m ( bottom ) are shown separately . arrowheads in a and b indicate a fibril that persists over > 2 m axially . scale bars 3 m ( a ) , 600 nm ( insets ) , 3 m ( b ) . reproduced from york et al . ( 2011 ) , with permission alternatively , photoswitchable fluorescent proteins and superimposed orthogonal standing light waves generating thousands of doughnuts can be used for live - cell super - resolution imaging according to the resolft principle ( standing for reversible saturable optical fluorescence transitions ) . relying on the use of fluorescent proteins , which can be photoswitched between a stable on- and off - state under substantially lower light intensities than sted microscopy , the method enables super - resolution imaging of large field of views ( > 100 m 100 m ) in less than 1 s with subdiffraction spatial resolution ( chmyrov et al . for sensitive fluorescence imaging of fixed whole organs , new sample preparation methods such as clarity ( chung and deisseroth 2013 ) and 3disco ( ertrk et al . 2012 ) have recently been developed that make organs transparent to light while keeping them intact , providing a detailed glimpse of their inner structure . for example , clarity works by removing the fatty tissue that surrounds cells and makes them opaque , while preserving the tissue s structure . importantly , the tissue can be labeled with different fluorescent probes after a washing step . the potential of the technique has been demonstrated by imaging the brain of an adult mouse allowing the visualization of neuronal connections and local circuitry deep inside the brain on a cellular level ( chung and deisseroth 2013 ) . congruously , the next step will be the visualization of neuronal connections inside the brain with molecular resolution applying the above - mentioned method . single - molecule localization microscopy techniques have the potential to substantially improve our understanding of how cellular structure and function is organized . currently , they can not compete with deterministic super - resolution imaging techniques such as sted and sim regarding imaging speed and temporal resolution in live - cell experiments , but they can provide single - molecule information about molecular distributions , even giving absolute numbers of proteins present in subcellular compartments . this ability provides the fascinating possibility to study encoding of cellular function at the molecular level . to produce faster image readout speeds over large fields of view , huang et al . used next - generation scientific cmos camera technology in combination with localization microscopy ( huang et al . this enabled an imaging speed of up to 32 frames per second applying an efficient noise correction algorithm . however , with increasing imaging speed , the photon output of fluorophores also has to be improved . therefore , new methods have to be developed that increase the localization precision by collecting more fluorescence photons per on - event . this can be achieved for example using two opposing objective lenses ( ram et al . alternatively , the photoswitching rates can be decelerated by chemical or physical means , allowing the detection of more fluorescence photons per switching event ( sauer 2013 ) . for example , triplet - state quenchers such as cyclooctatetraene ( cot ) ( altman et al . 2012 ) can be used to increase the number of fluorescence photons emitted per switching event by optimizing the cycling between the singlet and triplet states before the fluorophore eventually enters a long - lived off - state ( olivier et al . for some red - absorbing dyes , the fluorescence quantum yield and lifetime could be substantially increased exchanging water by heavy water ( d2o ) ( lee et al . the magnitude of this effect scales with the emission wavelength , reaching a particularly high value of 2.63-fold for the cyanine dye cy7 ( klehs et al . 2013 ) . alternatively , the already mentioned caged non - fluorescent organic dyes that can be activated upon irradiation with uv or ir light ( belov et al . 2010 ; grimm et al . 2013 ) could be used in combination with a reducing and oxidizing system ( roxs ) , e.g. , of 1 mm ascorbic acid and 1 mm methyl viologen under oxygen depletion ( vogelsang et al . roxs increases the photostability of fluorophores by efficient depopulation of the triplet state and recovery of the singlet state ( vogelsang et al .

super - resolution imaging by single - molecule localization ( localization microscopy ) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution . constant improvements in fluorescent probes , efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy . today , it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes . thus , it exhibits potential to address fundamental questions of cell and developmental biology . here , we briefly introduce the history , basic principles , and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy ( dstorm ) and summarize key developments and examples of two- and three - dimensional localization microscopy of the last 8 years .

Introduction Single-molecule localization microscopy with synthetic fluorophores Live-cell localization microscopy 3D super-resolution imaging Technical challenges of 3D localization microscopy Super-resolution imaging of tissue and organs Conclusion and perspective

PMC3132519

atherosclerosis is the major cause of cardiovascular morbidity and mortality , with underlying pathological processes that may begin during childhood [ 1 , 2 ] . although the exact causes of atherosclerosis are not clear , it is thought to involve lipoprotein influx into the wall , across the endothelium , and chronic inflammation . over time , lipids accumulate in the inner wall and plaques develop , resulting in reduction or blockage of blood flow . eventually , this condition can lead to heart attacks and strokes [ 3 , 4 ] . this is most evident in regions of branching and curvature and has therefore been attributed to spatial variation in mechanical forces , particularly the haemodynamic wall shear stress exerted on the endothelium by the flow of blood . near - wall blood velocity , on which shear stress depends , can not be accurately measured by direct techniques . however , ecs form a monolayer between the blood and arterial wall that is regulated by haemodynamic forces through flow - mediated signal transduction [ 3 , 5 ] . of relevance to the present study , endothelial cells and their nuclei align with the predominant flow direction and elongate in response to increased shear . their shape has been used to assess patterns of wall shear stress in previous studies , [ 6 , 25 ] , including our studies aimed at understanding why the pattern of disease around aortic branches changes with age [ 8 , 9 ] . in the present study , we developed methods for automated analysis of ecs morphology . the first step is to detect the boundaries of the cells against the background of stained images . this is difficult in cases where the noise level is high and the image contrast is poor . this has motivated us to use support vector machines ( svms ) as a classifier because recent work has shown this approach to outperform many conventional classifiers . in this paper , we describe the visual features and the subsequent application of the svm as a classifier to detect thin boundaries of endothelial cells . endothelial monolayers were stripped from the descending thoracic aortas of rabbits by a modification of the hutchen procedure of bond et al . and hirsch et al .. this involves pressing the endothelial surface of the opened aorta against double - sided adhesive tape adhered to the surface of a microscope slide . aortas were obtained from three male new zealand white rabbits ( harlan interfauna strain ) , one mature and two immature , that had been perfused in situ with 10% neutral buffered formalin at physiological pressure for 90 s followed by 20 ml of silver nitrate solution ( 2.5 mg / ml , sigma ) , followed by further formalin fixation for 30 mins . all animal procedures complied with the animals ( scientific procedures , uk ) act 1986 . cell boundaries were examined around the origins of seven intercostal arteries from the descending thoracic aortas of the three rabbits . a montage of images of the area around each branch mouth was obtained using a zeiss axioplan microscope . the spatially varying sensitivity of the microscope and camera system resulted in shading of the individual images ; in section 3 , we describe the correction of these distortions , though correction is not needed in our final system . each montage was then divided into subregions ; the subregions each corresponded to an arterial area of approximately 660 1100 pixels , and they were located in a 3 3 grid centred on the branch mouth . the central element of the 9-element grid was not used , since it was largely occupied by the branch mouth , giving eight regions at each of the seven branches , and hence 56 regions in total . the images corresponding to these 56 regions comprised the data set to which the current analysis was applied . one of the sample images ( with a size of 660 1100 pixels ) taken from one of the montages is shown in figure 1(a ) and its manually traced counterpart in figure 1(b ) . the cell boundary labelling or segmentation of ecs can be described as a binary classification problem in the sense that cell boundary pixels should be labelled as object or foreground and non - boundary pixels should be labelled as background . each of the 56 datasets contained more than 500 cells , giving > 25,000 cells requiring analysis . it is extremely tedious to trace the boundaries manually for tens of thousands of cells , and so a number of different methods was applied to attempt the segmentation of boundary pixels automatically . in order to assess the applicability of intensity thresholding to the task , we analysed the intensity distributions of image data after applying intensity correction based on background subtraction using a least - squares polynomial fit to a second - order spatial illumination model . for images that fell across boundaries of a montage , we also used the average values of intensity on either side of the boundary to perform additive compensation of intensity differences in different acquisitions . then , by using hand labelling of image and background data , we estimated the parameters of a standard mixture model : ( 1)p(i)=pop(i o)+pbp ( i b ) , where po and pb are the prior probabilities of the pixels and p(i | o ) and p(i | b ) are conditional probabilities of boundary and background , respectively . p(i | b ) and explains the histograms of all pixel intensities pooled together . as shown in figure 2 , the conditional probability density functions of background and cell boundary pixels overlap with each other strongly . it is thus hard to set a global threshold which distinguishes between background and boundary pixels ; we have previously described results of our initial work on the same data using adaptive thresholding following quadratic - based trend removal , a type of adaptive high - pass filtering . we therefore developed a technique with the help of a class of machine learning algorithms known as support vector machines ( svms ) to computationally detect the boundaries of the images . the method is attractive in that it is able to handle the weak boundary signals in our data , and does not require explicit edge - linking or explicit illumination correction these latter two modules of classical segmentation are implicit properties in the choice of features . once such labellings are obtained , it is fairly straightforward to apply a variety of shape analysis techniques on the boundary representations to study the morphology of cells under the effects of biological flow . the rest of the paper describes the method we have developed for automatically detecting the cell boundaries using svms and some evaluations of its performance . features are extracted from the cell boundary image and mapped into feature space . to train the classifier , then , using the input feature space generated from the training image and an svm kernel , a decision model is generated ; this model is then applied to segment other boundary images . the svm classifies image pixels into two different classes : boundary and non - boundary . it is useful to consider the complexity of this problem , and why the selection of patch - based features , though increasing feature space dimensions , helps solve the segmentation problem . figure 4 illustrates the complexity of learning to detect boundary pixels . since we can not rely purely on the image intensity of a single pixel in order to classify it the basis of thresholding approaches the decision - making process on the correct classification for each pixel needs to consider the intensity of its neighbours as well . figure 4 shows four different configurations of pixels , similar to the cliques of markov - random fields ( mrfs ) , and considers a subspace of the full 9-dimensional space of the 3 3 patch represented by the intensities along a horizontal line of three pixels centred on the pixel to be classified . three of these pixels are valid boundary points , and the fourth ( b ) is an isolated pixel that has a boundary - like intensity , but contains no neighbouring pixels to support it . the consideration of three pixels is not enough to allow a correct decision to be made concerning pixel a ( boundary ) and pixel b ( non - boundary ) , as they lie in the same place in 3-dimensional feature space . including all the eight nearest neighbours of a pixel to be classified provides a partial solution , but the separation of classes in a 9-dimensional space is more difficult , and almost impossible using a linear classifier such as a fisher criterion . this is the motivation for using svms . through the use of kernels that generate nonlinear combinations of variables , svms are able to find decision boundaries for the classes that are not easily separable in the original space . other approaches to this problem might include algorithms to perform boundary linking , based on directions of gradients . such approaches might work for images in which the object boundaries correspond to edges of regions which contain different average pixel intensities on either side of the boundary ; for the case of thin structures , second - order directional derivatives or phase - invariant measures of orientation are more appropriate for estimating structure direction to perform edge linking . even so , creating an edge - linking algorithm to correctly handle the very complex spatial patterns formed by junctions between cells is no trivial task , and it is better to pursue a training approach which can learn how to appropriately classify pixels based on spatial configurations of neighbours . however , given the nature of the poor signal - to - noise levels in the images , even a 3 3 neighbourhood of pixel values is not sufficient as a feature vector ; and whilst a 5 5 neighbourhood might provide more information that would allow , say , pixels lying on gaps in boundaries to be correctly classified , it would require an increase in the dimensions of the feature vector , more training and testing data , and also would incur higher computational cost in training and classification . instead , we resort to generating features that group information from a wider area , for example , through spatial filtering , to compactly capture information over a larger neighbourhood which explicitly describes local orientations . we have taken an intuitive approach to selecting features , but have validated it using a feature selection approach . we have explored a rich set of possible feature vector combinations , using up to 75 components in the feature vector , but do not reproduce them all here . rather , we take the strongest feature vector components to illustrate the process of feature selection using the principle of minimum redundancy , maximum relevance ( mrmr ) . this is illustrative , as it shows that some features are much more informative than others . although we have used feature selection to assess some possible features , one has to be careful in assessing features individually . as illustrated by figure 4 , some features provide strong discriminating power only when included as a collection over space a patch - based approach , whereby some components of the feature vector are taken from a property that is estimated at different locations over neighbourhood space . this is the case for the patch - based information such as the intensity and orientation dominance field . whilst linear classifiers can not learn the tortuous decision boundaries suggested by the training data in , say , 9-dimensional space that are required to satisfy orientation invariance , the svm is able to find separating hyperplanes which generalise well from relatively few examples . each pixel in the image is mapped to a 33-dimensional feature vector by extracting information from a 3 3 neighborhood of the central pixel : nine intensity elements extracted from 3 3 nearest neighbors ; eighteen features extracted from 3 3 nearest neighbors of an orientation dominance field ; the magnitude of the vectors in this field ranges from 0 to 1 . the magnitude of the values can be used as an indication of anisotropy , in which strongly isotropic neighbors will produce values near to 0 and strongly anisotropic neighbors will produce values near to 1 ; an example of this field over a 3 3 patch is shown in figure 5 . these features are quasi - invariant to slow background illumination changes because they are generated from band pass filters ; six statistical features generated from the 3 3 intensity neighborhood of a candidate pixel , given in table 1 . the 33-dimensional feature vector given in ( 2 ) is then described for each pixel in the image in both the training and testing phases of the svm ( 2)fvn=[i(33),o(33),med , range , e , m2,3,4 ] . the feature vector components are normalised by removing the mean of each feature and dividing the result by the standard deviation of each feature . we used the minimum redundancy , maximum relevance ( mrmr ) measure to evaluate the importance of , and thereby select , the components of the feature vector input to the svm . as implied by figure 4 , some features should not be separated or assessed on their own . the result of mrmr is not repeated here , because it is performed against a very large number of neighbourhood features , including edge filters , statistical moments of intensities , and order - statistic neighbourhood measures . as a general rule , we found that orientation dominance , pixel intensities , and statistical moments were the most discriminating , ( table 2 ) , and so we included these in the feature vector we have used for segmentation . a certain degree of caution is required in interpreting mrmr results : individual neighbourhood pixels may be split apart in ranking of mrmr methods . thus , for example , if pixels from an 3 3 patch are all included in the mrmr , they may be separated in the ranked list of relevance obtained from the standard mrmr algorithm . when such features , obtained from different pixel locations in a feature space image around the candidate pixel , are included in an mrmr analysis , one should note the frequency with which the different locations of one property occur in , say , the top k ranked relevance measures . if a large proportion of features of one property occur close to the top of the list , then all the neighbouring pixels over the 3 3 grid in that property should be included in the vector . this algorithm is trained with a sample image of size 62 62 pixels taken from one of the 56 regions , as illustrated in figure 6 . the 33-dimensional feature vectors generated over this region are used as an input to the svm that classifies image pixels into boundary and non - boundary pixels . a binary image is generated after classification with each black pixel corresponding to a boundary and each white pixel corresponding to a non - boundary pixel [ 13 , 18 ] . for training , we used the rbf kernel [ 19 , 20 ] with the maximum cost function / parameter set at 1 and = 0.14 . with these settings , 2144 the algorithm was written with the help of the libsvm package in matlab r2009b , and it took 13.38 seconds on a desktop pc with pentium(r)4 2.8 ghz processor running windows . the time taken to classify an image of size 512 the algorithm provided usable boundary data in 43 out of the 56 regions . in the remaining 13 regions , image data are very noisy . one such image is shown in figure 7 , and it may be noted that even discerning the boundaries by eye is difficult ; high - pass filtering such an image does not improve matters . the binary images in figures 8(b ) and 8(d ) show classifications of pixels into boundaries and background by the svm algorithm . the performance of the svm approach in segmenting cell boundaries was evaluated on four images containing manually traced boundaries ; one of these images is shown in figure 1(b ) . the manually traced boundaries provide ground - truth data that allows the accuracy of the segmentation to be evaluated on a pixel - by - pixel basis . four trained svm kernels were applied to each of these test images , and the results were used to determine performance . in total , the performance of the algorithm was assessed on 3.9 million pixels . the parameter , , was varied from 0.02 to 0.14 across all four svm kernels in step sizes of 0.01 . the rbf kernel reached its best accuracy of 93% at = 0.14 and the worst result it achieved on a single image was 80% . accuracy of segmentation was assessed by noting the number of correctly classified pixels ( either boundary or background ) and is defined by ( 3)accuracy%=(tp+tn)(tp+tn+fp+fn ) , where tp , fp , tn , and fn are true positive , false positive , true negative , and false negative , respectively . in another experiment , we compared ground truth , an svm with rbf kernel and two conventional segmentation algorithms , canny and kittler , in four image patches of size 128 128 pixels . different thresholds were set for the decision in order to obtain a number of points along a receiver operating characteristics ( roc ) curve , which describes the performance of a classification method and feature space in addressing a discrimination task as one alters the balance between type i and type ii errors . for the canny and kittler algorithms , 961 and 63 possible detection thresholds were applied to each image , respectively . the svm model accuracy is largely based on the selection of the model parameters so , to find points along a roc , a search algorithm was used to attempt training and classification as values of the most significant parameters are altered across a wide range . for example , as the rbf kernel depends on two main parameters ( c and ) , a range of 4 to + 4 with interval of 0.1 for c and from 0.1 to 2 with an interval of 0.001 for was selected . however , since each image to be assessed might have different statistical characteristics , generating an roc might require slightly different parameter settings . for a given image , the model was further evaluated with 5-fold cross - validation to find the best possible combination of parameters . table 4 and figure 9 present the classical segmentation technique results in comparison to the svm with rbf kernel , and the roc plots are given in figure 10 . table 4 also includes a comparison with the otsu method , which employs an automatic thresholding and is therefore not easily amenable to roc analysis . this paper presents a method for detecting ec boundaries that reduces the need for human intervention . previously , cell boundaries have been segmented manually [ 9 , 25 ] , since noise and unclear or faint boundaries would have led to the failure of traditional automated segmentation techniques . previous reports have described computational work on corneal endothelial cell boundary detection [ 2629 ] , but the detection of cell boundaries was not as challenging as the ec boundaries in our images . in our previous work , we suggested some approaches to automatic cell boundary detection . to address issues of accurately finding edge maps with this previous method , we developed the current method , which has successfully worked on image data with a poor signal - to - noise ratio ( snr ) . our algorithm uses small neighbourhood patches in intensity , and orientation dominance patches of each candidate pixel in the image , and successfully segmented 43 out of 56 available regions to an acceptable level of accuracy our method has some limitations : it does not perform well on image patches where the brightness variation is very large or where cell boundaries are not visible by eye . we have found that illumination correction , such as by high - pass filtering , does not improve results and may even worsen them slightly if pixel noise is amplified . the method requires good manual labelling of one example as input in the training process . this necessitates selecting a patch with a representative variation in intensity to yield a satisfactory generalised model . we have only worked on kernels already available in libsvm , but performance might be improved by developing a new kernel for such data , which was beyond the scope this research . tests in other types of biomedical image data , such as retinal images , have suggested that with a comparable training process and comparable features , similar promising segmentation results can be achieved with minimal effort . future work will seek to extend the segmentation method to other biomedical image problems . the method we developed will enable us to explore the relationships between blood flow and cell behavior , between cell nuclear shape and cell boundary shape , and between length : width ratio and cell orientation in different regions around arterial branches , by allowing large numbers of cells to be analysed .

this paper presents an automatic detection method for thin boundaries of silver - stained endothelial cells ( ecs ) imaged using light microscopy of endothelium mono - layers from rabbit aortas . to achieve this , a segmentation technique was developed , which relies on a rich feature space to describe the spatial neighbourhood of each pixel and employs a support vector machine ( svm ) as a classifier . this segmentation approach is compared , using hand - labelled data , to a number of standard segmentation / thresholding methods commonly applied in microscopy . the importance of different features is also assessed using the method of minimum redundancy , maximum relevance ( mrmr ) , and the effect of different svm kernels is also considered . the results show that the approach suggested in this paper attains much greater accuracy than standard techniques ; in our comparisons with manually labelled data , our proposed technique is able to identify boundary pixels to an accuracy of 93% . more significantly , out of a set of 56 regions of image data , 43 regions were binarised to a useful level of accuracy . the results obtained from the image segmentation technique developed here may be used for the study of shape and alignment of ecs , and hence patterns of blood flow , around arterial branches .

1. Introduction 2. Data Acquisition 3. Segmentation of Endothelial Cell Boundaries 4. A Segmentation Algorithm for Cell Boundaries 5. Results 6. Discussion

PMC3705897

epilepsy is the most common serious chronic neurological brain disorder afflicting humankind , with no racial , socioeconomic , national , or geographic predilections ; it affects more than 50 million people worldwide . the tendency of epilepsy to onset in children renders its socioeconomic impact disproportionate ; moreover , the disorder is also associated with high comorbidity and significant life - long stigmatization . since current anticonvulsant drugs are effective in less than 65% of people ( and their use is associated with side effects in more than 50% of people ) , the need for new , effective , and safe therapies for epilepsy is a continuing neuropharmacological priority . to address this need , ( i.e. , the brain 's own anti - seizure compound ) as a platform around which to develop new drugs . through this work , we have identified -alanine as one possible candidate ecm [ 2 , 3 ] . -alanine occurs within the human central nervous system ( cns ) and has been identified as both an inhibitory neuromodulator and neurotransmitter . -alanine is structurally intermediate between -amino acid ( glycine ) and -amino acid ( gaba ) inhibitory neurotransmitters ( figure 1 ) . -alanine satisfies the prerequisite classical criteria for being a neurotransmitter : -alanine occurs naturally in the cns , is released by electrical stimulation through a ca - dependent process , has binding sites , and inhibits neuronal excitability . -alanine has five recognized receptor sites : glycine coagonist site on the nmda ( n - methyl - d - aspartate ) complex ( strychnine - insensitive ) ; glycine receptor site ( strychnine sensitive ) ; gaba - a receptor ; gaba - c receptor ; and blockade of gaba - transporter ( gat ) protein - mediated glial gaba uptake . -alanine binding has been identified throughout the neocortex , hippocampus , and various limbic structures . despite being a simple amino acid , -alanine remains essentially unexplored as either a neurological drug or a drug design platform ( although it has been used as a dietary supplement ) . -alanine , found in both the cns and skeletal muscle , has been studied extensively for its role in biosynthesis of the carnosine dipeptide . it has been claimed that augmented levels of carnosine , achieved through -alanine dietary supplementation , may enhance exercise performance and delay exhaustion by increasing the buffering capacity of skeletal muscle . therefore , it has been suggested that the anaerobic working capacity of muscle tissue can be increased by ingesting -alanine [ 7 , 8 ] . in many countries , dietary supplement and is used widely by males and females of varying ages , from young adults to seniors [ 912 ] . the typical daily dose averages 26 grams , administered orally , with the main side effects reported to be temporary sensory paraesthesia and mild flushing . during initial toxicology studies to evaluate -alanine as a putative orally active ecm , we anecdotally noted a pronounced difference in the side - effects experienced by individuals of caucasian and asian descent . although individuals of asian descent did experience some paraesthesia it was markedly delayed , milder , and presented initially in different locations than the paraesthesia experienced by caucasian participants . since -alanine is an endogenous neurotransmitter substance within the human cns ( and is not an atypical or exogenous molecule ) , these differences were unexpected . accordingly , we have pursued a pilot study to assess different interindividual physiological responses to the orally administered neurotransmitter -alanine . our study design has addressed this previously unreported finding , which could have implications for the clinical use of -alanine as an ecm . this investigator - initiated clinical trial was conducted at a single study centre , without blinding , randomization , or crossover . the trial was designed to test the hypothesis that after 30 minutes individuals of asian descent experience different and/or delayed side - effects that are milder in nature and have a different pattern of distribution , from those observed in individuals of caucasian descent . for the purposes of this study we define asian descent to mean that both parents are of oriental heritage , including origins in south asia , southeast asia , or east asia . we define caucasian descent to mean that both parents are white individuals , regardless of heritage . secondary endpoints include the subjective side - effect profile described by study participants , aged 18 years and older . ten adult volunteers of each ethnicity were recruited and upon providing informed consent were given a single 3 g dose of -alanine , purchased commercially from allmax nutrition and verified for its purity and composition . the study drug was dissolved in 200 ml of an artificial fruit - flavoured drink ( tang ) , which participants were instructed to drink completely over a timed four - minute oral consumption period . participants remained for 30 minutes after taking the study drug for observation and discussion detailing any side - effects they experienced . after 30 minutes , participants were asked to complete a questionnaire to score their perception of the symptoms they experienced upon ingesting -alanine from 1 ( no symptoms ) to 5 ( extremely unpleasant symptoms ) . the questionnaire was comprised of nine symptom questions , which sequentially asked responders whether they experienced pins and needles ( paraesthesia ) and/or itching around their mouth , ears , arms , hands , or legs and , finally , whether or not they felt themselves to be flushing . analysis was based on intention to treat , comparing change from baseline in measures of symptoms between the two treatment groups by means of t - tests . characteristics were tested for significant differences between the two treatment groups by means of two - tailed paired t - tests . data were analyzed to address the hypothesis whether inherent differences exist between asians and caucasians in terms of reported side - effects following oral administration of -alanine . the average age of both asian and caucasian participants was 31 years . in both groups , the male / female ratio was 7/3 . the average weight of caucasians , at 170 pounds , was greater than in the average weight of asians , at 139 pounds ; however , the body mass index ( bmi ) was 24.9 for caucasians and 22.2 for asians ( not statistically significant difference ) . average responses in terms of perceived side - effect severity were lower in asians than caucasians in 6/9 instances , as shown in figure 2 . caucasian participants reported an average of 5.1 symptoms in the 30-minute observation period , while asians reported an average of only 2.2 . ( this could speak to personal attributes of participants ; that is , certain individuals are more willing to freely describe everything they experience , no matter how minor and considering many of the asian participants were communicating predominately in their second language ( english ) this may have been a hindrance to free and open communication . accordingly , a translator was available during data collection to minimize any language barrier issues . ) when the results of all nine symptom questions are summed , there is a statistically significant difference between asians and caucasians ( p < 0.05 ) . however , when each of the individual symptom questions is independently analyzed , only two demonstrate statistical significance ( p < 0.05 ) : ( i ) pins and needles sensation in arms / hands and ( ii ) pins and needles sensation in legs and feet . also evident when examining participant 's self - reported symptoms is that while caucasians averaged 8 minutes prior to their first reported symptom , asians experienced their first symptoms after 14 minutes or longer had elapsed ; furthermore , the onset of symptoms in caucasians was acute and relatively intense whereas symptom onset in asians was gradual and with low initial intensity . therefore , participants of asian descent had an average of six further symptoms free minutes prior to experiencing anything that differed from their baseline . additionally , there was a tendency for asians to report that they felt their first symptom in a unique bilateral retro - aural distribution ( over the mastoid process , posterior to the external pinna of the ear ) while caucasians reported to notice their first symptoms within an extremity ( particularly the anterior upper thigh ) . subsequently , we examined whether participant gender was contributing to data trends , rather than ethnicity alone . by considering the average and median responses to the questionnaire by asian males versus caucasian males and asian females versus caucasian females ; there was clear trend that was evident amongst male participants . for every question , caucasian males ranked their symptoms to be more severe than their asian counterparts . the same was true for the median responses in 8/9 instances , as represented by figure 3 . asian females reported their symptoms to be more bothersome than caucasian females ( the opposite of what was true in males ) and were relatively equal in reported severity when considering median answers . however , regardless of ethnicity , males seemed to be more bothered by their symptoms than females , see figure 4 . the fact that caucasian males reported more frequent and more intense side - effects seems peculiar as an increase in side - effects is expected with lower body mass , as it is the case with most pharmaceuticals . however , we noted the opposite : with caucasian males experiencing the most side - effects , yet having the highest body weight of participants . future investigation may be required with increased number of participants to explain the observed variances between ethnicities and to understand whether the mechanism of action is the same and what this means in terms of the interindividual efficacy of -alanine . future clinical research investigating -alanine will have to be sensitive to the fact that the side - effect profile can vary based on gender and ethnicity . this notion will have to be given careful thought considering the challenges of blinding a study drug that produces notable and distinctive side - effects within 30 minutes of ingestion . in conclusion , following oral administration of the -alanine neurotransmitter , there were definite differences in the timing , localization , and intensity of side - effect perception between asians and caucasians . asian participants had an average of six more symptom - free minutes prior to experiencing any side - effects . although participants of asian descent did experience paraesthesia , they were delayed with different initial side - effects and distribution , when compared to the effects experienced by caucasian participants . when asian participants did report an initial side - effect , it was predominately localized in a retro - aural distribution . lastly , we observed a statistically significant higher prevalence of pins and needles sensation in the upper and lower extremities in caucasians . this pilot study challenges the assumption that an amino acid with properties of a neurotransmitter should have similar side - effects in everyone , regardless of ethnicity . although racial and ethnic differences in response to synthetic drug molecules are well known , -alanine is an endogenous neurotransmitter substance within the human cns ( and is not an atypical or exogenous molecule ) , and thus these differences were unexpected . this study also foreshadows challenges in running a blinded clinical trial assessing the efficacy of -alanine versus a placebo in the treatment of epilepsy ( since participants receiving -alanine will experience a characteristic set of paraesthesia side - effects ) .

previously , we have identified -alanine as a potential endogenous anticonvulsant molecule . -alanine occurs within the human central nervous system and has been identified as both an inhibitory neuromodulator and neurotransmitter that is bioavailable to brain after oral administration . during preliminary compounding trials to ascertain dosing strategies for -alanine , we noted pronounced differences in the side effect profile experienced by individuals of asian and caucasian descent . to investigate whether ethnicity affects -alanine - induced side effects , we administered 3 g of -alanine in 200 ml of fruit drink to ten people of each ethnic background and observed them for 30 minutes . data collected included basic physical statistics ( height , age , and weight ) and descriptions of all side effects , as reported by participants . we found that participants of asian descent experienced paraesthesia , but significantly different in time of onset , intensity , and anatomical localization , as compared to the effects experienced by caucasian participants . since -alanine is an endogenous neurotransmitter substance within human brain , these side effect differences were unexpected .

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

PMC3540918

recent reports have highlighted white adipose tissue ( wat ) as one of the primary contributors to the chronic low - grade inflammation commonly observed in obesity and related complications ( e.g. , type 2 diabetes , metabolic syndrome ) . as wat expands , it has been hypothesized that microhypoxia results in adipocyte stress and , ultimately , death via aponecrosis . adipocyte death and extravasation of fat into the extracellular space provide strong signals for macrophage recruitment and inflammation within wat . several important cytokines are implicated in these processes , including monocyte chemoattractant protein-1 ( mcp-1 ) , tumor necrosis factor- ( tnf- ) , interleukin-6 ( il-6 ) , and vascular endothelial growth factor ( vegf ) . chemokines , including mcp-1 , are in part responsible for recruiting macrophages to the area of inflammation , which further contribute to local wat inflammation by releasing their own host of cytokines . vegf has been shown to be necessary for wat growth in order to provide microvascularization as the wat expands in order to prevent microhypoxia and resulting wat inflammation . we have demonstrated that after establishing obesity ( 6 wks of high - fat diet ( hf ) ) , groups of mice placed on either a 12-wk treadmill moderate exercise program or low - fat diet ( lf ) , or combination intervention were protected from the 35-fold increase in inflammatory markers in wat as compared with hf diet sedentary mice . additionally , groups of mice underwent the same treatments in a shorter time period ( 6 wks of treadmill training and/or lf / hf ) , with no differences found in these same markers . taken together , those findings suggest that a longer - term intervention ( i.e. , 12 versus 6 wks ) is necessary to protect against the inflammatory damage induced by long - term hf diet whereby mice exercised for 16 wks on an hf diet experienced reductions in macrophage and inflammatory markers without marked improvements in body weight or adipose tissue accumulation . whether or not exercise during the initial consumption of hf diet could have prevented or attenuated the deleterious inflammatory effects of hf diet could not be determined from these studies . with obesity on the rise in young people , it is important to understand if exercise plays an important role in either preventing or minimizing complications associated with obesity during this vulnerable age . therefore , the purpose of this study was to determine the effects of concomitant hf ( 45% fat ) feeding and 6 wks of exercise training on adipose tissue inflammation in young mice compared to young mice on a chow ( 17% fat ) diet . utilizing a 4-arm model ( e.g. , hf - sedentary ( hf - sed ) , hf - exercised ( hf - ex ) , normal chow - sedentary ( nc - sed ) , nc - exercised ( nc - ex ) ) , we hypothesized that diet - induced weight gain would be observed in both hf diet groups with a lesser degree found in the hf - ex group . further , we hypothesized that inflammatory markers would be upregulated with the hf , yet exercise training would attenuate these markers in the wat . mice were randomly assigned to one of 4 groups . these groups included ( 1 ) hf - sed , ( 2 ) hf - ex , ( 3 ) nc - sed , and ( 4 ) nc - ex . groups underwent simultaneous diet modification and exercise training for 6 wks , after which mice were euthanized for tissue collection and analyses . c57bl/6 male mice from jackson laboratories ( bar harbor , me ) arrived at 4 wks of age and were individually housed ( n = 40 ) . following 2 wks of acclimatization , 20 of the mice were randomized to an hf diet for 6 wks , with the remaining mice continuing their chow diet . the hf diet was a 45% fat diet purchased from research diets ( new brunswick , nj ) , with the chow diet provided by harlan ( 17% fat ) . animals were kept on a reverse light - dark cycle , with lights on at 2100 h and off at 0900 h. the animal protocol was approved by the university of illinois at urbana - champaign 's institutional animal care and use committee , and animal health and care guidelines were strictly followed . the mice were treadmill trained during their dark cycle for 40 min / d at 12 m / min , with 12% grade , 5 d / wk for 6 wks . training occurred on a motorized treadmill ( jog - a - dog ; ottawa lake , mi ) between 0900 and 1030 h , with a gradual increase in duration over the first week of training , so that all runners were trained at the intended intensity by the 6 - 7th session . all of the sedentary animals were exposed to the same treadmill noise and handling as the runners in attempt to control for stress associated with treadmill exposure . treadmill training was employed over wheel running in order to accurately deliver a specific volume of exercise at moderate intensity . animals were euthanized 48 h following their last exercise session in a fasted state ( 12 h fast ) using co2 inhalation . after sacrifice , livers were extracted , weighed , frozen on dry ice , and stored at 80c until analysis . liver tissue ( 50200 mg ) was homogenized for 710 min in 4 ml of isopropanol with a polytron disrupter , with the resulting homogenate centrifuged at 4,500 rpm for 10 min . the supernatant ( 5 l ) was used to determined triglyceride ( trg ) content ( l - type ) ( wako diagnostics ; richmond , va ) . epididymal fat pads were immediately harvested after euthanization , weighed , aliquoted , frozen on dry ice , and stored at 80c until analysis . rneasy mini kits ( qiagen ; valencia , ca ) were used for determination of total rna . rna was quantified and purity assessed using a nanodrop spectrophotometer ( thermo scientific ; wilmington , de ) . affinityscript ( stratagene , agilent technologies ; palo alto , ca ) reverse transcription was used to create cdna from the mrna according to the manufacturer instructions . lastly , an mx3000p real - time pcr machine was used to perform pcr using brilliant ii sybr green master mix kits ( stratagen , agilent technologies ; palo alto , ca ) . the thermal protocols consisted of ( 1 ) denaturing for 10 min at 95c , ( 2 ) 40 cycles of 95c for 30 s , and ( 3 ) annealing at 60c for 1 min and 72c for 30 s. the housekeeping gene was glyceraldehydes-3-phosphate dehydrogenase ( gapdh ) . the ct method was used to represent the data relative to gapdh expression as fold change from nc - sed . all duplicate ct ( critical threshold ) values were within 0.5 units from each other , and no group differences in ct were observed for gapdh among the runs of pcr ( p values ranged 0.600.96 among the different primers ) . primers were designed online by integrated dna technologies ( http://www.idtdna.com/ ) and verified by the national center for biotechnology information ( ncbi ) blast feature ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . whole blood was obtained from the mice immediately prior to sacrifice using the retro - orbital eye bleed technique . blood was centrifuged for 15 min at 2500 rpm at 4c with the plasma aliquoted and stored at 80c . plasma glucose was later determined on a ysi 2300 glucose analyzer ( yellow springs inc . a one - way analysis of variance ( anova ) with repeated measures was used to test for changes in body weight throughout the 6-week study period between groups ( week group ) . a two - way anova ( diet exercise ) was used to test for differences in baseline body weight , final body weight , change in weight , fat pad weight , liver weight , liver trg , glucose concentrations , food intake , and wat mrna gene expression data . following a significant finding , lsd post hoc tests were used to determine specifically where the differences were . the epididymal fat pad weight / body weight ratio was used as a covariate in determining the effects of epididymal adiposity on the exercise effects . figure 1 depicts the body weights of the mice over the 6-wk study period . differences in body weight were evident beginning at week 1 and continuing through to 6 wks , with the hf diet groups having greater body weights than the nc groups ( p < 0.05 ) . surprisingly , no differences in body weight between ex and sed , hf or nc , groups were observed at any time point . epididymal fat pad weight expressed in absolute grams or relative to body weight were lowest in the two nc groups , with the hf - ex group having the highest values , followed by the hf - sed ( p < 0.05 ) ( table 1 ) . lastly , total estimated caloric intake over the 6-wk period was only different between the two diet groups ( p < 0.05 ) , with no effect of exercise . no group differences were observed in liver weights ( table 1 ) . liver triglycerides were greater in the hf - sed group compared to all other groups ( p < 0.0001 ) , with no differences observed between the 3 remaining groups ( figure 2 ) . plasma glucose was not different between groups at the end of 6 wks ( table 1 ) . no difference between groups was observed for leptin , mcp-1 , and tnf- mrna gene expression data ( figures 3(a)3(c ) ) . however , f4/80 mrna gene expression was greatest in the hf - sed group ( p < 0.05 ) , with no differences in the remaining 3 groups ( figure 3(d ) ) . lastly , vegf mrna gene expression was greatest in the hf - ex group ( p < 0.05 ) , again with no differences among the 3 other groups ( figure 3(e ) ) . the main findings from this study were that exercise did not attenuate weight gain in either the chow or hf diet groups but did appear to normalize liver triglyceride content in the hf diet group compared to that of the chow groups . further , exercise training did not alter several potent inflammatory markers ( i.e. , tnf - alpha , mcp-1 , and leptin ) but did appear to have an effect within the hf diet group for f4/80 ( lowered ) and vegf ( increased ) expression . these data suggest that in the face of a fatty insult , before the onset of established obesity , moderate exercise training exerts beneficial effects at two locations known to be potent contributors to chronic inflammation the liver and wat . not surprisingly , the mice fed the hf diet gained more weight compared to the nc - fed mice . this body weight difference was evident as early as one week into the hf diet feeding . the exercise training protocol did not attenuate the hf diet - induced weight gain , nor did it reduce body weight gain in nc - fed mice . the fact that exercise training did not attenuate weight gain in either group suggests that the intensity was indeed moderate and coaligns with human data suggesting that moderate - intensity exercise alone may not be an effective weight - loss strategy . our protocol was unlike voluntary wheel running protocols , which generally result in higher exercise duration ( e.g. , 38 km / night ) and are known to induce a significant caloric deficit resulting in weight loss . moreover , the mice were fed ad libitum throughout this study , and training did not increase caloric intake within each diet . again , this validates the moderate intensity level of the exercise intervention , as exercise protocols that are strenuous enough to produce weight loss generally result in an increase in voluntary energy intake [ 12 , 13 ] . although the moderate exercise training protocol used did not result in a reduction in weight gain , it was sufficient to normalize hf diet - induced hepatic steatosis . this finding corroborates other work that has shown exercise to be an effective means to reduce hepatic steatosis [ 6 , 14 ] . these current findings suggest that exercise training , even in the absence of weight loss , prevents hf diet - induced hepatic steatosis , a characteristic of metabolic dysfunction , in a young mouse model obesity . although exercise did not reduce weight gain , regional body fat deposition was altered , as indicated by a training - associated reduction in epididymal fat mass in the mice on hf diet . as adiposity is closely related to the metabolic syndrome , which includes hepatic steatosis , future research will need to address whether the protective effect of exercise on liver triglyceride accumulation is mediated through a reduction in visceral fat per se or a reduction in adipose tissue inflammation . in support of this , johnson et al . demonstrated that aerobic exercise reduced hepatic and visceral fat in obese adults without reducing body weight . further , kanda et al . suggest an mcp-1-dependent connection between adipose tissue macrophage infiltration and hepatic steatosis . while we did not observe any group differences in leptin , mcp-1 , and tnf- , all of which are considered to be important markers of inflammation in obesity , we did observe elevated f4/80 mrna in wat only in the hf - sed group compared to hf - ex and chow - fed groups . this suggests that macrophage infiltration ( as indicated by f4/80 mrna ) precedes upregulation of inflammatory markers stemming from the wat early on in the development of obesity . more importantly , moderate exercise training normalized f4/80 mrna , comparable to the nc groups . it is also possible that the macrophages were not producing tnf- , contributing to our lack of findings on that front . , they reported that metabolic disturbances were evident after 6 wks of an hf diet ( 60% fat ) and that activated macrophage markers are also present at 6 and 8 wks ( f4/80 and cd11c ) , while interferon- ( ifn- ) and interleukin ( il)-12p40 were not significantly altered by the hf diet after 4 or 8 wks of feeding nor were tnf- or mcp-1 after 4 or 8 wks . our data supports the lack of an inflammatory response within epididymal adipose tissue after approximately 6 wks of exposure to an hf diet , while macrophage infiltration is suggested with the increase in f4/80 . kawanishi et al . also reported no weight loss or adipose tissue loss with a 5 d / wk , treadmill training protocol ( 60 min / d ) following a 16-wk intervention , yet tnf- and f4/80 gene expression were rescued by the training in their hf diet mice ( 60% hf diet ) along with a decrease in cd11c ( m1 macrophage marker ) . while we also report an attenuation in f4/80 in the hf - ex group , along with an increase in vegf , it is important to note that our study is one of the first to report such findings in a group of young mice exposed to an hf diet for a relatively short period of time while undergoing concomitant exercise training . this demonstrates that this time period of high - fat exposure is an important period for investigation , and efforts in this area may help improve health outcomes . our most salient finding is that moderate exercise training early on in the development of obesity normalizes macrophage infiltration gene expression in white adipose tissue , even in the absence of weight loss . this may be particularly important in relation to youth obesity . the mechanism for the anti - inflammatory effect of exercise needs further investigation . recent work has shown that hypoxia , resulting from hypertrophied adipocytes with the progression of obesity , is causatively related to the inflammatory milieu that develops in obese adipose tissue [ 1922 ] . moreover , diminished vascular expansion in the adipose tissue with obesity has been suggested to be a possible trigger of the hypoxia that ensues . interestingly , recent work has shown that exercise training increases endothelial cell density and vegf gene expression in the adipose tissue of rats fed chow diet . we report here that exercise training also increases vegf gene expression in hf fed young mice . one possibility is that exercise training facilitates an adipose tissue compensatory response , such that capillary density increases with an expansion of adipose tissue mass . this would then prevent the adipocyte hypertrophy - associated reduction in oxygenation , resulting in hypoxia ( and inflammation ) . oxygenation assessment of the adipose tissue was beyond the scope of this current study , but the hypothesis that exercise training facilitates the angiogenic response in adipose tissue to prevent hypoxia and the resultant exacerbation of inflammation needs further investigation . although the results of this study are suggestive of a weight - loss independent anti - inflammatory effect of exercise that occurs early on in the development of obesity , there are limitations of this study that need to be considered as the data are evaluated . insulin resistance was not assessed in this study , which is an important barometer of metabolic health and is known to affect even young individuals during the progression of obesity . thus , future studies should assess the effect of exercise training on insulin sensitivity in young mice on hf diet . other studies have examined the effects of exercise on insulin sensitivity while on an hf diet in rodent models [ 2527 ] , with these studies generally using older models or not specifically stating the age of the mice or rats ; therefore , using younger models is warranted . second , relative gene expression levels do not always correspond to protein expression ; thus , future studies should measure protein levels of inflammatory markers and quantify immune cells in order to more adequately assess the inflammatory profile of wat with exercise training . similarly , in order to more precisely assess the angiogenic potential of wat , future planned studies will measure capillary density of wat after exercise training , in addition to a better characterization of macrophage infiltration . in conclusion , in the absence of inflammation in the intraabdominal wat depot , as assessed by gene expression levels of inflammatory markers , moderate exercise training concomitant with hf feeding protected young mice against excess liver triglyceride accumulation . moreover , exercise was associated with reduced gene expression of f4/80 ( indicative of macrophage infiltration ) and increased expression of the angiogenic marker vegf within the wat . these changes were observed without exercise - induced differences in body weight , suggesting that the effect of exercise is independent of body weight loss . the mechanism may be related to a slight exercise - mediated reduction in epididymal fat pad mass , as observed in the hf - ex group .

we aimed to determine the effects of 6 wks of exercise on inflammatory markers in mice concomitantly fed either high - fat ( hf ) or normal chow ( nc ) diets in young mice . c57bl/6 mice were randomized into ( n = 10/group ) an nc / sedentary ( nc / sed ) , nc / exercise ( nc / ex ) , hf / sed , and hf / ex groups . treadmill exercise was performed 5 d / wk at 12 m / min , with 12% grade for 40 min / d . liver triglycerides and gene expression of f4/80 , mcp-1 , tnf- , leptin , and vegf in visceral white adipose were determined . nc groups had lower body weights after 6 wks versus the hf groups ( 22.8 0.2 versus 25.7 0.4 g ) ( p < 0.0001 ) . f4/80 gene expression ( indicator of macrophage infiltration ) and liver triglycerides were greatest amongst the hf / sed group , with no differences between the remaining groups . vegf ( indicator of angiogenesis ) was greatest in the hf / ex versus the other 3 groups ( p < 0.05 ) . exposure of an hf diet in sedentary young mice increased visceral adipose depots and liver triglycerides versus an nc diet . exercise training while on the hf diet protected against hepatic steatosis and possibly macrophage infiltration within white adipose tissue . this suggests that moderate exercise while on an hf diet can offer some level of protection early on in the development of obesity .

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

PMC3649170

a 60-year - old woman is presented who required resection of both pulmonary and adrenal metastases from cervical adenocarcinoma . the patient was diagnosed with stage ia adenocarcinoma of the cervix which was treated surgically . five years later she was diagnosed with recurrent , metastatic cervical adenocarcinoma to both lungs and her right adrenal gland . following multiple courses of chemotherapy over the next 2-years , pet - ct imaging failed to show any advancement of her disease . because of the stability of the metastatic disease and due to the fact that no futher metastases developed , a multidisciplinary team decided to consider surgical resection of her metastases . the decision was made to perform the surgery in two stages , first , a left thoracotomy for resection of the left pulmonary nodules , followed 3 months later by a right thoracotomy and a trans - diaphragmatic right adrenalectomy . this case report is unique as it involves simultaneous pulmonary and adrenal resection due to metastatic cervical cancer and that the operation required partial vena caval resection due to local spread of disease . a multi - disciplinary team approach was utilized consisting of general , thoracic and vascular surgery . the second stage of the procedure was carried out through double thoracotomy incisions a thoracotomy through the 5 intercostal space for resection of her pulmonary metastases and an additional thoracotomy through her 8 intercostal space to provide adequate exposure for retroperitoneal dissection of her right adrenal gland . following the wedge resections of her right lung metastases , dissection a firm mass was palpable prior to incising the diaphragm , and it appeared that the adrenal metastasis had invaded the diaphragm locally . a 3 cm x 3 cm portion of diaphragm was incised and dissection was carried down around the liver and the adrenal mass was excised from the caudate lobe . the cancer was noted to be invading the wall of the ivc , and therefore , a vascular surgeon was consulted to assist in completion of the resection . dissection of the caudate lobe of the liver to gain proximal and distal control of the ivc . the tumor is being retracted cephalad with a clamp . following careful dissection of the posterior ligamentous attachments of the ivc to the liver , a satinsky vascular clamp was placed and a 1 cm x 1 cm x 0.5 cm portion of the ivc was excised . the entire specimen was removed en bloc ( figure 2 ) consisting of diaphragm , right adrenal metastasis measuring 6 cm x 4 cm x 3 cm , a scant amount of normal adrenal tissue , the small patch of ivc and the right adrenal vein which was completely encased by tumor . a patch angioplasty of the ivc was then completed with a ptfe patch and sewn in with a running 5 - 0 prolene suture ( figure 3 ) . the diaphragm was closed primarily with pledgeted 1 - 0 ethibond mattressed sutures , two 28-french chest tubes were placed through separate stab incisions , and the lung was re - expanded under direct vision . the patient tolerated the procedure well and was discharged home on post - operative day 8 . the en bloc specimen with the ivc surface in the foreground and the left adrenal vein clearly visible ( arrow ) . this case is unique in that it discusses a transthoracic , transdiaphragmatic adrenalectomy for metastatic cervical adenocarcinoma and partial resection of the ivc to remove the tumor en bloc . al.(1 ) subsequently , this thoracoscopic technique has been reported in case reports , small institutional studies , and animal studies(2 ) . use of minimally invasive techniques in this case would not have been feasible or appropriate due to invasion of the ivc , but when certain criteria are met its use has been shown to be both safe and effective for what is otherwise considered to be a very morbid procedure . previously , if a patient was found to have concurrent pulmonary and adrenal lesions , both thoracotomy and laparotomy incisions would be required ; this approach is much less desirable due to reported success with the trans - diaphragmatic surgery . in 1998 , flores et . al.(3 ) described 2 cases of trans - diaphragmatic adrenalectomy with concurrent pulmonary lesions one of which consisted of a metastatic leiomyosarcoma . again in 2006 , a case report was published describing a transdiaphragmatic adrenalectomy in patients with non - small cell lung cancer and these authors(4 ) and others previously(5 ) have theorized that due to the high frequency of lung cancer with ipsilateral adrenal metastasis a direct lymphatic connection must exist between the lung and the retroperitoneum . , improved survival from combined resection of pulmonary and adrenal lesions in cases of lung carcinoma has also been shown(6 ) indicating that when this clinical situation exists simultaneous resection may be considered as a viable option . a more recent study in 2005 by mercier et . al.(7 ) , has also confirmed improved survival in patients with non - small cell lung cancer . a small case series out of bnai zion medical center in 1996 looking at 13 patients discussed using the transthoracic approach for removal of solitary adrenal masses irrespective of a concurrent pulmonary mass(8 ) . this case highlights the use of a multidisciplinary team to resect simultaneous metastases to the lung and adrenal gland from cervical adenocarcinoma through one skin incision and demonstrates the possibility of accomplishing this when the tumor is locally invasive into major structures such as the ivc . prolonged survival has been shown in small trials related to primary lung cancer with sole adrenal metastasis if both lesions are resected . simultaneous spread of cancer to the adrenal gland and to the lung should not by itself exclude complete resection . admittedly , the chance for curative resection in this patient is certainly smaller compared to her chances for cure at her initial resection , however , she had tolerated 2 years of extensive chemotherapy without any advancement in her metastases by our most sensitive screening methods . this report is meant to highlight the technical feasibility of the operation and not the rationale . the rationale for proceeding was made on an individual basis after multiple conversations between the patient and her surgeons .

we present a 60-year old woman with recurrent cervical adenocarcinoma who presented with metastasis to both lungs and to her right adrenal gland . a thoracotomy was performed for resection of her pulmonary metastasis and then the right adrenal gland was excised through a trans - diaphragmatic approach . the adrenal gland resection was more complex due to involvement of the tumor with the inferior vena cava ( ivc ) which was repaired with a ptfe patch graft . this case demonstrates both an interesting approach to surgical resection of multiple metastases as well as a safe , although more challenging , alternative to partially resect and repair the ivc .

INTRODUCTION CASE REPORT DISCUSSION

PMC4852541

hand disability limits a patient 's activities of daily life , leading to reductions in self - sufficiency and deterioration of quality of life . this condition can be caused by injuries , surgery and diseases . recovering hand function requires pharmacological treatment together with a rehabilitation protocol including therapeutic exercises ( kinesiotherapy ) adapted to the patient 's specific needs . according to the american kinesiotherapy association , kinesiotherapy is the application of scientifically based principles to design specific exercises aimed to enhance the strength , endurance , and mobility of individuals with functional limitations which require extended physical conditioning . a kinesiotherapic exercise program , prescribed by health professionals , is often a necessary step for individuals who have been inactive , suffer a reduction in joint motion or muscle strength , are experiencing joint pain or are recovering from active disease , . active kinesiotherapy , consisting of both flexibility and strengthening physical exercises adapted to the special needs of patients with rheumatoid arthritis ( ra ) and systemic sclerosis ( ssc ) , has been demonstrated to be useful in recovering , maintaining and improving hand function in these rheumatic diseases , . ra is a systemic chronic inflammatory disease , primarily affecting synovial joints , mostly at the hands , characterized by the development of fibrosis in joints and tendons and by the progressive erosion of cartilage and bone , which can lead to substantial loss of functionality and , in late stage , deformities . ra has a worldwide distribution with an estimated prevalence of about 1% and mainly affects 3050-year - old females . ssc is an autoimmune disease that targets the microvasculature and leads to fibrosis in the skin , the muscoloskeletal system and internal organs . thickening of the skin of the hand and tendon retraction can result in contractures of the fingers , determining hand impairment . in such diseases , kinesiotherapy aims to : ( i ) strengthen the muscles and provide greater joint support reducing load and stress through the affected joints , ( ii ) maintain or improve the flexibility in affected joints and surrounding tissues , ( iii ) help reduce bone loss related to inactivity , inflammation and the use of specific medications ( e.g. , corticosteroids ) , . active kinesiotherapy would require constant monitoring by health professionals but often it is impossible to closely assist every patient , because of the reduced availability of specialized facilities and qualified staff . therefore it is common practice that active kinesiotherapy is performed at home using common objects ( e.g. , elastic bands ) and self - managed by patients after an appropriate training . such an unsupervised scenario leads to the impossibility of assessing whether the protocol is correctly followed or not , which has implications on its effectiveness . in this paper a solution mixing on a single device both a local and a remote monitoring system for hand kinesiotherapy is presented and evaluated . the device has been designed thanks to a tight cooperation between a bioengineering unit and specialists in rheumatology , starting from the current state of the art and introducing novel experimental protocols . the proposed low - cost device allows a simple and objective monitoring of the patients ' rehabilitation sessions through a set of custom sensorized tools . it can guide the patient by means of a simple user interface for home sessions , whereas for outpatient clinic evaluations the same device operates as a pc peripheral . the remote operating mode is supported by an embedded gsm / gprs module able to transmit the main statistics about the rehabilitation session to a tcp / ip server for deferred analysis . from there , data can be retrieved as needed for local analysis : this functionality is supported by a custom software framework . a clinical trial involving 20 patients with ra and ssc has been used as testbench for the proposed approach . during a period of 12 weeks the patients have been monitored exploiting the telemonitoring option in order to provide a support of information for the patient - doctor interaction . the patients ' hand functional evaluation has been performed with traditional methods and with the proposed device , revealing the effectiveness of the approach and highlighting some aspects that can be further studied in order to improve the reliability and reproducibility of the system . ii a brief analysis of the state of the art in the field is provided . section iii presents the design and implementation of the proposed device , whose external telemonitoring infrastructure is briefly described in section iv . the results obtained in the first clinical trial and the conclusions are presented in sections v and vi respectively . the most frequent approach is the definition of a series of exercises the patient can perform at home using objects of daily life such as flexo - extending the fingers with a counter resistance applied ( using a rubber band ) , pressing each fingertip against the thumb , repeatedly screwing in and unscrewing a screw with a screwdriver , rolling a newspaper page into a ball , and others . by their very nature , these exercises can not be easily standardized in order to ensure the correct execution at home . furthermore , relying on unsensorized objects , they can not be quantitatively monitored in local or remote scenarios for the evaluation of the functional deficit . this evaluation is rather performed in a clinical setting using biomechanical measurements such as the range of movement ( rom ) , hand extension and strength . also questionnaires gathering the patient 's perception of his or her disability , e.g. , the dreiser test are used for this purpose . some systems , such as the jamar dynamometer ( isometric ) and the vigorimeter ( dynamic ) , allow the clinical evaluation of the grip strength , whereas some others also allow the evaluation of the single finger pinch force ( e.g. , jamar pinch gauge ) . some devices such as the h500 h and kit by biometrics ltd . allow monitoring both of them in multiple repetitions . these devices are only suitable for one - shot functional assessment , as they are not designed as tools for rehabilitation purposes . the commercial pablo system by tyromotion gmbh , designed for neurological rehabilitation , allows performance of several rehabilitation exercises but only grip strength , single finger pinch force and indirect rom of the wrist can be measured for functional assessment of the hand . nowadays the majority of studies on hand rehabilitation are focused on post - stroke patients , who usually need to relearn the movements . for this purpose , many prototypical robotic systems have been presented , such as the handsome device for the pinch - grasp movement , the actuated thumb exoskeleton ( atx ) or the handexos wearable multiphalanges device . also , they are unsuited for rheumatic patients , who frequently present hand deformity caused by the illness 's progress . there are also some commercial robotic devices such as the amadeo system ( tyromotion gmbh ) and the hand mentor ( kinetic muscles inc . ) but their applications are constrained to specific movements , often requiring the support of specialized nurses , representing a justified approach only for paralyzed patients . some systems in literature exploit sensorized tools to allow the monitoring of active rehabilitation exercises . these systems are usually low - cost and easy to transport , so they are suitable for a telemedicine approach . for example , both the devices presented in and exploit a tracking system for the upper limb monitoring and a graphical user interface on a pc to guide the post - stroke patient in the exercise execution and to send the monitored data to the physician . the system presented in enables the evaluation of the follow - up of hand - transplanted patients . it includes a commercial sensorized hand glove ( humanglove by humanware s.r.l . ) to perform kinematic assessment of the hand and the fingers , and two devices for the mechanical evaluation of the force of the hand and fingers , and it is managed by an acquisition software on a pc with data storing onto a web server . these exercises are not specifically designed for the described dysfunction and the use of these sensorized gloves is not suitable for patients with hand deformity . another example of a telerehabilitation system has been presented in the european project hellodoc for the recovery of the upper limb in neurological patients . it includes a portable unit based on a desk installed at the patient 's home providing a computer and a set of sensorized daily life objects for the monitoring of some parameters during their use . such a device is closer to the one presented in this paper . in the last decade virtual reality has been introduced in rehabilitation devices , improving the quality of the patient interaction with the rehabilitation system . such systems require the use of a pc or a game console , which manages the rehabilitation session and the communication from the patient 's home to the physician 's pc through a wired connection , thus requiring a physical installation in the patient 's home . the majority of them require a great effort for both the physician in interpreting the acquired data and the patient in learning how to interact with the system . the first step of this research has been the development of a set of adapted physical exercises able to improve both the strength and the agility of patients with ra and ssc . taking into account the common peculiarities of the two diseases , ra and ssc , the strength exercises were conceived and evaluated with the goal of avoiding joint stress and overload . to this aim , all of them require isometric muscle contraction , and should be tailored by the patients at a pain - free intensity . during isometric contraction the muscular contractile component shortens and stretches the elastic component , without changing the joint angles involved in muscle work , thus preventing relapse of inflammation in affected joints . conversely , the agility and stretching exercises , which are obviously dynamic , should present the minimum counter - resistance in order to emulate a tool - free exercise . overall , the wrist should not be used as a lever but only as a support for the hand , leaving to the fingers the exercise effort , in every exercise . seven different exercises have been conceived , all of them requiring multiple series , each one composed of multiple repetitions of the same gesture . among them hand extensione6 . rotation ( fast manipulation)e7 . finger tapping ( piano - like ) e3 . finger tapping ( piano - like ) despite the differences between the two rheumatic diseases , the proposed set of exercises is able to cope with the needs of both the populations of ra and ssc patients , producing significant improvements in their strength , agility and dexterity , as will be clear in section v. at the same time , they probably could be successfully applied to other pathological conditions that require the same characteristics of the rehabilitation exercises , which may , open the proposed device to a wider application scenario . the exercises have been engineered with the sensorized tools depicted in fig . 1 and embedded in a portable device : 1)a tool for hand grasping exercises in pinch and grip ( e1 and e2 ) , which exploits a low - cost flexiforce a201 force sensor by tekscan ( max force in linearity response : 440n ) , pressed between the two halves of a handle covered with neoprene;2)a tool made of an aluminum plate which sustains a flexiforce a201 force sensor ( 110n ) . its sensible area can be pressed against the plate through a silicon pad , with a finger acting in opposition to the thumb ( e3);3)a tool with an exposed 5-lobe nylon handle which can not spin but it is internally fixed to a bar which in turn presses one of two flexiforce a201 force sensors ( 110n ) as a torque is applied clockwise or counterclockwise respectively , using the fingers only ( e4);4)a tool based on a moving metal structure , made of two rollers ( ces3088-zz by rollon ) on a rail for linear movement ( tes30 - 1040 by rollon ) , connected by an analog draw wire position sensor ( lx - pa-15 by tme ) which provides information on the relative distance between the rollers . the rollers are actuated by the patient , who inserts the hand between the two metallic profiles attached to them , by opening and closing the hand ( e5 ) . a small counter - resistance ( about 4n ) needed for the rope winding is opposed to the extension movement;5)a tool consisting of a multi - turn precision potentiometer ( vishay 534 , 10 rounds ) with an aluminum knob attached to its shaft . the resistant torque of the potentiometer is negligible ( 0.006 nm ) , providing a support for a low - resistance dynamic rotation exercise . the patient is asked to spin the knob using the fingers only , both clockwise and counterclockwise ( e6);6)a capacitive touch board for a finger tapping test ( piano - like ) . the tool comprises 8 sensible areas which are scanned continuously by an msp430f2013 microcontroller unit ( mcu ) in order to detect which areas have been touched . the patient is asked to touch 5 of the 8 available sensible areas with the fingertips in order from the little finger to the thumb as fast ( and precisely ) as possible ( e7 ) . 1.the different exercises and the related tools ( plus the temperature sensor ) supported by the proposed device . a tool for hand grasping exercises in pinch and grip ( e1 and e2 ) , which exploits a low - cost flexiforce a201 force sensor by tekscan ( max force in linearity response : 440n ) , pressed between the two halves of a handle covered with neoprene ; a tool made of an aluminum plate which sustains a flexiforce a201 force sensor ( 110n ) . its sensible area can be pressed against the plate through a silicon pad , with a finger acting in opposition to the thumb ( e3 ) ; a tool with an exposed 5-lobe nylon handle which can not spin but it is internally fixed to a bar which in turn presses one of two flexiforce a201 force sensors ( 110n ) as a torque is applied clockwise or counterclockwise respectively , using the fingers only ( e4 ) ; a tool based on a moving metal structure , made of two rollers ( ces3088-zz by rollon ) on a rail for linear movement ( tes30 - 1040 by rollon ) , connected by an analog draw wire position sensor ( lx - pa-15 by tme ) which provides information on the relative distance between the rollers . the rollers are actuated by the patient , who inserts the hand between the two metallic profiles attached to them , by opening and closing the hand ( e5 ) . a small counter - resistance ( about 4n ) needed for the rope winding is opposed to the extension movement ; a tool consisting of a multi - turn precision potentiometer ( vishay 534 , 10 rounds ) with an aluminum knob attached to its shaft . the resistant torque of the potentiometer is negligible ( 0.006 nm ) , providing a support for a low - resistance dynamic rotation exercise . the patient is asked to spin the knob using the fingers only , both clockwise and counterclockwise ( e6 ) ; a capacitive touch board for a finger tapping test ( piano - like ) . the tool comprises 8 sensible areas which are scanned continuously by an msp430f2013 microcontroller unit ( mcu ) in order to detect which areas have been touched . the patient is asked to touch 5 of the 8 available sensible areas with the fingertips in order from the little finger to the thumb as fast ( and precisely ) as possible ( e7 ) . the different exercises and the related tools ( plus the temperature sensor ) supported by the proposed device . additionally , a skin temperature probe ( ntc thermistor ysi 400 compatible ) has been integrated to measure the temperature of the hand ( between thumb and index ) at the beginning and at the end of the session . taking into account the two rheumatic diseases , this sensor has been included to evaluate the effect of the exercise session in ssc patients , where one of the objectives is to increase the blood inflow to the affected tissues . the choice of the sensors and the consequent design of the tools have been driven by simplicity for the patients and a good trade - off cost / performance . each tool , except the one for e7 which provides a digital signal and is accurately described in , is interfaced to the mcu in charge of controlling the device functionality through a signal conditioning stage , before digitalization . signal conditioning consists of low - pass active filters based on the tlv2375 operational amplifier . since all the signals coming from the sensors are unipolar , a single supply configuration without any biasing circuitry can be employed , thus limiting the stage complexity and the number of resistive elements , also reducing the mains interference . a single pole rc cell sets the cut - off frequency at 48 hz , acting as anti - alias filter for the sampling , performed at 150 hz by the mcu through the internal a / d converter in order to guarantee an adequate time resolution for the signal processing algorithms . the temperature sensor is inserted in the filter feedback line of an operational amplifier , in a circuit calibrated to measure a skin temperature in a range from 20 to 40c . all the flexiforce sensors are inserted between ground and the inverting input of an operational amplifier , making the stage a variable gain amplifier . providing to the non - inverting input a fixed voltage , obtained by means of a 0.5 v reference and an rc cell , the output voltage varies linearly with the force applied to the sensor . both the vishay 534 potentiometer and the tme lx - pa-15 draw wire sensor are inserted in a voltage divider , with the wiper connected to the non - inverting input of an operational amplifier through an rc cell , so that the output value is proportional to the voltage at the wiper . the signals coming from the tools equipped with analog sensors are processed exploiting three different algorithms , whose use depends on the shape of the signal to be processed . such a processing is useful for the online extraction of the statistical parameters related to the execution quality , presented for the different exercises in table i. the extraction of these parameters from the raw signals is briefly described hereafter . table irelevant parameters for the statistical summary associated to the different exercisesexercisesextracted parameters and related statisticsel , e2 , e3 [ e4 , e5] force [ torque , extension ] : max , min , avg , std repetitions duration ( only the active part ) : avg and std number of repetitionse6 rotation angle and rotation speed : avg , std exercise duration number of repetitionse7 duration of correct / wrong sequences : avg , std number of touches per second number of correct / wrong sequencesavg : average ; std : standard deviation . all the isometric exercises ( e14 ) and the extension one ( e5 ) yield a peak - shaped waveform , where each peak represents the patient 's action on the device . a threshold - based algorithm detects the peaks , extracting their magnitude , duration and distance . the minimum detection threshold for e14 has been defined by the specialists for the different exercises whereas for e5 it has been imposed by the designers . for e5 , the system performs a zero - calibration with the closed hand of the patient so that the effective extension is measured . e6 yields a terraced waveform , where each plateau represents the release phase ( when the patient releases the knob for the next repetition ) and the edges represent the phase when the patient spins the knob with his fingers ( as depicted in fig . 1 ) . the algorithm detects the edges and extracts their amplitude and duration , respectively leading to the rotation angle and speed , and the plateau duration . the signal segmentation involves the identification of parts where the amplitude is substantially constant , after a moving average smoothing . the signal processing for the tapping exercise e7 is based on the analysis of the digital words coming from the sensor by a masking procedure in order to recognize a valid sequence , allowing the patient to perform the exercise either leaving in place the finger after a touch ( but detaching them after the sequence has been closed by the thumb ) or detaching them ( one or more at a time ) after a touch . for the temperature , the algorithm detects when the signal becomes stable ( i.e. , its variation is under an empirical threshold ) and then extracts a stable voltage value proportional to the resistance by averaging a few samples in order to smooth the noise . the proposed device embeds all the aforementioned tools , providing all the electronics for the signal acquisition , processing and transmission . in order to guarantee both robustness and portability , an ultra low - power mcu , the msp430fg4618 by texas instruments , is used for managing the whole system . the device handles only the integer values coming from the a / d converter , limiting the number of computations induced by the fixed - point numerical representation on the mcu . the conversion into the corresponding physical quantities exploiting the calibration values embedded into the device software is performed either on the device ( only on the statistical summary , at the end of the whole session , just before data transmission ) or on the pc when a therapist is locally controlling the device in real - time . the underlying design idea is that the same computerized device should be able both to guide the patient in the execution of the exercises when he performs the kinesiotherapic session at home ( deferred telemonitoring mode ) and to execute the commands issued by a pc under the local supervision of a therapist ( real - time control mode ) . the latter operating mode is useful either for rehabilitation sessions performed under the direct supervision of a therapist or for the functional evaluation of the hand in the context of a therapeutic exercise . such a research is the natural consequence of a previous work in the field , which was limited in the number of exercises and did not include any telemonitoring support . fig . 3 shows how the different elements of the proposed system mutually interact , highlighting the area of interest of each user in the different scenarios . the communication features are implemented by means of an internal gsm / gprs module ( sim900 by simcom embedded on the 8100-tdggmsm_900 board ) interfaced to the mcu usart port and an external bluetooth one ( wt11-a - ai by bluegiga ) interfaced to the uscia port . the choice of an external bluetooth module is dictated by the need of providing such a functionality only to the therapist . the connection mean is a 25 poles d - sub female connector hosted on the front panel of the device ( also enabling the mcus programming ) . a 2 m cable allows putting the gsm / gprs antenna far from the metal case of the device and from the patient . notably , the system is battery powered so also a power connector ( for charging only ) , a charge led and low - battery led are included . the device has been designed including several security mechanisms against macro - shocks ( e.g. , no operation when charging ) and mechanical injuries ( e.g. , soft materials , rounded edges ) . even though it is always difficult to evaluate the final cost of a device , when it is in its prototypical form , from a simple estimation of the manufacturing costs including the materials , a value of about 700 euros per device can be computed . this estimate does not include the researcher 's and designer 's wages . when the device operates in deferred telemonitoring mode ( no bluetooth module connected at boot ) , it launches a programmed sequence of rehabilitation exercises and collects , during the execution , the statistics representative of the patient 's performance , storing them until the end of the session . the mcu firmware controls the patient interface on the device in order to assist him during the exercise execution , enabling a simple training experience . it consists of an on / off switch and two push - buttons ( for starting a series and for skipping a single repetition ) for the input , a buzzer and several leds for the output ( one led for every exercise , in order to pinpoint the exercise to be performed , 2 leds to identify which hand must be used , 2 leds for the first and second series , one led blinking at 1 hz for a time reference in the sustained exercises ) plus a multi - purpose 7-segment display . the latter is used alternately to show the number of correct repetitions performed within a series , the percentage of effort related to the full sensor range , the elapsed time for the temperature , and other exercise - specific feedbacks . in this operating mode , the raw signals are used to extract the performance parameters in real - time , but then they are discarded . at the end of the rehabilitation session , a local vector is filled with the related statistical summary and it is sent to the remote server via a tcp / ip connection established by the embedded gsm / gprs module . the data is sent as a binary stream without encryption since no sensible data are sent and the patient is identified only by the unique international mobile subscriber identity ( imsi ) code of the sim card hosted by the gsm / gprs module . if the communication aborts , after a limited number of trials the data is stored into the mcu flash memory , informing the user through the 7-segment display , allowing him to safely turn off the device . at the next switch on , if data are present in the flash memory a further forwarding attempt is performed , overcoming a temporary network or server malfunction . the role of the different actors involved in this scenario is depicted in fig . when the system is in real - time control mode ( which happens when , at switch on , the mcu detects the bluetooth module ) , after a handshake including the calibration constants transmission to the pc , the device puts itself in stand - by . on the pc , a standalone c++ software application based on the qt framework v4.7 and exploiting the third party library qextserialport for handling the communication with the pc 's bluetooth adaptor , gives to the therapist the possibility of controlling the device . as soon as an exercise is chosen , all the on - board resources are initialized and , after the exercise is started by the patient , both the raw signal and a vector containing the running statistics on the extracted parameters ( once per second ) are sent to the pc in real - time . the real signal , scaled by the host taking into account the calibration constants , is shown on the therapist 's real - time control application along with the running statistics , until the exercise is either aborted or completed . the patient interface on the device operates in the same way as in the deferred telemonitoring mode . during the experimental trial , the real - time control mode has been used for training the patients and for periodic outpatient clinic evaluations . the role of the different actors involved in this mode is depicted in fig . in order to enable the telemonitoring feature , the system has been completed with a server application and the therapist 's client application for the patient 's performance evaluation . both software components have been developed in c++ exploiting the support of the qt framework . in this implementation , the data sent by the devices are interpreted on a positional basis in order to store them in the database . to access the data , the client can build requests to create queries ( actually formed by the server after request decoding ) . the retrieved data is organized in frames , containing the parameters relative to a rehabilitation session of a single patient . to protect the database integrity , data downloaded from the server are stored in a portable file format ( .csv ) for further analysis in external programs . the monitoring application offers a bird 's eye view of the patient 's compliance by a table clearly showing when the patients have performed the rehabilitation session and when not . in a second window , the performances relative to each exercise for a given patient can be graphically visualized . these quantities can be used to gather clues on the quality of the rehabilitation session . for instance , in strength exercises the force values are connected to the execution speed , since the faster the action the lower the force . a clinical trial with a non - ce marked medical device , reviewed and approved by the italian public health department , has involved 10 patients affected by ra and 10 by ssc . since established guidelines for rehabilitative exercises actively performed by patients are still lacking , the trial was not designed to compare the results of the patients using the presented device and methods against those performing a standard rehabilitation . the primary outcome of this pilot study is the evaluation of the degree of functional improvement with respect to the baseline of the patients . the two groups were selected in order to have similar characteristics . according to the epidemiology of the single diseases , ages were between 39 and 74 ( median 55.5 ) for ra and between 44 and 73 ( median 56.5 ) for ssc . all the patients were trained in the autonomous use of the proposed device by the specialists and then the devices were entrusted to them for 12 weeks in order to perform the kinesiotherapy at home . the kinesiotherapic protocol was approved by the ethical committee of the aou of cagliari ( italy ) where the trial took place . it defined the order and the number of series / repetitions for each exercise and the number of weekly rehabilitation sessions ( 5 per week ) . as explained above , the protocol was the same for both ssc and ra patients . the trial was performed according to the principles of good clinical practice and the declaration of helsinki . in clinical practice , hand disability is assessed through the hand algofunctional dreiser index , the grip strength is measured by asking the patient to grasp a sphygmomanometer cuff inflated at the standard pressure of 20 mmhg , the joint rom ( related to the wrist and fingers flexion - extension ) using a goniometer and the hand extension ability on the plane . patients were evaluated in the outpatient clinic , by the same health professional , at the beginning ( t0 ) and at the end ( t2 ) of the trial using both the described standard indexes and an unique device identical to the one used at home for the everyday training for a more effective comparison . 8 and 9 show the percentage variation ( with respect to t0 ) of these indexes at t2 , using the dominant hand ( the right for all the patients ) . the box plot highlights the median value and the 25th and 75th percentiles , whereas the whiskers span the range of the outermost samples not taken as outliers ( that in turn are identified as crosses in the graph ) . in order to highlight those parameters showing a significant within - subjects improvement throughout follow - up , the pre - test vs. post - test results were compared using the paired sample wilcoxon test . statistically significant values were considered for a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p$\end{document } value less than 0.05 and they have been marked in figures with an asterisk . 8.box plot of the percentage variation of some functional hand parameters at the end of the trial ( t2 ) with respect to the beginning of it ( t0 ) . romf : rom finger flexo - extension ; romw : rom wrist flexo - extension . 9.box plot of the percentage variation of some functional hand parameters at the end of the trial ( t2 ) with respect to the beginning of it ( t0 ) . ctorque : clockwise rotation torque ; atorque : anticlockwise rotation torque ; 1st4th force : fingers force in opposition to the thumb . box plot of the percentage variation of some functional hand parameters at the end of the trial ( t2 ) with respect to the beginning of it ( t0 ) . romf : rom finger flexo - extension ; romw : rom wrist flexo - extension . box plot of the percentage variation of some functional hand parameters at the end of the trial ( t2 ) with respect to the beginning of it ( t0 ) . ctorque : clockwise rotation torque ; atorque : anticlockwise rotation torque ; 1st4th force : fingers force in opposition to the thumb . the increase in the four indexes ordinarily used in clinical practice ( rom fingers , rom wrist , grip and extension ) , as depicted in fig . 8 , highlights the actual improvement of the patients ' physical performance at the end of the clinical trial . the same increasing trend is shown by the other parameters in the two figures , measured with the proposed device , proving the significance of the proposed method according to the standard evaluation methods . the discrepancies between the grip and extension results , with or without the device , can be ascribed to the different modalities that characterized the test execution . in fact , the device measures an isometric grip force whereas a non - isometric grip is performed during the traditional assessment . similarly , hand extension measurement with the device imposes a counter - resistance , not present in the traditional assessment . in order to use a typical clinical index for the evaluation of the patient 's health perception , the percentage reduction of this index for both ra and ssc patients is depicted in fig . it should be noted that the reduction of this index in t2 , with respect to t0 , corresponds to a better perceived hand functionality , so the result in fig . 8 is expressed as a positive percentage variation even though actually such an index decreased over time . the proposed device allows the evaluation of other strength / agility parameters not used in the clinical practice , whose percentage variations are shown in fig . the first three indexes highlight an increasing trend in clockwise / counterclockwise rotation torque ( e4 ) and in the finger tapping ( e7 ) speed . the improvement of the latter index is due both to the improvement of patients ' agility and to their ability in learning how to execute the exercise , proved by the correlated increase of the right number of touches . the last four indexes , representing the trend of the finger pinch exercise ( e3 ) , show worse results . these are supported by the telemonitoring data which show that the time spent in the exercise and the performance tend to decrease after the very first days , becoming stable , despite the overall improvement in the hand functionality . further investigations revealed that the exercise was perceived as being really difficult for almost every patient . since the proposed threshold technique gives the patient a feedback ( a chiming buzzer ) as soon as the minimum required effort is reached , the patients were prone to execute this exercise with the smallest effort , avoiding applying the maximum force . in a future trial such a feedback will be modified in order to avoid this issue , providing a goal for the patient . we also observed that the values of dynamic rotation angles ( e6 ) , not presented here , are not realistic , since the patients tend to cheat the exercise when rotating the knob , spinning it in order to exploit its inertia thus achieving very large angles , above physiological limits . this is completely perceivable from the remote monitoring and has been confirmed by the periodic evaluations of the patients . such a result highlights the limits of an autonomous home rehabilitation system which can be cheated by non - conscientious patients . the introduction of a counter resistance in the rotation knob would avoid the free rotation , thus reducing this problem . it will be interesting to see whether such a hardware control is able or not to provide the same results that a doctor - patient interaction would in the light of the telemonitoring data . although a study on acral temperature regulation would require taking into account several influencing factors , exploiting the embedded temperature sensor we observed an increasing trend of the mean temperature difference \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta t={\rm t}_{end}-t_{start}$\end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t_{end}$\end{document } is the temperature after the rehabilitation session and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t_{start}$\end{document } that before it . the results of our analysis are presented in table ii . as can be seen , the results suggest the efficacy of the kinesiotherapy approach implemented in the proposed device in increasing blood inflow to the hand , which was one of the goals of the therapeutic exercises for the ssc patients . they , starting from lower values of the temperature , achieve a larger improvement compared to the ra patients , who do not present such a microcirculation problem . table iiacral temperature analysisgrouphandpre - exercise [ c]post - exercise [ c]p valuesscdx\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 30.6 ( \pm 1.3)$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 32.3 ( \pm 1.6)$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ < 0.0007$\end{document}sx\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 30.9 ( \pm 1.6)$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 33.0 ( \pm 1.3)$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ < 0.0001$\end{document}ardx\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 32.5 ( \pm 2.3)$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 34.1 ( \pm 1.5)$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ < 0.0005$\end{document}sx\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 33.6 ( \pm 2.1)$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 34.9 ( \pm 1.3)$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ < 0.0057$\end{document}average ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \pm$\end{document } standard deviation ) average ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \pm$\end{document } standard deviation ) from a clinical perspective , the effectiveness of the kinesiotherapic sessions was demonstrated by the findings of a statistically significant improvement in both subjective ( dreiser 's index ) and objective ( e.g. , rom , strength ) parameters . at the end of the follow - up both ra and ssc patients showed an increased average amplitude of the movements ( in e5 , e6 ) , a higher speed of execution ( e5 , e6 , e7 ) and a better ability to perform fast and precise movements ( e7 ) . moreover , an increased endurance was developed , as demonstrated by the increased average duration of the strength exercises ( e1 , e2 , e4 ) despite a stable average of the amplitude . these satisfactory results might also be ascribed to a very high compliance of the patients to the exercise protocol . the exception represented by e3 may be in part related to the medical need to keep the exercise intensity at a pain - free level . it can be noticed that the patients enrolled in the ra and in the ssc group achieved different results . 8 and 9 , there is a difference in the between - subject results achieved by patients from the same group . this should not be surprising and must be attributed to the biological variability of the subjects and to the fact that the best results can only be achieved through a personalized rehabilitation program . however , for the purpose of this pilot uncontrolled clinical trial , aimed at evaluating the feasibility and effectiveness of the proposed approach , we chose to define the working parameters at the beginning and not change them over the duration of the trial in order to effect a more rigorous evaluation of the method . as a consequence , not enabling any protocol personalization , we could not ensure an equal level of improvement . starting from the achieved results , further studies , based on adjustable working parameters , will address the effectiveness of telemonitored personalized rehabilitation programs . in this paper we presented the conception and evaluation of a portable device for both local hand evaluation and remote monitoring of hand kinesiotherapic sessions . the device , expressly designed thanks to a tight cooperation between bioengineers and rheumatologists , aims at solving some problems of the current techniques in the kinesiotherapic treatment for rheumatic patients , in particular those affected by ra and ssc . the improvement of patients ' conditions was found in accordance with the standard clinical evaluation and the patients expressed their satisfaction in the quest questionnaire . 15 out of 20 patients indicated both efficacy and user - friendliness as the main positive aspects of the device , and the related quest scores were 4.22 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( { \pm}{0.81})$\end{document } and 4.90 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( { \pm}{0.32})$\end{document } , in a scale between 1 and 5 . about one half of the patients asked about the possibility of continuing with the device after the end of the clinical trial , and none of the enrolled patients reported augmented pain associated with the rehabilitation with the proposed device . from a health - care perspective , the experimental device represents an effective and safe tool for assessing hand disabilities in patients affected by rheumatic diseases with hand impairment and provides an innovative approach to monitor kinesiotherapic home exercise in those patients who do not have access , for various reasons , to specialized centers . in future studies we plan to address the issues that arose in the clinical trial , related to the autonomous use of the device , adding novel features and studying from different perspectives the approach to telerehabilitation in patients with these kinds of disabilities

current clinical practice suggests that recovering the hand functionality lost or reduced by injuries , interventions and chronic diseases requires , beyond pharmacological treatments , a kinesiotherapic intervention . this form of rehabilitation consists of physical exercises adapted to the specific pathology . its effectiveness is strongly dependent on the patient 's adhesion to such a program . in this paper we present a novel device with remote monitoring capabilities expressly conceived for the needs of rheumatic patients . it comprises several sensorized tools and can be used either in an outpatient clinic for hand functional evaluation , connected to a pc , or afforded to the patient for home kinesiotherapic sessions . in the latter case , the device guides the patient in the rehabilitation session , transmitting the relevant statistics about his performance to a tcp / ip server exploiting a gsm / gprs connection for deferred analysis . an approved clinical trial has been set up in italy , involving 10 patients with rheumatoid arthritis and 10 with systemic sclerosis , enrolled for 12 weeks in a home rehabilitation program with the proposed device . their evaluation has been performed with traditional methods but also with the proposed device . subjective ( hand algofunctional dreiser 's index ) and objective ( rom , strength , dexterity ) parameters showed a sustained improvement throughout the follow - up . the obtained results proved that the device is an effective and safe tool for assessing hand disability and monitoring kinesiotherapy exercise , portending the potential exploitability of such a methodology in clinical practice .

Introduction State of the Art Design of the Kinesiotherapy Device The External Telemonitoring Infrastructure The Clinical Trial Results Conclusion

PMC4285032

the rise in the prevalence of obesity over the past decade has become an increasingly relevant issue because this medical condition is an important risk factor for type 2 diabetes mellitus ( t2 dm ) and cardiovascular disease ( cvd ) , which may lead to a higher incidence of all - cause mortality . approximately 30% of obese individuals do not have metabolic syndrome and are thus at a lower risk of developing t2 dm and cvd ; these individuals are thought to exhibit metabolically healthy obesity ( mho ) , which is an obese phenotype free of any metabolic abnormalities . the phenotypes of mho , metabolically unhealthy obesity ( muho ) , and nonobesity have been recognized since the 1980s but between the years of 1980 and 2000 epidemiological research demonstrated that individuals who were overweight or obese did not always exhibit higher rates of cvd and mortality . subsequently , there has been an increase in the number of studies attempting to characterize the mho phenotype , which is characterized by the absence of metabolic parameters such as dyslipidemia , insulin resistance , hypertension , and unfavorable inflammatory profiles despite the presence of an elevated body mass index ( bmi ) . several diverse definitions of the mho phenotype have been proposed based on the presence of various metabolic abnormalities in a subject . accordingly , the prevalence rate of mho in obese individuals varies from 10% to 25% based on the criteria and populations that are used by a particular study . despite the enhanced interest and increasing number of studies investigating mho , there are no unique criteria that can be used to define mho and , as a result , the implications of the mho phenotype and even its very existence are still under debate . thus , the present review discusses the various definitions , pathophysiologies , and clinical implications of mho as well as the debate regarding its existence . individuals with mho display a favorable metabolic profile that is characterized by a high level of insulin sensitivity , a low incidence of hypertension , favorable lipid profiles , satisfactory fat distribution , and a low level of systemic inflammatory responses . it has been suggested that the mho phenotype exists , even though there are large variations in its prevalence rate , as a function of the criteria used to define it . several definitions are currently used to account for metabolic health ( table 1 ) and most authors define mho using measures of blood pressure , lipid status ( mainly high density lipoprotein cholesterol and/or triglycerides ) , glycemic and insulin resistance statuses ( fasting blood glucose levels or homeostasis model assessment [ homa ] ) , and systemic inflammation ( as defined by high - sensitivity c - reactive protein levels ) . however , it is difficult to compare the findings of various studies because there is no accepted standard definition of metabolic health and different studies use different inclusion criteria and/or cutoff points for metabolic measures . among the inclusion criteria used in various studies , there is a diversity of adiposity measures although they typically include waist circumference ( wc ) , bmi , and body fat percentage ( bf% ) . the prevalence of metabolic health was investigated in a population of swedish subjects using six sets of criteria for obesity , which included measures such as bmi , abdominal obesity , and bf% . based on the different sets of criteria , the prevalence of mho ranged between 3.3% and 43.1% in men and 11.4% and 57.5% in women . similarly , the proportions of mho individuals among obese participants were 6.8% when using the criteria of aguilar - salinas et al . , 14.2% when using the criteria of karelis et al . , 23.7% when using the criteria of wildman et al . , 30.2% when using the criteria of meigs et al . , and 36.6% when using homa . furthermore , the agreement of mho classifications among these studies was poor . likewise , the proportions of mho individuals in a korean population were 24.2% when using the criteria of meigs et al . 28.5% when using the criteria of karelis et al . , and 59.7% when using the criteria of wildman et al . . these comparative studies illustrate the considerable variability that exists in estimations of the incidence of mho when different sets of criteria are used to define metabolic health . despite the controversy surrounding these definitions and doubts concerning the existence of the mho phenotype , a characterization of mho individuals represents an important tool that can be used to evaluate the contribution of various types of fat distribution to the development of metabolic diseases and cvd . additional studies and a consensus regarding the mho phenotype are necessary in order to determine the manner in which this tool can be used to assess the cardiometabolic status of a patient in relation to appropriate fat accumulation . it is likely that he mho phenotype is the result of several underlying mechanisms and the interaction between genetic , environmental , and behavioral factors ( fig . 1 ) . each of these factors affects abdominal fat distribution , visceral and ectopic fat accumulation , and insulin resistance , which are all important causative factors that contribute to the development of unhealthy obesity . in some instances , the concept of the mho phenotype is used interchangeably with the notion of insulin - sensitive obesity because mho subjects display better insulin sensitivity than muho subjects . the plasticity of adipose tissue , which allows for the storage of the excessive fat that is accumulated by obese individuals , may be the primary factor that discriminates healthy obesity from unhealthy obesity . the amount of visceral fat mass in insulin - resistant obese individuals is independently associated with bmi and total body fat mass while an artificial increase in the subcutaneous fat mass of rodents results in positive metabolic effects . in muho individuals , the storage capacity of adipocytes may be exceeded and lipids can accumulate ectopically in visceral fat depots , liver and muscle cells , and pancreatic -cells whereas the subcutaneous adipose tissue of mho individuals possesses the ( intrinsic ) propensity to expand which , in turn , leads to a preservation of insulin sensitivity . additionally , mho subjects exhibit a greater proportion of subcutaneous fat compared with muho subjects . therefore , the plasticity of adipose tissue that allows for expansion in response to excess fat in obese individuals with proper angiogenesis is important for healthy obesity . the interaction between genes and the environment will also matter in the development of mho . fat mass , fat distribution and the number of adipocytes are heritable traits for which genes account for between 25% and 70% of the observed variability . a number of studies have attempted to determine whether there would be a common genetic influence of these two phenotypes in addition to their gene - specific effects associated with fat mass and visceral fat . developmental genes such as hoxa5 , gpc4 , and tbx15 exhibit changes in expression that are closely correlated with patterns of fat distribution , and b6j mice gain more weight , have higher levels of insulin and leptin , and show a greater degree of glucose intolerance than 129j mice on a normal diet . kulkarni et al . generated mice from three genetic backgrounds ( c57bl/6 [ b6 ] , 129sv , and dba ) that were double heterozygous knockouts of the insulin receptor and insulin receptor substrate-1 and found that 90% of b6j mice developed diabetes while less than 5% of 129j mice developed diabetes . additionally , a genome - wide scan of an intercross between these strains indicated that at least four loci on three different chromosomes are involved in this process . in a study of obesity - discordant monozygotic twins ( age range , 22.8 to 35.8 years ) , approximately half of the obese co - twins exhibited a typical response to obesity that was characterized by marked insulin resistance , dyslipidemia , and fatty liver tissue whereas the other half of the obese co - twins were as metabolically healthy as their lean co - twins . in the same study , the mho group exhibited a very low percentage of liver fat and the maintenance of mitochondrial function in conjunction with the absence of inflammation in subcutaneous adipose tissue . the mho phenotype may also be characterized by the presence of adipose tissue with reduced secretory capabilities or diminished responsiveness to the effects of adipokines . it has been shown that fat distribution and differences in the adipocytokine secretory properties of fat depots are likely play an important role in the outcomes associated with the mho phenotype . furthermore , changes during the adipose expansion process can influence the secretion of good adipokines , such as adiponectin , which promotes insulin sensitivity , decreases inflammation , and enhances cell survival . in fact , obese co - twins who are as metabolically healthy as their lean co - twins exhibit a disproportionate increase in circulating leptin levels and a lower expression level of adiponectin in adipose tissue . low systemic inflammation may be another key feature of the obesity status of an individual . for example , of 44 obese ( bmi > 30 kg / m ) patients who underwent laparoscopic bariatric surgery , the muho patients had a less favorable inflammatory profile in their visceral adipose tissue that resulted from the infiltration of proinflammatory macrophages , which exhibited increased nucleotide - binding oligomerization domain - like receptor family pyrin domain - containing 3 inflammasome activity and interleukin-1 production . adipose tissue also acquires immunological properties via the infiltration of activated macrophages , neutrophils , and t- and b - cells and these immunometabolic interactions lead to low - grade inflammation . in fact , the involvement of the immune system in obesity - related metabolic diseases in humans leads to a specific t - cell signature in adipose tissue . there are additional mechanisms and various differences that distinguish the mho and muho phenotypes , such as improved mitochondrial function , increased aerobic fitness , and a greater degree of insulin sensitivity ( fig . 2 ) , but the precise mechanisms supporting the mho concept remain to be elucidated . a number of studies have reported that the mho phenotype is associated with increases in mortality , cvd , t2 dm , and non - alcoholic fatty liver disease . in a prospective cohort study of 22,654 individuals between the ages of 20 and 59 years ( average duration , 13.4 years ; epic - morgen ) , the mortality risk of metabolically healthy abdominal obese ( mhao ) subjects was investigated using the criteria of the adult treatment panel iii report of the national cholesterol education program criteria and a definition of obesity based on wc . these authors found that the mortality risk of mhao individuals was approximately 40% higher than that of metabolically healthy non - abdominal obese individuals . however , regardless of the definition , mho and metabolically abnormal obese individuals have been found to have an increased risk of mortality relative to metabolically healthy normal - weight individuals . a 30-year follow - up cohort study of obese men with and without metabolic syndrome identified a 2.4- and 1.7-fold higher risk of mortality , respectively , compared to normal - weight subjects without metabolic syndrome . these studies suggest that the mho phenotype may not accurately predict increased mortality . there is also conflicting evidence regarding the association of the mho phenotype with subclinical markers of atherosclerosis . compared to mho subjects , muho subjects exhibit a significant increase in the odds ratio ( or ) for subclinical atherosclerosis as assessed by coronary artery calcium ( cac ) scores . in a similar population , mho subjects exhibited a significantly increased or for cac scores , which were attenuated when metabolic risk factors were adjusted for . these findings suggest that the definition of the mho phenotype should include the absence of any metabolic components . however , when mho individuals were followed up during longitudinal studies investigating increases in the risks of cvd and all - cause mortality , there were conflicting findings . a 7-year study found that mho subjects were not at an increased risk of cvd or all - cause mortality compared with healthy non - obese individuals while another study identified a higher prevalence and a greater severity of angiographic coronary artery disease among muho and normal - weight subjects , as compared to mho and normal - weight subjects . an extended follow - up period ( > 15 years ) revealed that obese participants without metabolic syndrome at baseline had an increased risk of major cvd events compared with non - obese subjects who were healthy at baseline . in a norwegian cohort of more than 60,000 people who were free of cvd , the findings of a 12-year follow - up period revealed that there was an increased risk of acute myocardial infarction among muho individuals and that obesity was more important than metabolic factors in the development of heart failure . a majority of the studies investigating the relationship between the mho phenotype and the risk of t2 dm are longitudinal . during a 20-year follow - up period conducted by the uppsala longitudinal study of adult men ( ulsam ) study , the risk of diabetes was significantly increased in normal - weight individuals with metabolic syndrome ( or , 3.28 ) , overweight subjects without metabolic syndrome ( or , 3.49 ) , overweight subjects with metabolic syndrome ( or , 7.77 ) , obese subjects without metabolic syndrome ( or , 11.72 ) , and obese subjects with metabolic syndrome ( or , 10.06 ) , as compared to normal - weight subjects without metabolic syndrome . similarly , another study found that mho individuals were at a threefold increased risk of developing diabetes but only if they progressed to an unhealthy phenotype during the follow - up period ( 5.5 to 10.3 years ) . in a study of mexican - americans and non - hispanic whites ( san antonio heart study ) , both metabolically unhealthy normal - weight and mho individuals had a 2.5-fold increased risk of developing diabetes . likewise , middle - aged asian mho individuals had a higher risk of developing t2 dm than their non - obese counterparts during a 5.4-year study . however , in a study of 6,748 koreans with a 4-year follow - up period , mho subjects did not have an increased risk of t2 dm compared with metabolically healthy non - obese subjects , which suggests that metabolic health is a more important determinant of the development of diabetes than obesity . as mentioned above , a number of studies have provided strong evidence supporting the existence of the mho phenotype . however , debate remains whether mho individuals are truly healthy , especially because no consensus has been reached regarding an accepted definition of mho or the influence of this phenotype on morbidity and mortality . nonetheless , the mho - like phenotype does not appear to be associated with a significant increase in the risk for cvd especially because , as in the ulsam study , an increased risk for cvd was observed in normal - weight subjects with metabolic syndrome , obese subjects without metabolic syndrome , and obese subjects with metabolic syndrome when compared to normal - weight individuals without metabolic syndrome . however , that study did not support the existence of an mho phenotype when defining this concept based on the absence of metabolic syndrome . very recently , a systematic review of eight studies ( n=61,836 subjects ) investigated the associations of bmi and metabolic status with total mortality and cardiovascular events and found that mho individuals have an increased risk of cardiovascular events , as compared to metabolically healthy normal - weight individuals . this study also revealed that all metabolically unhealthy subjects , including normal - weight , overweight , and obese individuals , had a similarly elevated risk of cardiovascular events . the researchers concluded that obese individuals are at increased risk for adverse long - term outcomes , even in the absence of metabolic abnormalities , compared with metabolically healthy normal - weight individuals and emphasized that there is no healthy pattern of increased weight . however , the findings of the present meta - analysis should be interpreted with caution as there are several limitations inherent in this study . the majority of studies included in this review provided relatively inadequate information regarding the health behaviors of the participants , they did not present data concerning weight gain in the subjects , they tended to focus only on total mortality and cardiovascular events , and they did not include older participants . according to a very recent editorial by hill and wyatt , along with the urgent focus to treat obesity regardless of the presence or absence of metabolic risk factors , it is important to reduce the long - term risk of death and cardiovascular events , as with any other chronic disease . however , the findings of the present meta - analysis indicate that any interventions or attempts to treat obesity should target not only the reduction of weight but also the type and proportion of fat that should be reduced . additionally , the manner in which this is achieved is important because the treatment of obesity without any consideration of the metabolic risk factors is not likely to improve the lives of patients . the implications and very existence of the mho phenotype are still associated with controversy due to the discrepancies among studies regarding the criteria used to define obesity , the experimental designs of these studies , and the ethnicity of the subjects included in these studies . although the clinical implications of mho , such as cardiovascular mortality , cvd , and the development of diabetes , can not be conclusively determined , the concept of a mho phenotype is important as a tool for the stratification of patients at a high risk for metabolic and cvds . however , a more unified set of criteria that can be used to define metabolic health needs to be established and clinical efforts aiming to treat obese patients should target reductions in body weight as well as improvements in metabolic risks . if this goal can not be accomplished , it would be like running from athens to marathon without carrying the message box .

metabolically healthy obesity ( mho ) is a new concept in which an individual may exhibit an obese phenotype in the absence of any metabolic abnormalities . there are a number of definitions of mho that utilize a variety of components . the findings of clinical and basic studies indicate that subjects with mho do not exhibit an increased mortality , an increased risk of cardiovascular disease , or an increased risk of type 2 diabetes mellitus , as compared to normal - weight controls . although these findings imply that metabolic health is a more important factor than obesity , several studies have shown that subjects with mho have a similar risk of metabolic or cardiovascular diseases as those with metabolically unhealthy obesity . thus , there is still debate regarding not only the implications of the mho phenotype but its very existence . accordingly , future studies should focus on developing a unified definition of mho and distinguishing subjects who will be at a high risk for metabolic and cardiovascular diseases .

INTRODUCTION THE MHO PHENOTYPE: DEFINITION MATTERS PATHOPHYSIOLOGY OF MHO CLINICAL IMPLICATIONS OF MHO: MORTALITY AND CARDIOVASCULAR DISEASE CLINICAL IMPLICATIONS: T2DM MYTH OF MHO CONCLUSIONS

PMC3346993

it is used for severe staphylococcal and streptococcal infections in patients intolerant of -lactam antibiotics , in methicillin - resistant staphylococcal infections , and against gram - positive organisms with multiple resistances to antibiotics . vancomycin has also been reported to cause concentration - dependent renal toxicities , and therefore monitoring of plasma vancomycin concentration is essential to obtain sufficient efficacy and to prevent toxic side effects . on the other hand , as a narrow spectrum antimicrobial the presence of significant in vitro synergy ( time - kill method ) between levofloxacin and vancomycin against clinical isolates of penicillin resistant pneumoniae was encouraging . this case demonstrates that high - dose levofloxacin in combination with vancomycin is well tolerated and appeared to be efficacious in a patient with refractory multiresistant pneumococcal pericarditis . vancomycin plus cephalexin [ 1 , 47 ] is also current standard therapy of skin and skin - structure infections ( cssis ) caused by gram - positive bacteria . chloramphenicol combining with vancomycin against vancomycin - resistant enterococci ( vre ) is also considered . monitoring of vancomycin and cephalexin concentration in human plasma is essential for obtaining sufficient efficacy as well as preventing side effects . various chromatographic methods have been developed for the determination of vancomycin individual in plasma samples . those include hplc with uv detection [ 914 ] , lc / ms [ 1517 ] , lc with electrochemical detection , and micellar electrokinetic capillary chromatography . furuta et al . reported the direct injection method of plasma sample using a semipermeable surface packing material column . however , these methods are relatively complicated and inconvenient , and the peak of vancomycin in the chromatogram is often not clearly separated from plasma blank peaks . in this case , quantification with univariate calibration will become biased and inaccurate . at the same time , simultaneous quantitative analysis of vancomycin and its combination therapy drug will make the analysis more difficult . however , by analyzing the hplc - dad data of unresolved peaks incorporating chemometric tools , it is possible to resolve those questions . ideally , each chromatographic peak corresponds to a single compound for hplc analysis in optimized separation conditions . generally , the retention factor ( k ) of the different analytes is between 1 and 20 allowing their complete separation . a peak with k equal to 0 is a component that does not interact with the stationary phase and elutes in the void volume . chromatographic separations can become a difficult task when the samples are environmental and biological ones which have a complex matrix effects . particularly , the chromatographic resolution generally becomes poorer and partially separated peaks often occur when the speed of chromatographic analyses is increased , for example , by using higher flows , shorter columns , and so forth . on the other hand , it will be time - consuming and material - costing when completely chromatographic separation is carried out for the complex samples . at this time , applying mathematical separation as a complementary of chromatographic separation [ 21 , 22 ] for resolving overlapping peaks is very promising . in chemometrics , three - way data analysis method can be used to resolve this question . for hplc - dad , dad can record the uv - vis spectra at every retention time , and a matrix ( elution time wavelength ) is obtained for every sample analyzed . the spectra of the calibration samples are measured at the different retention times of the peak of the analyte of interest and the retention times are selected by analyzing either a pure standard or a sample with a known added concentration of the analyte . the spectra of the predicted samples are measured at the different retention times of the peak from the unknown sample . then the concentration of the analyte of interest in the overlapping peaks can be quantified by applying second - order calibration algorithms to the cube matrix . in this paper , simultaneous and direct quantitative analysis of vancomycin and cephalexin in human plasma samples by using hplc - dad coupled with second - order calibration algorithms was developed . the calibration results obtained with parafac - als and self - weight alternative trilinear decomposition ( swatld ) were compared . these methods allow quantifying directly vancomycin and cephalexin concentration in complex human sample matrices without being influenced by the interference from the components . in the following part , bold capital letter for matrices , bold lowercase for vectors , and italics for scalars were used . the mathematical formulation of hplc - dad for n components samples can also be expressed as follows : ( 1)xijk=n=1nainbjnckn+eijk(i=1, ,i ; j=1, ,j ; k=1, ,k ) , where xijk is the intensity of the kth sample at ith chromatographic retention time and at jth wavelength . ain , bjn , and ckn are the elements of the loading matrices a , b , and c , respectively . a and b are the spectra and chromatographic profiles matrices for all n components , respectively . these can be expressed as a = ( a1 , a2 , , an ) , b = ( b1 , b2 , , bn ) , and c = ( c1 , c2 , , cn ) . eijk is the element of the measurement error matrix which contains the variation not captured by the model . it can be viewed as an extension of beer 's law to second - order data . this amounts to assuming that the measured peak is the sum of the individual peaks of each analyte and that the profile and the spectrum of one analyte are proportional in all the samples . as a consequence , the decomposition of a three - way data array built with response matrices measured for a number of samples is often unique , allowing chromatographic profile and spectral profiles , as well as relative concentrations of individual sample components to be extracted directly . they include the generalized rank annihilation method ( gram ) , direct trilinear decomposition ( dtld ) , parallel factor analysis ( parafac ) , alternating trilinear decomposition ( atld ) , coupled vectors resolution ( cover ) , and self - weighted alternating trilinear decomposition ( swatld ) , multivariate curve resolution coupled to alternating least squares ( mcr - alss ) . due to the possibility of making analytical determinations even in the presence of nonmodelled interferents ( this is known as the second - order advantage ) and to identify the analyte of interest , three - way algorithm is becoming increasingly important in routine analysis . in the second - order calibration algorithms that allow quantification even in the presence of noncalibrated components , the parafac - als and swatld methods [ 3234 ] parafac is a generalization of principal component analysis ( pca ) to higher orders . in parafac , each component is trilinear , in contrast with bilinear pca , where one score and one loading vector are obtained for each component . three loading vectors ( an , bn , cn ) are therefore given for each parafac component . the parafac model of a three - way array is found by minimizing the sum of the squares of the residuals eijk . each parafac component gives three loadings : one relates to the chromatographic profile ( an ) , one relates to the spectral profile ( bn ) , and one relates to the content of the samples ( cn ) . als successively assumes the loadings in two modes and then estimates the unknown set of parameters of the last mode . the algorithm converges iteratively until the relative change in fitting between two iterations is below a certain value ( the default is 10 ) . it is initialized by either random values or values calculated by a direct trilinear decomposition based on the generalized eigenvalue problem . constraining the parafac solution can sometimes be helpful in terms of the interpretability or the stability of the model . the resolution of spectra used to require the nonnegativity constraint since negative spectral parameters do not make sense . the parafac algorithm is based on a least - square minimization , whereas swatld uses a procedure known as alternating trilinear decomposition . , the swatld algorithm has the advantages of fast convergence and insensitivity to the excess factors used in calculations . according to some experience there is a more detailed explanation of the algorithm in [ 29 , 35 ] . all computer programs were written in the matlab ( mathworks ) programming environment , and all calculations were carried out on a personal computer ( pentium iv processor ) . cephalexin was purchased from national institute for the control of pharmaceutical and biological products in changsha . the stock solution was prepared by dissolving the vancomycin and cephalexin in the doubly distilled water , respectively . human plasma samples received from the center of blood in changsha obtained from the healthy people were used to prepare predicted samples . six of 2 ml samples of plasma were combined with various concentrations of the vancomycin and cephalexin stock solutions and ethyl acetate were added . thereafter , the mixture was shaken in a vortex for 30 s and centrifuged at 3500 rpm for 5 min , and the organic phase was evaporated , respectively . then , doubly distilled water was added to these flasks in order to obtain 10 ml solution . the plasma fraction was collected and stored at 4c until analyzed . throughout the analysis was carried out using an hplc system ( agilent series 1100 , agilent technologies , palo alto , ca , usa ) , which consists of vacuum degasser , autosampler , and a binary pump , equipped with a zorbax eclipse xdb - c8 analytical column ( 125 4.0 mm ; 5 um ) . theoretically , the analytes of interest can be determined precisely by changing the experimental conditions to achieve full resolution . this involves spending time and resources , and there is no guarantee that the separation will be complete . according to our experiments , the completely separation of vancomycin and cephalexin is difficult to carry out . it is also noticed that the chromatographic peak shape of vancomycin will become wide when we try to separate vancomycin and cephalexin by adjusting the mobile phase . mobile phases a and b were methanol and 0.1 m disodium hydrogen phosphate buffer ( 40/60 v / v % ) , respectively . the flow - rate used was kept at 1.0 ml / min in each study . the wavelength used was 200.0 nm to 380.0 nm with an interval of 2.0 nm . the run time used was in the range of 0 min~5 min ( 1/30 min intervals ) . the baseline effect is compensated by subtracting the measurement matrix of an average blank from the sample measurement . sixteen samples are divided into two sets ; that is , the first ten samples are the calibrated sets ( no human plasma ) and the remaining six are the predicted set . the human plasma was only added to six predicted samples ( drug - free plasma ) . after eliminating the noninformation data obtained with hplc - dad , three - way data arrays ( 250 50 10 ) were treated by using the parafac - als and swatld , respectively . the chemical rank is defined as the number of significant factors distinguishing from noise . when using parafac , an initial definition of the number of factors to build the model is necessary . this choice is of fundamental importance because all conclusions about the deconvolution and quantitation results will be related with this number of factors . , deciding the underlying species in the mixture is always the key step to further qualitative and quantitative analysis . some methods have been suggested to estimate the chemical rank of the three - way data arrays . bro and kiers suggested obtaining the number of responsive components ( n ) by consideration of the internal parameter known as core consistency diagnosis ( corcondia ) , which is a measure of how well a given model is able to reproduce the so - called tucker core of a cube of data . the core consistency is calculated as a function of a trial number of components . it remains near a value of 100 when the number is less than or equal to the optimum ; for exceeding component numbers it drops below 50% . unconstrained parafac - als models of this hplc - dad data were developed using one to ten components , and the percentage of fit is used as the initial approach to select the number of factors . figure 2(a ) shows the value obtained for the 16-sample cube when studying the human plasma sample . as can be seen , the core consistency drops to a very low value when using five components to model the cube , suggesting that n = 4 is a sensible choice . it shows that the human plasma introduces some interferences to the mixture system . on the other hand , anyone of these methods can not ensure to obtain the accurate result for a practical mixture system . two or more methods are often used to estimate the appropriate component number of the mixture to confirm the result . we also suggest a simple linear transform incorporating monte carlo simulation approach ( which names ltmc ) to determine the component number of the three - way data arrays . the newly proposed method decides the chemical rank through performing a simple linear transform procedure to the original cube matrix to produce two subspaces by singular value decomposition : one of two subspaces is derived from the original three - way data array itself and the other is derived from a new three - way data array produced by the linear transformation of the original one . projection technique incorporating the monte carlo approach acts as distinguishing criterion to chose the appropriate component of the mixture . the projection residuals for the former four factors are relatively small but a rapid increase for the later factors . because the first four factors represent the real factors spaces , the later factors represent the noise spaces . it indicates that the trilinear data arrays request to be fitted exactly with four factors . once the appropriate component number is correctly determined , three - way hplc - dad data for the sixteen samples were analyzed by parafac - als and swatld , respectively . specific details on the implementation of parafac and swatld methods will be given as follows . figures 3 and 4 show the chromatographic profile and uv - vis spectra profile obtained with swatld . the chromatography and spectra of pure vancomycin and cephalexin were obtained by measuring the pure vancomycin and cephalexin and decomposing them by singular value decomposition ( svd ) . we regarded these normalized profiles as the spectra of the pure and used them as reference spectra to evaluate the reliability of the models in the calibration . the correlation coefficients which calculate the pure spectrum and the ones resolved by swatld all exceed 0.998 for the each individual analyte in this paper . the result of the parafac - als is not given for its sameness with swatld . two means can be used to decide on the concentration of the predicted samples ( unknown sample ) . a and b denote the chromatographic profile and spectral profile , and xun denotes the data matrix which comes from an unknown sample . it is noticeable that the interference included in the unknown samples should be contained in matrices a and b. otherwise the result will be inaccurate . type two is often applied ; that is , the calibration step and predicted step are synchronous implemented . the relative concentration ckn can be acquired by running two algorithms with the other loading . the unknown concentration y^un of the nth component can be obtained by regressing it based on the known standard concentration y of the nth component : ( 3)k = y+[c1n c2n ckn],y^un = cunk . here the accuracy of the models was calculated by the root mean square error of prediction ( rmsep ) : ( 4)rmsep=i=1m(yiy^i)2 m , where yi and y^i are the added and predicted concentrations of the given vancomycin or cephalexin in the ith prediction sample , and m is the number of prediction samples . rmsep was 0.81% for vancomycin and 0.95% for cephalexin with swatld and 0.89% for vancomycin and 1.02% for cephalexin with parafac - als . it can be found that the root mean square error of prediction of two algorithms is very coincident . these values can also be expressed in terms of recovery ( as the percentage ratio between the predicted and the true concentration ) so that they could be compared with two algorithms ( table 1 ) . the average recoveries for the parafac - als procedure are 101 5% for vancomycin and 96 3% for cephalexin . for swatld , the average recoveries are 102 4% for vancomycin and 97 3% for cephalexin , respectively . so , in all the cases , recoveries are around the ideal 100% for both methods ; it shows that the dispersion of the results was lower for swatld than that for parafac - als . on the same time , the iterative numbers of swatld are shorter than parafac - als ( swatld only requests 15 times but 478 times for parafac - als ) and insensitive for the chemical rank of the mixture system . the most important process for comparison of analytical methods is the determination of figures of merit ( fom ) , such as sensitivity , selectivity , and limit of detection ( lod ) . in multivariate calibration , the net analyte signal ( nas ) calculation is strictly necessary for the fom evaluation . the nas for multiway data is analogous to those for first - order procedures , which is defined as the part of the signal that relates uniquely to the analyte of interest . in this case , as the data are bilinear , the nas is the pure analyte data obtained by parafac . the sensitivity is estimated as the nas at unit concentration , as shown in ( 5 ) , and the selectivity is the ratio between the sensitivity and the total signal , as shown in ( 6 ) : ( 5)sen = k{[(ata)1]nn[(btb)1]nn}1/2,(6)sel={[(ata)1]nn[(btb)1]nn}1/2 , where a and b are the matrices which collect spectral and chromatographic profiles for all n components , respectively . the limit of detection ( lod ) is calculated as ( 7)lod=3.3s(o ) , where s(o ) is the standard deviation from the concentration estimated for three different blank samples in the parafac and swatld models , respectively . the figures of merit of vancomycin and cephalexin for parafac - als and swatld are displayed in table 2 . as can be observed , the figures of merit for both parafac - als and swatld are very similar and only the sensitivity of swatld is greater than that of parafac - als . in terms of the figures of merit , cephalexin is more selective than vancomycin as its spectra are the most different in shape ( see figures 2 and 3 ) from the others and therefore the least correlated . this preliminary information suggests that cephalexin will be predicted at lower concentrations ( it is the more sensitive ) . simultaneous and direct determination of vancomycin and cephalexin in human plasma by coupling hplc - dad with second - order calibration algorithms was developed in this paper . this approach allows quantifying vancomycin and cephalexin under the incompletely chromatographic separation and presenting unknown inference in the prediction samples . slightly better results in the plasma samples analysis are obtained by application of swatld calibration compared to parafac - als . this work demonstrated that the use of hplc - dad coupled with second - order calibration algorithms is a powerful tool to quantify overlapped chromatographic profiles for complex analysis of drugs in plasma . at the same time , the methodology involving parafac and swatld did not require as many calibration samples as the pls models do and , what is more , would allow the determination of any of the vancomycin and cephalexin in the presence of unknown interferences ( second - order advantage ) even if they were not included in the model . extremely important issues such as reduction in the time of analysis and consequently costs and amount of contaminant solvents should also be considered . the figures of merit calculated for both parafac and swatld were very similar and the results should be considered satisfactory based on the complexity of the samples analyzed .

a simple , rapid , and sensitive method for the simultaneous determination of vancomycin and cephalexin in human plasma was developed by using hplc - dad with second - order calibration algorithms . instead of a completely chromatographic separation , mathematical separation was performed by using two trilinear decomposition algorithms , that is , parafac - alternative least squares ( parafac - alss ) and self - weight - alternative - trilinear - decomposition- ( swatld- ) coupled high - performance liquid chromatography with dad detection . the average recoveries attained from parafac - als and swatld with the factor number of 4 ( n = 4 ) were 101 5% and 102 4% for vancomycin , and 96 3% and 97 3% for cephalexininde in real human samples , respectively . the statistical comparison between parafac - als and swatld is demonstrated to be similar . the results indicated that the combination of hplc - dad detection with second - order calibration algorithms is a powerful tool to quantify the analytes of interest from overlapped chromatographic profiles for complex analysis of drugs in plasma .

1. Introduction 2. Theory 3. Experimental 4. Results and Discussion 5. Conclusion

PMC4356870

amphibians are the most threatened vertebrate group , suffering large - scale declines in species diversity since at least , according to historical data , the 1970s ( stuart et al . , 2004 ) . a decade ago the iucn 's global amphibian assessment indicated that a third of the estimated amphibian species had declined or become extinct ( stuart et al . , 2004 ; such declines may be attributed to a number of factors ranging from habitat destruction , pollution and exploitation , to climate change and disease ( beebee and griffiths , 2005 ) . the disease known as chytridiomycosis ( amphibian chytrid ) , caused by the fungal pathogen batrachochytrium dendrobatidis , has been responsible for major global amphibian declines ( readel and goldberg , 2010 ) . along with chytrid , amphibians are host to a wide variety of parasites ( du preez and carruthers , 2009 ; netherlands et al . , 2014a ) , including intraerythrocytic and extracellular haemoparasites ranging from protozoans , comprising both intracellular apicomplexans ( davies and johnston , 2000 ) and extracellular flagellates ( acosta et al . , 2013 ) , to extracellular nematode microfilariae ( baker , 2008 ) as well as those intracellular parasites of uncertain identity such as the viral and bacterial infections ( davies and johnston , 2000 ; davis et al . , the most attention , however , has been given to those parasites of the first three groups mentioned , most likely due to the frequent findings and thus greater basis of knowledge of these organisms in anuran hosts . furthermore , of these three groups , those of the protozoa , particularly the apicomplexans , would appear to be the most studied of all ( see davies and johnston , 2000 ; netherlands et al . however , since few parasite surveys on frogs have been carried out in sub - saharan africa , the degree of this haemoparasite diversity remains unknown ( readel and goldberg , 2010 ; netherlands et al . yet , such diversity of knowledge regarding these parasites is necessary before further studies can be done on elucidating the effects that these parasites may have on their natural hosts , and the role these parasites may have in amphibian conservation . southern africa currently boasts 159 known species of frogs in 33 genera and 13 families ( du preez and carruthers , 2009 ; channing and baptista , 2013 ; channing et al . this study presents the results of a haemoparasite survey of frogs from three localities in kwazulu - natal , south africa ( see fig . 1 ) . localities include the formally protected ndumo game reserve ( ngr ) and the reserve 's anthropogenically impacted surrounds , as well as kwa nyamazane conservancy ( knc ) . all three sampling areas fall within a sub - tropical region known for its biological richness and as such the province , kzn , boasts the highest diversity of frog species in south africa ( see du preez and carruthers , 2009 ) . the following study thus aimed to determine and record , through a multispecies haemoparasite survey on frogs , if this parasite diversity paralleled that of its rich frog diversity . ndumo game reserve ( ngr ) ( 265200.0s 321500.0e ) is situated in the west of the maputaland bioregion , close to the borders of south africa , swaziland and mozambique . the maputaland bioregion , located in northern kwazulu - natal ( kzn ) and crossing into southern mozambique , is one of the most biologically rich areas in southern africa ( haddad , 2003 ) and has a sub - tropical climate . ndumo is a large reserve , holding 10,117 ha of diverse habitats , including floodplains , sub - tropical bush , savannah and woodland , to riparian forest ( wesoowska and haddad , 2009 ) . the area directly surrounding the ngr ( 270013.8s 321649.9e ) is not formally protected and thus is covered in rural tribal villages , causing the vegetation to be heavily impacted by the villagers ' livestock and subsistence farming practices . approximately 80 km to the south lies the kwa nyamazane conservancy ( knc ) ( 272334.9s 320840.8e ) , a small conservation area running along the phongola river and surrounded by large sects of agricultural land , most of it utilised for sugar cane farming . these localities were specifically chosen as all three are located on , and are thus supplied by a permanent water source , the phongola river ( see fig . 1 ) . frogs were collected via active sampling at night in all three localities as mentioned above . all these sites were visited during the warmer and rainy months of february and november 2012 , april and november 2013 and february 2014 . during collection captured frogs were held in disposable plastic bags and transported back to a field laboratory either at the ngr or knc , where they were identified to species level using du preez and carruthers ( 2009 ) . a drop of blood was collected from each frog via cardiac or femoral venipuncture using a sterile heparinised insulin syringe . a portion of this blood was used to prepare a thin blood smear , which once air - dried in a dust - proof container was fixed immediately using absolute methanol and stained thereafter using a modified solution of giemsa stain ( fluka , sigma - aldrich , steinheim , germany ) ; the other portion was dropped into a microcentrifuge tube with an equal volume of 70% ethanol for future molecular analysis . all frogs were processed the morning after collection and were released within 24 h of capture . smears were screened using a 100 immersion oil objective on a nikon eclipse e800 compound microscope ( nikon , amsterdam , netherlands ) , and images were captured with an attached nikon digital camera . the average parasitaemia was calculated per 100 erythrocytes , with ~10 erythrocytes examined per blood smear following cook et al . the estimated average parasitaemia for extracellular parasites were calculated as number of parasites / per - slide ( ps ) with an approximate field of 20,000 blood cells examined . this study received the relevant ethical approval ( north - west university ethics approval no . : nwu-0000514-s3 ) , as well as approval to do research from the appropriate conservation authorities ( ezemvelo kzn wildlife , permits : op 674/2012 , op 5139/2012 , op 526/2014 , and op 839/2014 . ) . the monte carlo variant of the fisher 's exact test , set to 10,000 replicates with a confidence interval of 99% , was employed to investigate significance in variation of prevalence between species , families , habitat types and sampling periods . the habitat types were established based on those described by du preez and carruthers ( 2009 ) . frog species were classified as terrestrial ( those species thriving and breeding away from a permanent water source for most of their lives ) , semi - terrestrial ( species thriving away from a permanent water source , but needing such a source to breed ) , semi - aquatic ( species requiring a position near a permanent water source for most of their lives in order to survive and breed ) and aquatic ( species permanently living and breeding in a water source , rarely leaving that source ) . the kruskal wallis test was used since it is suitable for non - parametric data and does not assume normal data distribution and equal sample size . it was applied to determine significance levels ( p < 0.05 ) of variation between infection intensity across species , families , habitat types , and sampling periods . it was further employed to determine significance of variation of the overall intensity of hepatozoon and trypanosoma across frog species , families , habitats and sampling periods . a non - parametric levenes 's test was used to verify the equality of variances in the samples ( homogeneity of variance , p > 0.05 ) ( nordstokke and zumbo , 2007 , 2010 ) . ndumo game reserve ( ngr ) ( 265200.0s 321500.0e ) is situated in the west of the maputaland bioregion , close to the borders of south africa , swaziland and mozambique . the maputaland bioregion , located in northern kwazulu - natal ( kzn ) and crossing into southern mozambique , is one of the most biologically rich areas in southern africa ( haddad , 2003 ) and has a sub - tropical climate . ndumo is a large reserve , holding 10,117 ha of diverse habitats , including floodplains , sub - tropical bush , savannah and woodland , to riparian forest ( wesoowska and haddad , 2009 ) . the area directly surrounding the ngr ( 270013.8s 321649.9e ) is not formally protected and thus is covered in rural tribal villages , causing the vegetation to be heavily impacted by the villagers ' livestock and subsistence farming practices . approximately 80 km to the south lies the kwa nyamazane conservancy ( knc ) ( 272334.9s 320840.8e ) , a small conservation area running along the phongola river and surrounded by large sects of agricultural land , most of it utilised for sugar cane farming . these localities were specifically chosen as all three are located on , and are thus supplied by a permanent water source , the phongola river ( see fig . 1 ) . frogs were collected via active sampling at night in all three localities as mentioned above . all these sites were visited during the warmer and rainy months of february and november 2012 , april and november 2013 and february 2014 . during collection captured frogs were held in disposable plastic bags and transported back to a field laboratory either at the ngr or knc , where they were identified to species level using du preez and carruthers ( 2009 ) . a drop of blood was collected from each frog via cardiac or femoral venipuncture using a sterile heparinised insulin syringe . a portion of this blood was used to prepare a thin blood smear , which once air - dried in a dust - proof container was fixed immediately using absolute methanol and stained thereafter using a modified solution of giemsa stain ( fluka , sigma - aldrich , steinheim , germany ) ; the other portion was dropped into a microcentrifuge tube with an equal volume of 70% ethanol for future molecular analysis . all frogs were processed the morning after collection and were released within 24 h of capture . smears were screened using a 100 immersion oil objective on a nikon eclipse e800 compound microscope ( nikon , amsterdam , netherlands ) , and images were captured with an attached nikon digital camera . the average parasitaemia was calculated per 100 erythrocytes , with ~10 erythrocytes examined per blood smear following cook et al . the estimated average parasitaemia for extracellular parasites were calculated as number of parasites / per - slide ( ps ) with an approximate field of 20,000 blood cells examined . this study received the relevant ethical approval ( north - west university ethics approval no . : nwu-0000514-s3 ) , as well as approval to do research from the appropriate conservation authorities ( ezemvelo kzn wildlife , permits : op 674/2012 , op 5139/2012 , op 526/2014 , and op 839/2014 . ) . the monte carlo variant of the fisher 's exact test , set to 10,000 replicates with a confidence interval of 99% , was employed to investigate significance in variation of prevalence between species , families , habitat types and sampling periods . the habitat types were established based on those described by du preez and carruthers ( 2009 ) . frog species were classified as terrestrial ( those species thriving and breeding away from a permanent water source for most of their lives ) , semi - terrestrial ( species thriving away from a permanent water source , but needing such a source to breed ) , semi - aquatic ( species requiring a position near a permanent water source for most of their lives in order to survive and breed ) and aquatic ( species permanently living and breeding in a water source , rarely leaving that source ) . the kruskal wallis test was used since it is suitable for non - parametric data and does not assume normal data distribution and equal sample size . it was applied to determine significance levels ( p < 0.05 ) of variation between infection intensity across species , families , habitat types , and sampling periods . it was further employed to determine significance of variation of the overall intensity of hepatozoon and trypanosoma across frog species , families , habitats and sampling periods . a non - parametric levenes 's test was used to verify the equality of variances in the samples ( homogeneity of variance , p > 0.05 ) ( nordstokke and zumbo , 2007 , 2010 ) . blood smears were collected from 436 frogs of 29 species , 6 genera and 11 families ( table 1 ) . of these 15/29 ( 52% ) of the frog species were infected with haemoparasites , making up 87/436 ( 20% ) of the total number of frogs ( table 2 ) . five groups of haemoparasites were recorded including intraerythrocytic haemogregarine and haemogregarine - like species of the genus hepatozoon and dactylosoma respectively , and intraerythrocytic organisms of a viral or bacterial nature , the species of which could not be identified ; furthermore , extracellular flagellate parasite species of the genus trypanosoma and microfilarid nematode species were observed . hepatozoon species accounted for most of the infections at 59/436 ( 14% ) , followed by trypanosoma species at 46/436 ( 11% ) ; viral or bacterial infections , microfilarid infections and dactylosoma species , accounted for 6/436 ( 1% ) , 2/436 ( 0.5% ) and 13/436 ( 3% ) of the overall prevalence respectively ( table 2 ) . as for the intensity of the groups , hepatozoon showed an overall ( all infected frogs pooled ) intensity of 5% , the dactylosoma an overall intensity of 1% , the viral or bacterial infections an overall intensity of 87% , the trypanosoma an overall intensity of 11 per blood slide , and the microfilarid nematode infections an overall intensity of 15 per blood slide ( table 2 ) . the overall prevalence of haemoparasites ( all parasite groups pooled ) varied significantly by frog species ( = 163.475 , p < 0.01 ) . ptychadena anchietae demonstrated the highest prevalence at 47/78 ( 60% ) and chiromantis xerampelina the lowest at 1/44 ( 2% ) ( table 2 ) . upon division of the frog species into groups including aquatic ( two species ) , semi - aquatic ( 17 species ) , terrestrial ( one species ) and semi - terrestrial ( nine species ) ( see table 1 ) , it was observed that only the semi - aquatic and semi - terrestrial groups contained infected species ( table 2 ) . these two groups varied significantly in prevalence of infection ( = 87.000 , p < 0.01 ) , with 79% of the infected individuals from the semi - aquatic group and only 21% from the semi - terrestrial group . of the semi - aquatic group , the genus ptychadena had the highest diversity of haemoparasites , infected with all types as recorded in table 2 . furthermore , p. anchietae , of all the infected frog species , revealed the highest prevalence of parasites , making up 47/87 ( 54% ) of the total with 47/78 ( 60% ) of the p. anchietae themselves found to be infected . hepatozoon species accounted for most of the infections followed by trypanosoma species , significance of intensity calculated via the use of the kruskal = 0.028 ) , across families ( = 11.717 , p = 0.006 ) , and across the different habitat types ( = 7.227 , p = 0.007 ) showed a significant difference . hyperolius marmoratus , in the semi - aquatic group , and amietophrynus maculatus , in the semi - terrestrial group , accounted for the highest intensities ( table 2 ) . hepatozoon intensity across the different sampling periods , however , showed no significant variance ( = 4.177 , p = 0.552 ) . trypanosoma intensity across frog species ( = 11.919 , p = 0.028 ) showed a significant difference ; however , across families ( = 3.802 , p = 0.664 ) , habitat types ( = 0.330 , p = 0.585 ) and sampling periods ( = 6.675 , p = 0.147 ) , no significant difference was observed . in this case hyperolius tuberilinguis , in the semi - aquatic group , and chiromantis xerampelina , in the semi - terrestrial group , accounted for the highest intensities ( table 2 ) . per locality , it was observed that the ngr , with 26 species examined , showed a prevalence of 77/360 ( 21% ) as compared with outside the ngr , with eight species examined and a prevalence of 0/54 ( 0% ) , and the knc , with seven species examined and a prevalence of 10/22 ( 45% ) . furthermore , the ngr had a higher diversity of haemoparasites , including 50/360 ( 14% ) infected with hepatozoon species , 11/360 ( 3% ) with dactylosoma species , 5/360 ( 1% ) with viral or bacterial organisms , 46/360 ( 13% ) with trypanosoma species and 2/360 ( 0.6% ) with microfilaria as compared with the knc frogs that were only infected with hepatozoon . on the whole , 20% ( 87/436 ) of the frogs in this study were infected with at least one haemoparasite group , some infected up to five . this was similar to previous comparable studies such as that of readel and goldberg ( 2010 ) in western uganda documenting a 17% ( 30/180 ) prevalence , even though this was found to be approximately half that of other studies in africa ( see mohammed and mansour , 1959 ; ball , 1967 ; readel and goldberg , 2010 ) . in this study , hepatozoon species accounted for most of the infections at 14% , which was equally true for the survey done in uganda by readel and goldberg ( 2010 ) . on the contrary , ball ( 1967 ) , during a survey completed in tanzania and kenya , found a considerably higher prevalence of 29% . readel and goldberg ( 2010 ) suggested this may be attributable to availability of insect vectors . trypanosome species were the second most common parasite infecting frogs in this study at 11% , just slightly higher than the 6% reported by readel and goldberg ( 2010 ) . both the results of the present and the readel and goldberg ( 2010 ) studies ' conducted in africa are in contrast to what has been recorded in other similar studies but on different continents ( see barta and desser , 1984 ; barta et al . , 1989 ) , in which the trypanosoma demonstrate a higher prevalence to that of hepatozoon or any other haemoparasite groups ( see werner , 1993 ) . microfilarid infections from the south african frogs studied here , were also seen to occur in low numbers , similar to readel and goldberg ( 2010 ) . infections not reported by readel and goldberg ( 2010 ) and ball ( 1967 ) , but reported in this study , were a dactylosoma species and viral or bacterial infections . the only other study in africa to report on parasite intensities was that by readel and goldberg ( 2010 ) , in which hepatozoon species contained an average intensity of 2.3% , trypanosoma species had an average intensity of 7.2 parasites , and microfilariae had an average intensity of 11.2 parasites . in comparison the total parasite intensity for the current study was 5.3% for hepatozoon species , 10.6 for trypanosoma and 14.5 for microfilariae . similar to readel and goldberg ( 2010 ) significant differences ( p < 0.01 ) in the prevalence of parasites among frog species were recorded during the current study , with p. anchietae showing the highest prevalence and c. xerampelina the lowest . both frog species prefer habitats close to water ( classified in this study as being semi - terrestrial ) ( du preez and carruthers , 2009 ) . ptychadena anchietae is a grass frog and is often found around the water 's edge whilst c. xerampelina is an arboreal frog species . since the abundance of possible vectors associated with water , such as mosquitoes and leeches , would be high in such habitats , it may explain the high prevalence of haemoparasites recorded from p. anchietae . the reason for such a low prevalence in c. xerampelina , particularly for hepatozoon species , which may be mosquito transmitted in such an environment ( desser et al . , 1995 ; davies and johnston , 2000 ) , can not be explained . this result is particularly peculiar since both frog species were infected with trypanosomes ( only a single individual of c. xerampelina ) , which are mosquito and leech transmitted ( barta and desser , 1984 ) . one of the only possibilities could be , since hepatozoon is transmitted via the ingestion of the infected invertebrate or vertebrate ( davies and johnston , 2000 ) , that c. xerampelina prefers a diet not inclusive of mosquitoes and other frogs . division of the frog species into groups showed that only the semi - aquatic and semi - terrestrial groups contained infected species , these two groups varying significantly in prevalence of infection ( p < 0.01 ) . the semi - aquatic group had the highest prevalence , likely attributable to the ptychadena species , one of them p. anchietae . the ptychadena species also showed the highest diversity of haemoparasites , infected with all five recorded groups . furthermore , of all the frog species , p. anchietae was the only species to be parasitised with a species of dactylosoma . these parasites are closely associated with water and thus are suggested to be transmitted by a leech vector ( barta , 1991 ) . reports of dactylosoma parasitising frogs in africa are numerous , accounts of these organisms from at least five countries and approximately eight species of frog ( see barta , 1991 ) . one such report was from south africa from the bufonid a. regularis ( most likely amietophrynus gutturalis ) by fantham et al . are referred to as a single species dactylosoma ranarum ( see barta , 1991 ) ; however , only future molecular work will be able to clarify if the species here is one of the same . furthermore , the ptychadenidae were the only frogs found infected with viral or bacterial organisms . viral or bacterial infections have been recorded from a cosmopolitan distribution of amphibians ( see desser , 1987 ) . unfortunately , very little is known about the identity , classification and effect of these organisms ( see desser , 1987 ; davies and johnston , 2000 ; davis et al . , alves de matos and paperna ( 1993 ) presented the most recent study of uncertain erythrocyte virus infections from p. anchietae in south africa . these virus or bacterial infections were found to be similar to several different viruses of the frog erythrocytic virus ( fev ) group such as toddia , pirhemocyton and other rickettsiales . in contrast to the above findings in both the semi - aquatic and semi - terrestrial groups , the frog species from the terrestrial as well as aquatic groups were not observably parasitaemic . since b. adspersus ( terrestrial ) spends most of its life underground ( du preez and carruthers , 2009 ) , contact with vectors would be rare . however , since the semi - aquatic group had the highest prevalence of parasites , most likely due to the frequent contact with vectors , it was expected that those of the aquatic group would be equally parasitised . yet , as in readel and goldberg ( 2010 ) , the species of xenopus ( aquatic ) here were found to be uninfected . such a finding is surprising as it would be expected that xenopus should contain a rather high prevalence of parasites , particularly as it is well known that leeches feed on these frogs ( see badets and du preez , 2014 ; kruger and du preez , 2015 ) . possible explanations for this could be that haemoparasites infecting xenopus are extremely host specific or were simply not present in the area sampled . intensity of hepatozoon species across frog species and families , as well as across habitat types were found to be significant ( p = 0.007 ) . hyperolius marmoratus ( hyperolidae ) of the semi - aquatic group , and a. maculatus ( bufonidae ) from the semi - terrestrial group , had the highest intensity . hyperolius marmoratus may be found permanently on the edges of water , where vector abundance and contact rates are likely to be high , thus accounting for this high intensity . amietophrynus maculatus appears to favour more static , shallow water bodies , which are also favoured by mosquito species , the high contact rates with possibly hepatozoon infected mosquitoes would thus be high . hepatozoon species have been reported and described from a few hyperolius species in africa , though the majority have been reported from bufonid species such as a. maculatus ( see netherlands et al . trypanosoma species intensities varied significantly only across species ( p = 0.028 ) , being highest in the semi - aquatic h. tuberilinguis and the semi - terrestrial c. xerampelina . these two species are permanently associated with water , and thus always in close association with an abundance of possible vectors . a plethora of trypanosome species have been described and reported from numerous african frog species , unfortunately many reports contain inadequate taxonomic descriptions and in numerous cases they are simply referred to as a trypanosoma sp . without any morphological data provided on the specific parasite ( see bardsley and harmsen , 1973 ; telford , 2009 ) . furthermore , since trypanosomes are known to be pleomorphic , the true diversity seen in this study can not be realised , and thus future molecular work along with morphological description is intended in order to differentiate between species and life stages of these organisms . intensity across sampling periods for both hepatozoon and trypanosoma species was insignificant ( p = 0.552 and p = 0.147 respectively ) , likely due to sampling occurring only during the wet seasons . parasites have always been seen in a negative light , especially with regards to human and livestock health . however , within the natural environment parasites may be seen as a crucial part of a functional and healthy ecosystem . parasites make a profound impact on the biodiversity of an ecosystem by influencing aspects such as host competition , migration , speciation and stability ( combes , 1996 ) . furthermore , parasites reflect their host species ' environmental interactions , revealing feeding behaviour , geographical ranges and social systems ( dobson et al . , 2008 ) . in a stable and healthy natural ecosystem parasites and their hosts have had the opportunity to co - evolve , the parasite causing few pathogenic effects in a healthy host animal . if however , this well established co - existence is disturbed by , for example , habitat destruction or the indiscriminate movement of host animals between habitats ; pathogenic effects may become apparent resulting in the destabilisation of the host population ( see combes , 1996 ) . additionally , in light of the above , the loss of parasite diversity would very likely have unforeseen , but grave consequences , especially with respect to regulation of host populations and their abundance within the communities ( dobson et al . , 2008 ) . ( 2008 ) , if the main aim of conservation biologists is to conserve fully functional food webs , it is imperative that parasites are thus also included within biodiversity conservation . ndumo game reserve was found to harbour the highest diversity of both frog species and haemoparasites as compared with the other two sites ( see table 1 ) , which are impacted by rural village settlements and peripheral commercial sugar cane agriculture respectively . anthropogenic impacts , as found in readel and goldberg ( 2010 ) , may account for the lack of diversity in these two sites , affecting vector distributions and contact rates between frog hosts and vectors . it is anticipated that through future work , including both morphological and molecular descriptions of the parasites reported in this study , that the biodiversity of this region will be elucidated . furthermore , it is hoped that with this biodiversity knowledge and the identification of potential vectors , the effects human activities may have on frog haemoparasite life cycles and as such their biodiversity will be clarified .

highlightsfirst frog ( 11 families , 6 genera , 29 spp . ) haemoparasite survey in south africa.showing a higher biodiversity as compared with similar research in africa.intraerythrocytic and extracellular haemoparasites of five groups found.hepatozoon and trypanosoma spp . were the most prevalent.protected areas boast higher parasite diversity compared with human impacted sites .

Introduction Materials and methods Study area and frog collection Preparation and screening of frog blood Statistical analysis Results Discussion Conflict of interest

PMC3359713

studies have shown that 30 minutes of moderate intensity exercise can confer health benefits if performed on most days of the week ( 5 days / wk ) [ 1 , 2 ] . these benefits include weight maintenance , weight loss , cardiovascular risk management [ 4 , 5 ] , and the avoidance of metabolic dysfunction such as type 2 diabetes . further , more than 30 min of physical activity a day are necessary for formerly obese individuals to maintain weight loss . success in obtaining improvements in these health outcomes often occurs because the exercise regimen is carefully controlled in a supervised setting where specific directions are provided [ 79 ] . as the population has become more obese , numerous guidelines have been established to help the lay public understand the amount of activity that should be incorporated into their lifestyle . for example , the 2008 physical activity guidelines for americans states that people should participate in 150 minutes of moderate - intensity aerobic activity every week ( p. vi ) , which would be approximately 30 minutes of fast walking five days a week [ 2 , 10 ] . sedentary individuals , however , may misinterpret the term moderate intensity activity , and their efforts to increase their physical activity levels may result in insufficient workloads for cardioprotection , glucose control , and weight loss and maintenance . previous research concerned with self - selected exercise pace has used descriptors such as walk briskly or preferred pace [ 1114 ] but have not examined the pace selected when subjects are told to walk at a moderate intensity . yet , moderate intensity is the terminology of the physical activity guidelines . thus , the purpose of this study was to determine the workload sedentary overweight and obese individuals would self - select when asked to walk at a moderate intensity . since exercise at intensities greater than 60% peak oxygen consumption ( vo2 peak ) is associated with more health benefits , we compared the self - selected pace to a prescribed pace ( 60% vo2 peak ) . we selected this workload because it was at the low end of the vigorous exercise range , is known to confer health benefits beyond just weight loss , and we anticipated that this intensity would be tolerable by our sedentary participants . we examined the potential differences in heart rate ( hr ) , ratings of perceived exertion ( rpe ) , and energy expenditure at the prescribed versus self - selected moderate intensity exercise . further , we determined if there was a relationship between the self - selected exercise intensity and adiposity or aerobic fitness . we hypothesized that most individuals would self - select a workload at the low end of the moderate intensity exercise pace , when required to walk for 30 min . we further hypothesized that this workload would result in a lower energy expenditure and lower rpe than when the exercise was at an exercise intensity know to confer health benefits . thirty - three overweight and obese individuals ( age 3050 y ) were recruited . inclusion criteria for the study were a body mass index ( bmi ) greater than 25 kg / m , nondiabetic status , not on -blockers , no known cardiovascular disease , no metabolic disorders , no orthopedic limitations and currently not participating in a regular exercise program or coached / guided physical training . all subjects were initially screened by a detailed medical history questionnaire before consideration for the study . all participants were required to complete an informed consent document , approved by the university institutional review board . all subjects completed 3 visits which included an exercise stress test , a 30 min submaximal walk at a self - selected moderate intensity pace and a 30 min submaximal walk at a 60% of vo2 peak ( prescribed pace ) . this workload was selected because it is at the low end of the vigorous exercise intensity workload and is known to provide cardioprotective benefits . the submaximal visits were not presented in a randomized fashion because of the potential for learning the ideal walking intensity during the prescribed exercise intensity condition . these visits were a minimum of 72 h apart and no longer than 2 weeks apart . each subject walked on a quinton treadmill ( bothell , washington ) , and a modified walking protocol was utilized that started at 2.5 mph and 0% grade . briefly , an increasing workload was administered by initially increasing speed in increments of 0.5 mph per stage until 3.5 mph was achieved . thereafter , increases in grade of 2.5% per stage were administered until volitional fatigue . at the end of each stage , a rating of perceived exertion ( rpe ) on a scale of 620 was collected from the subject . the american college of sports medicine ( acsm ) guidelines were followed to establish if a physician needed to be present . during exercise , expired gases were collected and analyzed for volume , o2 and co2 content using a cosmed quark b4 metabolic analyzer ( rome , italy ) that was calibrated prior to each test . oxygen consumed ( vo2 ) and co2 produced ( vco2 ) were calculated . heart rate ( hr ) was collected continuously with a polar heart rate monitor ( polar electro , lake success , ny ) throughout the study . concorde , ca ) and the test was administered according to the manufacture 's guidelines . height and weight were measured without shoes on prior to testing using a healthometer ( sunbeam products inc , boca raton , fl ) . subjects were asked to wear the same clothing on each visit . during the second visit , subjects were instructed to walk at a moderate intensity around an indoor track for thirty minutes . subjects self - selected the pace they wanted to utilize for the 30 minutes of exercise and received encouragement to keep walking but were given no further directions than to walk at a moderate intensity . time per lap was monitored with a stopwatch ( timex ironman marathon stopwatch , timex , n. little rock , ar ) . speed varied somewhat in a given lap , but there were no significant differences in the lap times documented during the walk for each lap ( data not shown ) . in addition , no significant change in vo2 was observed between laps . during this test , the subject donned a harness that carried the k4 cosmed portable metabolic analyzer ( rome , italy ) which was calibrated before each test . ratings of perceived exertion were collected every five minutes of exercise . at the end of thirty minutes , total distance covered was measured in order to calculate average speed . the exercising pace was selected based on the performance on the vo2 peak test from the first visit . wearing the same k4 apparatus as the track visit , the subject walked on a quinton treadmill while expired gases were collected . heart rate was measured continuously , and again rpe was ascertained at the end of every five minutes . the vo2 peak was considered the highest value achieved at the end of the test while meeting all other acsm guidelines for a successful stress test . the calculated vo2 from the track and treadmill evaluations was averaged into one - minute intervals and from these data energy expenditure per minute was calculated [ 2326 ] , as well as per kg fat free mass ( ffm ) . total expenditure for the duration of exercise of both visits was then calculated as the sum of the one - minute averages . steady state values were used for the calculation of average vo2 and hr responses to exercise in order to accurately represent level of workload accomplished . also from the vo2 data the met level was calculated with 3.5 ml / kg / min equaling 1 met . a paired t - test was performed to compare variables from the track and treadmill visit . when comparing for gender differences , a 2 ( male versus female ) x treatment ( self - selected versus prescribed ) anova with repeated measures or 2 ( obese versus overweight ) x treatment ( self - selected versus prescribed ) anova with repeated measures was employed . using the statistical package for the social sciences ( spss ) for windows ( chicago , il ver . a pearson correlation was used to determine the relationship between the differences in energy expenditure and other measured variables , as well as between walking speed and descriptive subject variables . those descriptive variables that were positively associated with walking speed were put into a linear regression analysis using the enter method to find the best predictor of walking speed . this method allows the entry of variables into the analysis and allows the program to select the order of entering the variable into the model . the bmi ranged from 2740 kg / m , and of the 33 subjects 21 subjects were obese and 12 were overweight ( table 1 ) . the men and women were similar in age ( ~42.5 y ) but the men were significantly ( p < 0.05 ) heavier , taller , and had a greater percent body fat . the men were slightly more fit ( p < 0.05 ) than the women . total energy expenditure for the prescribed walking was significantly greater than the energy expenditure for self - selected walking pace ( 955.7 39.2 kj versus 872.7 39.9 kj , p < 0.01 , resp . ) , resulting in 83 kj more being expended at the prescribed pace than in the self - selected pace . the men had a greater energy expenditure than the women ( self - selected : males 1057.7 51.0 kj and females 803.4 kj 26.4 kj - prescribed : males 1218.6 35.1 kj and females 857.1 22.3 kj , p < 0.01 ) , and there were no differences in total energy expenditure between the obese and overweight subjects . adjusting the data for fat free mass did not alter the findings , and the prescribed walking pace ( 10.4 0.3 kj / kg ffm / min ) elicited a higher rate of energy expenditure than the self - selected pace ( 9.4 0.3 kj / kg ffm / min , p < 0.05 ; figure 1 ) . however , men had a higher energy expenditure ( kj / kg ffm / min ) than the women ( p < 0.01 ) , and the overweight subjects had a slightly higher energy expenditure than the obese subjects ( p < 0.05 ) . self - selected exercise resulted in a workload of 52% of vo2 peak versus 58% of vo2 peak for the prescribed exercise ( p < 0.05 ) . the mean prescribed % vo2 is lower than we had targeted as many of the subjects could not complete 30 min of exercise at ~60% vo2 peak . the overweight subjects walked at a lower percentage of vo2 peak than the obese subjects ( p < 0.05 ) , regardless if the exercise pace were self - selected or prescribed . the % hr max for the 30 min of prescribed and self - selected walking were not significantly different ( self - selected : 75.1 11.2 b / min ; prescribed : 75.5 7.9 , table 2 ) . mean rpe values for the prescribed walking was slightly greater than for the self - selected walking pace ( self - selected : mean 11.3 0.2 , range 813 versus prescribed : mean 12.5 0.3 , range 1115 , table 2 ) ; no sex differences were observed . converting the work intensity to mets revealed no differences until 15 minutes of exercise where the met levels significantly increased and the self - selected intensity was less than the prescribed intensity , and remained lower through the remaining 30 minutes ( self - selected 4.2 0.2 , prescribed 4.9 0.2 mets , p < 0.01 ) . an association was found between the difference in energy expended between the prescribed and self - selected walking and vo2 peak ( r = 0.53 , p < 0.01 , figure 2(a ) ) , and adjusting for sex did not alter this finding . the best predictor of the self - selected walking speed in this cohort of subjects was fat mass which explained 32% of the variability ( coefficient = 0.564 , r = 0.32 , p < 0.001 , figure 2(b ) ) . fitness , age , height , and sex were not significant predictors of walking speed in this cohort of subjects . although the current physical activity recommendation for health is to accumulate at least 30 min of moderate intensity physical activity daily , the interpretation of moderate can vary considerably between individuals . a misconception of the term moderate may potentially result in the selected physical activity workload not being in an appropriate intensity range to be cardioprotective . the present study revealed the following : ( 1 ) the self - selected pace ( 52% vo2 peak ) was in the range that is defined as moderate ( 4059% vo2 peak ) , ( 2 ) the hr response was similar between experimental conditions yet , ( 3 ) rpe was greater in the prescribed bout than when the exercise was self - selected , and ( 4 ) a lower energy expenditure occurred with the self - selected workload than with the prescribed workload . these findings indicate that when participants are allowed to self - select exercise intensity , the workload would meet the acsm standards and potentially would be effective in increasing cardiorespiratory fitness ; however , the workload may not be adequate to provide other health benefits . our subjects were able to select a workload in the range of moderate intensity when asked ; however , when the subjects were asked to increase the workload slightly to the high end of the moderate intensity range ( ~58% of vo2 peak ) , 68% of the subjects could not complete our prescribed workload , and the workload had to be reduced after 1520 min of exercise . in addition , this reduction in workload may have resulted in slightly smaller differences in energy expenditure between the self - selected and prescribed pace . further the obese individuals worked at a higher % vo2 peak than the overweight subjects , in agreement with findings by mattsson et al . . possibly , obese individuals receive more information on the appropriate exercise pace or possibly they have a smaller vo2 reserve than an overweight individual thus working at a higher percentage of their aerobic capacity . the inability to complete 30 min at the higher intensity parallels the findings of ekkekakis and lind who noted that imposing a speed that is just 10% higher than what overweight women self - selected led to a decline in reported pleasure and decreased affective responses . this may explain why studies in controlled settings obtain more dramatic effects on health variables [ 29 , 30 ] than when subjects participate in unsupervised exercise . moreover , other studies have shown that subjects told to exercise at a brisk pace and not at their preferred pace the exercise intensity increased to levels well above the minimum of the recommended range [ 11 , 12 ] . hence , this provides further evidence that the descriptor used in the physical activity recommendation must be carefully considered . although there was no difference in the exercise hr between the self - selected and prescribed trial , rpe increased with the prescribed workload , indicating cardiophysiological tolerance but increased perception of the workload . this finding highlights that there may be a mismatch between perceived - exertion and physiological responses of borg 's model of effort continua in sedentary obese individuals . more specifically , a higher intensity of exercise is less pleasurable and enjoyable , thus perceived as greater exertion . lind et al . have shown that declines in affective valence and consistent ratings of perceived exertion are found when subjects are at an exercise intensity that exceeds the transition across the lactate threshold . we did not measure the lactate threshold in the present study , but a workload at 60% vo2 peak most likely would have been at or above their lactate threshold , based on other studies in sedentary individuals . an rpe of 13 is considered to be approximately the lactate threshold so our subjects as well as those subjects from the other studies kept their self - selected paces below the lactate threshold . further it was also observed that as subject fitness increased , as represented by vo2 peak , there was an increase in the difference in energy expended between walking trials ( figure 2 ) . the workload selected in the moderate range may reduce chd risk factors or all - cause mortality , but this workload may be inadequate for weight loss or to improve other health outcomes . from a meta - analysis conducted by swain and franklin greater cardioprotective benefits in fact , a faster walking speed was associated with a reduced chd risk , independent of the total energy expenditure [ 15 , 34 ] . one study found that only physical activities at intensities > 4.5 mets were associated with a decreased incidence of hypertension and reduced all - cause mortality . our self - selected pace was below this met value while the prescribed pace was above this value . additionally more favorable risk profiles occur with vigorous activity compared to moderate - intensity physical activity . swain and franklin reported that 3 studies indicated that groups exercising at the highest intensity ( 6575% of vo2 max ) experienced a decrease in diastolic blood pressure , but the group that exercised at lower intensities ( 4557% vo2 max ) did not . likewise improvements in glucose control and insulin sensitivity only occurred at vigorous intensities ( 6570% vo2 max ) but not at moderate intensities ( 4055% vo2 max ) . in general , greater relative intensities result in greater improvements in aerobic fitness and selected chd risk factors . other epidemiologic studies have shown that each 1-met increases in exercise capacity confers an 817% reduction in cardiovascular and all - cause mortality [ 15 , 36 , 37 ] . a review of the literature has shown that in low - fit subjects a minimal intensity of 30% vo2 reserve is needed , while in high - fit subjects exercise above 40% vo2 reserve is needed to improve cardiovascular fitness . in the present study only the prescribed workload was adequate to provide some of these health benefits and still many subjects could not complete this workload ; this agrees with previous research . in the self - selected walking pace , our subjects walked at a lower perceived exertion ( ~11 ) than when the exercise was prescribed . this rpe was slightly higher than that reported by pintar et al . , who noted that both normal weight and overweight women ( ~20 yr old ) selected a preferred walking intensity that resulted in the selection of a similar rpe ( ~10 ) for a 15 minute trial . the women had a slightly higher rpe for the task than men , and more closely obtained the desirable exercise intensity . mattsson et al . reported that normal weight subjects use about 36% vo2 max when walking at a self - selected comfortable pace . similarly huebschmann et al . reported that during cycle ergometer exercise that individuals with type 2 diabetes perceived the work to be more difficult ( higher rpe ) than obese individuals , even when adjusted for the relative work intensity . the higher self - selected rpe in our study may be because the self - selected pace on the track was their first testing day and the subjects may have been slightly anxious . in contrast to our finding , dasilva et al . reported a higher perceived exertion and a less positive affective valence in treadmill walking than during over ground walking . the best predictor of walking speed in the present study was fat mass which explained 32% of the variability in self - selected speed ; fitness did not significantly explain the variability in walking speed . these data support an inverse relationship between adiposity and self - selected walking speed and suggested that obese individuals may choose lower walking speed in their daily exercise regimens as compared to lean individuals . discrepancies in these findings are most likely due to the fact that they used college - aged women , while our subject pool was more middle - aged and both men and women were included . furthermore , it is well established in the literature that the addition of nonmetabolically active weight increases energy expenditure proportional to the weight . thus individuals carrying more body fat would most likely perceive the exercise to be more difficult . a potential limitation of the present study is that visits two and three could not be randomized due to the learning effect that the prescribed exercise visit may have had on the self - selected walking pace . by allowing our subjects to walk on a track , this enabled the subject to have normal variance in speed and be blinded to pace by walking on the track during the second visit . all visits were separated by at least three days to avoid the influence of fatigue and muscle soreness ; no subject was tested a second time if they still had muscle soreness following the first study day . there is also the possible confounding factor that the self - selected pace was measured on a track , while the prescribed pace was on the treadmill . we have previously shown that energy expenditure does not differ when walking on the track or treadmill at similar speeds ; however other investigators have found a higher metabolic cost of treadmill walking [ 40 , 43 ] and a higher perceived exercise and a less positive affective valence . in this paper we have only cited the acsm and aha guidelines of 150 minutes of moderate intensity aerobic activity every week ( p. vi ) , which would equal 30 minutes of walking five days a week . however , it should be acknowledged that there are other guidelines that have been published ( e.g. , the institute of medicine , world health organization , usda , etc . ) that recommend for the prevention of weight gain at least 60 min of moderate physical activity daily is needed and for weight loss / weight maintenance this may need to be as high as 6090 min daily . individuals wishing to lose weight may have difficulty not only completing 30 minutes of exercise but also completing longer durations which have been shown to be necessary for successful weight loss . further it should be noted that although we collected the data as one exercise bout for 30 minutes , there has been considerable research that has indicated that the 30 minutes ( or 60 minutes ) can be accumulated using multiple short bouts of exercise , which may be a more efficient strategy for individuals who are incorporating higher intensity physical activity into their day or need to put in 6090 minutes / day . according to the recommendations from acsm and the american heart association , individuals should participate in at least 150 minutes a week of moderate intensity cardiovascular exercise or 75 minutes a week of vigorous - intensity aerobic physical activity . previous literature has indicated that more vigorous exercise is needed for many of the health - related benefits . despite moderate exercise being a nebulous term , which can be widely interpreted , these preliminary data show that subjects self - selected a workload in the middle of the moderate range , but less than the workload ( > 60% vo2 peak - high end of the moderate intensity range ) that may be necessary to confer health benefits beyond just weight loss . this finding potentially has broad implications to practitioners in giving physical activity recommendations , as workload recommendations at the high end of the moderate range may result in a decreased in adherence . further men and overweight subjects tended to select lower workloads when asked to exercise at a moderate pace .

the purpose of this study was ( 1 ) to determine if overweight / obese individuals ( age 2650 y ) would self - select moderate exercise intensity when asked to do so and ( 2 ) to determine how this self - selected workload compared to exercising at a workload ( 60% peak aerobic capacity ) that is known to provide cardioprotective health benefits . oxygen consumption ( vo2 ) and energy expenditure were measured in 33 men / women ( bmi 27 kg / m2 ) who completed two 30 min walking bouts : ( 1 ) self - selected walking pace on an indoor track and ( 2 ) prescribed exercise pace ( 60% vo2 peak ) on a treadmill . the data revealed that ( 1 ) the prescribed intensity was 6% higher than the self - selected pace and elicited a higher energy expenditure ( p < 0.05 ) than the self - selected pace ( + 83 kj ) ; ( 2 ) overweight subjects walked at a slightly lower percentage of vo2 peak than the obese subjects ( p < 0.05 ) ; ( 3 ) men walked at a lower percentage of vo2 peak than the women ( p < 0.05 ) . in conclusion when asked to walk at a moderate intensity , overweight / obese individuals tended to select a lower workload in the moderate intensity range which could be maintained for 30 min ; however , a higher intensity which would be more cardioprotective could not be maintained for 30 min by most individuals .

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

PMC3658827

suid herpesvirus 1 ( suhv-1 ) is the causative agent of aujeszky 's disease , a disease of great impact on the swine industry . due to the important economic losses caused by this disease , notification of any outbreak infected young pigs suffer from neurological damage , whereas infected adults suffer from abortion and respiratory signals . suhv-1 has only one serotype , and virus differentiation is made by molecular techniques , in particular by the restriction fragment length pattern ( rflp ) , allowing the differentiation of circulating viral strains . the genotyping of samples by rflp was fundamental to understand the epidemiology of suhv-1 . despite the development of other molecular typing techniques , the main method of characterizing the suhv-1 is still digesting the full genome by the enzyme bamhi ( bamhi - rflp ) [ 48 ] . this methodology was used by several authors to characterize suhv-1 isolates in europe , japan , argentina , and brazil [ 716 ] . genotypes i and ii are distributed worldwide , and genotypes iii and iv , originally described in denmark and thailand , respectively , were no longer reported . currently , genotype i is prevalent in populations of wild boars in europe . the disadvantages of bamhi - rflp are the need for virus isolation and a time - consuming electrophoresis . the small amount of viral dna and the lack of facilities for virus isolation are also critical in the implementation of this technique . the aim of this work was to develop a fast multiplex pcr followed by enzymatic restriction analysis using bamhi enzyme that allows the characterization of suhv-1 without the need of virus isolation , even in samples with small viral dna amounts . primers were designed based on bamhi restriction sites found in the complete genome of suhv-1 available in genbank ( bk001744 ) , using the software webcutter ( http://rna.lundberg.gu.se/cutter2/ ) . after detection of the best regions , the sequences were submitted to the program pimer3plus . the amplicon sizes were chosen according to the restriction fragments that would be generated after enzymatic digestion and the possibility of their use in a multiplex pcr ( tables 1 and 2 ) . eleven isolates of suhv-1 ( five of genotype i and six of genotype ii ) , previously characterized in other works [ 16 , 19 ] , the vaccine strain bartha , standard sample shope , and attenuated isolate nia-4 , were used for pcr development . twenty clinical brain samples of pigs positive for suhv-1 by virus neutralization and virus isolation , collected from outbreaks in the state of santa catarina , brazil , in the years of 2002 and 2003 were used to standardize the technique in tissue sample . ten negative samples of the same tissue were also used to determine whether the pcrs would be able to detect the virus without unspecific reactions . total dna was extracted by commercial kit wizard genomic dna purification ( promega , usa ) and stored at 20c for later genotyping . the reactions were developed by testing different parameters such as annealing temperature , concentration of the reagents ( primers , dna polymerase , mgcl2 ) , and duration of each stage . the first tests were performed with separate primers for subsequent use in the multiplex . the multiplex pcrs were standardized using two sets of primers : mix a , which contained bamhi-1 - 738 , bamhi-3 - 256 , and bamhi-6/7 - 637 , and mix b , which contained primers bamhi-4 - 357 , bamhi-19/20 - 519 , and bamhi-16/17 - 799 , both of the mixes generate three bands prior to restriction enzyme . a 20 l reaction was standardized with the following concentrations of reagents : 10 pmol of each primer , 4 l of 5x buffer gotaq green ( promega , usa ) , 2.0 mmol / l magnesium chloride , 6% dmso , 300 mmol / l dntp ( invitrogen , usa ) , and 3 u gotaq hotstart ( promega , usa ) . the conditions used for both pcr were as follows : a denaturing stage at 95c for 5 min . , 35 cycles at 95c for 50 s , 60c for 50 s , 72c for 1 min . , and a final stage extension at 72c for 7 min .. positive and negative controls were used in all the pcr mixes to check for contamination and unspecific amplifications . enzymatic restriction of the mpcr with bamhi was performed according to the manufacturer 's instructions ( new england biolabs , usa ) . the gels were analyzed on 2.5% agarose gel stained with ethidium bromide at 0.5 mg ml after one hour of running . the primers chosen for pcr target nine restriction sites for bamhi detected in the in silico analysis of complete genome ( table 1 ) . the regions were chosen because they are the key points used to differentiate genomic types . among the chosen primer pairs , bamhi-1 - 738 and bamhi-4 - 357 are most important for the differentiation of all genotypes . genotype ii has no restriction site in bamhi-1 - 738 , and genotype iii has no restriction site in bamhi-4 - 357 ( table 2 ) . these same areas offer the opportunity to subtyping with the enzyme bsteii , which has a restriction site in bamhi-4 - 357 for genotype i. the other primers were selected because the regions can be used to differentiate some suhv-1 strains , like the standard strain shope , used in our laboratory for virus neutralization tests . the bamhi - mpcr was first developed with a set of six primer pairs in one reaction , but good visualization of the results could only be possible after two hours of electrophoresis . it was possible to differentiate genotypes i and ii using enzymatic restriction after dna amplification with mix a ( figure 1(a ) ) . mix b was used to differentiate specific strains like standard strain shope ( figure 1(b ) ) . among the twenty clinical samples , seventeen amplified as expected . three samples required the addition of a larger amount of dna for better visualization of bands in agarose gel . all isolates previously characterized in other studies [ 15 , 16 ] showed the expected restriction patterns for genotype ii ( figure 1 ) . the bamhi - rflp is one of the most used methods for molecular characterization of suhv-1 . although sequencing methods are considered more accurate [ 8 , 18 ] , the restriction enzyme analysis is still important for genotyping of suhv-1 . the biggest problems of rflp are the need of ultracentrifuges , the necessity of virus isolation and the electrophoresis that takes over eight hours under controlled temperature [ 14 , 16 ] . the multiplex pcr followed by restriction with bamhi ( bamhi - mpcr ) developed in this study allows the analysis of restriction sites for genotyping of suhv-1 directly from clinical samples without the need of any equipment other than that of a basic molecular biology laboratory . the bamhi - mpcr was able to detect the virus in all the clinical samples , which demonstrated its importance for genotyping suhv-1 when there is a shortage of equipment or isolation is not possible . we have previously developed a nested pcr for the same purpose in our laboratory , but it is a more laborious and time - consuming technique and very prone to contamination . pcr followed by restriction enzyme has been used characterize other viruses of the herpesviridae family , such as bovine herpesvirus 1 , 2 , and 5 , herpes simplex 1 [ 22 , 23 ] , varicella - zoster virus , and human cytomegalovirus . these techniques are simple and easy to perform in molecular biology laboratories and are very useful in epidemiological studies . genotyping of suhv-1 allows a better understanding of its epidemiology and helps to weigh the impacts of this infectious agent and , ultimately , to reduce or even avoid the losses it produces . molecular techniques have been used with many objectives such as to verify the introduction of new genotypes in a country , to characterize field isolates derived from vaccines samples and even to identify suhv-1 isolates from wild animals , such as the wild boar . the bamhi - mpcr is a fast and useful method for genotyping that allows for comparison with results from the published data of the last twenty - five years since the beginning of the use of bamhi - rflp technique .

suid herpesvirus 1 ( suhv-1 ) is the causative agent of aujeszky 's disease . the infectious agent has only one serotype , but it was classified by restriction enzyme analysis of the whole genome into four genotypes , named i to iv . the aim of this study was to standardize a rapid method for genotyping suhv-1 without virus isolation , using a multiplex - pcr followed by enzymatic restriction analysis . the complete genome of the virus was analyzed in silico to determine the restriction sites for the enzyme bamhi . primers were designed to flank sites with emphasis on certain points of differentiation of genotypes . the standard pcrs were able to detect the suhv-1 and also to differentiate genotypes from brain tissue of infected pigs . the bamhi - pcr is a rapid , practical , and sensitive way to genotype suhv-1 .

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

PMC3749605

over the past decade the ability to engineer and produce materials at the nano- or near - atomic scale has triggered rapid product development due to their new interesting properties that were not previously seen at scales above the micrometer . industrial applications using nanoparticles have resulted in an almost exponentially growing demand for nanosized materials . due to increasing use of nanoparticles in variety of consumer goods , humans are constantly exposed to such nanomaterials besides exposure at production sites [ 15 ] . unintended exposure to nanomaterials may occur via inhalation , dermal exposure , or gastrointestinal tract absorption and may pose a great risk [ 6 , 7 ] . despite their wide application , little zinc oxide ( zno ) is among the most commonly utilized group of nanomaterials and has wide - ranging applications . as a well - known photocatalyst , zno has received much attention in the degradation and complete mineralization of environmental pollutants [ 9 , 10 ] . zno nanoparticles ( zno - nps ) are used in industrial products including cosmetics , paints , and medical materials . zno - nps have external uses as antibacterial agents in ointments , lotions , mouthwashes , and surface coatings to prevent microorganism growth . zno - nps have also been used as a dietary supplement in human and livestock because zinc can stimulate the immune system and act in an anti - inflammatory way [ 12 , 13 ] . many in vitro studies demonstrated that zno - nps are toxic to mammalian cells and are even more toxic than other nanoscale structures of metallic oxide [ 1416 ] . in combination with uv exposure , zno - nps are known to generate reactive oxygen species ( ros ) like hydroxyl radicals or hydrogen peroxide in aqueous solutions leading to efficient decomposition of organic compounds . brunner et al . showed that a three - day exposure of human mesothelioma and rodent fibroblast cell to zno - nps ( 19 nm ) caused dna and mitochondrial damages . the human lung is a vulnerable organ for nanoparticle invasion as there is approximately 2300 km of airways and 300 million alveoli , giving rise to a large surface area , which is in contact with the environmental atmosphere and the ultrafine particulate pollutants present in it [ 19 , 20 ] . adverse systemic reactions have been observed after inhalation of zno fumes or accidental ingestion of large amounts of the metal [ 2123 ] . inhalation of zno has been found to compromise pulmonary function in pigs and caused pulmonary impairment and metal fume fever in humans [ 24 , 25 ] . karlsson et al . found that zno - nps ( 71 nm ) decreased cell viability and caused oxidative dna damage in human alveolar carcinoma epithelial cells ( a549 ) . despite the wide inhalational exposure of zno - nps , there are very few studies to demonstrate its toxicity on normal human lung cells and the possible mechanism of toxicity . therefore , the present study evaluated the toxicity of 50 nm zno - nps in human lung epithelial cells ( l-132 ) and tried to elucidate involvement of oxidative stress therein . as zinc - mediated metallothionein ( mt ) upregulation has been found to protect against oxidative stress - induced cellular injury , attempts were also made to study the effect of zno - nps on expression of mt in l-132 cells . dulbecco 's modified eagle 's medium ( dmem ) , penicillin , streptomycin , fetal bovine serum ( fbs ) , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , 2,7-dichlorofluorescin diacetate ( dcfh - da ) , metaphosphoric acid ( mpa ) , dimethyl sulfoxide ( dmso ) , o - phthaldialdehyde ( opt ) , and 5,5-dithiobis(2-nitrobenzoic acid ) were purchased from sigma chemical company ( st . the zno - nps ( 50 nm ) used in this study were synthesized in drde ( defence research and development establishment , gwalior , india ) , by sol gel method . the human lung epithelial cell line ( l-132 ) was purchased from national centre of cell science ( nccs , pune , india ) . cells were cultured in dmem medium containing 10% fbs and 100 u / ml penicillin - streptomycin - amphotericin mixture and incubated at 37c with 5% co2 . physicochemical properties of particles were analyzed using transmission electron microscopy ( tem ) , dynamic light scattering ( dls ) , and zeta potential analyzer . the morphology and size of nanoparticles in the stock dispersion were determined by transmission electron microscopy ( tem ) . dry powder of particles was suspended in cell culture medium at a concentration of 1 mg / ml and then sonicated at room temperature for 10 min at 40 w to form a homogeneous suspension . after sonication and stabilization , the tem samples were prepared by drop coating of the stock suspension on carbon - coated copper grids . tem measurements were performed at an accelerating voltage of 120 kv ( model 1200ex , jeol ltd . , tokyo , japan ) . zetapals ( brookhaven instruments corporation , holtsville , ny ) was used to determine the hydrodynamic size and zeta potential of particle suspension in cell culture medium . the sonicated dispersion of zno - nps was used to expose the cells at 5 , 25 , 50 , and 100 g / ml for 24 h. based on the results of previous study done ( data not shown ) with different doses of nanoparticles , a dose range was selected which is also studied by ahamed et al . . exposure of cells was performed with 80% confluence of cell in 25 cm flasks and 24-well plates in a humidified atmosphere at 37c and 5% co2 . cells free of zno - nps were used as control cells throughout each assay . l-132 cells were plated into a 24-well plate at a density of 50,000 cells / well . cells were grown overnight in the full medium followed by exposure to zno - np . following the exposure of 24 h , the cells were incubated with mtt ( 5 mg / ml ) for 4 h. the medium was then removed , and 200 l of dmso was added into each well to dissolve formazan crystals , the metabolite of mtt . after thorough mixing , the plate was read at 570 nm for optical density that directly correlates with cell quantity . survival rate was calculated from the relative absorbance at 570 nm and expressed as the percentage of control . cells were plated into a 6-well culture plate at a density of 75000 cells / well ( in 2 ml growth medium ) . after overnight growth , supernatants from the culture plates were aspirated out , and fresh aliquots of growth medium containing zno - nps in desired concentrations ( 5100 g / ml ) were added . upon incubation , cells were washed with phosphate buffered saline ( 0.1 m pbs , ph 7.4 ) , and the morphological changes were observed using leishman 's stain under an inverted phase contrast microscope at 200x magnification . the production of intracellular reactive oxygen species ( ros ) was measured using dcfh - da . dcfh - da passively enters the cell , where it reacts with ros to form a highly fluorescent compound , dichlorofluorescein ( dcf ) . l-132 cells were plated into a 24-well plate at a density of 50,000 cells / well . the cells were incubated at 37c for 30 min with dcfh - da working solution ( 100 m in methanol ) to yield a 10 m into each well . then cells were washed twice with pbs and exposed to different concentrations of zno - np for 6 , 12 , and 24 h. after exposure to zno - np , the fluorescence was observed at 485 nm excitation and 525 nm emission using a bio - tek synergy ht - i plate reader ( bio - tek instruments , usa ) . the method is based on a reaction between gsh and o - phthaldialdehyde ( opt ) which gives the fluorescence . thus gsh concentration in a sample solution can be determined by observing the fluorescence at 360 nm excitation and 420 nm emissions . l-132 cells were seeded into a 25 cm flask at a density of 1.0 10 cells . after 24 h exposure to zno - nps , the cells were scraped and pelleted by centrifugation at 5000 rpm for 5 min , then washed in pbs . the cells were homogenized in 200 l of phosphate - edta buffer ph 8.0 and 80 l of 20% metaphosphoric acid . the cell homogenate was centrifuged at 16000 rpm at 4c for 30 min . the assay was performed by taking 100 l supernatants and mixing it with 800 l of phosphate - edta buffer containing 100 l opt ( 10 mg / ml in methanol ) . after thorough mixing and incubation at room temperature for 15 min , fluorescence was measured at 360 nm excitation and 420 nm emission using bio - tek synergy ht - i plate reader ( bio - tek instruments , usa ) . results were calculated as nmol of glutathione per mg of protein and presented as a percentage of the control group . protein assays in the cell lysate were performed using a bca protein assay kit ( pierce , usa ) . l-132 cells ( 5 10 cells ) exposed to zno - nps ( 5 , 25 , 50 , and 100 g / ml ) for 48 h were collected into tubes and washed with pbs . the cells were incubated for 3 h in lysis buffer ( 20 mm tris - hcl , ph 8.0 , 5 mm edta , 0.1 m nacl , 0.5% sds , and 100 g / ml rnase ) at 37c . after incubation , phenol : chloroform ( 1 : 1 ) mixture was used to extract dna . by adding an equal volume of ice - cold absolute isopropanol , dna was precipitated . dna was dissolved in 50 l of 1x te ( 10 mm tris , 1 mm edta , ph 8.0 ) . twenty g of dna was loaded onto 1.5% agarose gel ; electrophoresis was carried out at 60 v for 2 h with tbe as the running buffer . the fluorescent probe hoechst 33342 stains nuclei specifically , with little or no cytoplasm labeling . cells exposed to different concentrations of zno - nps ( 50 nm ) were collected and sequentially washed by pbs . then , the cells were kept in 1 g / ml hoechst working solution for 15 min in the dark at room temperature . finally , the cells were washed twice with pbs to remove excess stain and examined under fluorescent microscope ( leica - dmlb , germany ) with excitation wavelength of 403 nm . human metallothionein ( mt ) messenger rna ( mrna ) expression was determined by reverse transcriptase polymerase chain reaction ( rt - pcr ) . cells were exposed to 5 , 25 , 50 , and 100 g / ml of zno - np for 24 h , and total rna was isolated using rneasy mini kit ( qiagen , usa ) . the concentration and integrity of rna were measured using nanodrop spectrophotometer prior to the experiment . the enhanced avian hs rt - pcr kit ( sigma , usa ) was used for the amplification of human mt-1 and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) gene , according to the manufacturer 's instructions . the sequences of primer sets used were mt-1 forward , 5-ccactgcttcttcgcttctc-3 and reverse , 5-aggagcagcagctcttcttg-3 ; gapdh forward , 5-ggcgctgagtacgtcgtggagt-3 and reverse 5-cgcctgcttcaccaccttcttg-3. all data were reported as mean standard deviation ( sd ) . statistical analysis was performed for the experiments conducted in at least triplicate using one - way anova followed by dunnett test . results with p other physicochemical characteristics are detailed in table 1 . mtt assay showed that exposure of human lung epithelial cells ( l-132 ) to 50 nm zno - nps at concentrations of 25 , 50 , and 100 g / ml for 24 h significantly reduced the cell viability in a concentration - dependent manner . however , reduction in cell viability at lower concentration of 5 g / ml was not significant . as the concentrations of zno - nps increased from 25 to 100 g / ml , the cell viability significantly decreased from 55% to 25% ( figure 1 ) . the morphology of human lung epithelial cells was examined after zno - nps exposure using phase contrast microscopy . cells exposed to 50 nm zno - nps for 24 h showed a round shape morphology than control cells ( figure 2 ) . at higher concentration cells appeared to be strongly damaged with a shrunken morphology and detached from the substrate indicating an almost complete destruction of the cells exposed to zno - nps ( figure 2 ) . the ability of 50 nm zno - np to induce intracellular oxidant production in l-132 cells was assessed by measuring dcf fluorescence as a marker of ros generation . with the increase of exposure time , a significant ros formation was observed . zno - nps significantly induced the formation of ros from 16% to 25% at concentration of 25 g / ml to 100 g / ml after 24 h of exposure ( figure 3 ) . the intracellular gsh measurement has shown decrease in the intracellular gsh level with the increasing concentrations of zno - nps after 24 h exposure . the significant difference in intracellular gsh level was observed at 50 and 100 g / ml with remaining gsh of about 14% and 4% , respectively , compared to control ( figure 4 ) . l-132 cell exposed to 50 nm zno - nps for 48 h showed dna damage ( figure 5 ) , while 24 h of exposure did not show any damage . extended exposure of zno - nps caused concentration - dependant dna damage which was observed in the form of ladder . the nuclei of exposed cells appeared to be fragmented , smaller , and rougher , with condensed nuclear material ( figure 6 ) . metallothionein is known to facilitate metal detoxification and is involved in scavenging of free radicals . after 24 h exposures of l-132 cells to 50 nm zno - nps , upregulation in mt gene expression was observed . although the expression was increased initially from concentration of 5 to 50 g / ml , later it decreased at 100 g / ml ( figure 7 ) . although the beneficial effects of zno - nps have attracted considerable attention in terms of nanomedicine [ 29 , 3638 ] , potential biological and environmental hazards should also be taken into account . in the present study , 50 nm zno - nps exposure significantly reduced cell viability of human lung cells starting at approximately 25 g / ml concentration . the lowering of cell density and the rounding of cells observed suggest that 25 , 50 , and 100 g / ml nanoparticle concentrations induced substantial cell death . the cytotoxicity results are in accordance with previous studies [ 16 , 3941 ] . oxidative stress as a common mechanism for cell damage induced by nanoparticles is well documented . a wide range of nanomaterial species have been shown to generate ros both in vivo and in vitro [ 2 , 4244 ] . similarly , in the present study , 50 nm zno - nps showed generation of ros with significant depletion of reduced glutathione store . it is well - known that oxidative stress leads to cell death , either by apoptosis or necrosis depending on its extent of severity . severe oxidative stress to cells causes necrosis while the moderate one causes apoptosis . in the present study , the type of cell death ( apoptosis / necrosis ) after 50 nm zno - nps exposures was evaluated by dna damage analysis and hoechst staining . formation of the larger dna fragments has been shown to occur in the absence of oligonucleosome formation . moreover , cleavage of dna into the larger fragments is sufficient to allow chromatin condensation and subsequent apoptosis in the absence of oligonucleosome formation . in agreement to this , exposure of 50 nm zno - nps produced large fragments of dna in the range of 100 to 850 bp showing ladder formation in agarose gel . hoechst staining also showed chromatin condensation and apoptotic body formation at higher concentration of zno - nps , suggesting apoptotic type of cell death . thus , it is possible that the zno - nps might have induced apoptosis in human lung cells . since metallothionein ( mt ) is considered as one of the essential biomarkers in metal - induced toxicity facilitating metal detoxification and protection from free radicals , the ability of zno - nps to modulate the expression of mt gene was also assessed . in present study , 50 nm zno - np exposed cells showed upregulation of the mt gene . at lower concentrations of zno - nps , mt gene expression study indicates that , during the cellular stress - induced by 50 nm zno - nps , initially cells were able to protect themselves by upregulating mt gene using its antioxidant and metal detoxifying properties but at higher concentrations of zno - nps ; same phenomenon did not work because of greater extent of cell stress showing decrease in mt expression . in summary , 50 nm zno - nps induced cytotoxicity in cultured human lung epithelial cells ( l-132 ) by elevating oxidative stress in a concentration - dependant fashion . zno - nps also induced dna damage characterized by chromatin condensation and dna ladder pattern illustrating apoptotic type of cell death . therefore , oxidative stress - induced apoptosis can be considered as one of the pathways of toxicity by zno - nps . hence , care has to be taken while processing and formulating the nanoparticles till its final finished product .

zinc oxide nanoparticles ( zno - nps ) are increasingly used in sunscreens , biosensors , food additives , pigments , rubber manufacture , and electronic materials . with the wide application of zno - nps , concern has been raised about its unintentional health and environmental impacts . this study investigates the toxic effects of zno - nps in human lung cells . in order to assess toxicity , human lung epithelial cells ( l-132 ) were exposed to dispersion of 50 nm zno - nps at concentrations of 5 , 25 , 50 , and 100 g / ml for 24 h. the toxicity was evaluated by observing changes in cell morphology , cell viability , oxidative stress parameters , dna damage analysis , and gene expression . exposure to 50 nm zno - nps at concentrations between 5 and 100 g / ml decreased cell viability in a concentration - dependent manner . morphological examination revealed cell shrinkage , nuclear condensation , and formation of apoptotic bodies . the oxidative stress parameters revealed significant depletion of gsh level and increase in ros levels suggesting generation of oxidative stress . zno - nps exposure caused dna fragmentation demonstrating apoptotic type of cell death . zno - nps increased the expression of metallothionein gene , which is considered as a biomarker in metal - induced toxicity . to summarize , zno - nps cause toxicity in human lung cells possibly through oxidative stress - induced apoptosis .

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

PMC3587394

type 2 diabetes is a worldwide epidemic associated with obesity and a sedentary lifestyle.1 the estimated lifetime risk of developing diabetes for a person born in the united states in 2000 is 32.8% for males and 38.5% for females.2 diabetes increases morbidity and mortality due to heart disease , stroke , blindness , kidney failure , foot problems , and periodontal disease,3 and has a significant impact on quality of life.4 in 2010 it accounted for us$376 billion or 12% of the global health expenditure . this is approximately us$1330 per person per year.5 treatment goals for patients with diabetes include achieving and maintaining optimal blood glucose , blood pressure , and lipid levels in order to prevent or delay the progression of chronic complications.6 exercise , along with diet and weight control , is considered essential for the prevention and management of diabetes . epidemiological studies suggest that physical activity can reduce the risk of type 2 diabetes by 30% to 50% in the general population.7 exercise helps treat the glucose , blood pressure , and lipid abnormalities often found in people with diabetes , and assists with weight loss maintenance.8 in the united states , only 39% of adults with diabetes are active compared to 58% of those without the condition.9 vo2max , the maximum amount of oxygen in milliliters that can be used in one minute per kg of body weight , is a measurement of cardiovascular fitness . it correlates with insulin sensitivity in people at risk of developing type 2 diabetes.10 moderate aerobic exercise requires 40% to 60% of vo2max or 50% to 70% of the maximum heart rate . aerobic exercise is considered vigorous when it requires > 60% vo2max or > 70% of the maximum heart rate.11 for many persons with diabetes , moderate aerobic exercise would be the equivalent of brisk walking . endurance aerobic exercise is usually performed continuously over a prolonged period of time at submaximal intensity . most recommendations are for 150 to 210 minutes per week of moderate - intensity endurance aerobic exercise , plus some resistance exercise , spread over three to five sessions.8,1214 this time commitment is in addition to all of the other self - care activities recommended for people with diabetes , and a lack of time is often cited as a reason for not exercising.15 a cardiac evaluation may be required especially when vigorous physical activity is being contemplated and in the presence of additional risk factors for coronary artery disease.16 meta - analyses on the effects of exercise have estimated that for people with type 2 diabetes , both aerobic and resistance exercise improve glycemic control to an extent comparable to some oral antidiabetic drugs.1723 exercise should theoretically be an attractive option for people who prefer not to use drugs , or wish to obtain additional blood glucose control benefits . a meta - analysis of randomized controlled trials of at least 12 weeks in duration concluded that structured exercise training of more than 150 minutes of exercise per week resulted in greater hba1c reductions ( 0.89% ) , than those with less weekly exercise time ( 0.36%).18 another meta - analysis of aerobic exercise studies concluded that not only did higher exercise intensity tend to produce larger improvements in vo2max , but that exercise intensity predicted postintervention hba1c ( r = 0.91 , p = 0.002 ) better than exercise volume ( r = 0.46 , p = 0.26 ) . workouts were , on average , 49 minutes ( including 10 to 15 minutes of warm - up and cool - down ) , with a mean of 3.4 sessions per week for 20 weeks.24 however , only one study included in the meta - analysis approached high - intensity at 75% of vo2max.25 in another meta - analysis for studies involving aerobic , resistance , and combined training , the overall reduction in hba1c was 0.8% ( 90% ci 0.3 ) with the effect of exercise intensity being unclear.20 skeletal muscle is responsible for most of the uptake of glucose after a meal , and transport of glucose into the muscle is considered the limiting step in glucose disposal.26,27 glucose transport occurs primarily by diffusion utilizing glucose transporter carrier proteins ( glut ) . both exercise and insulin regulate glucose transport mainly by the translocation of the glut4 isoform from an intracellular compartment to the plasma membrane and transverse tubules.8,28 glut4 levels are considered an important determinant of insulin sensitivity26,27 at rest and postprandially , glucose uptake is insulin - dependent , with the major purpose being the replenishment of muscle glycogen stores.8 insulin - stimulated glut4 translocation is generally impaired in type 2 diabetes.28 during exercise , muscle utilizes glucose made available by intramuscular glycogenolysis and by increased glucose uptake . both aerobic and resistance exercises increase glut4 abundance and translocation , and hence blood glucose uptake by a pathway that is not dependent on insulin.8 glucose uptake into contracting muscle is therefore normal even in the presence of type 2 diabetes.8,28,29 following exercise , glucose uptake remains elevated with the contraction - mediated pathway remaining active for several hours.8 during moderate - intensity exercise ( 60% vo2max ) of short duration in persons without diabetes , increased glucose uptake by muscle is balanced by an equal rise in hepatic glucose production , and blood glucose levels remain unchanged.8,30 there is a decrease in insulin level , which sensitizes the liver to glucagon , thus increasing glucose production.30 catecholamines play a role in increasing glucose production only during moderate - intensity exercise greater than 2 hours duration . with type 2 diabetes , blood glucose uptake by muscles usually increases more than hepatic production.31 this is also normally accompanied by a decline in plasma insulin levels , greatly reducing the risk of hypoglycemia in diabetics not using insulin or insulin secretagogues.8 the effects of aerobic exercise vary with duration and intensity , but following a single exercise session there is generally an increase in insulin action and hence glucose tolerance for between 24 and 72 hours.8 skeletal muscle is responsible for most of the uptake of glucose after a meal , and transport of glucose into the muscle is considered the limiting step in glucose disposal.26,27 glucose transport occurs primarily by diffusion utilizing glucose transporter carrier proteins ( glut ) . both exercise and insulin regulate glucose transport mainly by the translocation of the glut4 isoform from an intracellular compartment to the plasma membrane and transverse tubules.8,28 glut4 levels are considered an important determinant of insulin sensitivity26,27 at rest and postprandially , glucose uptake is insulin - dependent , with the major purpose being the replenishment of muscle glycogen stores.8 insulin - stimulated glut4 translocation is generally impaired in type 2 diabetes.28 during exercise , muscle utilizes glucose made available by intramuscular glycogenolysis and by increased glucose uptake . both aerobic and resistance exercises increase glut4 abundance and translocation , and hence blood glucose uptake by a pathway that is not dependent on insulin.8 glucose uptake into contracting muscle is therefore normal even in the presence of type 2 diabetes.8,28,29 following exercise , glucose uptake remains elevated with the contraction - mediated pathway remaining active for several hours.8 during moderate - intensity exercise ( 60% vo2max ) of short duration in persons without diabetes , increased glucose uptake by muscle is balanced by an equal rise in hepatic glucose production , and blood glucose levels remain unchanged.8,30 there is a decrease in insulin level , which sensitizes the liver to glucagon , thus increasing glucose production.30 catecholamines play a role in increasing glucose production only during moderate - intensity exercise greater than 2 hours duration . with type 2 diabetes , blood glucose uptake by muscles usually increases more than hepatic production.31 this is also normally accompanied by a decline in plasma insulin levels , greatly reducing the risk of hypoglycemia in diabetics not using insulin or insulin secretagogues.8 the effects of aerobic exercise vary with duration and intensity , but following a single exercise session there is generally an increase in insulin action and hence glucose tolerance for between 24 and 72 hours.8 high - intensity interval training ( hit ) consists of brief bursts of very vigorous exercise separated by brief recovery periods . while there is no universal definition of hit , it often refers to exercise performed with an all out effort , or at least to an intensity that approaches vo2max ( 90% vo2max).32 the classic form of all out hit is the wingate test . after about 3 to 5 minutes of warm - up the subject cycles for 30 seconds at maximum effort against a standardized resistance . typically four to six wingate tests are performed separated by 4 minutes of rest , for a total of 2 to 3 minutes of maximal exercise spread over 15 to 30 minutes.33 this all out cycle ergometer form of hit is also referred to as sprint interval training ( sit ) . when used in this paper , sit will refer only to wingate tests as just described . because of the intensity and short duration of the wingate test , most of the power generated represents anaerobic as opposed to aerobic power , with an aerobic power contribution of between 16% and 19.5%.3436 the primary energy source is glucose derived from muscle glycogen , and as aerobic capacity the initial 30-second wingate test can use almost a quarter of the stored muscle glycogen , and although the rate of glycogenolysis is reduced in subsequent bouts , significant amounts of lactate accumulate.37,38 the exercise is extremely stressful with the perceived exertion being very high . reports of nausea and light - headedness are not uncommon.39 it requires a high level of motivation , and often sessions are supervised , with significant verbal encouragement to exert maximal effort.40 the average person with diabetes is not likely to tolerate this well . while time spent exercising intensely is very short , training time commitment is longer as it will include warm - up , cool - down , and rest periods . other forms of hit may not require all out effort and may be performed at only half the intensity of an all out sit protocol but with longer sessions , more repetitions , and shorter rest periods , and are tolerated much better.41,42 high - intensity exercise may also be performed on a continuous basis , but even very fit persons can usually maintain an intensity of 80% vo2max for only 10 to 15 minutes.30 the exercise load needed depends on the individual s exercise capacity . for people with a low vo2max of 20 ml / kg per minute , the necessary exercise load may be equivalent to walking up a slight grade at 3 mph.42 in intense exercise ( > 80% vo2max ) , unlike at lesser intensities , glucose is the exclusive muscle fuel.30 catecholamine levels rise markedly , causing glucose production to rise seven- to eightfold while glucose utilization is only increased three- to fourfold . in people without diabetes there is a small blood glucose increase during intense exercise that increases further immediately at exhaustion and persists for up to 1 hour . all out sit sessions over 2 weeks improved the mean cycle endurance time to fatigue while cycling at approximately 80% of pretraining vo2max by 100% ( from 26 to 51 minutes).43 this required a total high - intensity exercise time of only 15 minutes with a total training time commitment of approximately 2.5 hours . in another study , a less intense version of hit ( 610 cycling bouts of 30 seconds each at 125% of the power at vo2max with 2 minutes recovery ) produced a similar improvement in vo2max after 4 weeks of training , as was seen in the more intense sit group ( three to five all out 30-second cycling bouts with 4 minutes of recovery).41 the less intense hit required only half the intensity but double the repetitions of the sit , and may be more practical for the nonathlete . the aim of this paper is to review the impact of high - intensity exercise of short duration on blood glucose levels in diabetic and nondiabetic people . in intense exercise ( > 80% vo2max ) , unlike at lesser intensities , glucose is the exclusive muscle fuel.30 catecholamine levels rise markedly , causing glucose production to rise seven- to eightfold while glucose utilization is only increased three- to fourfold . in people without diabetes there is a small blood glucose increase during intense exercise that increases further immediately at exhaustion and persists for up to 1 hour . hit is effective in improving aerobic endurance . in one study six all out sit sessions over 2 weeks improved the mean cycle endurance time to fatigue while cycling at approximately 80% of pretraining vo2max by 100% ( from 26 to 51 minutes).43 this required a total high - intensity exercise time of only 15 minutes with a total training time commitment of approximately 2.5 hours . in another study , a less intense version of hit ( 610 cycling bouts of 30 seconds each at 125% of the power at vo2max with 2 minutes recovery ) produced a similar improvement in vo2max after 4 weeks of training , as was seen in the more intense sit group ( three to five all out 30-second cycling bouts with 4 minutes of recovery).41 the less intense hit required only half the intensity but double the repetitions of the sit , and may be more practical for the nonathlete . the aim of this paper is to review the impact of high - intensity exercise of short duration on blood glucose levels in diabetic and nondiabetic people . pubmed was searched in july 2012 using the following search terms : high - intensity interval training , sprint interval training , and high - intensity exercise each combined with glucose and/or diabetes . to be included : ( 1 ) exercise intensity had to be at least 80% vo2max or 90% maximum heart rate , or include maximal cycle ergometer sprints ; ( 2 ) the duration was no more than 15 minutes of high - intensity exercise and 30 minutes of total exercise time per session ; and ( 3 ) glycemic control was assessed . six studies with a total of 51 male and 14 female participants in the high - intensity exercise groups are reviewed . four of these used sit ( maximal effort cycle ergometer ) exercise protocols of 2 to 6 weeks duration,39,4446 and two used near maximal running as the intervention ( table 1).47,48 although near maximal running would not be as intense as the cycle ergometer , it was not surprising that in the study with the heavier subjects,47 overuse shin splint injuries caused three out of eight participants in the intense running group to miss between one and four training sessions . three studies divided participants into an intervention and comparison group,39,47,48 with two stating that allocation was random.39,48 both richards et al39 and babraj et al44 studied young healthy subjects who were sedentary or recreationally active and found that 2 to 3 days after a 2-week exercise program consisting of six sessions of sit , insulin sensitivity but not fasting blood glucose improved compared to baseline . whyte et al46 studied overweight and obese sedentary men , and after a similar sit protocol found no improvement in fasting blood glucose ; moreover , insulin sensitivity was improved compared to baseline at 24 but not 72 hours . richards et al39 randomized subjects to one of three groups : ( 1 ) six sessions of sit ; ( 2 ) a single session of sit ; and ( 3 ) no exercise . insulin sensitivity was estimated before and 72 hours after the intervention by means of a hyperinsulinemic euglycemic clamp , considered the gold standard test . it increased in the group that did six sessions of sit ( mean change of glucose infusion rate : + 1.7 0.6 mg / kg per minute , p = 0.04 ) but not in the single session sit and no exercise groups . the intervention had no effect on the thermogenic response to beta - adrenergic receptor stimulation , which is considered an important determinant of energy expenditure and by extension a major regulator of energy balance and body mass . babraj et al44 estimated insulin sensitivity before and 48 to 72 hours after the intervention by means of oral glucose tolerance tests and the cederholm index . while fbg and fasting insulin levels were unchanged , both glucose area under the curve ( auc ; 12% ) , and insulin auc ( 37% ) , were significantly reduced during the oral glucose tolerance tests . in addition , aerobic cycling performance was improved by about 6% ( p < 0.01 ) compared to baseline . endurance aerobic and strength training studies of up to 16 months duration have generally demonstrated only a reduction in insulin auc in response to a glucose load following training , without a concurrent reduction in glucose auc.44 burgomaster et al45 demonstrated that sit increased muscle glut4 content , a determinant of insulin sensitivity , by 20% compared to baseline after 1 week of exercise , and that the levels remained elevated over the remaining 5 weeks of training and a subsequent 6 weeks of detraining . muscle oxidative capacity , as estimated by the protein content of cytochrome c oxidase subunit 4 ( cox4 ) also increased by 35% after 1 week of hit , and remained higher compared with baseline after 6 weeks of detraining ( p < 0.05 ) . nybo et al47 found that 20 minutes of near maximal running ( 40 minutes of total exercise time ) per week for 12 weeks was as effective as 150 minutes of running at 65% vo2max per week over the same period , in improving both fasting blood glucose and blood glucose 2 hours after the ingestion of 75 g of glucose . for the latter , blood glucose was improved from a mean of 6.1 ( standard error ( se ) 0.6 ) mmol / l to 5.1 ( se 0.4 ) mmol / l ( p < 0.05 ) in the maximal running group and from 5.6 ( se 1.5 ) mmol / l to 4.9 ( se 1.1 ) mmol / l ( p < 0.05 ) in the prolonged running group . sandvei et al48 had similar findings , with only 7.5 to 15 minutes of near maximal running per week , but a longer total exercise time when warm - up and rest periods were included . these studies therefore demonstrate that in young nondiabetic adults , as little as 15 minutes of high - intensity exercise spread over 2 weeks is enough to improve insulin sensitivity without a change of body weight . in contrast , a typical aerobic training program consumes 2000 to 3000 kcal / week with guidelines recommending 150 minutes of training per week . it was postulated that despite the negligible energy expenditure , hit improved insulin action by depleting muscle glycogen stores.44 six studies with a total of 51 male and 14 female participants in the high - intensity exercise groups are reviewed . four of these used sit ( maximal effort cycle ergometer ) exercise protocols of 2 to 6 weeks duration,39,4446 and two used near maximal running as the intervention ( table 1).47,48 although near maximal running would not be as intense as the cycle ergometer , it was not surprising that in the study with the heavier subjects,47 overuse shin splint injuries caused three out of eight participants in the intense running group to miss between one and four training sessions . three studies divided participants into an intervention and comparison group,39,47,48 with two stating that allocation was random.39,48 both richards et al39 and babraj et al44 studied young healthy subjects who were sedentary or recreationally active and found that 2 to 3 days after a 2-week exercise program consisting of six sessions of sit , insulin sensitivity but not fasting blood glucose improved compared to baseline . whyte et al46 studied overweight and obese sedentary men , and after a similar sit protocol found no improvement in fasting blood glucose ; moreover , insulin sensitivity was improved compared to baseline at 24 but not 72 hours . richards et al39 randomized subjects to one of three groups : ( 1 ) six sessions of sit ; ( 2 ) a single session of sit ; and ( 3 ) no exercise . insulin sensitivity was estimated before and 72 hours after the intervention by means of a hyperinsulinemic euglycemic clamp , considered the gold standard test . it increased in the group that did six sessions of sit ( mean change of glucose infusion rate : + 1.7 0.6 mg / kg per minute , p = 0.04 ) but not in the single session sit and no exercise groups . the intervention had no effect on the thermogenic response to beta - adrenergic receptor stimulation , which is considered an important determinant of energy expenditure and by extension a major regulator of energy balance and body mass . babraj et al44 estimated insulin sensitivity before and 48 to 72 hours after the intervention by means of oral glucose tolerance tests and the cederholm index . while fbg and fasting insulin levels were unchanged , both glucose area under the curve ( auc ; 12% ) , and insulin auc ( 37% ) , were significantly reduced during the oral glucose tolerance tests . in addition , aerobic cycling performance was improved by about 6% ( p < 0.01 ) compared to baseline . endurance aerobic and strength training studies of up to 16 months duration have generally demonstrated only a reduction in insulin auc in response to a glucose load following training , without a concurrent reduction in glucose auc.44 burgomaster et al45 demonstrated that sit increased muscle glut4 content , a determinant of insulin sensitivity , by 20% compared to baseline after 1 week of exercise , and that the levels remained elevated over the remaining 5 weeks of training and a subsequent 6 weeks of detraining . muscle oxidative capacity , as estimated by the protein content of cytochrome c oxidase subunit 4 ( cox4 ) also increased by 35% after 1 week of hit , and remained higher compared with baseline after 6 weeks of detraining ( p < 0.05 ) . nybo et al47 found that 20 minutes of near maximal running ( 40 minutes of total exercise time ) per week for 12 weeks was as effective as 150 minutes of running at 65% vo2max per week over the same period , in improving both fasting blood glucose and blood glucose 2 hours after the ingestion of 75 g of glucose . for the latter , blood glucose was improved from a mean of 6.1 ( standard error ( se ) 0.6 ) mmol / l to 5.1 ( se 0.4 ) mmol / l ( p < 0.05 ) in the maximal running group and from 5.6 ( se 1.5 ) mmol / l to 4.9 ( se 1.1 ) mmol / l ( p < 0.05 ) in the prolonged running group . sandvei et al48 had similar findings , with only 7.5 to 15 minutes of near maximal running per week , but a longer total exercise time when warm - up and rest periods were included . these studies therefore demonstrate that in young nondiabetic adults , as little as 15 minutes of high - intensity exercise spread over 2 weeks is enough to improve insulin sensitivity without a change of body weight . in contrast , a typical aerobic training program consumes 2000 to 3000 kcal / week with guidelines recommending 150 minutes of training per week . it was postulated that despite the negligible energy expenditure , hit improved insulin action by depleting muscle glycogen stores.44 there has been little testing of brief high - intensity exercise in either type 1 or type 2 diabetic patients . in addition to maximal cycle ergometer sit , hit protocols included a less intense form of hit not requiring all out effort , and protocols requiring very short bursts of exercise ( as little as 4 seconds ) interspersing up to 30 minutes of moderate exercise . noninterval high - intensity exercise protocols included continuous exercise at 80% to 110% vo2max for up to 13 minutes . six studies measured the effects of single exercise sessions , and the other two studies were of 2 and 7 weeks duration . all involved small numbers of subjects ( up to eight different subjects in an intervention group ) , and except for three studies the mean age was < 35 years ( table 2 ) . a single session of continuous high - intensity exercise resulted in 60 minutes of postexercise hyperglycemia,49 while both a single session of hit,50 and a 2-week training program51 have been shown to improve postprandial glucose control over a 24-hour period following exercise . little et al51 evaluated the effects of six sessions of hit over 2 weeks on glucose regulation 48 to 72 hours after the last training session in people with type 2 diabetes . most participants engaged in 60 minutes or less of exercise per week prior to entering the study . the hit protocol required only 30 minutes of high - intensity exercise per week , with a total time commitment ( including warm - up , cool - down , and rest ) of 75 minutes . the exercise intensity was less and may be more acceptable than all out sit protocols . when asked how enjoyable would engaging in hit three times per week for the next 4 weeks be , the mean response was 7.9 1.0 on a scale ranging from 1 ( not enjoyable at all ) to 9 ( very enjoyable ) . additionally , it elicited ratings of perceived exertion of 4 to 8 on a 10-point scale . before training and from 48 to 72 hours after the last training session , glucose regulation was assessed using 24-hour continuous glucose monitoring under standardized dietary conditions . the average 24-hour blood glucose concentration was reduced by 13% , from 7.6 mmol / l ( sd 1 ) to 6.6 mmol / l ( sd 0.7 ) after training ( p < 0.05 ) . the sum of the 3-hour postprandial glucose auc for breakfast , lunch , and dinner was reduced by 30% ( p < 0.05 ) . gillen et al,50 studying 7 of the 8 individuals who participated in the study by little et al,51 and using an identical exercise protocol , demonstrated that a single exercise session also reduced the sum of the 3-hour postprandial glucose auc ( p = 0.01 ) and the proportion of time spent above 10 mmol / l in the 24-hour postexercise period when compared to a nonexercising control day . average 24-hour blood glucose was , however , not significantly reduced ( p = 0.16 ) . the results of the two studies might be clinically significant , as controlling postprandial hyperglycemia is a treatment goal with type 2 diabetics . kjaer et al49 investigated the effect of 5 minutes of high - intensity exercise on blood glucose control during and for 3 hours immediately following exercise in type 2 diabetic patients ( two on a sulfonylurea and five on diet only ) . there was a greater and more sustained rise in glucose levels in type 2 diabetics compared to controls . in type 2 diabetic subjects , blood glucose increased from a pre - exercise level of 147 mg / dl ( sd 21 ) to a peak 30 minutes this value was maintained until 60 minutes postexercise , and then plasma levels decreased over the remainder of the 180-minute recovery period . for the controls , blood glucose increased from a pre - exercise level of 90 mg / dl ( sd 4 ) to a peak at 10 minutes postexercise at 100 mg / dl ( sd 5 ) . glucose concentrations at 60 minutes postexercise did not differ significantly from pre - exercise levels . in both groups , plasma insulin levels increased after exercise above pre - exercise levels , and returned to baseline about 120 minutes postexercise . plasma epinephrine and glucagon responses to exercise were higher in type 2 diabetics than in control subjects ( p < 0.05 ) . however , 24 hours after exercise in the type 2 diabetic group and not the controls , there was an increased effect of insulin on glucose uptake compared to the pre - exercise state as estimated by the insulin clamp technique . other studies have found similar increases in insulin - mediated glucose disposal after short - term high - intensity exercise in insulin - resistant subjects.52,53 it was concluded that because of exaggerated counter - regulatory hormonal responses , maximal dynamic exercise results in a 60-minute period of postexercise hyperglycemia and hyper - insulinemia in type 2 diabetics.49 the high - intensity studies involving type 1 diabetics mainly investigated blood glucose control during and in the 2 hours after exercise . the number of cycle bouts per training session was increased from four in week 1 , to six in week 2 , eight in week 3 , and ten in weeks 47 . sit resulted in a greater rise in plasma glucose during and immediately after exercise ( a 20-minute period ) in diabetics compared to nondiabetic controls . guelfi et al55 studied the effects of hit ( repeated 4-second cycle ergometer efforts separated by rest ) on blood glucose during exercise and in the immediate 1-hour postexercise period . after the postprandial peak in blood glucose , on alternate days , participants either exercised or rested . during exercise blood glucose declined more rapidly as compared to the nonexercising controls , indicating that high intensity exercise may increase the risk of hypoglcemia . however during the recovery period blood glucose levels continued to decline in the controls while remaining stable in the exercise group suggesting a decreased risk of postexercise hypoglycemia . guelfi et al56 also compared a hit protocol that was combined with moderate - intensity exercise ( repeated 4-second cycle ergometer efforts over 30 minutes separated by cycling at 40% vo2max ) to moderate exercise only ( cycling at 40% vo2max ) for 30 minutes . blood glucose fell to a greater extent in the moderate exercise group compared to the hit group , and remained stable in the hit group in the 1-hour recovery period while continuing to fall in the moderate - exercise group . blood glucose at 1-hour postexercise was 3.3 mmol / l lower than the pre - exercise level in the hit group , and 6.3 mmol / l lower in the moderate exercise group ( p = 0.021 ) . however , maran et al,56 using a similar exercise protocol , demonstrated that following morning hit , blood glucose was significantly lower between midnight and 6 am the next day compared to when only moderate - intensity exercise was done.57 mitchell et al58 showed that continuous noninterval exercise at 80% vo2max until exhaustion ( approximately 10 to 13 minutes ) increased blood glucose during and in the 2-hour postexercise period . thus , unlike moderate - intensity exercise , hit is unlikely to cause hypoglycemia during or immediately postexercise in type 1 diabetics , but 14 to 20 hours later it may result in lower glucose levels . a single session of continuous high - intensity exercise resulted in 60 minutes of postexercise hyperglycemia,49 while both a single session of hit,50 and a 2-week training program51 have been shown to improve postprandial glucose control over a 24-hour period following exercise . little et al51 evaluated the effects of six sessions of hit over 2 weeks on glucose regulation 48 to 72 hours after the last training session in people with type 2 diabetes . most participants engaged in 60 minutes or less of exercise per week prior to entering the study . the hit protocol required only 30 minutes of high - intensity exercise per week , with a total time commitment ( including warm - up , cool - down , and rest ) of 75 minutes . the exercise intensity was less and may be more acceptable than all out sit protocols . when asked how enjoyable would engaging in hit three times per week for the next 4 weeks be , the mean response was 7.9 1.0 on a scale ranging from 1 ( not enjoyable at all ) to 9 ( very enjoyable ) . additionally , it elicited ratings of perceived exertion of 4 to 8 on a 10-point scale . before training and from 48 to 72 hours after the last training session , glucose regulation was assessed using 24-hour continuous glucose monitoring under standardized dietary conditions . the average 24-hour blood glucose concentration was reduced by 13% , from 7.6 mmol / l ( sd 1 ) to 6.6 mmol / l ( sd 0.7 ) after training ( p < 0.05 ) . the sum of the 3-hour postprandial glucose auc for breakfast , lunch , and dinner was reduced by 30% ( p < 0.05 ) . gillen et al,50 studying 7 of the 8 individuals who participated in the study by little et al,51 and using an identical exercise protocol , demonstrated that a single exercise session also reduced the sum of the 3-hour postprandial glucose auc ( p = 0.01 ) and the proportion of time spent above 10 mmol / l in the 24-hour postexercise period when compared to a nonexercising control day . average 24-hour blood glucose was , however , not significantly reduced ( p = 0.16 ) . the results of the two studies might be clinically significant , as controlling postprandial hyperglycemia is a treatment goal with type 2 diabetics . kjaer et al49 investigated the effect of 5 minutes of high - intensity exercise on blood glucose control during and for 3 hours immediately following exercise in type 2 diabetic patients ( two on a sulfonylurea and five on diet only ) . there was a greater and more sustained rise in glucose levels in type 2 diabetics compared to controls . in type 2 diabetic subjects , blood glucose increased from a pre - exercise level of 147 mg / dl ( sd 21 ) to a peak 30 minutes postexercise at 169 mg / dl ( sd 19 ) . this value was maintained until 60 minutes postexercise , and then plasma levels decreased over the remainder of the 180-minute recovery period . for the controls , blood glucose increased from a pre - exercise level of 90 mg / dl ( sd 4 ) to a peak at 10 minutes glucose concentrations at 60 minutes postexercise did not differ significantly from pre - exercise levels . in both groups , plasma insulin levels increased after exercise above pre - exercise levels , and returned to baseline about 120 minutes postexercise . plasma epinephrine and glucagon responses to exercise were higher in type 2 diabetics than in control subjects ( p < 0.05 ) . however , 24 hours after exercise in the type 2 diabetic group and not the controls , there was an increased effect of insulin on glucose uptake compared to the pre - exercise state as estimated by the insulin clamp technique . other studies have found similar increases in insulin - mediated glucose disposal after short - term high - intensity exercise in insulin - resistant subjects.52,53 it was concluded that because of exaggerated counter - regulatory hormonal responses , maximal dynamic exercise results in a 60-minute period of postexercise hyperglycemia and hyper - insulinemia in type 2 diabetics.49 the high - intensity studies involving type 1 diabetics mainly investigated blood glucose control during and in the 2 hours after exercise . the number of cycle bouts per training session was increased from four in week 1 , to six in week 2 , eight in week 3 , and ten in weeks 47 . sit resulted in a greater rise in plasma glucose during and immediately after exercise ( a 20-minute period ) in diabetics compared to nondiabetic controls . guelfi et al55 studied the effects of hit ( repeated 4-second cycle ergometer efforts separated by rest ) on blood glucose during exercise and in the immediate 1-hour postexercise period . after the postprandial peak in blood glucose , on alternate days , participants either exercised or rested . during exercise blood glucose declined more rapidly as compared to the nonexercising controls , indicating that high intensity exercise may increase the risk of hypoglcemia . however during the recovery period blood glucose levels continued to decline in the controls while remaining stable in the exercise group suggesting a decreased risk of postexercise hypoglycemia . guelfi et al56 also compared a hit protocol that was combined with moderate - intensity exercise ( repeated 4-second cycle ergometer efforts over 30 minutes separated by cycling at 40% vo2max ) to moderate exercise only ( cycling at 40% vo2max ) for 30 minutes . blood glucose fell to a greater extent in the moderate exercise group compared to the hit group , and remained stable in the hit group in the 1-hour recovery period while continuing to fall in the moderate - exercise group . blood glucose at 1-hour postexercise was 3.3 mmol / l lower than the pre - exercise level in the hit group , and 6.3 mmol / l lower in the moderate exercise group ( p = 0.021 ) . however , maran et al,56 using a similar exercise protocol , demonstrated that following morning hit , blood glucose was significantly lower between midnight and 6 am the next day compared to when only moderate - intensity exercise was done.57 mitchell et al58 showed that continuous noninterval exercise at 80% vo2max until exhaustion ( approximately 10 to 13 minutes ) increased blood glucose during and in the 2-hour postexercise period . thus , unlike moderate - intensity exercise , hit is unlikely to cause hypoglycemia during or immediately postexercise in type 1 diabetics , but 14 to 20 hours later it may result in lower glucose levels . clinical practice guidelines typically recommend that people with type 2 diabetes perform moderate to vigorous aerobic exercise and resistance exercise three to five times per week for a total of at least 150 to 210 minutes per week . many persons do not achieve the recommended amounts of exercise with lack of time being cited as a reason.15 meta - analyses have shown that non - hit programs can produce hba1c improvements between 0.6% and 0.89% , an amount that is clinically significant and comparable to some medication regimens . classic sit protocols can require as little as 2 to 3 minutes of maximal exercise spread over 15 to 30 minutes . with healthy nondiabetic subjects , 2-week protocols have resulted in improvements in muscle glut4 content , insulin sensitivity , and fbg . improvements in vo2max were similar to that achieved by much longer sessions of endurance aerobic exercise . in type 2 diabetic patients , a low - volume 2-week hit program increased glut4 protein , a marker of insulin sensitivity , and decreased average blood glucose 48 to 72 hours postexercise . however , unlike with moderate - intensity exercise , blood glucose levels tend to be higher in both type 1 and 2 diabetic patients during and in the 2 hours immediately following intense exercise due to rising catecholamine levels promoting glycogenolysis , and these levels may remain high in the 2-hour post - exercise period . hit depletes muscle glycogen and it is possible that after catecholamine levels decrease in the post - exercise phase , a period of increased peripheral uptake of glucose follows as glycogen stores are replenished . the optimal exercise strategy has not been determined , but low volume sit with as little as 7.5 minutes of high - intensity exercise per week may be a time - efficient exercise strategy to help control blood glucose in diabetic patients and improve insulin sensitivity in nondiabetic adults . unlike moderate - intensity exercise , high - intensity exercise decreases the risk of hypoglycemia during and immediately after exercise in diabetic patients . therefore , there may be no need for well controlled patients on insulin or insulin secretagogues to eat or decrease medication dosage shortly before high - intensity exercise . however , the perceived exertion associated with the all out version of hit is very high and the acceptability , feasibility , and safety for the sedentary diabetic and nondiabetic population are in doubt . the cost of exercising and the provision of facilities ( equipment , supervision , and gyms ) also have to be taken into account if this form of exercise is to have a mass impact . the less strenuous version of hit used by little et al51 might be preferred to all out sit as it was well accepted while still being of short duration . studies on the effect of high - intensity exercise on blood glucose have been few and of short duration , and have involved a small number of patients who were probably not representative of the general diabetic population . with diabetics , it is therefore uncertain if any improvements in blood glucose achieved by a brief intervention would be sustained over a longer period , reduce hba1c levels , improve health outcomes , and can be replicated in the general diabetic population . similarly , in the nondiabetic population it is not known whether improvements in insulin sensitivity would be sustained and result in a clinically important endpoint such as diabetes prevention . further studies are needed to determine whether hit programs , perhaps in the less intense form or as an adjunct to moderate - intensity exercise , would be effective in the long - term and have a high enough adherence rate to be efficacious . large scale randomized trials lasting years may be necessary to show whether hit can prevent diabetes , and such trials may be impractical .

backgroundmoderate - intensity exercise improves blood glucose ( bg ) , but most people fail to achieve the required exercise volume . high - intensity exercise ( hie ) protocols vary . maximal cycle ergometer sprint interval training typically requires only 2.5 minutes of hie and a total training time commitment ( including rest and warm up ) of 25 minutes per session . the effect of brief high - intensity exercise on blood glucose levels of people with and without diabetes is reviewed.methodshie ( 80% maximal oxygen uptake , vo2max ) studies with 15 minutes hie per session were reviewed.resultssix studies of nondiabetics ( 51 males , 14 females ) requiring 7.5 to 20 minutes / week of hie are reviewed . two weeks of sprint interval training increased insulin sensitivity up to 3 days postintervention . twelve weeks near maximal interval running ( total exercise time 40 minutes / week ) improved bg to a similar extent as running at 65% vo2max for 150 minutes / week . eight studies of diabetics ( 41 type 1 and 22 type 2 subjects ) were reviewed . six were of a single exercise session with 44 seconds to 13 minutes of hie , and the others were 2 and 7 weeks duration with 20 and 2 minutes / week hie , respectively . with type 1 and 2 diabetes , bg was generally higher during and up to 2 hours after hie compared to controls . with type 1 diabetics , bg decreased from midnight to 6 am following hie the previous morning . with type 2 diabetes , a single session improved postprandial bg for 24 hours , while a 2-week program reduced the average bg by 13% at 48 to 72 hours after exercise and also increased glut4 by 369%.conclusionvery brief hie improves bg 1 to 3 days postexercise in both diabetics and non - diabetics . hie is unlikely to cause hypoglycemia during and immediately after exercise . larger and longer randomized studies are needed to determine the safety , acceptability , long - term efficacy , and optimal exercise intensity and duration .

The impact of brief high-intensity exercise on blood glucose levels Effect of aerobic and resistance exercise training on glycemic control Glucose metabolism during moderate-intensity exercise High-intensity exercise Glucose metabolism during high-intensity exercise Aerobic endurance and high-intensity exercise Methods Results High-intensity interval training and insulin sensitivity in healthy nondiabetic adults High-intensity training and glucose regulation in people with diabetes Effects of high-intensity exercise on blood glucose in type 2 diabetic patients Effects of high-intensity exercise on blood glucose in type 1 diabetic patients Summary Conclusion

PMC4054066

polymerization shrinkage still harms the interfacial quality . when the shrinkage takes place under confinement , due to bonding to cavity walls , stresses on the bonding interface the presence of gaps on the tooth / resin interface is considered as the first sign of restoration failure . although polymerization shrinkage is considered the main cause of the adaptation problems of composite restorations , it is an inherent characteristic of the composite and directly related to its composition . moreover , the photoinitiator concentration can influence the shrinkage stress and consequently , the gap formation . however , the effect of photoinitiator concentration in gap formation of composite restorations has not been evaluated . the most frequently employed photoinitiator on dental composites is camphorquinone ( cq ) , which is a type ii photosensitizer ( it needs a reducing agent to produce free radicals ) with a light absorption peak near 468 nm . when cq is photoactivated , in the excited form , a pair of free radicals is produced by proton abstraction . such a process can be made highly efficient by the formation of a complex between the photoexcited sensitizer and a reducing agent , such as a tertiary amine . a higher concentration of cq can induce a quick and high generation of free radicals , yielding the production of a faster polymerization kinetics reaction and higher degree of conversion . as a result , mechanical properties of the material may also be enhanced . however , studies have shown that there is an ideal level for the increase of cq concentration and above this level , the increase in photoinitiator concentration does not benefit the final degree of conversion . conversely , if the concentration of photoinitiators were kept too low , resin composite could not be satisfactorily cured , which has been associated with poor biocompatibility , color stability , physical and mechanical properties , wear resistance and potentially early failure of the restoration . in addition , a substantial problem is that cq is inherently yellow and has poor photobleaching , which means the yellow color remains after light irradiation . it may cause problems in color matching to natural dental substrate and limits the quantity of cq that can be incorporated in dental composites . a slow polymerization reaction allows a slow development of the composite stiffness and leads to better flow that can reduce the shrinkage stress . studies have d emonstrated that the marginal adaptation of resin composites can be improved by light curing with low power density . conversely , the application of an adequate energy dose for composite curing is necessary to achieve deep and complete polymerization of the material . thus , modulated curing methods , such as soft - start ( ss ) and pulse - delay ( pd ) have been proposed . the ss curing method enhances marginal sealing of cavo - surface margins of composite restoratives , especially due to initial lower viscosity of the composite , allowing a better material flow during the earlier stages of curing . ss also leads to shrinkage , surface hardness and residual monomer concentration similar to conventional photoactivation , in situations with similar radiant exposure . moreover , the pd photocuring method , which is a variation of the ss method , can also decrease the intensity of polymerization stress . this curing method consists of an initial low irradiance exposure followed by a lag period in the absence of the curing light from 10 s to 5 min before a final high irradiance exposure . the initial low energy density is enough to allow the start of the polymerization reaction of the composite . the higher irradiance performed on the last stage of the curing method is responsible for ensuring similar physical and mechanical properties to the composite polymerized using the continuous light method . some studies have associated this method with reduced gap formation , caused by the reduction of shrinkage stress . however , it was also showed that pd method yields the formation of polymers with increased susceptibility to softening in ethanol , even though with similar monomer conversion in comparison with that produced using the continuous light curing technique . the effects of modulated photoactivation method have been related to the composition of the resin material . however , it is important to consider that most studies evaluating modulated photoactivation methods were conducted using commercial composites where fillers , resin matrix and photo initiation system differ considerably . therefore , it seems to be more appropriate to use a resin composite model in which the variables could be controlled . thus , the aim of this study was to determine the influence of photoinitiator concentration on superficial and internal adaptation of experimental composites photocured by different methods . the first tested hypothesis was that a higher concentration of photoinitiator can increase gap formation . monomer mixtures consisting of 65 wt% of bisgma ( bisphenol - a - glycidyl methacrylate ) and 35 wt% of triethylene glycol dimethacrylate ( tegdma ) were prepared . the light - curing initiator system selected was cq as photoinitiator and dimethylaminoethyl methacrylate ( dmaema ) as the co - initiator ( proportion 1:1 by weight ) . the light - curing initiator system ( cq / dmaema ) was thoroughly dissolved in the monomer matrix in the following concentrations : 0.5 wt% ( c1 - 0.25% cq/0.25% dmaema ) , 1.0 wt% ( c2 - 0.5% cq/0.5% dmaema ) and 1.5 wt% ( c3 - 0.75% cq/0.75% dmaema ) . furthermore , the inhibitor butylated hydroxytoluene was added to the organic matrix in a concentration of 0.1 wt% to avoid spontaneous polymerization of the monomers . the organic matrix was reinforced with silanized barium aluminum silicate glass fillers ( baalsi average size : 0.5 m ) and silica ( sio2 average size : 0.04 m ) . the fillers were gradually added in the resin matrix and mixed homogeneously to a 65 wt% loading . considering this filler content , 80 wt% were baalsi and 20 wt% were sio2 . the formulation of the experimental composites used in this study was selected based on previous studies . a total of 120 bovine incisors were selected , cleaned and stored in a 0.5% chloramine t solution at 4c for no more than a week . roots were sectioned 1 mm below the cement - enamel junction using a double - face diamond saw ( k. g. sorensen , sao paulo , sp , brazil ) . the buccal surface was ground on a water - cooled mechanical polisher ( metaserv 2000 , buehler , uk ltd , lake bluff , il , usa ) using 80 , 180 , 320 and 600 grit silicon carbide ( sic ) abrasive paper ( carbimet disc set , # 305178180 , buehler , uk ltd , lake bluff , il , usa ) in order to expose a flat enamel area of at least 36 mm . these teeth were then observed on a stereomicroscope ( zeiss , manaus , am , brazil ) at 25 magnification , to investigate whether the enamel was completely removed . a cavity with 3 mm width , 3 mm length and 2 mm deep was prepared on the flattened surface using cylindrical diamond tips ( # 56 , kg . sorensen , so paulo , sp , brazil ) mounted in a standard preparation device . in this condition , the cavity walls had a 90 angle with the dentin surface plane , while the internal cavity angles were rounded due to the design of the diamond tip used . if any sign of pulp exposure was noticed during the cavity preparation , the specimen was discarded . after cavity preparation , each specimen was restored using an etch - and - rinse adhesive system ( adper single bond 2 , batch # 3hr , 3m / espe , st . paul , mn , usa ) , applied in accordance with manufacturer 's instructions : cavity was etched with 35% phosphoric acid gel ( scotchbond etchant , 3m / espe , st . paul , mn , usa ) for 15 s , rinsed for 10 s and blot - dried . the adhesive system was applied twice with a 5 s interval in between , dried carefully with air for 15 s in order to remove residual solvent ( observing a glossy surface ) and light cured for 20 s using a quartz tungsten halogen ( qth ) light ( xl 2500 , 3m / espe , st . afterward , the teeth were randomly assigned into six groups ( n = 10 ) , according to the type of composite ( c1 , c2 and c3 ) and curing method ( high - intensity ; low - intensity ; ss ; pd ) . paul , mn , usa ) was used for all curing methods using radiant exposure around 28 j / cm . after the light curing procedures , the specimens were stored in distilled water at 37c for 24 h. after that , the specimens were finished under running water using 600- and 1200- grit sic abrasive paper ( carbimet disc set , # 305178180 , buehler , lake bluff , il , usa ) to expose and polished with 1 m and 0.5 m diamond pastes in a polish cloth under water . specimens were ultrasonically cleaned for 5 min between finishing and polishing steps . in order to evaluate the marginal adaptation of the restorations , impressions were taken with a low viscosity polyvinyl siloxane material ( aquasil , dentsply detrey , konstanz , germany ) and poured using an epoxy resin ( buehler , lake buff , il , eua ) to obtain replicas . afterward , the replicas were gold - sputter coated ( balzers - scd 050 sputter coater , liechtenstein ) and observed by sem ( jeol , jsm-5600 lv , sem , japan ) for evaluation , measurement and classification of the cavity margins . the classification of the margins was made at 200 magnification directly on the microscope monitor and the measurements were made by using a multi - point measuring device that allowed the observation of the entire perimeter of the restoration at 25 . the perimeter of the restorations was measured and margins were recorded and classified according to the morphologically defined parameters previously described by kemp - scholte and davidson . ( 1 ) perfect margin : defined as a continuous , gap - free transition between filling and tooth substrate and ( 2 ) marginal gap : observed as gap formation with the loss of interfacial adhesion . marginal gap formation was calculated and expressed as a percentage of the cavity perimeter of each specimen . the length of the gap formed was calculated as a percentage of the entire margin length . after the evaluation of superficial adaptation , the restorations were cut in slices ( 1 mm thick ) in the buccal - lingual direction , using a cutting machine ( isomet 1000 , buehler , lake bluff , il , usa ) to obtain three slices of each restoration for internal adaptation measurements . thus , the sectioned slices were replicated and the internal margins of the restorations evaluated according to the procedures described for the superficial adaptation protocol . for this reason , data were transformed after transformation , data showed normal distribution according to kolmogorov - smirnov test . transformed data ( gap % ) were subjected to two - way analysis of variance ( anova ) , considering the factors composite and photoactivation method and tukey 's test at a significance level of 5% . all statistical analysis was executed in assistat beta 7.5 software ( assistat statistic assistance by prof . dr . francisco de assis santos e silva , deag - ctrn university of campina grande pb monomer mixtures consisting of 65 wt% of bisgma ( bisphenol - a - glycidyl methacrylate ) and 35 wt% of triethylene glycol dimethacrylate ( tegdma ) were prepared . the light - curing initiator system selected was cq as photoinitiator and dimethylaminoethyl methacrylate ( dmaema ) as the co - initiator ( proportion 1:1 by weight ) . the light - curing initiator system ( cq / dmaema ) was thoroughly dissolved in the monomer matrix in the following concentrations : 0.5 wt% ( c1 - 0.25% cq/0.25% dmaema ) , 1.0 wt% ( c2 - 0.5% cq/0.5% dmaema ) and 1.5 wt% ( c3 - 0.75% cq/0.75% dmaema ) . furthermore , the inhibitor butylated hydroxytoluene was added to the organic matrix in a concentration of 0.1 wt% to avoid spontaneous polymerization of the monomers . the organic matrix was reinforced with silanized barium aluminum silicate glass fillers ( baalsi average size : 0.5 m ) and silica ( sio2 average size : 0.04 m ) . the fillers were gradually added in the resin matrix and mixed homogeneously to a 65 wt% loading . considering this filler content , 80 wt% were baalsi and 20 wt% were sio2 . the formulation of the experimental composites used in this study was selected based on previous studies . a total of 120 bovine incisors were selected , cleaned and stored in a 0.5% chloramine t solution at 4c for no more than a week . roots were sectioned 1 mm below the cement - enamel junction using a double - face diamond saw ( k. g. sorensen , sao paulo , sp , brazil ) . the buccal surface was ground on a water - cooled mechanical polisher ( metaserv 2000 , buehler , uk ltd , lake bluff , il , usa ) using 80 , 180 , 320 and 600 grit silicon carbide ( sic ) abrasive paper ( carbimet disc set , # 305178180 , buehler , uk ltd , lake bluff , il , usa ) in order to expose a flat enamel area of at least 36 mm . these teeth were then observed on a stereomicroscope ( zeiss , manaus , am , brazil ) at 25 magnification , to investigate whether the enamel was completely removed . a cavity with 3 mm width , 3 mm length and 2 mm deep was prepared on the flattened surface using cylindrical diamond tips ( # 56 , kg . sorensen , so paulo , sp , brazil ) mounted in a standard preparation device . in this condition , the cavity walls had a 90 angle with the dentin surface plane , while the internal cavity angles were rounded due to the design of the diamond tip used . if any sign of pulp exposure was noticed during the cavity preparation , the specimen was discarded . after cavity preparation , each specimen was restored using an etch - and - rinse adhesive system ( adper single bond 2 , batch # 3hr , 3m / espe , st . paul , mn , usa ) , applied in accordance with manufacturer 's instructions : cavity was etched with 35% phosphoric acid gel ( scotchbond etchant , 3m / espe , st . paul , mn , usa ) for 15 s , rinsed for 10 s and blot - dried . the adhesive system was applied twice with a 5 s interval in between , dried carefully with air for 15 s in order to remove residual solvent ( observing a glossy surface ) and light cured for 20 s using a quartz tungsten halogen ( qth ) light ( xl 2500 , 3m / espe , st . afterward , the teeth were randomly assigned into six groups ( n = 10 ) , according to the type of composite ( c1 , c2 and c3 ) and curing method ( high - intensity ; low - intensity ; ss ; pd ) . cavities were bulk filled with one of the tested experimental composites . curing methods are described in table 1 . the same qth curing unit ( xl 2500 , 3m / espe , st . paul , mn , usa ) was used for all curing methods using radiant exposure around 28 j / cm . after the light curing procedures , the specimens were stored in distilled water at 37c for 24 h. after that , the specimens were finished under running water using 600- and 1200- grit sic abrasive paper ( carbimet disc set , # 305178180 , buehler , lake bluff , il , usa ) to expose and polished with 1 m and 0.5 m diamond pastes in a polish cloth under water . specimens were ultrasonically cleaned for 5 min between finishing and polishing steps . in order to evaluate the marginal adaptation of the restorations , impressions were taken with a low viscosity polyvinyl siloxane material ( aquasil , dentsply detrey , konstanz , germany ) and poured using an epoxy resin ( buehler , lake buff , il , eua ) to obtain replicas . afterward , the replicas were gold - sputter coated ( balzers - scd 050 sputter coater , liechtenstein ) and observed by sem ( jeol , jsm-5600 lv , sem , japan ) for evaluation , measurement and classification of the cavity margins . the classification of the margins was made at 200 magnification directly on the microscope monitor and the measurements were made by using a multi - point measuring device that allowed the observation of the entire perimeter of the restoration at 25 . the perimeter of the restorations was measured and margins were recorded and classified according to the morphologically defined parameters previously described by kemp - scholte and davidson . ( 1 ) perfect margin : defined as a continuous , gap - free transition between filling and tooth substrate and ( 2 ) marginal gap : observed as gap formation with the loss of interfacial adhesion . marginal gap formation was calculated and expressed as a percentage of the cavity perimeter of each specimen . the length of the gap formed was calculated as a percentage of the entire margin length . after the evaluation of superficial adaptation , the restorations were cut in slices ( 1 mm thick ) in the buccal - lingual direction , using a cutting machine ( isomet 1000 , buehler , lake bluff , il , usa ) to obtain three slices of each restoration for internal adaptation measurements . thus , the sectioned slices were replicated and the internal margins of the restorations evaluated according to the procedures described for the superficial adaptation protocol . in order to evaluate the marginal adaptation of the restorations , impressions were taken with a low viscosity polyvinyl siloxane material ( aquasil , dentsply detrey , konstanz , germany ) and poured using an epoxy resin ( buehler , lake buff , il , eua ) to obtain replicas . afterward , the replicas were gold - sputter coated ( balzers - scd 050 sputter coater , liechtenstein ) and observed by sem ( jeol , jsm-5600 lv , sem , japan ) for evaluation , measurement and classification of the cavity margins . the classification of the margins was made at 200 magnification directly on the microscope monitor and the measurements were made by using a multi - point measuring device that allowed the observation of the entire perimeter of the restoration at 25 . the perimeter of the restorations was measured and margins were recorded and classified according to the morphologically defined parameters previously described by kemp - scholte and davidson . ( 1 ) perfect margin : defined as a continuous , gap - free transition between filling and tooth substrate and ( 2 ) marginal gap : observed as gap formation with the loss of interfacial adhesion . marginal gap formation was calculated and expressed as a percentage of the cavity perimeter of each specimen . the length of the gap formed was calculated as a percentage of the entire margin length . after the evaluation of superficial adaptation , the restorations were cut in slices ( 1 mm thick ) in the buccal - lingual direction , using a cutting machine ( isomet 1000 , buehler , lake bluff , il , usa ) to obtain three slices of each restoration for internal adaptation measurements . thus , the sectioned slices were replicated and the internal margins of the restorations evaluated according to the procedures described for the superficial adaptation protocol . , data were transformed after transformation , data showed normal distribution according to kolmogorov - smirnov test . transformed data ( gap % ) were subjected to two - way analysis of variance ( anova ) , considering the factors composite and photoactivation method and tukey 's test at a significance level of 5% . all statistical analysis was executed in assistat beta 7.5 software ( assistat statistic assistance by prof . dr . francisco de assis santos e silva , deag - ctrn university of campina grande pb superficial gap formation ( % ) means and standard deviations for all photoactivation methods are listed in table 2 . according to two - way anova , for superficial marginal adaptation , only the factor the factor photoactivation method as well as the interaction between the two factors was not statistically significant . in addition , the composite c1 presented higher gap formation when compared to c2 or c3 . superficial gap formation ( % ) means and standard deviations for all photoactivation methods internal gap formation ( % ) means and standard deviations for all photoactivation methods are listed in table 3 . as observed for superficial marginal adaptation , only the factor composite showed a significant difference , regardless of the photoactivation method . in the accompanying results of marginal gap formation , there was no difference between the curing methods , regarding the composite used . in addition , the composite c1 presented higher gap formation when compared to c2 or c3 . internal gap formation ( % ) means and standard deviations for all photoactivation methods photoinitiator concentration is a fundamental parameter that determines the polymerization characteristics of a resin composite . this concentration varies among the commercial composites and its effects on marginal and internal adaptation are not still completely elucidated . taira et al . found cq concentrations that range from 0.17 to 1.03 wt% of the resin and shintani et al . reported a range of 0.03 - 0.09 wt% of the dental composite . in this study , the concentration of cq ranged from 0.25 to 0.75 wt% of the resin matrix ( which corresponds to 0.087 - 0.26 wt% of the composite ) and it has affected superficial and internal gap formation . however , this effect was opposite that expected : the composite with lower concentration of initiators ( c1 ) presented a higher gap percentage then composites with a higher concentration ( c2 and c3 ) , rejecting the first hypothesis . alonso et al . , using the same composite formulation as the present work , found a significant reduction of monomer conversion when c1 ( 0.5% of cq ) was compared to c3 ( 1.5% of initiator ) , showing that the higher the initiator concentration , the higher the conversion degree . in this sense , it was expected that c3 would present higher gap formation , since a higher degree of conversion means higher shrinkage , higher elastic modulus and higher stress at the bonding interface . the poorly polymerized composite would not adequately bond to the adhesive resin , allowing gap formation , as observed in figure 1 . the failure occurs between the restorative composite and the adhesive layer , what could be related to the poor polymerization of the composite . rc : restorative composite , ad : adhesive layer photoinitiator concentration of a resin composite must be sufficient to allow an adequate polymerization . an insufficient polymerization would affect biocompatibility of the composite , due to residual monomer release . showed that cq and tegdma are released to the aqueous substrate by unsatisfactory polymerized resin materials and can damage oral tissue when presented in high concentration . in addition , bonding problems , like the ones demonstrated in the present study [ figure 1 ] , can also occur in composite restorations with inadequate polymerization . however , it is important to consider that cq / amine concentration should be as low as possible due to its yellowing effect , which can cause difficulty in matching dental color and also the tendency to internal discoloration yielded by amine . so , considering the composites evaluated in this study , 1% of cq / amine ( c2 ) could be considered to be adequate since it is sufficient to guarantee a satisfactory polymerization and marginal seal , since c2 and c3 present similar gap formation [ tables 2 and 3 ] and a similar degree of conversion , according to alonso et al . esthetics and bond durability of these restorations are directly influenced by the marginal seal . in general , the origin of gaps has been related to three main factors : contraction stress of composite restoratives , bonding failures , and external mechanical or thermal stress . in the present study , specimens were not submitted to any mechanical or thermal stress and bonding procedures were standardized for all groups . this condition enabled the evaluation of gap formation as a result of contraction stress of the experimental composites with different photoinitiator concentrations and different curing methods . therefore , it is important to state that besides the care taken during specimen preparation , gap formation studies usually show high variability of the data , as inside each group some specimens present a perfect seal while others present a high percentage of gaps . data variability in gap formation studies can be attributed to the great variability of the dental substrate , especially considering bonding to dentin . for this reason , it is important to use a minimum of 10 specimens per group . the majority of the specimens showed a perfect seal of the superficial enamel margins in the present study , as observed in figure 2 . enamel presents high mineral content , allowing good and stable bond strength when the etch - and - rinse technique is used . these results are in accordance with the studies of correr et al . and alonso et al . regarding internal adaptation , gap formation was more frequently observed at the axiopulpal angle and pulpal wall . misfit at the angle may be attributed to the more complex accommodation of the composite in this region , as observed in figure 3 . in addition , stress concentration on the cavity angle and on defects at the adhesive layer contribute to misfit . on the pulpal wall , gaps can be attributed to the greater complexity of adhesion due to the higher density and diameter of dentin tubules , decreasing the intertubular area that is essential for bonding . d : dentin , rc : restorative composite another studied factor was the curing protocol . it was hypothesized that modulated photoactivation methods could reduce gap formation ( second hypothesis ) . modulated curing methods did not improve marginal or internal adaptation of composite restorations , corroborating lopes et al . and amaral et al . irradiance can directly influence the polymerization rate , which is related to shrinkage stress development . the higher polymerization rate decreases composite flow , leading to more frequent gap formation on the tooth / restoration interface due to the fast development of rigidity , reducing the viscoelastic period . thus , the increase of elastic modulus added to polymerization shrinkage yields stress development at the bonding interface , which has been related to marginal adaptation failure of composite restorations . in this sense , the low irradiance method or the modulated ones can be convenient by reducing the polymerization rate , which favors a slower contraction stress development . on the other hand , this effect was not able to reduce gap formation in the present study . this suggests that the effects of reduced curing rates on contraction stress are limited and significant reductions in stress can be verified only after the curing rate drops below a certain threshold , as stated by braga et al . in this sense , it could be speculated that the determinant factor for gap formation would be the energy dose applied , so that composites photocured with similar energy density presents similar gap formation . this speculation is supported by others studies considering that the total volumetric contraction of a composite and maximum stress of this system depend on monomer conversion , which is energy - dose - dependent . alonso et al . showed that the degree of conversion of the composites tested here was similar for all photoactivation methods as the same energy dose was applied . some studies have shown that the efficacy of modulated methods vary according to composite formulation . in this way another disadvantage of these methods is the decrease of cross - link density on polymer network , caused by the lower irradiance application , yielding the composite matrix to be more susceptible to degradation . in accordance with the results of the present study , resin composites with different photoinitiator concentration show different marginal and internal adaptation features , perhaps due to the polymerization behavior . however , it is not affected by modulation of light intensity ; composite restoration photocured by modulated methods presents similar marginal quality as the energy dose was kept constant . thus , only the understanding of the problem statement , allied to the development of techniques to reduce their consequences , would help clinicians to obtain the maximum benefits of resin composite restorations in clinical practice . therefore , higher photoinitiator concentrations in composite allow better marginal seal for the tested conditions .

objective : the aim of this study was to evaluate the influence of photoinitiator concentration on marginal and internal adaptation of composites photocured by modulated methods.materials and methods : composites based on bisgma / triethylene glycol dimethacrylate and 65 wt% of filler were prepared with different concentrations of camphorquinone / amine ( c1 - 0.5% , c2 - 1% , c3 - 1.5% ) . cavities were prepared ( 3 mm 3 mm 2 mm ) on the buccal surface of 120 bovine incisors and the adhesive system adper single bond 2 was applied following manufactures instruction . specimens were then distributed according to type of composite ( c1 , c2 , c3 ) and photoactivation method ( high - intensity 750 mw / cm2 for 40 s ; low intensity 150 mw / cm2 for 200 s ; soft - start 150 mw / cm2 for 10 s + 750 mw / cm2 for 38 s ; pulse - delay 150 mw / cm2 for 10 s + 3 min dark + 750 mw / cm2 for 38 s ) . superficial and internal margins were analyzed by scanning electron microscopy , using the epoxy replica technique . the length of gaps was expressed as a percentage of the total length of the margins . data were submitted to two - way analysis of variance and tukey 's test ( = 0.05).results : modulated curing methods did not influence gap formation regarding both superficial and internal adaptation . the composite with the lower initiator concentration ( c1 ) presented higher gap formation when compared with those with higher concentrations ( c2 and c3).conclusion : modulated photoactivation methods did not reduce gap formation for the experimental composite restorations evaluated . however , higher photoinitiator concentrations promote better marginal seal .

INTRODUCTION MATERIALS AND METHODS Formulation of experimental composites Specimen preparation Evaluation of marginal and internal adaptation by scanning electron microscopy Statistical analysis RESULTS DISCUSSION CONCLUSION

PMC5090127

consequently , the wine is one of the liquid products that are subjected to falsification or adulteration regarding the variety , vintage , and geographical region . the wine origin has been considered to be a quality indicator and wine consumers often require information on the provenance . in the literature , various classifications of wines based on their variety , vintage , and geographical origin using different criteria , such as the phenolic compounds [ 2 , 3 ] , combination of polyphenols and antioxidant activity , isotope ratios [ 5 , 6 ] , volatile aroma compounds [ 7 , 8 ] , and amino acids , were reported . the composition and concentration of phenolics in wine depend on the type of grape used for wine production , the procedures employed for winemaking , and the chemical reactions that occur during the aging of wine . polyphenols control the color , aroma , bitterness , and taste , acting as photoprotective pigments and antioxidants and playing an essential role in wine quality . also , phenolic composition of wines influenced their color stability and browning reactions and higher polyphenolic content helps to stabilize the wine against detrimental temperature effects . phenolic compounds can be successfully used for wine authenticity assessment as they are characteristic for the type of wine and can provide information on geographical origin . polyphenolic compounds are classified as flavonoids ( flavanols , flavonols , dihydroflavonols , and anthocyanins ) and nonflavonoids ( hydroxybenzoic and hydroxycinnamic acids , stilbenes , and phenolic alcohols ) , the last class representing the majority of polyphenolic compounds of white wines . nevertheless , flavonoids have a greater impact on the structure and color of wine compared to nonflavonoids . the flavonoids are found in skins , seeds , and stems of white grapes and represent about 25% of total phenolic content ( tpc ) in white wines . for european white wines grown under cool climate conditions , the presence of flavonoids the polyphenolic compounds show antioxidant effect that is related to the health benefit of moderate wine consumption . the antioxidant activity depends on the phenolic profile of wine because each polyphenol contributes differently to the wine 's activity . during storage , oxidation of polyphenolic compounds leads to changes in the levels of antioxidants in wine , as a consequence of changes in the redox equilibrium , being essential because of its influence on the organoleptic characteristics and because of its importance regarding its antioxidant potential . several in vitro and in vivo methods have been used to measure the antioxidant activity in wines , such as oxygen radical absorbance capacity ( orac ) method , 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ) ( abts ) , 1,1-diphenyl-2-picrylhydrazyl ( dpph ) , trolox equivalent antioxidant capacity ( teac ) , ferric reducing ability of plasma ( frap ) , and n , n - dimethyl - p - phenylenediamine ( dmpd ) [ 17 , 18 ] . besides polyphenolic compounds , sugars are of great importance for the organoleptic quality of wines . the positive effect of total sugars content ( tsc ) is due to the changing gustatory structure and fullness and body and softening astringency of wines . the influence of sugars on wine characteristics depends on their total quantity , structure , composition , and distribution , appearing as relevant variables differing among segments and showing some differences between varieties . one of the most often used parameters for monitoring the maturation of wines is ph , which is a measure of the likelihood and speed of occurrence of ph - dependent reactions . the major roles of ph regarding the quality of wine are as follows : microbial stability of wines and perception of acidity and its impact on fruit flavor and acid taste and sugar - acid balance of wines . winemakers can adjust the ph of the wine by organic acids or ion exchange materials or by use of rootstock , but at increased cost of input . another marker for vintage and geographical origin is the stable oxygen isotope ratios ( o / o ) of wine water . the main factor that affects the oxygen isotopic ratio from plant water is the available water source which , in most cases , is the groundwater that basically results from precipitations . additionally , the climatic conditions , which already influenced the isotopic composition of precipitations , and , also , the evaporation and transpiration processes always produce enrichment in heavy isotopes of plant water in comparison with groundwater . beside these , other factors that affect the oxygen isotopic ratios of wine water are as follows : meteorological conditions , soil type , and date of harvesting . the aim of this study was to evaluate the influence of different characteristics of wines ( variety , vintage , and vineyard ) on their properties . chemometric techniques such as analysis of variance ( anova ) , cluster analysis ( clu ) , and principal component analysis ( pca ) , which provide the possibility of systematizing the obtained data from different analytical techniques [ 26 , 27 ] , were used in order to investigate the differentiation of selected wines with respect to the type , the geographical origin , and the production year . on the basis of our knowledge , this is the first study of the influence of these characteristics on the quality of romanian white wines . twenty - seven commercial wine samples from different vineyards of romania , produced in 2008 , 2009 , and 2010 vintages , were investigated in this study . the chosen wine cultivars for this work were feteasc alb , sauvignon blanc , and riesling . all measurements and analyses were carried out in triplicate and the data were presented as the means standard deviations . first , 0.3 ml of sample was mixed with 1.5 ml of folin - ciocalteu reagent and , after 5 minutes , 1.2 ml of sodium carbonate 0.7 m was added . sample was incubated at room temperature , in dark place for 2 h , and the absorbance was measured at 760 nm . the results were expressed in g of gallic acid / ml of wine on the basis of calibration curve obtained by the same procedure using standard solution of gallic acid ( 10250 g / ml ) . first , 1.2 ml of wine was mixed with 0.6 ml of nano2 ( 5% ) and , after 5 min , 1.2 ml of alcl3 ( 10% ) was added . then , after 5 min , 2 ml of naoh 0.1 m was added . standard solution of rutin ( 10125 g / ml ) was used for the calibration curve and the results were expressed in g of rutin / ml of wine . aliquots of 0.25 ml of wine diluted three times with distilled water were added to 3.0 ml of 0.09 mg / ml methanolic solution of dpph . the absorbance of the reaction mixture was measured at 517 nm , after 20 min . calibration was performed using vitamin c as standard , in the concentration range of 0.1500.275 mg / ml , following the same procedure . the obtained calibration curve ( y = 1.471x , r = 0.9912 ) was used for antioxidant activity calculation . the dry and semidry wines were diluted 50 times and the semisweet wines were diluted 300 times . 0.1 ml of diluted wine was mixed with 0.5 ml of 5% aqueous solution of phenol , and then 2.5 ml of concentrated sulfuric acid was added . the total sugar content was calculated on the basis of calibration curve obtained by the same method using d - glucose as standard . the ph of each wine was measured using a ph - meter inolab ph 7110 . the equilibration of co2 with the wine water was carried out by introducing 5 ml of the wine sample into a calibrated sample bottle and cooling it down to 80c . after venting the bottle , the carbon dioxide was introduced at a pressure value of about 600 torr , the bottle being placed in the thermostatically controlled water bath at 25c . the isotopic equilibrium is reached overnight ( around 16 h ) . before measuring o , after the equilibration step , the carbon dioxide contained in the bottles was extracted and cryogenically purified . the measurements of o / o isotopic ratio were performed by delta v advantage isotope ratio mass spectrometer ( thermofinnigan , bremen , germany ) by dual inlet method . o / o isotope ratios determinations were performed versus laboratory standard water calibrated using reference material ( rm ) water , puerto rico laboratory reference , w-39500 , iaea , vienna , with ovsmow = 1.52 0.07. o value is expressed according to the relation(1)18o=1000rsamplerrefrref , where rsample and rref are o / o isotope ratios of the sample and of the carbon dioxide used as the reference gas . the reproducibility of the measurements was 0.2. the experimental results were subjected to anova , cluster analysis , and principal component analysis using statistica 10 software ( statsoft inc . the anova analysis was performed in order to reveal the differences between geographical region , production years , and wine variety and to determine which variables affect the response of the investigated problem . the differences were considered to be significant at the level of p < 0.05 for 95% probability . according to these p values , the characteristics of wines were ranked , the best rank being given by the lowest p value . the cluster analysis ( clu ) the clu is used as a feature for clustering variables that identifies the key variables which explain the principal dimensionality in the data . basic chemometric characterization of the investigated wine samples is made by principal component analysis ( pca ) , which depicts a natural grouping of the studied objects as well as the variables ( descriptors ) in multidimensional space without forcing the objects or variables to be organized according to some classification principle . pca is a powerful technique that reduces the dimension of original data matrix by retaining the maximum amount of variability . before carrying out the pca two important tests are made : kaiser - meyer - olkin ( kmo ) measure of sampling adequacy and bartlett 's test of sphericity . bartlett 's test measures the null hypothesis that the original correlation matrix is an identity matrix , being significant for p values less than 0.05 . three white wine sorts were investigated in this study , namely , feteasc alb ( traditional romanian wine sort ) , sauvignon blanc , and riesling . all experimental results are presented in table 1 as means standard deviations ( n = 3 ) . in the case of sauvignon blanc wines , a difference between the two vineyards from oltenia region is observed . thus , tpc for 2009 is 174.8 g / ml in the case of vineyard i and 257.4 g / ml of wine from vineyard ii . the polyphenols are present in the solid part of grapes and their extraction is determined by winemaking conditions , such as grapes squeezing procedure , ph , and temperature . ( 2007 ) confirming that winemaking techniques have a high influence on phenolic content . regarding feteasc alb wines , the highest amount of tpc is found in wines from region of moldavia ( 413.3 g / ml in 2009 ) , while , for feteasc alb wines from muntenia region , in the same year , tpc was 230.7 g / ml and 260.0 g / ml , respectively . in the case of wines made by wineries from muntenia region , no notable differences in the tpc are observed , the tpc values being comparable for the same production year and the highest tpc value being for 2010 . concerning riesling wines , a descending trend from 2008 to 2010 is observed , the tpc values decreasing from 445.2 g / ml for 2008 to 276.3 g / ml for 2010 . the biosynthesis of polyphenols is mostly influenced by sun exposure and temperature , so the wines produced from the grapes cultivated in warm and sunny areas will have the highest quantity of polyphenolics . thereby , the low tpc value for 2010 could be due to the fact that this year was a rainy year in transilvania region . there are differences between the regions , oltenia , muntenia , transylvania , and moldavia . thereby , in 2008 the highest tpc value is found in wines from the region of transylvania ( 445.2 g / ml ) that is almost twice as high as those from oltenia and muntenia regions . in 2009 , the highest tpc value is found in moldavia region ( 413.3 g / ml ) , while in 2010 the tpc values are not very different . these differences could be explained by different weather conditions in these regions , because the tpc increases during the maturation of grapes , especially in warm and sunny areas . the analysis of climatologic condition from 2008 and 2009 shows that during the maturation of grapes the temperatures were higher and sunny period was longer in transylvania and moldavia than in oltenia and muntenia , while the 2010 was a rainy year in all regions . statistical data analysis proves that the vineyard has a significant influence on the tpc ( p = 0.003 ) , while the varieties and vintage do not affect considerably the quantity of polyphenols , but a significant interaction effect between year and vineyard ( p = 0.014 ) is observed . these results allow concluding that the polyphenolic content depends primarily on vineyard and winemaking technology , which is in agreement with other reported results . the experimental values of tfc varied between 12.6 g / ml and 48.2 g / ml , being significantly influenced by variety ( p = 0.032 ) and vineyard ( p = 0.002 ) , while the year does not have an important contribution to the variation of tfc ( p = 0.977 ) . in the case of sauvignon blanc wines , the tfc values are different for oltenia and muntenia regions , the highest differences being between the two vineyards from oltenia . the tfc is lower in vineyard i as compared to vineyard ii , following the same trend during all three years . the highest tfc value for feteasc alb wines was obtained for the sample from moldavia region in 2010 ( 41.68 g / ml ) . differences between the two vineyards from muntenia and , also , between muntenia and moldavia region were observed . the lowest tfc values correspond to wines from muntenia vineyard ii , followed by those from vineyard i whereas the highest tfc value belongs to moldavia wines . a descending trend of tfc values was observed over the years 20082010 for the riesling wines from transylvania and oltenia regions , these values being higher than those for wines from muntenia . moreover , in the case of wines from muntenia the situation is opposite ; the tfc values increase from 2008 to 2010 . the tfc value of romanian riesling wines is lower than the levels mentioned in the literature for this type of wine from other countries [ 3638 ] ; they even have the highest tfc content among the three studied wine types . the average percent of flavonoids amount in total phenols was 9.38% being below the set level by 20% of tpc and proving the quality of the analyzed wines . the highest percentage is obtained for riesling from oltenia , 2008 ( 16.75% ) , and for feteasc alb from moldavia , 2010 ( 16.52% ) , while sauvignon blanc from oltenia , 2010 , has the smallest quantity of flavonoids in total phenols ( 5.06% ) . the experimental aoa values ( table 1 ) show that significant differences appear only from one vineyard to another ( p = 0.000 ) and between varieties ( p = 0.001 ) . the year does not show significant effect on the aoa ( p = 0.683 ) , but a statistical interaction effect between year and vineyard ( p = 0.002 ) , respectively , and between year and variety ( p = 0.035 ) is observed . for example , in the wines produced by the two vineyards from oltenia region the aoa are ranging from 2.39 mol / ml to 3.43 mol / ml . the differences between vineyards could be due to the additional treatments used in the controlled fermentation of grapes that reduce the antioxidant activity as a result of chemical transformations , such as catechin derivatives oxidation . also , the medium values of aoa show that sauvignon blanc wines have the smallest antioxidant activity ( 3.41 mol / ml ) , the highest activity being found for riesling wines ( 4.65 mol / ml ) . analyzing the tpc and aoa values , it can be observed that a correlation between these two properties does not always exist . thus , the wines from muntenia region , produced in 2008 and 2009 , and the wines produced in 2010 in the vineyard from moldavia have a higher antioxidant activity comparing with polyphenolics content . in the case of wines from transylvania a linear correlation between tpc and aoa is observed ( r = 0.9992 ) proving that the antioxidant activity is determined only by polyphenolic compounds . the antioxidant activities are not correlated with polyphenolic content in the case of vineyards from oltenia region . these behaviors are explained by the fact that the antioxidant activity depends more on the type of phenolic compounds from wine compared to their total quantity due to the following considerations : the relation structure - activity of antioxidant compounds ; the contribution of each polyphenolic compound to the total antioxidant activity according to the number of oh and och3 groups and their position on the ring ; the polymerization degree of phenolics ; and the ratio between monomeric and polymeric forms because the inhibition of free radicals tends to increase with polymerization degree [ 18 , 39 ] . the tsc is an important regulatory parameter that is used to classify wine styles and to determine the endpoint of fermentation , the sugars being responsible for the formation of ethanol as well as a number of secondary products . experimental results show that feteasc alb wines from the region of moldavia are semisweet wines . in 2008 , the potential of sugars accumulation is higher than that in 2009 and 2010 , the tsc being highest in this year ( 49.01 mg / ml ) . this fact is due to climatologic conditions , on that year being low precipitation and high temperature during ripening , which have led to a high accumulation of sugars . this type of wines originating from the muntenia region is found to have a high content of sugars ; the quantity is different among vineyards , being higher in vineyard i than in vineyard ii . this behavior could be explained by differences in the winemaking technology , particularly fermentation process that was used in the vineyards . regarding the sauvignon blanc wines , the trend of tsc is different among oltenia and muntenia region . in oltenia region , the highest value is obtained in the 2008 year in both vineyards , while the tsc values for 2009 and 2010 are almost equal . on the other hand , in muntenia region the highest tsc value is obtained in 2010 , whereas for 2008 and 2009 the obtained tsc values are very similar . in the case of riesling wines , the highest tsc corresponds to the year 2008 for all three regions , being much higher compared to 2009 and 2010 . in the oltenia region , the year 2008 proved to be the most favorable one regarding climatic conditions for the accumulation of sugars ( 7.86 g / l ) . in the following years , the trend is descending , the tsc values significantly decreasing until 5.63 g / l in 2009 , respectively , and 4.52 g / l in 2010 . on the other hand , in transylvania and muntenia , the situation is opposite ; the lowest tsc values correspond to 2009 and slow increase of tsc is observed in 2010 . from anova analysis it can be seen that the variety and the vineyard have a significant influence on the tsc ( p = 0.024 , p = 0.000 ) , while the vintage does not affect considerably the quantity of sugars , but a significant interaction effect between year and vineyard ( p = 0.005 ) is observed . one of the factors that could be responsible for the tsc modification of tsc from one region to another is the water deficit , which is one of the main components of the so - called terroir effect . the water deficit leads to a low dimension of berry , thus influencing the sugar content due to the limited carbon assimilation . however , a slight - to - moderate water deficit has a positive effect on the berries and wine quality because the content of sugars is higher in smaller berries than in bigger ones , where a dilution of sugars has been produced . the determined ph values are between 3.03 and 3.46 being strongly influenced by variety of wine ( p = 0.002 ) , whereas the vineyard has a weak influence on it ( p = 0.049 ) . the ph values of sauvignon blanc wines are different in the two regions , oltenia and muntenia . also , the ph of the wines from oltenia is higher for those from vineyard i compared to those from vineyard ii . regarding feteasc alb wines , no large variations in ph were observed for 2009 and 2010 , but some differences were observed between vineyards and regions . moreover , even in the same region , namely , muntenia , the ph values varied between vineyards being lower in vineyard i than in vineyard ii where the wines have the highest ph values . in the case of riesling sort , a descending trend of ph was observed over the years 20082010 for the wines from transylvania region . over against transylvania , the ph values of wines from muntenia showed an ascending tendency over these three years . the ph has an effect on stability of wine , low ph inhibiting microorganism growth . as a result , white wine with a ph above 3.4 and red wine above 3.5 may have stability problems . the experimental results show that two wines , namely , feteasc alb from vineyard ii , muntenia , 2009 and 2010 vintage , exceed slightly the value of 3.4 and , consequently , could have some problems regarding their stability . from the isotopic values of investigated wines presented in table 1 , a statistical interaction effect between year and vineyard was observed ( p = 0.004 ) . the isotopic differences which appear in the same wine sort , produced in the same area but in different production years , are due to the different meteorological condition that prevails in the above - mentioned years . for instance , in the case of sauvignon blanc wine from oltenia , produced in the same vineyard in 2008 , 2009 , and 2010 , the oxygen isotopic value is varying from o = 3.7 ( for 2008 vintage ) to o = 1.1 ( for 2009 vintage ) and o = 1.6 ( for 2010 vintage ) , respectively . these differences arise mainly from the different quantities of precipitation that had been fallen in these years , rather than important differences in temperature among these years . thus , the annual mean temperature of all three years in this area was about 12c with slightly higher temperature in 2008 during the maturation period ; meanwhile , the quantity of precipitation in 2008 was 592 mm / year comparative with 2009 and 2010 vintages when the quantity of precipitations was 674 mm / year and 708 mm / year , respectively . even if 2009 was a warm and dry year in transylvania region , o value of the wine that was produced in this year is the lowest one , o = 1.2 , as compared with those from 2008 and 2010 , respectively ( table 1 ) . in this case , it is necessary to take into account the fact that during the harvesting period there were significant precipitations falls , which can affect drastically the isotopic ratio of oxygen . in this sense , important variation of oxygen isotopic ratios as function of harvesting date was previously reported by . from muntenia region , the investigated wine sorts were as follows : sauvignon blanc , feteasc alb , and riesling . in this region , the lowest o values were obtained for the wines produced in the rainy year 2010 . beside this , the warm and dry year 2009 was characterized by higher values of o from wine water especially in comparison with those from 2008 harvest . the investigation of two wine sorts from the same vineyard from oltenia region presents the same trend of o variation from one year to another ; moreover o values of the wine produced in the same year are very similar . this can be explained by the fact that o is a better indicator for geographical origin rather than for identification of wine sort . the most important factor that affects wine water o value is the isotopic composition of the water which is available to plant . in most cases , this available plant water is the rain water , which is directly related to the meteorological particularities of a specific year . it was previously reported that the degree of isotopic enrichment among different fruits ( or sorts of the same fruits ) is species dependent . on the other hand , different harvest periods , specific for each grape sort , will generate specific o values for each wine type . thus , we can explain the interdependence between the production year and the wine sort . the differentiation and classification of wine samples on the basis of their chemical composition , geographical origin , variety , or quality belong to the basic applications of chemometric methods in enology , methods that offer the possibility of a fast and efficient extraction of the information originating from large sets of data . the anova is the initial step in the identification of some parameters that are statistically contributing to the data sets variability providing information on the differences among wine varieties or wines from different regions . also , the importance of each characteristic of wine can be ranked on the basis of p values . the most important parameter that can distinguish between the wine varieties is the aoa followed closely by ph . tsc and tfc have a moderate influence , while tpc can not be used for differentiation of wines . regarding the vineyard the best variables to discriminate between wine samples were aoa and tsc followed closely by tfc and tpc , whereas the ph has a moderate significance in the wine discrimination . however , unlike varieties and region , the vintage can not distinguish the wine samples , all p values being greater than 0.05 . it is also clear that o values are not among the significant characteristics for wines differentiation according to variety , vineyard , or vintage . in conclusion , anova enabled the discrimination among origins of wines , without being able to conclude which parameters are the best descriptors . cluster analysis is an unsupervised pattern recognition that involves trying to determine relationships between objects ( samples ) without using any prior information about these relationships . hierarchical agglomerative cluster analysis is the most common approach and its result is typically illustrated by a dendrogram . generally , the objects have similar properties within a cluster and different proprieties between clusters . as a clustering criterion ward 's method the twenty - seven wine samples were divided into three main clusters . in order to find out the differences between the three clusters , anova was run again , this time having as independent variable the grouping cluster variable , obtained from previous analysis . from the six parameters used in statistical analysis , only tpc , tfc , and cluster 1 comprises wine samples from muntenia and oltenia region ; meanwhile , in cluster 2 an overlay appears from all three wine regions . finally , cluster 3 comprises only one sample from transylvania region , drj 2008 , with the highest tpc and tfc values . the obtained value of kmo test is 0.581 , which indicates that pca is suitable for carrying out the analysis of our data set . the value obtained for bartlett test ( 0.001 ) also indicates that some relationship between the variables exists and the pca is appropriate for data analysis . the correlation matrix ( table 2 ) was used for analysis , where the values greater than 0.3 representing a strong correlation between variables are marked with italics . significantly positive correlations were found between tfc and aoa , tfc and tpc , aoa and tpc , respectively , and tsc and tfc . some negative correlations were also observed between o and tpc , o and tfc , respectively , and ph and tfc . these strong correlations , which were observed between analyzed variables , are another indicator that pca is appropriate and might provide reliable and distinct principal components . pca was running using the following parameters : extraction method , principal component , and eigenvalues , and values greater than 1 were retained . the results indicate that only two components have eigenvalues bigger than 1 ( 2.462 and 1.400 , resp . ) . the first component explains 41.04% from the total variance , while the second component has a variance of 23.33% . the first principal component has strong positive loading of tpc and tfc ( values > 0.75 ) , moderate loading on tsc ( values between 0.5 and 0.75 ) , and weak loading on aoa ( values of 0.30.5 ) , while the second one has strong positive loading of aoa and ph and weak loading of isotope ratios o / o . as a result , the most important pca graph is obtained ( figure 2(a ) ) , where the natural grouping of the analyzed wine samples can be observed . most of the sauvignon variety samples are well separated from riesling and feteasc and had negative pc1 and pc2 values . the majority of riesling samples have negative values for pc1 and positive values for pc2 . some of feteasc samples had different pc values , compared to other samples of the same variety . the variability among the same - sort wine samples is due to the fact that wines come from different regions of origin , thus not only variety but also region affects the positioning of the wine samples . in contrast , the ph and isotope ratio o / o exhibit negative values for pc1 . with regard to the pc2 , only tsc display slightly negative value , while all other parameters have large positive values . the analyzed white wines present differences with regard to cultivars , vineyard , and vintage . the results show that the tested parameters together with chemometric techniques have a significant potential to be used to discriminate white wines . some of the tested parameters of wines ( tpc , tfc , aoa , and tsc ) are significantly influenced by the vineyard , whereas cultivars exerted significant influence on tfc , aoa , tsc , and ph . the vintage has no influence on the wines characteristics , but it presents a significant interaction with vineyard in the case of tpc , aoa , tsc , and o and with cultivars in the case of aoa . also , it has been demonstrated that chemometric methods used in wine samples interpretation are very useful in order to highlight the essential parameters and to classify the sample after a predetermined criterion .

the wine is one of the most consumed drinks over the world , being subjected to falsification or adulteration regarding the variety , vintage , and geographical region . in this study , the influence of different characteristics of wines ( type , production year , and origin ) on the total phenolic content , total flavonoids content , antioxidant activity , total sugars content , ph , and 18o/16o isotopic ratio was investigated . the differentiation of selected wines on the basis of tested parameters was investigated using chemometric techniques , such as analysis of variance , cluster analysis , and principal component analysis . the experimental results are in agreement with other outcomes and allow concluding that variety and vineyard have the major influence on the studied parameters , but , also , statistical interaction effect between year and vineyard and year and variety is observed in some cases . the obtained results have demonstrated that these parameters together with chemometric techniques show a significant potential to be used for discrimination of white wines .

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

PMC4678919

rift valley fever virus ( rvfv ) is an arthropod - borne phlebovirus in the family bunyaviridae , with widespread epidemiology throughout sub - saharan african regions first reported in the early 1900s by a veterinary surgeon in kenya . the virus is maintained and transmitted by mosquitoes , mainly aedes and culex species , . though the virus has been shown to be maintained in mosquito populations through vertical and horizontal transmission , there has also been evidence of its maintenance in mammalian reservoirs , and there remains the possibility that there are other uncharacterized wildlife reservoirs which allow transmission at the wildlife / domesticated livestock interface via mosquito . the epidemiologic range of the virus has been increasing , with reported outbreaks in west africa , saudi arabia , north africa and southern africa , , , , . there is evidence that rvfv is capable of transmission through a varied repertoire of diverse anthropophilic mosquito species , primarily aedes and culex species . indications of increasing diversity of vector competence may lead to rapid expansions in the future , which may pose a risk to countries geographically farther from traditional areas considered endemic . increasing circulation of rvfv in competent mosquito populations is a common observation in endemic areas after increased rainfall ; it is usually followed by increased transmission to domesticated animals , where it is capable of causing severe animal health problems such as abortion , , . this can result in a significant economic burden on the affected communities , particularly in arid areas that rely on geographically limited agriculture . an increased prevalence in human disease is also observed , particularly amongst agricultural workers , who are at a higher risk of exposure via mosquito bite , and abattoir workers or butchers , who are at risk of exposure via contaminated blood when preparing meat from infected animals . exposure via the bodily fluids of infected animals and also via aerosolization of the virus during butchering has been previously suggested as a major route of exposure for high - risk groups and may put an individual at higher risk of developing more severe clinical symptoms , such as haemorrhagic manifestations due to exposure to higher viral titres . rift valley fever outbreaks are regularly reported in east africa and have also been reported in mauritania . one recent study identified tunisia as a high - risk country ; the authors inferred that environmental conditions and the presence of the vector meant that an epizootic occurrence was possible if the virus was present a constant and considerable risk throughout the year , with a particularly high probability in july , after the wettest months . previous studies looking for serologic evidence of exposure to rift valley fever in 20062007 found no positivity in the studied population . indeed , as a result of anthropogenic influences such as irrigation and well drilling , surface water levels in several arid regions have increased over the years , allowing the emergence of vector - borne diseases due to the wider distribution of competent mosquitos throughout the country , thus increasing potential risk since previous studies were performed in both human and animal populations . on the basis of mathematical modelling tunisia covers a wide climatic range , from a mediterranean climate , with its rainy winter , in the north to a saharan climate in the south ( fig . 1 ) . the northern part of the country is separated from the south by the tunisian ridge , a range of hills running northeast to southwest for approximately 220 km , marking the climatic boundary between the mediterranean sea to the north and the dry steppes of central tunisia . between the northern slopes of the tunisian ridge and the chains of hills bounding it on the south are extensive plateaus , called the high tell . the sahara is separated from the central steppe land by a series of salted areas called chotts . samples were collected from patients seeking care at hospitals who had reported unexplained acute fever ( n = 181 ) . samples were also actively taken from abattoir workers ( n = 38 ) in order to survey evidence of previous exposure amongst groups designated to be at high risk of infection . most participants originated from the governorates of sousse , sfax and mahdia ( fig . 1 ) , and samples were collected during the summer of 2014 . samples were collected by vacutainer , and plasma was separated , frozen at 20c and transported to public health england porton down , uk , for analysis . the study was conducted after receipt of ethical approval from the ethical committee ( hhs - irb 00008931 ; farhat hached university hospital , protocol reference date 8 april 2013 ) . nucleic acid extraction was performed using a qiaamp viral rna mini kit ( qiagen ) according to the manufacturer 's instructions . amplification was performed using a previously published assay as follows : forward : 5-aaaggaacaatggactctggtca-3 ; reverse : 5-cacttcttactaccatgtcctccaat-3 , probe 5-6fam aaagctttgatatctctcagtgccccaa bhq1 - 3. reverse transcriptase ( rt)-pcr assays were conducted in one step using the superscript iii quantitative real - time pcr ( qrt - pcr ) kit ( life technologies ) according to the manufacturer s instructions with cycling conditions of 50c for 10 minutes and 95c for 2 minutes , followed by 40 cycles of 95c for 10 seconds and 60c for 40 seconds , followed by holding at 40c for 2 minutes . data acquisition was performed using the abi 7500 real - time pcr machine ( applied biosystems ) with 45 analysed cycles with a threshold of 0.05 and data analysed using the abi 7500 on - board software . sera were tested for the presence of antibodies reactive to rvfv using commercially available indirect immunofluorescence testing kits ( euroimmun ) . all serum samples were tested for the presence of rvfv by qrt - pcr before serologic testing under containment level 2 conditions . in brief , sera samples were diluted 1/100 and incubated on irradiated / fixed rvfv infected and noninfected vero cells for 60 minutes at room temperature before washing five times for 5 minutes in sample buffer including 0.1% tween 20 . antibodies binding to the infected cells were detected and measured through a secondary antibody labelled with fluorescein isothiocyanate ; 1/100 was set as the minimum cutoff . for immunoglobulin ( ig ) m measurement , the process was modified by initial dilution of sera in eurosorb igg removal reagent ( euroimmun ) before commencing the protocol as before . slides were mounted in mounting media and viewed under a fluorescent microscope at excitation / emission peaks of 495/517 nm , respectively . positive and negative controls provided with the commercial kit were tested to provide a benchmark to which samples were compared . samples collected from patients with acute fever were initially tested according to local protocols for serologic reactivity to brucella , rickettsia and west nile virus and the presence of these agents through blood and urine cultures . all samples in this study were shown to be negative during this testing phase and were sent to public health england porton down to be analysed for rift valley fever by both molecular and serologic testing . in total 219 samples were tested by qrt - pcr and indirect immunofluorescence to ascertain the level of exposure in the study population . commercial immunofluorescence controls were tested to provide a benchmark to which samples could be compared . the commercial immunofluorescence assay test kit utilized in this study is european community ( ce ) marked as a diagnostic kit and has been validated in house for diagnostic use against a panel of positive and negative samples . the presence of rvfv was not detected by qrt - pcr in either cohort ; however , 7.8% of the tested nonfebrile cohort showed igg serologic reactivity ( n = 3 ) to rvfv at the screening dilution , a reaction shown to be repeatable on retesting ( fig . 2 ) . furthermore , 8.3% of febrile patients were found to have circulating igm reactive to rvfv ( n = 15 ) . samples which tested positive by igg and igm were titrated to 1/10 000 dilutions in phosphate - buffered saline to evaluate the abundance of reactive antibody . the 18 serologically reactive samples were derived from patients and participants in the governorates of sousse , mahdia and sfax , areas highlighted in the bioclimatic map comprising fig . in contrast , three seropositive samples drawn from afebrile farmers and abattoir workers showed only igg reactivity , with igm absent ( table 1 ) . clinical details were taken for all samples collected during this study , in line with normal clinical practice in tunisia , including travel history . sample 48 showed some fluorescence at 1/1000 dilution ; however , on retest , it failed to titrate beyond 1/100 . sample 42 titrated beyond this , to 1/10 000 , making this the strongest igm reaction detected in this study . our results strongly suggest the presence of active rvfv circulation in tunisia , confirming our expectation from a recent appraisal of north africa for the relative risk of harbouring circulating rvfv that was recently undertaken . this finding is not unexpected , with rvfv also being found in northwestern africa , . tunisia is notable for its close proximity to european mediterranean countries and the migratory patterns of birds through the country leading northwards into europe . there is also evidence of rvfv exposure in bird populations , which are a potential zoonotic risk for vector - borne disease . for example , birds infested with hyalomma marginatum , the principal vector of crimean - congo haemorrhagic fever virus , have been detected as far north as the united kingdom . accumulation of surface water and changing surface topology affect bird migratory routes and vector population size ; both factors result in a high risk of an increase in prevalence of zoonotic disease . the potential for cross - border transmission through the exchange of economically important animal species from neighbouring countries may also result in the importation of zoonotic pathogens at the wildlife domesticated livestock interface . circulation of west nile virus in the region is recognized , and investigations have suggested an increasing prevalence which may be attributable to anthropogenic environmental changes . these events illustrate the need for improved and comprehensive surveillance in areas where environmental change is recognized to track relative risk to human and animal health to arthropod - borne diseases such as rvfv and west nile virus . rift valley fever , while primarily being transmissible through the bite of the mosquito vector species , may also be transmitted through direct contact with blood . there are several anecdotal reports of veterinary workers engaged in animal necropsies being exposed to high levels of rvfv during the course of their duties , and surveillance activities have detected prevalence of reactive antibody and the virus itself in abattoir and slaughterhouse workers , . critical to the control measures imposed during the outbreak in saudi arabia in 2000 was the education of agricultural workers to avoid close contact sick animals and consumption of diseased meat in order to reduce risk of spread through direct contact . this risk may be substantially increased during certain religious festivals in tunisia , where the slaughter of domestic ruminants by individual families disseminates the risk to a much wider population . it is notable that in our study no abattoir or agricultural workers sampled tested positive for igm , which is indicative of recent exposure ; instead , they only showed signs of previous historical exposure . however , this may be an artefact of our results resulting from the small numbers of samples collected from these populations . other countries perceived as being at high risk of rvfv outbreaks are neighbouring algeria and libya . the strong igm response detected in the course of this study is indicative of a recent and strong reaction to exposure to rvfv or a serologically related virus . it is notable that there was a lack of igg response seen in any sample from febrile patients , suggesting collection soon after exposure before detectable igg appearing or nonspecific reactivity with a closely related phlebovirus . additionally , the lack of a detectable virus genome complicates confirmatory testing , something which may be attributed to sampling after the viraemic stage of disease has occurred . detailed medical records were collected for all patients and high - risk participants in this study . of the 18 seroreactive cases , records indicated that none had travelled abroad in the 2 months preceding sampling . additionally there was no identifiable link between seropositive abattoir workers and the importation of livestock from abroad for slaughter , as the peak of importation occurred after the sampling period . even so , the possibility remains of a case introduced through travel or movement of livestock in a country with optimal conditions for an outbreak . this study provides evidence to support more detailed and thorough surveillance activities for rvfv in order to conclusively demonstrate whether these cases are autochthonous findings and to survey its prevalence in the region s indigenous mosquito populations . because of the potential for rvfv to create a devastating agricultural burden , its control becomes critically important when it is discovered to be circulating in a region . a campaign of active surveillance , prevention strategies , increased research and vaccination of livestock all play a role in the control of rvfv and serve not only to reduce the potential economic burden but also , by interrupting the virus transmission amongst livestock , to interrupt transmission across the human livestock interface , thus significantly reducing the risk to public health . active collaboration between public health and animal health institutions of tunisia is critical , as is increasing cohesive planning with the institutes of neighbouring countries . though our study is limited in scope , we hope that it provides a foundation for further surveillance and investigation of rvfv in tunisia .

rift valley fever virus ( rvfv ) is capable of causing dramatic outbreaks amongst economically important animal species and is capable of causing severe symptoms and mortality in humans . rvfv is known to circulate widely throughout east africa ; serologic evidence of exposure has also been found in some northern african countries , including mauritania . this study aimed to ascertain whether rvfv is circulating in regions beyond its known geographic range . samples from febrile patients ( n = 181 ) and nonfebrile healthy agricultural and slaughterhouse workers ( n = 38 ) were collected during the summer of 2014 and surveyed for exposure to rvfv by both serologic tests and pcr . of the 219 samples tested , 7.8% of nonfebrile participants showed immunoglobulin g reactivity to rvfv nucleoprotein and 8.3% of febrile patients showed immunoglobulin m reactivity , with the latter samples indicating recent exposure to the virus . our results suggest an active circulation of rvfv and evidence of human exposure in the population of tunisia .

Introduction Materials and Methods Results Discussion Conclusion

PMC5028868

by 2050 , the number of people over the age of 65 is projected to increase from an estimated 524 million to nearly 1.5 billion , representing 16% of the world 's population . many of these individuals and their families will experience challenges as older adults learn how to manage and cope with chronic health problems such as cancer , heart disease , and diabetes that require long - term care . over the past decade , significant increases in healthcare costs have led to greater utilization of and increased need for informal caregiving from family members who provide emotional support , assistance with activities of daily living ( e.g. , bathing , grooming , and feeding ) , and instrumental support ( e.g. , paying bills , shopping , and assistance with medication ) for individuals with chronic illness and disabilities [ 2 , 3 ] . trends of older adults living longer with chronic illnesses have coincided with younger adults ( i.e. , 1825-year - olds ) assuming caregiving responsibilities for family members , especially since marriage and childbearing have been delayed and college enrollment overall in the young adult population has increased . young adults comprise 1218% of adult caregivers , and the average age of a young adult caregiver is 21 years , suggesting that many young caregivers are college - age . although young adult caregivers can not be automatically equated with college students , as social selection processes may predispose certain young adults to take on a family caregiving role , the national center for education statistics estimates that 40 percent of the us population between the ages of 18 and 24 is enrolled in degree - granting postsecondary institutions , representing a sizeable group of young adults who are or could become caregivers . one study of young adult caregivers , for example , found 38 percent of participants caring for grandparents reported completing some college . although little is known about undergraduate students acting as family caregivers , research has found that caregiving by college students has been associated with increased burden in transitioning to college , reduced social engagement , and greater stress and depression [ 6 , 7 ] . similarly , college caregivers may experience additional stress because they may be relatively inexperienced with providing care , which may pose more challenges for them in fulfilling their duties . the more general literature outside of college students has shown that factors such as caregiving history , ses , and the availability of support programs , along with mediators such as social support systems and personal coping responses , may influence an individual 's ability to handle the stressors that accompany transitioning to the caregiving role . there is considerable variety between families and individual members in their ability to recognize and plan for the caregiving needs of others . individuals who anticipate and plan for the needs of older adult family members are more satisfied with the amount of family discussion and planning than those who ignore those needs . beliefs and personality characteristics play a meaningful role in an individual 's anticipation of and decision to adopt a caregiving role . for example , family members with an internal locus of control tend to anticipate and prepare for the caregiving needs of their parents and grandparents . those with an avoidant attachment style perceive caregiving as a burden and are less willing to provide future care for parents . in contrast , adult children who possess more filial obligation and display attachment to parents are more likely to commit to future help . despite the research examining predictors of adjustment to becoming a caregiver and the more specific research beginning to identify the widespread nature of caregiving duties among college students , no research has examined the personality characteristics that might predispose college students to assuming a caregiving role . this is unfortunate because the more general research has identified personality variables as playing a major role in shaping caregiving experiences [ 14 , 15 ] . a well - established measure of personality is the five - factor model , which includes the dimensions extraversion , agreeableness , conscientiousness , neuroticism , and openness to experience . the big five personality traits have been found to be related to caregiver evaluation of care tasks , use of coping strategies , and evaluation of the care recipient 's impairment [ 17 , 18 ] . extraversion in particular has been associated with positive experiences in providing care and a greater ability to recognize the benefits of caregiving [ 19 , 20 ] . also , caregiver agreeableness and conscientiousness have been related to a better relationship with the care recipient , as well as to increased benefit finding . caregiver neuroticism , on the other hand , has been associated with more mental health problems , increased susceptibility to care - related stressors [ 19 , 22 ] , and decreased salutogenic behaviors . there is also evidence suggesting extraversion and neuroticism may predict caregivers ' level of optimism concerning their future health . finally , caregiver openness has been related to awareness for future care needs and more gathering of care - related information . outside of the big five model , caregivers with greater empathy report higher life satisfaction and less depression , as well as appraising caregiving as less threatening and stressful . broadly defined , empathy is the ability to correctly perceive another 's feelings . a proposed model of caregiver compassion suggests personality characteristics such as empathy , level of intimacy , and attachment style moderate the level of compassion people feel in response to another 's suffering , resulting in ( or inhibiting ) various helping behaviors . as individuals develop empathy , they learn to recognize and feel what others feel before developing the capacity to maintain emotional distance from another 's internal experience while retaining the ability to recognize another 's feelings . caregivers in the early stages of empathy development may quickly feel overwhelmed by the emotional experience they share with the recipient . in contrast , greater empathy has been associated with better provision of support by caregivers and reduced anxiety . given the aging demographic trends in the us and the increasing likelihood that today 's college - age young adults will one day provide care for their relatives with a chronic health condition , it is extremely important to examine the factors that might predict their willingness to provide care . while previous studies have examined the relationship between a number of personality traits and different aspects of caregiving , including how personality traits are associated with assuming and maintaining a caregiving role , limited research to date has investigated the relationship between personality traits and one 's willingness to provide care for a family member with a chronic health condition , especially in a young adult population that will be increasingly likely to do so the purpose of the current study is to examine whether the big five personality traits and empathy are associated with college students ' willingness to provide care for a family member with a chronic condition . first , it is hypothesized that empathy , agreeableness , and conscientiousness will be positively associated with willingness to provide emotional care . research has suggested that agreeableness and conscientiousness are both related to a better relationship with the care recipient , as well as to increased benefit finding . second , it is hypothesized that openness will be positively associated with willingness to provide instrumental care . previous research has shown that openness is related to awareness of future care needs and more gathering of care - related information . third , it is hypothesized that neuroticism will be negatively associated with willingness to care more generally . prior research indicates caregiver neuroticism is associated with more mental health problems , increased susceptibility to care - related stressors [ 19 , 22 ] , and decreased salutogenic behaviors . and finally , it is hypothesized that extraversion and empathy will be positively associated with general willingness to care . research has shown that extraversion is related to positive experiences in providing care and to recognizing the benefits of caregiving [ 19 , 20 ] . all participants were over age 18 and enrolled in a psychology course at the university . initially , 343 students completed the study . only participants who answered at least five of seven ( 71% ) reliability - check items correctly were included in the current study with 13 respondents omitted for this reason ( see table 1 for a description of participant demographics ) . additionally , one participant was excluded who was identified as intersex in order to be able to include gender as a covariate in our analyses , yielding a final sample size of 329 . participants indicated social class by answering , what is your family 's social class ? upper class : $ 200,000 & up ( ceos , politicians ) ; upper middle class : $ 60,000199,999 ( professionals ) ; lower middle class : $ 30,00059,999 ( professional support & sales ) ; working class : $ 15,00029,999 ( clerical , service , & blue collar ) ; lower class : $ 7,00014,999 ( part - time & unemployed ) . responses were coded 5 , 4 , 3 , 2 , and 1 respectively . given that income was the primary component assessing social class , it was treated as a continuous variable in all analyses . participants answered yes or no to the following question , in the past ( but no longer ) , did you provide unpaid informal care to a relative or friend with a chronic health condition lasting three months or longer ? no responses were coded as 0 while yes responses were coded as 1 . participants indicated gender by answering , what gender label best describes you ( select one ) ? responses were coded as 1 , 2 , 3 , 4 , or 5 , respectively . all but one participant were identified as either man or woman and as such gender was treated as a dichotomous variable . participants reported their age in response to how old are you ( in years ) ? the wtc scale is a 30-item measure assessing an individual 's attitude toward providing instrumental , nursing , and emotional support for individuals with a chronic condition . the original scale was intended to assess willingness to care for a person with hiv / aids ; thus the directions were modified such that person with hiv / aids was replaced by a family member that you might have to provide care for at some point because they have a chronic illness , injury , or disability . individual caregiving items within the questionnaire are general in nature ( e.g. , providing encouragement , cleaning up , and paying for medicines ) and not specific to persons with hiv / aids , so no further modifications were necessary . in order to prevent alternative explanations that may explain nonwillingness of participants to take over care responsibilities , such as an inability or financial circumstances , we instructed participants to assume that you are able to provide the following types of care . select the response that best shows how willing you are to do each one . additionally , participants were told that a chronic health condition is a disease or a disability that lasts for three months or longer . the wtc has four scores : a global willingness - to - care score as well as three mean subscale scores including emotional care ( providing emotional support such as encouraging someone who feels hopeless , e.g. , to listen to someone 's concerns about death or dying ) , instrumental care ( assisting with everyday tasks such as preparing meals for someone , e.g. , to bring home groceries for someone ) , and nursing care ( measuring physical caretaking activities such as helping someone take medicine , e.g. , to clean up after someone who has lost bowel or bladder control ) . the wtc score has high reliability for each subscale ( alphas range : .84.91 ) . the bfi-10 is a 10-item scale with an optional 11th item derived from the big five inventory-45 measuring the big five factors of personality : neuroticism , extroversion , openness , agreeableness , and conscientiousness . the 11-item version was used for the current study , producing subscales composed of two items except for agreeableness , which was composed of three items . pearson product moment correlations were calculated for the extraversion ( r = .391 ) , conscientiousness ( r = .362 ) , and neuroticism ( r = .348 ) items , which were all significant ( p < .001 ) , as were the correlations among the three items composing the agreeableness subscale ( r : .213.256 ; p < .001 ) . the bivariate correlation for the items assessing openness was not significant ( r = .058 , p = .290 ) suggesting a potential issue with the internal consistency for this subscale . the teq is a 16-item scale that assesses the ability to perceive the emotional state of another person and respond sympathetically . the teq has good internal consistency ( cronbach 's alpha = .85 ) and convergent validity . instructors who were willing to assist provided students with study information and a link to the survey . interested students reviewed and completed an online consent prior to participation and completed the survey by submitting their responses using the online platform . participants received extra credit points at the instructor 's discretion , but not totaling more than 1% of their overall grade . this study received institutional review board approval by the authors ' institution prior to recruitment . preliminary analyses examined correlations within each of the wtc subscales , the bfi subscales , and the teq . three hierarchical multiple regressions investigated the extent to which personality traits ( neuroticism , openness , extraversion , agreeableness , and conscientiousness ) and empathy were associated with each of the willingness - to - care subscales ( emotional care , instrumental care , and nursing care ) . prior to running primary analyses , a series of bivariate correlations between the three types of willingness to care and demographic characteristics including age , gender , family socioeconomic status , and employment status were conducted to identify important covariates to include in the models , as demographics have previously been shown to be associated with care provision [ 32 , 33 ] . as prior work has found that individuals with less time available due to their employment status ( e.g. , families with dual earners compared to single earners ) provide less care , we treated employment status as a continuous variable to assess if this phenomenon extended to willingness to provide care . given that much of the prior work has not been conducted with college - age samples , only demographic characteristics that were significantly associated with outcome variables in the current sample would be included in the final analyses . age , gender , and employment status were shown to be associated with the outcomes in the bivariate correlations and were included as covariates in all analyses ( table 2 ) . in each of the three regressions , demographics were entered in the first step , with all of the personality variables as well as empathy entered in the second step . the three willingness - to - care subscales were entered as the dependent variable in each regression . to explore if age , gender , or employment status interacted with personality traits , a series of exploratory analyses were conducted . interactions between each personality trait and age and each demographic characteristic were included in the third step predicting each outcome variable and are summarized following the primary results for each criterion variable . normality assumptions were assessed prior to running analyses . for all five personality subscales ( neuroticism , openness , extraversion , agreeableness , and conscientiousness ) , empathy , and nursing care , normality assumptions were met . however , emotional and instrumental care had skewness of 3.82 and 1.46 and kurtosis of 18.62 and 2.60 , respectively , and thus did not meet the assumption of normality . to address these violations , a reflection of emotional and instrumental care data was performed via log transformation and produced new skewness values of .70 and .40 and kurtosis values of .29 and 1.07 for emotional and instrumental care , respectively . an assessment of vif and tolerance statistics indicated no multicollinearity was present among the variables . additionally , an examination of bivariate correlations indicated no correlations greater than .70 . a series of correlations were conducted to examine the bivariate relationships between all variables ( table 3 ) . all willingness - to - care variables were positively associated with each other , as well as being positively related to empathy and personality traits of agreeableness and conscientiousness . willingness to care extraversion was positively related to agreeableness and empathy but negatively associated with neuroticism , while agreeableness was positively related to conscientiousness and empathy . additionally , neuroticism and conscientiousness were negatively related , while empathy was positively related , to all personality traits except for neuroticism and openness . in the first hierarchical multiple regression ( table 4 ) , neo personality traits and empathy were regressed onto willingness to provide emotional care after accounting for age , gender , and employment status . demographics were entered into the first step , for which the overall model was significant , f(3,325 ) = 3.21 , p < .05 , and r = .03 . no demographic characteristics produced any unique effects ( all p > .053 ) . all personality variables as well as empathy were entered in the second step which was also significant , f(9,319 ) = 5.41 , p < .001 , and r = .13 , with a significant increase in the amount of variance explained in emotional care , f(6,320 ) = 6.35 , p < .001 , and r = .10 . the personality trait of agreeableness as well as empathy was uniquely and positively associated with emotional care . all other personality variables were not independently related to emotional care ( all p > .184 ) . this suggests that greater agreeableness and empathy are associated with greater willingness to provide emotional care . to explore if age , gender , or employment status interacted with each personality trait , interaction terms with each of these demographic variables and the personality traits were included in step three . the overall model was significant , f(27,301 ) = 2.13 , p < .01 , and r = .16 ; however , there was no significant increase in explainable variance f(18,301 ) = 0.55 , p = .931 , and r = .03 . there were also no significant interactions between personality traits by age ( all p > .257 ) , gender ( all p > .240 ) , or employment status ( all p > .186 ) . in the second hierarchical multiple regression ( table 5 ) , demographics were entered in the first step , yielding a significant test , f(3,325 ) = 6.21 , p < .001 , and r = .05 . both age and employment status were uniquely and positively associated with instrumental care . in the second step , empathy and all personality variables were entered , producing an overall significant model , f(9,319 ) = 4.81 , p < .001 , and r = .12 . there was also a significant increase in the amount of variance explained in instrumental care , f(6,319 ) = 3.94 , p < .001 , and r = .07 . after accounting for demographics , empathy was independently positively associated with instrumental care with age maintaining significance . no neo personality traits were associated with instrumental care ( all p > .187 ) and employment status dropped in significance in follow - up analyses ( p = .090 ) . this finding suggests that greater empathy and older age are related to greater willingness to provide instrumental care . as part of the exploratory multiple regressions , separate interaction terms between age , gender , and employment status with personality characteristics the overall model was significant , f(27,301 ) = 1.990 , p < .01 , and r = .15 ; however , there was no significant increase in explainable variance f(18,301 ) = 0.631 , p = .874 , and r = .03 . there was a significant interaction of employment status by neuroticism ( = .44 , p = .030 ) , such that individuals who were more neurotic and were employed full - time were less likely to provide care than unemployed or part - time employed individuals ( figure 1 ) . all other interactions with personality traits ( all p > .380 ) as well as interactions between personality traits by age ( all p > .267 ) and by gender ( all p > .089 ) were not significant . in the third regression ( table 6 ) , demographics were entered in the first step , which was not significant , f(3,325 ) = 2.45 , p = .063 , and r = .02 . all personality traits and empathy were entered into the second step , which was significant , f(9,319 ) = 3.61 , p < .001 , and r = .09 , with a significant increase in the amount of variance explained , f(6,319 ) = 4.12 , p < .01 , and r = .07 . no neo personality traits or demographic characteristics were uniquely related to willingness to provide nursing care ( all p > .08 ) . similar to the third step in the first two exploratory multiple regressions , separate interaction terms between age , gender , and employment status with personality traits were included in step three . the overall model was significant , f(27,301 ) = 1.74 , p < .05 , and r = .14 ; however , there was no significant increase in explainable variance f(18,301 ) = 0.82 , p = .672 , and r = .04 . all interactions of personality traits by age ( all p > .366 ) , by gender ( all p > .100 ) , and by employment status ( all p > .182 ) were not significant . with more adults living to an older age and with chronic health conditions , younger adults are assuming greater roles as family caregivers . though many young caregivers are in college , no studies have examined the association between personality traits and willingness to provide care in a college - age population . as such , the purpose of this study was to investigate the relationship between the big five personality traits and empathy and willingness to provide care for a relative with a chronic health condition in a college - aged sample . it was hypothesized that empathy , agreeableness , and conscientiousness would be positively associated with willingness to provide emotional care , that openness would be positively associated with willingness to provide instrumental care , that neuroticism would be negatively associated with willingness to care more generally , and that extraversion and empathy would be positively associated with general willingness to care . a series of hierarchal multiple regressions found that after controlling for demographics , personality traits explained 10% of the variance in willingness to provide emotional care , 7% in instrumental care , and 7% in nursing care . within these models , greater empathy was uniquely associated with willingness to provide emotional , instrumental , and nursing care for a family member in the future . similarly , participants with high agreeableness were more willing to provide emotional care , and older age was a unique predictor of instrumental care . the finding that college students who exhibit a greater level of empathy are more willing to care for a family member with a chronic health condition is in line with previous research indicating empathy is a source of altruistic motivation . according to the empathy - altruism hypothesis , the motivation to help is altruistic to the degree that it is evoked by an empathic emotional response , such that empathic concern can lead to altruistic motivations to help others [ 34 , 35 ] . by extension this suggests that those who exhibit more empathic characteristics may have a greater tendency for empathic responding and subsequently are more motivated to help or care for a family member , as found in the current study . given that empathy was uniquely predictive of all three willingness - to - care subscales , participants who were more empathic may have been more willing to help or provide care irrespective of the type of care , which is supported by numerous studies linking empathic concern to various helping behaviors [ 37 , 38 ] . agreeableness was also identified as being uniquely and positively associated with willingness to provide emotional care for a family member with a chronic health condition . broadly , agreeableness has been argued to be representative of more humanitarian aspects of personality including nurturance and altruism and has long been identified as personality disposition positively related to volunteering and prosocial behaviors . this suggests that people who are more agreeable may be better able to provide emotional support such as being encouraging , caring , and comforting , as well as more inclined to engage in helping behavior . this is in line with prior research outlining that nurses with greater levels of agreeableness are likely to utilize positive emotional social support . additionally , previous work indicates that those who exhibit greater levels of agreeableness are more likely to perceive the caregiving role as meaningful and positive . within the context of the previous literature , in the current study , those scoring higher on agreeableness may have been better able to find the caregiving role as important and beneficial and therefore were more willing to provide care . female gender , older age , and a higher level of employment were associated with more willingness to provide various types of care in the bivariate correlation matrix . although these findings are generally in line with the previous literature , it is important to note that all of these effects were small - sized in the matrix , with the statistically significant correlations indexing 1.2% ( e.g. , age and emotional care ) to a maximum of 2.9% ( e.g. , age and instrumental care ) of shared variance . and when entered into the multiple regressions along with the personality predictors in each model 's step two , none of these demographics remained significant predictors except for age predicting willingness to provide instrumental care ( = .14 ) , which just surpassed the = .05 threshold . as a result , these demographic effects should largely be seen as negligible and only statistically significant in the bivariate correlations because of the current study 's large sample size . however , a significant interaction of employment status by neuroticism emerged , whereby college students who were more neurotic and were employed full - time were less likely to be willing to provide instrumental care than unemployed or part - time employed individuals . a potential interpretation of this effect is that college students who are working full - time , in combination with being high in neuroticism , may feel particularly overwhelmed by their employment demands and as a result may not have the personal resources necessary to be devoted to providing instrumental care for a family member with a chronic condition . however , this effect should be interpreted with caution given that 54 demographics by personality interactions were examined in an exploratory fashion as predictors ( three demographics by six personality traits , for each of the three willingness - to - care criterion variables ) , and this was the only interaction to reach statistical significance . this exploratory approach was highly prone to familywise error , so this effect should be examined in future studies before definitive interpretations are made . although results did not support our hypotheses of positive relationships between willingness to care with extraversion and openness , it is possible that the substantial amount of variance accounted for by empathy eclipsed the effect these personality aspects had on willingness to care . additionally , it is worth noting that the correlation of the two items composing the openness subscale was relatively low and not significant , calling into question the internal consistency of this subscale in the current sample . while this scale has been previously validated among a college - aged sample and the subscale intercorrelations from the current sample are in line with those from the validated study , it is possible that there are additional characteristics that may have impacted the potential reliability of the openness subscale in particular and prevented it from showing an effect on willingness to care . though individuals with higher levels of extraversion have been shown to recognize the benefits of caregiving and tend to report positive experiences in providing care [ 19 , 20 ] , these rewards may not be apparent or compelling enough for extraverted college students . research indicates potential caregivers perceiving fewer gains than losses as a caregiver may be less likely to adopt a caregiving role . for extraverted college students in particular ( and perhaps in comparison to caregivers from other age groups or population segments ) , the losses to one 's social life or losses to the progression in one 's academic pursuits may be fairly dramatic , and as a result some extraverted college students may be less willing to make sacrifices in their social lives in order to provide care given the extremely social nature of college settings the results of this study suggest that research on interventions attempting to increase empathy or to make it more salient may be particularly useful in exploring how to increase college students ' willingness to provide care . in an intervention aimed to improve peer counseling skills in students , hatcher et al . provided instruction in nonjudgmental and empathic listening , facilitative feedback , and self - observation through role play . college students in the intervention group showed significant increases in empathic concern and perspective taking suggesting this type of intervention that targets empathic communication may be especially helpful with college students , as they may become better able to identify as the potential family member with a chronic health condition and thus more willing to provide care . in terms of the current study 's finding that agreeableness was associated with willingness to provide emotional care , intervention research making salient for college students the values of being cooperative , kind , and considerate ( all facets of agreeableness ) in the context of the family system may be a helpful direction . such research could involve interventionists designing motivational interviewing techniques that attempt to help college students identify discrepancies between these aspects of their personality and their actual behaviors of providing care for their family member with a health condition . additionally , research on training that target daily life skills such as food preparation , financial management , and home maintenance may help the field better learn how to improve college student 's self - efficacy in the provision of care and perhaps resultantly willingness to do so in the future . similarly , college students who are better equipped with the necessary skills to care for themselves may be better positioned and more willing to care given greater personal resources ( e.g. , energy , social support ) to enact informal care . there are some limitations that should be considered when interpreting the findings of this study . first , participants were asked to anticipate their willingness to provide care for a relative but were not asked to identify the person , the likelihood of actually becoming a caregiver , or the quality of their relationship to this family member . the relationship of a participant to particular family members ( e.g. , niece / nephew , child , and cousin ) , the quality of that relationship , and the likelihood of actually taking on a caregiving role with that person could indeed influence one 's self - reported willingness to care . research should ask participants specifically whom they are thinking about providing care for and what the quality of that relationship is , as these variables could make one more or less apt to provide care for the family member . additionally , data on the severity of the illness , injury , or disability imagined by the participant were not collected and may impact willingness to provide care . related to this , participants were asked to imagine someone that they might have to provide care for at some point because they have a chronic illness , injury , or disability . the definition of chronic illness , injury , or disability encompasses a wide range of issues and differing care demands and at some point covers a wide time - frame . as a result , responses may vary depending on what a respondent imagined based on this prompt . future research may benefit from including more specific language regarding the potential provision of care by participants in the future . family members with more severe injuries or illnesses , for instance , dementia , require greater care on behalf of the caregiver than others do , which may impact how much and what type of care they are willing to provide . students who have a greater sense of obligation and/or lack of choice to care for those in the family network may be more or less willing to care than those who perceive fewer obligations . finally , the sample consisted entirely of psychology students and was overrepresented by participants from middle - class families and women and thus may not generalize to all college students . future studies should assess relationship quality between the potential caregiver and future care recipient as well as familial responsibility in an effort to better understand the effects of personality on willingness to provide care for a relative with a chronic health condition . this study adds to the growing body of research assessing willingness to provide care for future family members by outlining specific personality traits among college students that may be influential for future care . with a growing population of older adults , young adults the results of this study identifying agreeableness , empathy , and age as being important for one 's potential for future caregiving can help shape future interventions especially those that incorporate perspective taking and training in life skills as a means of boosting empathic concern and subsequently greater willingness to care .

the purpose of this study was to investigate among college students the relationship between personality traits and willingness to care for a relative with a chronic health condition . 329 undergraduate students completed an online questionnaire . hierarchical multiple regressions found that after controlling for demographics personality traits explained 10% of the variance in willingness to provide emotional care , 7% in instrumental care , and 7% in nursing care . within these models , greater empathy was uniquely associated with willingness to provide emotional , instrumental , and nursing care for a family member in the future . similarly , participants with high agreeableness were more willing to provide emotional care , and participant older age was a unique predictor of instrumental care . the results can help shape research on interventions that incorporate perspective taking , motivational interviewing , and training in life skills as a means of boosting college students ' willingness to provide care for a relative with a chronic health condition .

1. Introduction 2. Method 3. Results 4. Discussion 5. Clinical Research Implications 6. Limitations and Future Directions 7. Conclusions

PMC5153494

temporal lobe epilepsy ( tle ) is the most common drug resistant epilepsy in adults . the majority of seizures in tle are associated with hippocampal sclerosis ( hs ) or other temporal lobe abnormalities , which can reliably be detected in vivo by mri [ 2 , 3 ] . patients with resection for tle generally do not report comprehension difficulties through either clinical reports or formal testing but complain of significant amnesia and anomia which reflect a semantic weakness [ 57 ] . a systematic review calculating pooled estimates of neuropsychological outcomes reported a 44% risk of decline in verbal memory and 34% risk of decline in naming after left - sided surgery . it reflects that left and right hs patients may experience distinctive functional reorganization in the nonepileptic temporal lobe under distinctive compensatory mechanisms to sustain key cognitive functions , such as language . semantic cognition can be decomposed into three interactive principal components implemented by separable neural networks : ( 1 ) semantic entry / exit or conceptualization ( translation between sensation / motor representations and semantic knowledge ) ; ( 2 ) the long - term representation of concepts / semantic memory ; and ( 3 ) semantic control ( mechanisms that interact with our vast quantity of semantic knowledge in order to generate time- and context - appropriate behavior ) [ 10 , 11 ] . by means of activation likelihood estimate ( ale ) technique , binder et al . reported a distinct , left - lateralized network specialized for storage and retrieval of semantic knowledge . the related areas included posterior inferior parietal lobe ( angular gyrus , ag ; supramarginal gyrus , smg ) , middle temporal gyrus ( mtg ) , fusiform , parahippocampal gyrus , dorsomedial prefrontal cortex ( dmpfc ) , inferior frontal gyrus ( ifg ) , ventromedial prefrontal cortex ( vmpfc ) , and posterior cingulate cortex ( pcc ) . it is proposed that concepts are formed through the convergence of sensory , motor , and verbal experience via the left anterior temporal lobe ( atl ) , a transmodal representational hub which primarily links pertinent semantic / conceptual information into the language system to produce a specific name . the conclusion that the atl is a crucial component in semantic cognition has been bolstered by contemporary basic neuroscience studies utilizing magnetoencephalography , distortion - corrected functional mri , pet , or repetitive transcranial magnetic stimulation . meanwhile , the claim that posterior temporoparietal areas are associated with semantic control has been demonstrated in both semantic aphasia patients and healthy people [ 11 , 16 ] . concerning the common phenomenon that left hs patients , especially those after surgical resection of atl , can perform within the normal accuracy range on standard semantic assessments but show measureable anomia [ 6 , 1720 ] , we posited that a semantic - lexical disruption in the intermediate processing step that relayed retrieved semantic information on to the language system resulted in the primary problem in naming . since temporal lobe epilepsy is considered as a network disease , its pathological feature requires us to examine the abnormal function of a whole network rather than a single epileptogenic region . to explore dysfunction of neural networks , functional connectivity changes in epilepsy patients have been tested by different neuroimaging modalities , such as repetitive transcranial magnetic stimulation ( rtms ) , corticocortical evoked potentials ( ccep ) , and eeg . although some of these techniques have the noninvasive advantage , resting state fmri ( rsfmri ) analysis possesses additional gains : resting state networks ( rsns ) are highly organized in space , are reproducible from subject to subject , and differ with aging and between genders . in addition , it also allows the search for significant baseline fluctuations to obtain task - free functional network information and identify epileptic circuits by providing clinicians and neurosurgeons with clues about where new or secondary epileptic foci may form , where seizures place most burden on the brain , and where are new core functional regions . fmri functional connectivity describes brain function and cooperation at a network level by identifying regions that make up a network of interest . it reflects the degree of signal synchrony between anatomically distant brain regions during resting state or tasks . however , these linear correlations do not provide information on the direction of influence between regions . coefficient - based gca is a directed functional connectivity method . given that imbalance of excitatory and inhibitory effect is a fundamental change in epilepsy , the gca technique has a special advantage for investigating the pathophysiological mechanism of hs patients by means of quantifying the magnitude and direction of influence of one region time series on another [ 28 , 29 ] . in sum , previous studies reported that left and right hs patients are differently impaired in semantic cognition . these patients offer the opportunity to study different impacts of focal structural lesions on functional connectivity within the semantic network . the objectives of this study were ( 1 ) to evaluate and contrast the occurrence of gray matter ( gm ) atrophy in patients with left and right hs in the semantic cognition network and ( 2 ) to quantify direction of influence between these anatomical regions using granger causality analysis . we hypothesized that the presence of hs is consistent with more pronounced , diffuse gm atrophy with the semantic atrophy the most severely damaged ; left hs patients ' classical anomia ( i.e. , can provide good information about unnamed items ) were caused by the unique deficit pattern of disrupted functional connectivity of the left anterior superior temporal lobe and other regions underlying semantic memory . twenty - four right - handed tle patients ( 17 females , 7 males ; age range 1648 years ; mean age 29.00 9.57 years ; epilepsy onset 12.46 9.06 years ; epilepsy durations 15.86 7.43 years ) with unilateral hs ( 13 left hs and 11 right hs ) were recruited from xuanwu hospital capital medical university . all patients underwent a comprehensive clinical evaluation and fulfilled the following inclusion criteria : ( 1 ) typical symptoms of tle as complex partial seizures , accompanied , or not , by simple partial seizures ; some patients had aurae , like epigastric rising , hallucination , and so on ; the seizure frequency was 4 - 5 times per day at most and 1 time per month at least ; ( 2 ) standard mri criteria for hs ( hippocampal atrophy , increased t2 signal , and loss of internal hippocampal architecture ) which were finally confirmed by histopathology ; ( 3 ) typical eeg findings ( interictal spike or sharp waves at the anterior temporal area in both wakefulness and/or sleep , various ictal rhythms including background attenuation , start - stop - start phenomenon , irregular 25 hz lateralized activity , and 510 hz sinusoidal waves or repetitive epileptiform discharges ) ; ( 4 ) no other neuropsychopathic diseases like intracranial tumor , cerebral hemorrhage , infarction , trauma , schizophrenia , affective psychosis , and so on . healthy adult controls ( hc ) without neurological or psychiatric medical history or medication known to impair memory were recruited . hc group consisted of 24 age and gender matched healthy controls ( 17 females , 7 males ; mean age 29.50 10.18 years ) . there was no difference between the three groups of left hs , right hs , and normal controls ( f = 0.499 , p = 0.611 ) . the local ethics committee approved the study and all participants gave written informed consent according to the declaration of helsinki prior to the study . mri images were acquired during interictal stage with a 3.0 t scanner ( magnetom tim trio , siemens healthcare , erlangen , germany ) using the 12-channel phased - array head coil supplied by the vendor . structural images were acquired with a sagittal mp - rage three - dimensional t1-weighted sequence ( tr = 1600 ms , te = 2.15 ms , flip angle = 9 , thickness = 1.0 mm , and fov = 256 mm 256 mm ) . functional images were acquired using the gradient echo - planar pulse sequence ( tr = 3000 ms , te = 30 ms , flip angle = 90 , and thickness = 3 mm ) . participants were instructed to stay awake with eyes closed . in accordance with previous studies mentioned in the introduction part , all rois were defined in accordance with the aal template , such as temporal pole of superior temporal gyrus ( tpstg ) and temporal pole of middle temporal gyrus ( tpmtg ) that functions in conceptualization ; mtg , fusiform , parahippocampal gyrus , dmpfc ( medial superior frontal gyrus in aal template ) , ifg , vmpfc ( medial orbitofrontal gyrus in aal template ) , and pcc that functions in memory storage ; angular gyrus ( ag ) and supramarginal gyrus ( smg ) that function in semantic control . given that gm volume decrease has been reported in both ipsilateral and contralateral temporal neocortex [ 3133 ] , and mtle patients always show atypical language lateralization , the current study selected 12 rois in each hemisphere ( 24 rois in all ) . volumetric data for cortical and subcortical structures were analyzed by optimized vbm and first , parts of fsl tools , separately . the principal focus of the current study was to contrast different structural damages to the semantic cognition network in both the left and right hs patients . to make sure the severe damage to semantic cognition network was not a by - product of overall gray matter volume ( gmv ) loss the initial stages of vbm analysis included removing nonbrain tissues by brain extraction tool and tissue - type segmentation with fast4 . the resulting gm partial images were then aligned to the mni 152 template by affine - registration . a symmetric study - specific gm template was created by averaging images and flipping along the x - axis . next , all the gm images which were nonlinearly registered to the study - specific gm template were modulated and smoothed by gaussian kernels with a sigma of 3 mm . regions within the semantic cognition network from wfu atlas were selected as rois in further two - sample t - test by randomization ( 5000 permutations ) with tfce implemented , between controls and left or right hs subgroups , respectively . in order to rule out the possibility that group difference was caused by the different pattern of whole brain atrophy among left and right hs patients , we added the remaining rois from wfu atlas in additional analyses to examine volumetric changes as aforementioned . all the volumetric results were considered statistically significant after fwe - correction at p < 0.05 , with cluster including more than 10 continuous voxels . first was used to segment the subcortical structures , including bilateral thalamus , hippocampus , amygdala , caudate nucleus , putamen , and globus palladium . we calculate the normalized volume of subcortical structures by multiplying the scaling factor obtained from sienax . it included the following steps : ( 1 ) slice timing correction ; ( 2 ) trilinear sinc interpolation for alignment ( motion correction ) ; ( 3 ) spatial normalization based on the mni space and resampled at 3 mm 3 mm 3 mm ; ( 4 ) band - pass filter ( 0.01~0.08 hz ) spatially smoothed with a 6 mm full - width - at - half maximum ( fwhm ) gaussian kernel ; and ( 5 ) head motion and ventricular and white matter signal regression . the multivariate gca ( mgca ) has been proved to be an optimal candidate to investigate the causal networks for its data - driven nature . we performed mgca on the time series of bold signal intensities from selected rois in both groups . the entire time series were averaged across voxels within each roi picked in each group and were then normalized across subjects to form a single vector per roi . the mgca detected causal interactions by computing directed transfer function ( dtf ) from a multivariate autoregressive model of the time series . we also adopted weighted dtf with partial coherence in order to emphasize direct connections and deemphasize mediated influences [ 36 , 37 ] . surrogate data were generated by randomizing the phase of the original time series spectrum while retaining its magnitude . a null distribution was obtained by generating 2500 sets of surrogate data and calculating the direct directed transfer function ( ddtf ) from these 2500 datasets . the ddtf value obtained from the original time series was verified using a null distribution for the one - tailed test with the significant p value of 0.01 . in addition , a difference of influence ( doi ) term was used to assess links that showed a dominant direction of influence , which limits potentially spurious links caused by hemodynamic blurring . the effective connectivity network of the 9 rois was constructed by visualizing the significant ddtf ( p < 0.01 , fdr corrected for multiple comparisons ) obtained after running the statistical significant test . the high degree nodes were considered to be the hubs of network . ( number of granger causal efferent connections to a node ) to find the central targets of network , and out - degree ( number of granger causal afferent connections from a node ) to find the central sources [ 40 , 41 ] . further , hubs of the network were defined if the sum of in - degree and out - degree of a node was at least 1.96 standard deviations ( sd ) greater than the average of in- + out - degree of all nodes in the semantic cognition network . we calculated ddtf values in all connections for every subject to explore the difference in the intensity of effective connectivity between groups , particularly with volumetric value of relevant roi as a regressor . the links that showed between - group changes in the strength of causal influence were those whose difference in the doi term significantly differed between groups by a paired t - test ( p < 0.05 , fdr corrected ) . volumetric comparison for normalized subcortical structures was conducted by two - sample t - test ( p < 0.05 ) . for each participant , a laterality index ( li ) of hippocampus volume ( hv ) was computed using the formula [ ( left hv right hv)/(left hv + right hv ) ] . a one - way anova was used to compare lis of left hs , right hs , and controls . pearson 's correlations were then used to examine the relationships between li and factors such as age at epilepsy diagnosis , years since diagnosis , seizure frequency , and linking strength between rois in each group . group comparisons between controls and patients with left or right hs identified gmv loss confined to bilateral hemispheres ( fwe corrected , p 0.05 ) . specifically , in the left hs subgroup , gmv loss was found in 12 ipsilateral rois and 7 contralateral rois ( figure 1 , left column ) ; in the right hs subgroup , gmv loss was in 2 ipsilateral rois and 1 contralateral roi ( figure 1 , right column ) . within the semantic cognition network , left hs patients showed atrophy in 4 areas of ifg , vmpfc , hippocampus , and mtg in the ipsilateral lobe , and 7 areas of ifg , vmpfc , parahippocampus , fusiform , ag , tpstg , tpmtg , and mtg in the contralateral lobe . meanwhile , right hs patients showed only ipsilateral mtg atrophy and no contralateral atrophy ( see details in table 2 ) . further analysis showed significant difference of atrophy severity in the left and right hs patients ( 19/90 : 3/90 ; atrophy area number / network number ; p < 0.001 ) . in particular , the left hemisphere was more severely damaged in the left hs patients than the right ones ( 12/45 : 1/45 ; p < 0.005 ) ( figure 2(a ) ) . in addition , atrophy severity of the semantic cognition network in the right hemisphere was more severe in the left hs patients than the right ones ( 7/12 : 1/12 ; p < 0.05 ) , and patients ' atrophy difference of the left nonsemantic cognition network also reached a significant level ( 8/33 : 0/33 ; p < 0.01 ) ( figure 2(b ) ) . even though , in the left hs patients , there was no significant differences of atrophy between left and right hemispheres ( 12/45 : 7/45 ; p = 0.20 ) or between the left and right semantic cognition network ( 4/12 : 7/12 ; p = 0.22 ) , their nonsemantic networks were more severely damaged in the left hemispheres ( 8/33 : 0/33 ; p < 0.01 ) ( figure 2(c ) ) . a one - way anova revealed significant differences between groups for hippocampal laterality index ( f = 12.70 , p < 0.001 ) . the li was highest ( most left - lateralized ) in the right hs patients ( mean = 0.13 , sd = 0.05 ) , followed by the healthy controls ( mean = 0.01 , sd = 0.03 ) and the left hs group ( mean = 0.21 , sd = 0.05 ) . subsequent contrasts revealed significant differences between controls and patients with left hs ( t = 3.31 , p = 0.002 ) , between controls and right hs patients ( t = 2.80 , p < 0.01 ) , and between groups of left and right hs ( t = 4.27 , p < 0.001 ) . a causal connectivity graph was constructed using the thickness of connecting lines to indicate the strengths of causal influences ( see figure 3 ) . for left hs patients , right hs patients , and healthy controls , causal influences within the semantic cognition network presented strongly covarying relations ( figures 3(a ) , 3(b ) , and 3(c ) ) . overall , connection density among the three groups was not significantly different ( f = 0.03 , p = 0.97 ) . but interconnection patterns between atl subregions were different in the three groups : all the four subregions ( left / right tpstg and tpmtg ) were significantly connected with each other in normal controls ; no causal influence of the areas was found in the left hs patients ; little causal influence remained in the right hs patients ( colored check - boards at the bottom of each part in figure 3 ) . meanwhile , node degree analysis yielded more differences between groups . in the normal controls , the only hub of semantic cognition network was the right tpmtg . specifically , the flow - in hub was the right dmpfc , while the right tpmtg was the only flow - out hub . in the left hs patients , specifically , the flow - in hub was the right dmpfc , while the right pcc was the only flow - out hub . however , there was no hub node in the semantic cognition network of right hs patients . further , by comparing node degree between the left hs , right hs , and controls , we found that node degree of the right pcc was significantly different between left hs patients and controls ( t = 2.23 , p < 0.05 ) , and node degree of the right mtg was significantly different between right hs patients and controls ( t = 3.42 , p < 0.002 ) . by comparing in - degree and out - degree between the left hs and controls , we found that the out - degree of right pcc ( t = 2.25 , p < 0.05 ) , right ifg ( t = 2.67 , p < 0.02 ) , left mtg ( t = 2.20 , p < 0.05 ) , and left tpmtg ( t = 2.07 , p < in contrast , by comparing in - degree and out - degree between the right hs and controls , we found that both the in - degree and out - degree of right mtg ( t = 3.03 , p < 0.005 ; t = 2.63 , p < 0.02 ) were significantly different . between - group analysis showed increased driving effect between nodes in ipsilateral structures and decreased driving effect between nodes of contralateral structures in left hs patients compared with normal controls . in detail , increased causal effects were found in the interactions from right ag to right parahippocampal gyrus , from right ag to right smg , from left mtg to left parahippocampal gyrus , from left tpmtg to left pcc , and from left tpmtg to left mtg ; decreased causal effects were found in the interactions from right pcc to left vmpfc , from right fusiform to left fusiform , and from left tpstg to right tpstg . there were also 3 exceptions where the causal effect from right vmpfc to right pcc and from left hippocampus to left parahippocampal gyrus decreased and where from right tpmtg to left mtg increased ( see figure 4(a ) ) . by contrast , directional interaction weight changes between right hs patients and the controls seemed rather systematic . increased interaction between ipsilateral rois originated from right vmpfc to right mtg ; decreased intrahemisphere interaction originated from right hippocampus to right dmpfc . increased interaction between contralateral rois originated from right ag to left ag , from left vmpfc to right mtg , and from right hippocampus to left dmpfc ; decreased interhemisphere interaction originated from left fusiform to right mtg , from right fusiform to left ifg , and from right mtg to left mtg ( see figure 4(b ) ) . there was significant correlation between hippocampus li and changed path weights of effectively interconnected rois . the linking intensity from right ag to right parahippocampal gyrus in the left hs patients was negatively correlated with hippocampal li ( r = 0.56 , p < 0.05 ) , while the linking intensity from right ag to right smg was positively correlated with hippocampal li ( r = 0.61 , p < 0.05 ) . no significant correlation was found between hippocampus li and epilepsy onset time , duration , and frequency in both groups . to explore disrupted conceptualization in mtle patients with hs , the current study focused on identifying structural and effective connectivity changes of the semantic cognition network . we found that the gray matter was significantly reduced in in both left and right hs patients . even though the two hemispheres were equally damaged in mtle patients with left hs , all the 7 regions that showed atrophy in the contralateral hemisphere were semantic cognition network components . meanwhile , significant increased linking intensity changes between ipsilateral regions and decreased linking intensity changes between contralateral regions ( particularly in the atl area ) were only found in the left mtle group . the consistent anatomical and functional connectivity changes suggested that the breakdown of effective connectivity between left and right hemispheres , possibly caused by the severely damaged contralateral hemisphere , was the reason of more severe language impairment of left hs patients . previous quantitative mri volumetric and voxel - based morphometry ( vbm ) studies have identified atrophy of the hippocampus along with distributed abnormalities in neighboring and distant structures including the entorhinal cortex [ 4345 ] , parahippocampal gyrus , basal ganglia , lateral temporal cortex , frontal lobe , and cerebellum . our findings were consistent with previous findings concerning the effect of epilepsy duration on gray matter volume in vbm studies [ 48 , 49 ] . the altered topologies can be attributed to the seizure - dependent reinforcement of an epileptogenic configuration of the brain network . our findings also revealed different seizure propagation effects in the two patient groups . in the left hs patients , it was manifested that the left hs patients were more easily affected and may experience more serious hippocampal injuries , as their hippocampal li varied more from normal controls . one theory about mechanisms underlying brain damage in mtle hypothesizes that seizure propagation determines the distribution of damage . our findings matched such a theory , since left hs patients were more easily affected . in addition , we specifically found that all the 7 atrophy regions in the right hemisphere in left hs patients were within the semantic cognition network . it indicated that left hs patients also displayed a higher vulnerability to seizure attacks in the potential compensatory semantic networks . we postulated that more serious anatomical changes in the left semantic cognition network and a disrupted compensatory mechanism in the contralateral hemisphere , which received targeted attack with higher vulnerability to disease , lead to more severe language impairment in the left hs patients . the results of the granger causality analyses using functional rois showed no significantly different connection density among the left hs patients , right hs patients , and normal controls . however , node degree analysis revealed that hubs of patients ' semantic cognition network changed . in contrast , the atl was not the longer semantic cognition network hub in both left and right hs patients . in particular , subregions of the atls ( tpstg and tpmtg ) in the left and right hemispheres were causally affected by each other in normal controls . it was in stark contrast with semantic cognition networks in patients with left and right hs , in which connections between atl subregions were all disrupted in left hs patients while only the bidirectional causal connection between left and right tpmtg remained in right hs patients . propose that bilateral anterior temporal lobes are amodal , abstract conceptual hubs that bind modality - specific properties , which are grounded in the sensory - motor system . pobric et al . used repetitive transcranial magnetic stimulation ( rtms ) to disrupt neural processing temporarily in the left or right temporal poles and reported that rtms disrupted semantic processing for words and pictures with the same degree . their work illustrated that left and right anterior temporal lobes are critical in forming concepts of both words and pictures . our findings suggested that even the residual weak interhemisphere interactions can sustain a relatively normal semantic network ( in the right hs patients ) . moreover , the integrating role of the semantic network hub in atl ( the right tpmtg ) can not be compensated by other region ( such as the right pcc in the left hs patients ) , even though it may also be effectively connected with many areas in the network . it implicated that left and right atls functioned as a transmodal hub via mutual interconnections . changes in interregional functional coupling are thought to represent compensatory mechanisms secondary to structural pathology and seizure - related activity . in terms of compensation strategies , patients with left hs showed that causal linking between nodes in ipsilateral structures increased , while causal effects between nodes of contralateral structures decreased . in contrast , patients with right hs showed a balanced change where the number of significantly increased interhemisphere and intrahemisphere causal interactions was the same as that of decreased interhemisphere and intrahemisphere causal interactions . however , it also indicates that if effective connections between ipsilateral and contralateral regions were damaged , patients with right hs were able to form a compensatory one to sustain a relatively normal semantic competence . in other words , the severe targeted atrophy in the contralateral hemisphere in patients with left hs caused disrupted interconnection between hemispheres and can not be substituted for by intensified connection between regions in the same hemisphere . thus , the breakdown of interhemisphere connections , especially those across left and right atls , leads to naming disability . the two significant correlations between hippocampus li and the path weights of the semantic cognition network both originated from the right ag in left hs patients . since gray matter of the right ag was also significantly reduced , the structural change of the right ag may influence the power flowing out from it . it was consistent with the conclusion that the ag occupies a position at the top of a processing hierarchy underlying concept retrieval and conceptual integration . as impaired semantic control was associated with deregulated access to knowledge , patients may have difficulty directing activation towards the target and away from irrelevant prepotent associations . by comparing structural changes of left and right mtle patients with healthy controls , the current study suggested that left hs patients had a higher vulnerability to seizure attacks , which may affect their compensation strategy . the interrupted effective connectivity between subregions of the atl across hemispheres , which performs a unitary , homogeneous , transmodal representation for conceptual information , may be the primary reason why left hs patients displayed severe name finding difficulties but relevant good comprehension ability . in sum , our study revealed that the severe and targeted anatomical changes resulted in failed compensatory strategy in left hs patients , which was characterized by increased intrahemisphere causal interaction but decreased interhemisphere causal links . the primary limitation in the current study is the small number of left and right hs patients . our intention was to maintain uniformity across patients , even if it means sacrificing sample size . however , it may also lead to no significant correlation between the magnitude of granger causality interaction and patients ' clinical data , such as disease onset time , duration , or seizure frequency .

occurrence of language impairment in mesial temporal lobe epilepsy ( mtle ) patients is common and left mtle patients always exhibit a primary problem with access to names . to explore different neuropsychological profiles between left and right mtle patients , the study investigated both structural and effective functional connectivity changes within the semantic cognition network between these two groups and those from normal controls . we found that gray matter atrophy of left mtle patients was more severe than that of right mtle patients in the whole brain and especially within the semantic cognition network in their contralateral hemisphere . it suggested that seizure attacks were rather targeted than random for patients with hippocampal sclerosis ( hs ) in the dominant hemisphere . functional connectivity analysis during resting state fmri revealed that subregions of the anterior temporal lobe ( atl ) in the left hs patients were no longer effectively connected . further , we found that , unlike in right hs patients , increased causal linking between ipsilateral regions in the left hs epilepsy patients can not make up for their decreased contralateral interaction . it suggested that weakened contralateral connection and disrupted effective interaction between subregions of the unitary , transmodal hub of the atl may be the primary cause of anomia in the left hs patients .

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

PMC4327190

o efeito antiplaca e antigengivite de um gel contendo extrato de rom a 10% foi avaliado utilizando um modelo de gengivite experimental parcial em humanos . 23 indivduos participaram voluntariamente deste estudo cruzado , duplo - cego , compreendendo dois perodos de 21 dias cada um . uma moldeira de acrlico foi confeccionada para cada participante , a qual foi utilizada como carreadora dos gis sobre a rea a ser avaliada ( hemiarco inferior esquerdo ) . os sujeitos foram aleatoriamente designados para usar o gel placebo ( grupo controle ) ou o gel teste ( grupo experimental ) , sendo instrudos a colocar o gel na moldeira e esta sobre os dentes teste , escovando os outros normalmente trs vezes ao dia . no dia 0 e dia 21 os ndices de placa visvel ( ipv ) e ndice de sangramento gengival ( isg ) foram registrados . os resultados no demonstraram diferena estatstica significante entre os grupos controle e experimental para nenhum dos ndices . o gel contendo extrato de rom a 10% no foi eficiente em evitar a formao de placa bacteriana supragengival e prevenir a inflamao gengival . gingivitis is a chronic inflammatory process limited to the gingiva and without either attachment or alveolar bone loss . it is one of the most frequent oral diseases , affecting more than 90% of the population , regardless of age , sex or race . the earliest clinical sign is the bleeding caused by a vasodilatory effect caused by an inflammatory response29 . the prevention of gingivitis by daily and effective supragingival plaque control using toothbrushing and dental floss is necessary to arrest a possible progression to periodontitis3,11 . although mechanical plaque control methods have the potential to maintain adequate levels of oral hygiene , clinical experience and population - based studies have shown that such methods are not being employed as accurately as they should by a large number of people . therefore , several chemotherapeutic agents such as triclosan , essential oils and chlorhexidine have been developed to control bacterial plaque , aiming at improving the efficacy of daily hygiene control measures6,8,10,15 - 17,23,26 . the interest in plants with antibacterial and antiinflammatory activity has increased as a consequence of current problems associated with the wide - scale misuse of antibiotics that induced microbial drug resistence7,18 . natural products such as astronium urundeuva , calendula , aloe vera , curcuma zedoaria and other herbal products have been tested with effective results12,19,25,29 . punica granatum linn ( family punicaceae ) , mostly known as " pomegranate " , is a shrub or small tree native from asia where several of its parts have been used as an astringent , haemostatic as well as for diabetes control5,13,24 . in brazil , the fruit of this tree is known as " rom " and is commonly used for treatment of throat infections , coughs and fever due to its antiinflammatory properties13,14 . in the only study available evaluating the effects of pomegranate on gingivitis , pereira and sampaio21 ( 2003 ) showed a significant reduction of gingival bleeding after using a dentifrice containing pomegranate extract . however , a control group was not included in that study . therefore , the purpose of the present investigation was to assess the effects of punica granatum linn extract on supragingival plaque formation and development of experimental gingivitis in comparison to a control formulation . twenty - five dental students from the university of fortaleza ( unifor ) ( 12 male and 13 female aged 22 to 28 years ) were enrolled in this study . all subjects had at least 20 natural teeth , among which the 4 posterior teeth in the lower left quadrant ( experimental teeth ) . all participants , randomly screened , were informed about the nature of the study and signed an informed consent form in compliance with the guidelines of the brazilian health council . students with medical disorders , severe periodontal disease and under antimicrobial therapy , as well as smokers , pregnant women and individuals presenting a probing depth 3 mm associated with any mandibular teeth were excluded from the trail . the protocol was approved by the university 's ethics committee ( report cotica / unifor ) an alginate impression of the experimental teeth was taken and poured in die stone to obtain casts . on each stone cast , a 0.3-mm - thick thermoplastic mouthguard material spacer was made using a vacuum former . upon the spacer , an individual toothshield was made of a 2-mm - thick thermoplastic mouthguard material , using the same vacuum former . the toothshield was trimmed 2 mm beyond the gingival margin to assure that gel would be in contact with the gingival margin of the experimental teeth during toothbrushing of the remaining teeth . the control and experimental gels were formulated and packed into tubes in a commercial drugstore . the tubes were previously coded to warrant that neither the examiner nor the volunteers knew their content , which was revealed by the pharmacist only after the study was completed . all students used both gels in alternate periods , according to a cross - over model . an infusion was prepared with powdered material at a ratio of 100 g powder to 1000 ml distilled water , cooled at room temperature and filtered . thereafter , 50 g of carboxymethylcellulose was added to the infusion ( 1000 ml ) and the mixture was kept boiling until complete dissolution to obtain the 10% gel concentration . the pomegranate extract concentration used in this work was based on the findings of previous in vitro studies13,20 that tested the gel at different concentrations and found that the 10% concentration yielded the most favorable results . a glycerin / ethanol mixture ( 50 ml:50 ml ) was added and the solution was vigorously stirred during 15 minutes until gel formation . a very small amount of menthol ( flavoring ) and a conserving agent were then added . this study was a randomized , double - blind comparison of 2 crossover groups of dental students performed in 2 experimental phases of 21 days each with a 1-month washout interval between them . to standardize the groups , the students were submitted to a meticulous evaluation ( pre - experimental phase ) to score the visible plaque index ( vpi ) and the gingival bleeding index ( gbi)1 of each tooth . all teeth of each subject were polished and flossed by the examiner to eliminate plaque remnants . thirty days after the initial phase , the volunteers were randomly assigned to 2 groups and the experimental phase began . on day 0 of both experimental periods , vpi and gbi were recorded . a personal " kit " containing a toothshield , a tube with 90 g of control or experimental gel and a commercial dentifrice with no antigingivitis properties ( sorriso , kolynos do brazil ltda . , osasco , sp , brazil ) was given to all students . during each 21-day experimental period , the subjects were instructed to fill the toothshield with the gel prior to insertion in the mouth and seat it over the experimental teeth for at least 1 min . the volunteers refrained from brushing the test quadrant , while the other teeth were normally brushed three times a day using the dentifrice ( same to all students ) . in addition to verbal instructions , the subjects were given written recommendations to follow at home . on the last day of each period ( 21st day ) , vpi and gbi indexes were recorded by the same examiner on the mesiobuccal , buccal , distobuccal , mesiolingual , lingual and distolingual surfaces of the experimental teeth . the gingival tissues were inspected for the presence of bleeding , recorded 10 seconds after running the tip of a who probe along the gingival margin ( 0.5-mm penetration into the sulci ) . the values of six sites of each tooth were recorded to obtain the vpi and gbi means . then , vpi and gbi means for the four test teeth were calculated to determine vpi and gbi means of each volunteer . intra - examiner agreement for vpi and gbi was calculated by repeating the measurements in 10 patients , with at least 1 hour of interval4 . twenty - five dental students from the university of fortaleza ( unifor ) ( 12 male and 13 female aged 22 to 28 years ) were enrolled in this study . all subjects had at least 20 natural teeth , among which the 4 posterior teeth in the lower left quadrant ( experimental teeth ) . all participants , randomly screened , were informed about the nature of the study and signed an informed consent form in compliance with the guidelines of the brazilian health council . students with medical disorders , severe periodontal disease and under antimicrobial therapy , as well as smokers , pregnant women and individuals presenting a probing depth 3 mm associated with any mandibular teeth were excluded from the trail . the protocol was approved by the university 's ethics committee ( report cotica / unifor ) an alginate impression of the experimental teeth was taken and poured in die stone to obtain casts . on each stone cast , a 0.3-mm - thick thermoplastic mouthguard material spacer was made using a vacuum former . upon the spacer , an individual toothshield was made of a 2-mm - thick thermoplastic mouthguard material , using the same vacuum former . the toothshield was trimmed 2 mm beyond the gingival margin to assure that gel would be in contact with the gingival margin of the experimental teeth during toothbrushing of the remaining teeth . the control and experimental gels were formulated and packed into tubes in a commercial drugstore . the tubes were previously coded to warrant that neither the examiner nor the volunteers knew their content , which was revealed by the pharmacist only after the study was completed . all students used both gels in alternate periods , according to a cross - over model . an infusion was prepared with powdered material at a ratio of 100 g powder to 1000 ml distilled water , cooled at room temperature and filtered . thereafter , 50 g of carboxymethylcellulose was added to the infusion ( 1000 ml ) and the mixture was kept boiling until complete dissolution to obtain the 10% gel concentration . the pomegranate extract concentration used in this work was based on the findings of previous in vitro studies13,20 that tested the gel at different concentrations and found that the 10% concentration yielded the most favorable results . a glycerin / ethanol mixture ( 50 ml:50 ml ) was added and the solution was vigorously stirred during 15 minutes until gel formation . a very small amount of menthol ( flavoring ) and a conserving agent were then added . this study was a randomized , double - blind comparison of 2 crossover groups of dental students performed in 2 experimental phases of 21 days each with a 1-month washout interval between them . the students were submitted to a meticulous evaluation ( pre - experimental phase ) to score the visible plaque index ( vpi ) and the gingival bleeding index ( gbi)1 of each tooth . all teeth of each subject were polished and flossed by the examiner to eliminate plaque remnants . thirty days after the initial phase , the volunteers were randomly assigned to 2 groups and the experimental phase began . on day 0 of both experimental periods , vpi and gbi were recorded . a personal " kit " containing a toothshield , a tube with 90 g of control or experimental gel and a commercial dentifrice with no antigingivitis properties ( sorriso , kolynos do brazil ltda . , osasco , sp , brazil ) was given to all students . during each 21-day experimental period , the subjects were instructed to fill the toothshield with the gel prior to insertion in the mouth and seat it over the experimental teeth for at least 1 min . the volunteers refrained from brushing the test quadrant , while the other teeth were normally brushed three times a day using the dentifrice ( same to all students ) . in addition to verbal instructions , the subjects were given written recommendations to follow at home . on the last day of each period ( 21st day ) , vpi and gbi indexes were recorded and the teeth were polished with pumice . vpi and gbi indexes were recorded by the same examiner on the mesiobuccal , buccal , distobuccal , mesiolingual , lingual and distolingual surfaces of the experimental teeth . the gingival tissues were inspected for the presence of bleeding , recorded 10 seconds after running the tip of a who probe along the gingival margin ( 0.5-mm penetration into the sulci ) . the values of six sites of each tooth were recorded to obtain the vpi and gbi means . then , vpi and gbi means for the four test teeth were calculated to determine vpi and gbi means of each volunteer . intra - examiner agreement for vpi and gbi was calculated by repeating the measurements in 10 patients , with at least 1 hour of interval4 . the mann - whitney non - parametric test was used to evaluate statistical differences between control and experimental groups on days 0 and 21 . in each group , the mean scores of vpi and gbi were compared between baseline and the end of the trial by the wilcoxon test . two students were excluded from the study during the experimental phase due to third molar extraction and restorative needs . the experimental gel had good acceptance and did not show adverse effects , such as abscess , ulcerations or allergic reactions . on day 0 , in both experimental periods , the control and experimental groups did not show statistically significant difference to each other with respect to vpi ( p=0.5385 ) and gbi ( p=0.3392 ) means ( p>0.05 ) . these results indicated that both groups were well balanced at baseline ( table 1 and 2 ) . at the 21st day , plaque ( p=0.4354 ) and gingival bleeding ( p=0.4685 ) were present in both groups , but the difference between them was not statistically significant ( table 1 and 2 ) . * means followed by the same uppercase letters on day 0 and day 21 do not differ statistically ( p>0.05 ) * * means followed by different lowercase letters in a same column differ statistically ( p<0.05 ) * means followed by the same uppercase letters on day 0 and day 21 do not differ statistically ( p>0.05 ) * * means followed by different lowercase letters in a same column differ statistically ( p<0.05 ) comparing the means between day 0 and day 21 in each group , there was a statistically significant difference in the vpi ( p=0.0000 ) and gbi ( p=0.0001 ) indexes . the inability of adult population to perform adequate mechanical toothcleaning has stimulated the search for chemotherapeutic agents added to dentifrices to improve plaque control and prevent gingivitis16 . the antibacterial activity of punica granatum linn has been evaluated in previous studies with good results . trivedi and kazmi28 ( 1979 ) , using extracts of fruit barks have observed an antibacterial activity of pomegranate extract against bacillus anthracis and vibrio cholerae while machado , et al.14 ( 2003 ) showed similar effect against staphylococcus aureus , in agreement with prashant and asha22 ( 2001 ) . considering that flavoring agents can promote a moderate antiplaque effect16 and that the contents of test and control gels were different only with respect to the presence of pomegranate extract , this agent did not have additional effect in reducing dental plaque formation . these data are not in agreement with those of a previous in vitro study20 that showed that a bacterial strain present in supragingival plaque , namely streptococcus sanguis , was sensitive to different concentrations of pomegranate extract , which demonstrated an inhibitory action similar to that of chlorhexidine2,9 . it should be highlighted , however , that in vitro studies do not reproduce exactly the oral conditions . in the present study , the punica granatum linn extract gel did not avoid plaque formation during the trial , as suggested by kakiuchi , et al.9 ( 1986 ) and pereira , et al.20 ( 2001 ) . as the gel was placed into on the toothshield in a non - diluted form , it may be speculated that gel solubilization by saliva would be necessary for its antimicrobial action to take place17 . the antibacterial agents present in pomegranate - the hydrolysable tannins - form complexes of high molecular weight with proteins soluble , increase bacterial lysis and moreover interfere with bacterial adherence mechanisms to tooth surfaces9,13 - 14 . there was no significant difference between the experimental and control groups in relation to gbi at the end of the trial . these results are not consistent with those reported by pereira and sampaio21 ( 2003 ) , who showed a significant reduction on gingivitis in patients using a dentifrice containing pomegranate extract . nevertheless , it is noteworthy that a control group was not included in that study , which does not allow accessing the actual gingivitis reduction rate related exclusively to mechanical plaque control . according to ross , et al.24 ( 2001 ) the antiinflammatory effect of pomegranate may be attributed to its considerable immunoregulatory activity over macrophages and t and b lymphocyte subsets . however , in both experimental and control groups , there was a significant increase in marginal bleeding , showing that pomegranate did not avoid the development of gingivitis . the binary score used in this study only evaluate the presence or absence of bleeding ; it does not allow the assessment of other characteristics of the inflammatory process , such as edema , changes in gingival contour and loss of tissue attachment . therefore , the possible effect of punica granatum linn extract on controlling the severity of gingivitis can not be discussed . due to the lack of clinical trials with a similar design investigating the effect of pomegranate on gingivitis , the data of this study were evaluated by parallel inferences , taken off from non specialized articles . therefore , more controlled trials using different concentrations of punica granatum linn extract are necessary to verify its action upon supragingival microflora in vivo and established gingivitis . further research will be important to identify the real benefits of pomegranate as a therapeutic and preventive agent for gingivitis , in addition to its common use in popular medicine . within the limits of this clinical study , it may be concluded that the gel containing 10% punica granatum linn extract was not efficient in preventing supragingival dental plaque formation and gingivitis .

objectives : the antiplaque and antigingivitis effects of a gel containing 10% punica granatum linn extract were evaluated using a 21-day partial - mouth experimental model of gingivitis.methods:23 volunteers participated in this cross - over , doubleblind study , carried out in 2 phases of 21 days each . for each period of the experiment , an acrylic toothshield was made for each volunteer to carry the test or placebo gel as well as to avoid brushing of the 4 experimental teeth ( posterior teeth in the lower left quadrant ) . the subjects were randomly assigned to use either the placebo gel ( control group ) or the test gel ( experimental group ) and were instructed to brush the remaining teeth normally 3 times a day . on days 0 and 21 , the visible plaque index ( vpi ) and gingival bleeding index ( gbi ) were recorded.results:the results did not show statistically significant difference between control and experimental groups for either of the indices ( vpi and gbi).conclusion : the gel containing 10% punica granatum linn extract was not efficient in preventing supragingival dental plaque formation and gingivitis .

Objetivos: Mtodos: Resultados: Concluses: INTRODUCTION MATERIAL AND METHODS Subjects Toothshield fabrication Test and control products Preparation of Punica granatum Linn extract Clinical design Clinical assessment STATISTICAL ANALYSIS RESULTS DISCUSSION CONCLUSION

PMC3162762

it is comprised of nonfunctioning primitive lung tissue , having no communication with the tracheobronchial tree , and contains a systemic arterial supply . pulmonary sequestration has been found to be associated with other congenital anomalies like congenital diaphragmatic hernia , congenital heart diseases , and gastrointestinal malformations . vomiting and repeated chest infections in patients of cerebral palsy ( cp ) are usually attributed to the reflux and aspiration of the secretions in these patients . rarely , other causes of vomiting and repeated chest infections are found in these patients . we report a patient of cp who presented with vomiting and repeated chest infections , having causes other than cp for vomiting and repeated chest infections . an 18-month - old male baby was presented to the medical emergency department of our institution with complaints of fever , cough , respiratory distress , and vomiting for 10 days . the baby was a patient of cp and did not achieve developmental milestones with respect to the age . he was febrile with a temperature of 100f ; pulse , 110/min ; respiratory rate , 40/min ; and bp within normal limits . at inspection of the respiratory system on auscultation of the chest , there were course crepts on the left side along with reduced air entry on the same side . there was a bronchial breathing on the lower zone of the left half of the chest . the baby was admitted in the medical emergency as a case of aspiration pneumonia and empirical antibiotics along with nebulization and chest physiotherapy instituted . a chest x - ray was requested , which revealed an impression of some cystic lesion in front of cardiac shadow , along with pneumonic patch , on left side of the chest [ figure 1 ] . his cbc report revealed mild anemia ( hb,9g / dl ) and raised tlc indicating acute infection . a computed tomography ( ct ) scan of chest was requested to delineate the cystic shadow in the chest . a call was sent to the surgical unit for management of hiatus hernia . at the end of treatment for the bronchopneumonia , patient was shifted to our department and an exploratory laparotomy was planned to treat the hiatus hernia . after optimizing the patient for surgery , following were the operative findings : chest x - ray showing left - sided bronchopneumonia and a shadow of cystic mass overlapping the cardiac location on opening the peritoneal cavity , cecum and appendix ( loaded with fecalith ) were present in the epigastrium with a band of ladd running toward the duodenum.there was widened hiatus , and stomach was present in the chest.after delivering stomach back into the abdominal cavity , a cystic mass was present in the chest . that mass was not having any communication with the normal tracheobronchial tree and had its own pleural covering . the blood supply was identified to come from thoracic aorta [ figure 2].a meckel 's diverticulum with a thickened base was found as a coincidental anomaly . on opening the peritoneal cavity , cecum and appendix ( loaded with fecalith ) were present in the epigastrium with a band of ladd running toward the duodenum . there was widened hiatus , and stomach was present in the chest . after delivering stomach back into the abdominal cavity , a cystic mass was present in the chest . that mass was not having any communication with the normal tracheobronchial tree and had its own pleural covering . note that the stomach ( s ) had been taken down from the hiatus and liver ( l ) was present on the opposite side we identified the cystic mass as pulmonary sequestration cyst and sent for histopathology after surgical resection . hiatus hernia was repaired and meckel 's diverticulectomy performed in view of having heterotrophic mucosa in it . at the end the histopathology of the submitted tissue revealed a pulmonary sequestration cyst enclosed in a pleural covering . the patient was on regular follow up and showed a remarkable improvement in episodes of repeated chest infections . there are two types of pulmonary sequestrations , identified on the basis of its anatomical location , intrapulmonary sequestration , and extrapulmonary sequestration . intrapulmonary sequestrations are surrounded by normal lung parenchyma without separate pleura , whereas extrapulmonary sequestration is enclosed completely in its own pleural sac . in our case , the sequestration was lying outside the main pleural cavity and had its own pleural investment . there are two commonly accepted schools of thought regarding the etiology of the pulmonary sequestrations . according to one theory ( the accessory lung bud theory ) , there developed an accessory lung bud from the ventral aspects of the primitive foregut . the accessory lung bud received its blood supply from vessels that connect to the aorta and that cover the primitive gut . if the event of accessory lung bud development occurred early in the course of embryogenesis , it would give intrapulmonary variant ; on the contrary , later development of the accessory lung bud would result in the extrapulmonary type . according the other school of thought , the necrotizing pneumonia causes obliteration of lower zone bronchi that may lead to the development of sequestration . we favor the first theory because the second theory can not explain the systemic arterial supply of the sequestration , which can only be acquired during developmental phase of life . both of the varieties differ in location , venous supply , and age of presentation . the venous drainage of intrapulmonary variety is pulmonic , whereas that of extrapulmonary variety is systemic . intrapulmonary sequestration presents in adulthood ; on the other hand , most of the cases of extrapulmonary sequestration present in the initial few years of life.[124 ] the presentation of pulmonary sequestration is mostly as an incidental anomaly in patients operated for some other surgical disease . the main presentation is that of repeated chest infections.[14 ] in our case , the patient was a diagnosed case of cp and had been admitted multiple times for repeated chest infections and vomiting . vomiting and repeated chest infections in these patients are usually considered the sequel of the cp itself , but in our case , vomiting and repeated chest infections were due to the hiatus hernia and pulmonary sequestration cyst . ct scan can identify such lesions , but as in our case , overlapping of stomach and sequestration might preclude the radiologist in delineating the sequestration cyst . we had the suspicion of some other associated anomaly in this patient due to a cystic shadow on the chest radiograph . extrapulmonary sequestration is found to be associated with congenital diaphragmatic hernia in about 30% cases . the extrapulmonary sequestration was present along with with hiatus hernia , malrotation of intestine , meckel 's diverticulum , hypospadias , and hydrocele . the most common approach for surgical resection is left posterolateral thoracotomy ; however , vats ( video - assisted thoracoscopy ) has also been advocated by some authors . during the operation , phrenic nerve should be carefully identified and saved ; otherwise , a diaphragmatic palsy might occur . , there developed a diaphragmatic eventration after excision of pulmonary sequestration ; and patient had another operation for the iatrogenic eventration of diaphragm.[47 ] we excised the sequestration cyst successfully through the transhiatal route . to conclude , the association of other anomalies like hiatus hernia and pulmonary sequestration as a cause of vomiting and recurrent chest infections in a patient of cp is very rare and a high index of suspicion is required to diagnose it . vomiting and repeated chest infections are not always the characteristics of the cp itself , other causes like pulmonary sequestration should also be considered in these patients .

pulmonary sequestration cyst is a rare entity in pediatric patients . most of the time , it is diagnosed as an incidental finding . it is associated with other congenital anomalies , especially congenital diaphragmatic hernia . we report a patient of cerebral palsy presented with vomiting and recurrent chest infections . he was diagnosed to have hiatal hernia on computed tomography scan of chest . at operation , a pulmonary sequestration cyst along with hiatal hernia , malrotation , and meckel 's diverticulum was encountered . the sequestration cyst was managed through transhiatal approach .

INTRODUCTION CASE REPORT DISCUSSION

PMC5369290

this clinic has been set up in a multispecialty tertiary care hospital in south india that already has a specialized unit for developmental pediatrics , pediatric neurology , and pediatric psychiatry . the author underwent specialized cvi vision assessment training abroad . the training involved vision assessment and rehabilitation of children with cvi in hospital and community setup . our cvi clinic was set up in the eye department of the parent institution in india in february 2011 . assessment to identify and characterize cvi requires dedicated space , time , and an interdisciplinary team approach . our existing examination rooms have been modified to reduce surrounding noise and to make it more appealing to these children . the clinic waiting area has walls painted with cartoon characters , space to run around , play with toys , and drawing materials to keep the child entertained . the examination rooms have been modified to be more child - friendly and are clutter and noise free . resources required for setting up cerebral visual impairment clinic all children presenting to our hospital , < 18 years of age , with cp , learning disability , autism , neurodegenerative diseases , and brain trauma are referred to the clinic . each child is referred to the cvi clinic by his / her pediatrician , neurologist , psychiatrist , occupational therapist , or ophthalmologist . the child 's referring clinician provides the necessary information regarding the neurological and developmental / cognitive assessment and radiological input . the pediatric ophthalmologist gathers this information from the hospital 's electronic medical records ( emr ) . in the cvi clinic this begins when the child comes into the waiting room of the clinic accompanied by the parents / caregivers and includes observing the children 's activities in the waiting area . the child is observed for what he / she is looking at , and the size and nature of the object that gains and maintains the child 's attention , as well as the distance the child can appreciate the object of regard . initial identification of visual - perceptual disorders is carried out , based initially on in - depth history taking , first eliciting spontaneously volunteered information about visual behaviors , then using a structured clinical question inventory as a guide to ensure that key issues are not omitted . visual acuity is evaluated using the best - corrected dioptric correction by the pediatric orthoptist . depending on the age and severity of physical disability , different tests are used . for children with severe cp , in whom visual acuity can not be quantified , we look for responses to light and high contrast pictures / toys , such as movement of limbs or heading toward the visual target , postural reactions , avoiding reactions , and changes in facial expression . eliciting optokinetic nystagmus also provides information on the visual potential in the child . children with mild - to - moderate cp are assessed with age and ability appropriate visual acuity tests . these include lea grating paddles , cardiff visual acuity cards , kay pictures , and snellen visual acuity chart . oculomotor functions including fixation , saccadic eye movements , accommodation , visual axis alignment , and stereopsis are also assessed . the visual field in children is assessed based on the behavioral responses , reflecting the child 's ability to locate targets presented in different areas of the visual field . neuropsychological tests were not employed in our subset of patients due to their multiple neuropsychological disabilities , rendering their administration impossible . all children presenting to our hospital , < 18 years of age , with cp , learning disability , autism , neurodegenerative diseases , and brain trauma are referred to the clinic . each child is referred to the cvi clinic by his / her pediatrician , neurologist , psychiatrist , occupational therapist , or ophthalmologist . the child 's referring clinician provides the necessary information regarding the neurological and developmental / cognitive assessment and radiological input . the pediatric ophthalmologist gathers this information from the hospital 's electronic medical records ( emr ) . in the cvi clinic , the trained pediatric ophthalmologist performs the neuro - ophthalmological assessment . this begins when the child comes into the waiting room of the clinic accompanied by the parents / caregivers and includes observing the children 's activities in the waiting area . the child is observed for what he / she is looking at , and the size and nature of the object that gains and maintains the child 's attention , as well as the distance the child can appreciate the object of regard . initial identification of visual - perceptual disorders is carried out , based initially on in - depth history taking , first eliciting spontaneously volunteered information about visual behaviors , then using a structured clinical question inventory as a guide to ensure that key issues are not omitted . visual acuity is evaluated using the best - corrected dioptric correction by the pediatric orthoptist . depending on the age and severity of physical disability , different tests are used . for children with severe cp , in whom visual acuity can not be quantified , we look for responses to light and high contrast pictures / toys , such as movement of limbs or heading toward the visual target , postural reactions , avoiding reactions , and changes in facial expression . eliciting optokinetic children with mild - to - moderate cp are assessed with age and ability appropriate visual acuity tests . these include lea grating paddles , cardiff visual acuity cards , kay pictures , and snellen visual acuity chart . oculomotor functions including fixation , saccadic eye movements , accommodation , visual axis alignment , and stereopsis are also assessed . the visual field in children is assessed based on the behavioral responses , reflecting the child 's ability to locate targets presented in different areas of the visual field . neuropsychological tests were not employed in our subset of patients due to their multiple neuropsychological disabilities , rendering their administration impossible . from the inception of the clinic in february 2011 to september 2015 , 1478 patients have been referred . the international patients have come from bangladesh , nepal , sri lanka , uae , and kuwait . of the patients referred into the clinic , 61% had cp , 28% had seizure disorders , autism was seen in 9.5% , and learning disabilities , neurodegenerative conditions , and brain injury together constituted 1.5% . of the 902 children with cp , 39% had severe cp ( gmfcs level 5 , macs level 5 , and cfcs level 5 ) , 45% had moderate cp , and 16% had mild cp . the most common perinatal cause of cvi in our clinic was birth asphyxia ( 40% ) , but there were about 20% of cases where a cause of the cvi could not be established . 1 . there was a mixed picture of higher visual dysfunction similar to that previously reported in the literature . visual acuity assessment ( number of patients ) t2 axial and t1 sagittal images showing gliosis in bilateral parietal lobe , thinning of the gray matter and volume loss in the white matter ( arrowhead ) , marked thinning involving the body ( posterior portion ) , splenium of the corpus callosum ( long arrow ) of the 1478 patients seen in the clinic , 295 ( 20% ) of the patients have returned for review . of these , 70% ( 206 patients ) were utilizing strategies recommended in the cvi clinic , and all reported significant progress in their child 's visual abilities . this was corroborated on clinical examination where the children were found to have gained improved eye contact , greater interest in toys and puppets , and enhanced interest in their surroundings . of the remainder , 5% had carried out the visual strategies suggested for a few months but had discontinued the training as they did not find any improvement in their child 's condition . a description of one of the cases presented to our clinic is described in box 1 . clinical presentation of a case seen in cerebral visual impairment clinic liaison between team members was conducted through telephonic conversations and hospital emr . as the clinical service has expanded , another pediatric ophthalmologist and pediatric orthoptist have gained in - house training in cvi assessment . the visual brain reconstructs a dual internal emulation of the visual scene , with the ventral stream : temporal lobes creating the imagery and the dorsal stream ; posterior parietal lobes mapping it in space and time . disorders of this process are unique to each child and involve incomplete mapping of the scene , which necessitates a dedicated quiet environment for optimum assessment . vision fundamentally facilitates learning throughout a child 's development , resulting in knowledge , skills , and traits that will shape the child 's personality and cognition . cvi is the preferred term as it is the result of damage to both gray and white matter of the brain . despite its absence from icd 10 , cvi is one of the leading causes for visual impairment in children both in the developed and developing world . visual dysfunctions are one of the main features of cp and are on a par with the motor disorders . up to 60%70% of children with cp in many western countries , such as the united kingdom , italy , the netherlands , germany , and sweden , this specialized service is routinely available for affected children . there is also involvement of governmental and nongovernmental organizations in their rehabilitation and follow - up . such services can also help maintain a database of visual impairment in cp children for the nation . in india , such specialized services or databases have yet to be developed . according to the published literature from india , the estimated incidence of cp is 3 per 1000 live births and cvi in cp is reported at 28% , this is just the tip of the iceberg . it is likely to be higher if visual perceptual dysfunction is included in the study . training in cvi is not yet part of postgraduate ( pg ) ophthalmology training program in india , neither is there any fellowship offering this facility . development of this specialized care will improve pg training and care for these neglected children . of late , there has been a growing awareness of cvi among pediatric ophthalmologists in the country . in spite of the availability of all the personnel in our hospital , the combined travel and discussion time currently renders setting up a clinic where all the multidisciplinary team ( mdt ) specialists are physically present is a challenge . currently , the pediatric ophthalmologist assessing cvi has access through hospital 's emr to the child 's systemic condition , intelligent quotient scoring , medication , physiotherapy and occupational therapy , and also neuroimaging . the primary clinician and the therapist also have access to the cvi assessment reports for further management of the child . we are developing a regular fortnightly meeting of the mdt to discuss and manage difficult cases . examination of a child with cvi is time consuming , and for any clinical examination to be fruitful , it is important that the child is comfortable throughout . children with cvi are at ease in a quiet environment with fewer distractions , which is not possible in a regular busy pediatric ophthalmology clinic . the examination rooms are accordingly designed to be spacious , uncluttered , and child - friendly with minimum background auditory noise . making the required modifications to our examination rooms was a challenge as the existing concrete structure needed to be refurbished with limited funds . it is important to remember that the needs and well - being of children with cvi and their families are central to establishing such a service . for appropriate service delivery to provide accurate and useful vision assessment of the child cvi assessment necessitates giving an assessment report to the referring clinician and therapists / teachers [ table 2 ] that includes the information needed by teachers to ensure that their teaching is accessible to the child . key links to the cvi clinic include community health - care workers , special educators , voluntary organizations , and parents / caregivers support groups . our limitation is a poor rate of return to the clinic , principally owing to low socioeconomic background and distant locations from where these children come , rendering regular review impracticable , and necessitating the development of alternative approaches to follow - up . assessment in cvi clinics is fruitless if recommended strategies are not understood nor implemented at home . the support groups have an important role to address the following issues : report of cerebral visual impairment clinic assessments and strategies for habilitation for mast x spread education and awareness about cviit is a daunting task for parents not only to accept the physical condition of their wards but also to carry out the strategies suggested . they need constant motivation , with regular encouragement and support . engaging with support groups and voluntary organizations proves beneficialthere is a constant need for economically viable tools and equipment to be developed because affected families are commonly under financial strainan important role of these organizations is to give feedback on services provided by the cvi clinic to assist in service improvements . spread education and awareness about cvi it is a daunting task for parents not only to accept the physical condition of their wards but also to carry out the strategies suggested . engaging with support groups and voluntary organizations proves beneficial there is a constant need for economically viable tools and equipment to be developed because affected families are commonly under financial strain an important role of these organizations is to give feedback on services provided by the cvi clinic to assist in service improvements . there is a need to set up specialized clinics in all parts of the country for patient care and training . for a holistic and successful management of cvi

aim : the aim of this study is to describe the setting up of a cerebral visual impairment ( cvi ) clinic in a tertiary care hospital in south india and to describe the spectrum of cases seen.materials and methods : the cvi clinic , set up in february 2011 , receives interdisciplinary input from a core team involving a pediatrician , neurologist , psychiatrist , occupational therapist , pediatric ophthalmologist , and an optometrist . all children , < 18 years of age , with cerebral palsy ( cp ) , learning disability , autism , neurodegenerative diseases , and brain trauma are referred to the clinic for functional vision assessment and opinion for further management.results:one thousand four hundred and seventy - eight patients were seen in the cvi clinic from february 2011 to september 2015 . eighty - five percent of the patients were from different parts of india . in the clinic , 61% had cp , 28% had seizure disorders , autism was seen in 9.5% , and learning disability , neurodegenerative conditions , and brain injury together constituted 1.5% . most of the children ( 45% ) had moderate cp . forty percent of cvi was due to birth asphyxia , but about 20% did not have any known cause for cvi . seventy percent of patients , who came back for follow - up , were carrying out the habilitation strategies suggested.conclusions:average attendance of over 300 new patients a year suggests a definite need for cvi clinics in the country . these children need specialized care to handle their complex needs . although difficult to coordinate , an interdisciplinary team including the support groups and voluntary organizations is needed to facilitate the successful implementation of such specialized service .

Materials and Methods Selection and description of the participants Running of the clinic Results Discussion Conclusions Financial support and sponsorship Conflicts of interest

PMC3336936

passive transport of drugs through membranes is the main process limiting their penetration into cells ( in the absence of a specific active transporter ) and thus a key step in their administration to the bodies of humans ( and animals ) . diffusion through membrane and partitioning between water and membrane phases are the key properties for this passive transport affecting kinetics and thermodynamics of permeation process , respectively . further , the equilibrium position of specific drugs in target membranes also affects their metabolism and transport ( both active and passive ) . the composition of biological membranes is complex and diverse , varying substantially among the outer and inner leaflets of both organelles and organs . they consist of proteins and lipids , in approximately equal mass proportions . while proteins are responsible for active transport and signaling , lipids pose the main barrier to passive membrane transport . the most important membrane for drug administration is the plasma membrane , through which drugs must penetrate to reach the internal milieu of target cells . however , mitochondrial and endoplasmic reticulum membranes are also involved in drug metabolism because they accommodate various drug - metabolizing enzymes ( e.g. , cytochromes p450 and udp - glucuronosyltransferases ) . the most abundant lipids in mammalian membranes are phosphatidylcholines ( pc ) , although phosphatidylserines , phophatidylethanolamines , sphingomyelins , and cholesterol are also present , thus pc bilayers are commonly used as simple membrane models . however , it must be remembered that in vivo membranes are much more complex , so results obtained using such simple models should be interpreted cautiously . several structural frameworks of lipid bilayers have been proposed , including the four - region model of marrink and berendsen and others presented by neale et al . and orsi et al . the four - region model , applied in the study presented here , describes the physicochemical properties and densities of lipids in the following four regions along a bilayer s normal axis ( figure 1):(i)the low headgroup density region ( hereafter region 1 ) , a polar zone with similar transport conditions to water , from the point where head groups are first encountered ( at minimal density ) and ending where the densities of head groups and water are comparable.(ii)the highly structured high headgroup density region ( region 2 ) , from the point where region 1 ends to the point closer to the bilayer center where the density of water decreases to below 1% and bulklike water disappears . strong coulombic interactions between polar groups keep polar molecules in the first two regions.(iii)the high density of acyl chains region ( region 3 ) is hydrophobic . double bonds of unsaturated lipids are typically localized in this region.(iv)the fourth , low density of acyl chains region ( region 4 ) , resides in the middle of the bilayer and terminal methyl groups are primarily located in this region . here , the two hydrophobic acyl chain regions are believed to form the main barrier for most druglike molecules , which are often water - soluble . the low headgroup density region ( hereafter region 1 ) , a polar zone with similar transport conditions to water , from the point where head groups are first encountered ( at minimal density ) and ending where the densities of head groups and water are comparable . the highly structured high headgroup density region ( region 2 ) , from the point where region 1 ends to the point closer to the bilayer center where the density of water decreases to below 1% and bulklike water disappears . strong coulombic interactions between polar groups keep polar molecules in the first two regions . the fourth , low density of acyl chains region ( region 4 ) , resides in the middle of the bilayer and terminal methyl groups are primarily located in this region . here , the two hydrophobic acyl chain regions are believed to form the main barrier for most druglike molecules , which are often water - soluble . upper left panel ( a ) : density profile of dopc bilayer along the normal to the lipid bilayer plane showing densities of the complete system ( black ) , water ( blue ) , dopc ( red ) , phosphates ( magenta ) , cholines ( cyan ) , carbonyls ( green ) , and terminal carbons ( dark blue ) . lower left panel ( b ) : free energy profile of coumarin along the dopc bilayer normal calculated from constraint simulation with initial structures obtained by free simulation ( cf ) . the calculated bilayer center penetration barrier , g , and water / lipids barrier , g , are labeled . the free energy profile was calculated for one bilayer leaflet and was symmetrized to the other one , the densities of the system were symmetrized along the middle of the bilayer . the vertical bins labeled by numbers denote four bilayer regions : 1 low density of head groups ( 2.22.9 nm ) , 2 high density of head groups ( 1.452.2 nm ) , 3 high density of acyl chains ( 0.51.45 nm ) , and 4 low density of acyl chains ( 00.5 nm ) . right panel ( c ) : structure of dopc bilayer , together with snapshots of coumarin initial structures . molecular dynamics ( md ) simulations can be used to estimate the equilibrium position of a drug in a lipid bilayer , its partition coefficients and diffusion coefficients simultaneously at subpicosecond and atomic resolution . the partitioning and mean position can be well described by a free energy ( g ) profile along the normal to the lipid bilayer , also known as a potential of mean force ( pmf ) . in principle , such free energy profiles can be calculated from partitioning values obtained by long unbiased simulation . however , this approach only provides reliable free energy profiles when all states along the g profile are thoroughly sampled . this is challenging for unbiased md simulations , because they usually do not sample adequately at the available simulation time scales . the sampling problem is based on the dependence of probability of drug crossing a membrane on the energy barrier for this phenomenon . if a barrier for a drug crossing a membrane is higher than 10 kcal / mol , the statistical probability of spontaneous membrane crossing is very low within typical time scales ( hundreds of nanoseconds ) accessible by unbiased atomistic md simulations . therefore during unbiased md simulations , the polar molecules do not usually enter freely the deeper parts of bilayer and the nonpolar molecules do not sample enough of the area of bulk water . great advances have been made in this field in recent years , and numerous methods for obtaining free energy profiles have been developed , including umbrella sampling , z - constraint method , metadynamics , adaptive biasing force , particle insertion , and others . however , although these techniques undoubtedly enhance sampling , all of them have drawbacks for estimating free energy profiles along bilayer normals . for example , in an analysis of interactions between charged and neutral forms of ibuprofen and aspirin with a dipalmitoylphosphatidylcholine ( dppc ) bilayer , boggara and krishnamoorti noted that the drugs ( especially charged forms ) caused deformations of the lipid bilayer in z - constraint simulations . similarly , maccallum et al . observed water defects at the water lipid interface in umbrella simulations applied for calculating free energy profiles of amino acids along the normal of a dioleoylphosphatidylcholine ( dopc ) bilayer . such deformation of lipid bilayer in simulations was recently analyzed by neale et al . neale and co - workers stressed that this systematic sampling error complicated the convergence of free energy profiles of druglike molecules along lipid bilayer normals , especially for charged molecules . as this systematic error may also originate during the generation of starting structure sets used for biased md simulations , this issue has been addressed in several previous studies using various strategies . neale et al . used an inflategro procedure , in which ( briefly ) a pre - equilibrated bilayer is expanded , a molecule of interest is inserted , and the bilayer is compressed and re - equilibrated . boggara and krishnamoorti inserted molecules into a lipid bilayer manually using the vmd visualization program . other approaches for generating starting structures have included growing the molecule inside the bilayer from zero size , pulling simulation , snapshots from unbiased simulation , and estimation of reaction coordinates using metadynamics . however , no methodological analyses of this problem have been previously published . here , based on an examination of the embedding of a nonpolar druglike molecule ( coumarin ; 1,2-benzopyrone ) in dopc bilayer , we show that systematic sampling error is difficult to avoid , but it can be reduced by using appropriate biased simulation and initial structure set generation method . coumarin naturally occurs in diverse plants , including tonka beans ( dipteryx odorata ) , vanilla grass ( anthoxanthum odoratum ) , sweet woodruff ( galium odoratum ) , sweet clover ( meliotus l. ) , sweet grass ( hierochloe odorata ) , and cassia cinnamon ( cinnamomum aromaticum ) . it is absorbed by humans both orally from food and through the skin from perfumes . further , it is a valuable test substance because it is a small , planar , rather rigid , nonpolar ( logpoct / wat 1.39 ) , biologically significant druglike molecule , as its skeleton can be recognized in many drugs ( e.g. , the anticoagulant warfarin and antispasmodic / insecticide hymecromone ) and other biologically active compounds ( e.g. , scopoletin ) . therefore , coumarin is an ideal model for assessing the quality of various methods for calculating g profiles of small low - polar druglike molecules along normals of lipid bilayers . dopc bilayer has been previously used as a model of endoplasmic reticulum membrane , in which coumarin is metabolized by membrane - anchored cytochrome p450 2a6 . the main aim of the study was to identify the mean position of coumarin in dopc bilayer from calculations of free energy profiles using different biased md simulations and different sets of initial structures . we also discuss convergence , advantages and disadvantages of z - constraint , and umbrella sampling methods using starting structures obtained by pulling and unbiased simulations . we focus on the systematic bias caused by choice of the initial structure set and the possibilities of avoiding this bias . the effect of choice of partial charges is also analyzed and results of biased and unbiased md simulations ( 3 s in total ) are compared . finally , a robust simulation protocol for obtaining a convergent free energy profile along a bilayer normal is suggested and tested against available experimental data for two coumarin derivatives embedded in dimyristoylphosphatidylcholine ( dmpc ) bilayer . the structure and topology of coumarin ( 1,2-benzopyrone ; cas number 91 - 64 - 5 ) was generated by the prodrg2 beta server using the gromos 53a6 forcefield . however , partial charges assigned by prodrg have been found to lead to unrealistic partitioning between water and cyclohexane phases . as the partial charges used can introduce another systematic error into free energy calculations , we addressed this problem by also using mulliken partial charges and restrained fit of electrostatic potential ( resp ) partial charges . the resp partial charges were successfully adopted by the second generation of amber family force fields . the electrostatic potential ( esp ) and esp partial charges were calculated by applying b3lyp / cc - pvdz method to coumarin geometry optimized at the same level of theory in gaussian 03 . mulliken partial charges , that were adopted by berger lipid force field , were calculated at the hf/6 - 31 g * level in gas phase . hereafter , all mentioned coumarin charges are resp charges , except those explicitly named as prodrg or mulliken charges . the lipid bilayer , as prepared and equilibrated by siu et al . , contained 128 dopc molecules , 64 in each leaflet , with a structure generated by the lipidbook server . the bilayer was oriented perpendicularly to the z - axis of the simulation box and equilibrated for another 10 ns by a free md simulation . water and salt ( nacl ) were added to give a physiological concentration , of 0.154 m , of salt in the aqueous phase ( excluding the lipid bilayer from the volume calculation ) . the equilibrated box contained 5,188 molecules of flexible simple point charge ( spc ) water , 19 na and 19 cl ions . the equilibrated surface area per lipid was 0.638 nm , and the start of the z - axis was set in the middle of the bilayer . the gromacs 4.0.7 package and united atom berger lipid force field were used for md simulations . the latter reduces the number of atoms in simulations , as it merges nonaromatic and nonpolar hydrogens with their carbons . this simplification likely results in higher diffusion coefficients than those observed in all - atom model simulations . berger lipid force field uses the mulliken partial charges calculated at the hf/6 - 31 g * level ( in gas phase ) . simulations were taken with 2-fs integration time steps under periodic boundary conditions in all directions , with particle - mesh ewald ( pme ) electrostatics , a van der waals cutoff at 1 nm , bond constraints determined by the lincs algorithm , v - rescale temperature coupling to 310 k , and berendsen anisotropic pressure coupling to 1 bar with 10 ps time constant and compressibility of 4.5 10 bar . a coumarin molecule was placed at the top of the simulation box , a 0.5 ns md simulation was executed to pre - equilibrate the system , then five independent md simulations with a total time of 3 s were generated . from the pre - equilibrated simulation two sets of starting frames for biased md simulations were generated : one by pulling coumarin to the bilayer center and the other from unbiased md simulation . the first set of starting frames for biased md simulations was obtained by pulling the center - of - mass ( com ) of coumarin against that of the lipid bilayer ( in its center ) . coumarin was pulled along the bilayer normal ( the z - axis ) for 6 ns using a pulling force constant of 10,000 kjmolnm ( 2,390 kcalmolnm ) and pulling rate of 1 nmns . pulling applies a harmonic potential on molecule and moves the center of this potential with a given pull rate . starting positions were collected as snapshots from the pulling simulation , spaced 0.1 0.02 nm apart along the z - axis from the area of bulk water ( 4 nm from the bilayer center ) to the middle of the bilayer . from the structures at one distance bin , the structure with the lowest potential energy was chosen as the starting frame for biased md simulations at a given distance from the center of the lipid bilayer . hereafter , constraint and umbrella simulations with initial structures generated by pulling simulations are referred to as constraint - pulling ( cp ) and umbrella - pulling ( up ) , respectively . in the other approach for generating starting frames , in which free unbiased simulation ( 20 ns long ) starting frames ( spaced 0.1 0.02 nm apart along the z - axis with the lowest potential energy of the structure at the respective points ) for umbrella and constraint simulations were chosen as described above . simulations with these starting structures are henceforth referred to as umbrella - free ( uf ) and constraint - free ( cf ) , respectively . with both sets of starting frames , umbrella sampling and constraint simulations ( 164 simulation windows in total ) were carried out as described below for 30 ns per simulation window , except for simulation bins in the 1.02.5 nm z - axis region ( for which simulation was prolonged for 50 ns , or up to 100 ns for cp and cf simulations , giving in the latter cases up to 6.9 s of biased simulation in total ) . in umbrella sampling a harmonic potential is applied between coms of two groups of molecules , here the drug coumarin and dopc lipid bilayer . the distance between coms of coumarin and dopc was restrained by a harmonic force constant of 2,000 kjmolnm ( 477.9 kcalmolnm ) . the force applied on coumarin was proportional to the square of the displacement from its original position , and a free energy profile was calculated from eq 1(4,55)1where p(z ) and u(z ) are the coumarin distribution and biasing potential along the bilayer normal , respectively . the force constant and distance between simulation windows were chosen to achieve equal sampling , as the presence of regions with low sampling density increases the error of umbrella sampling . forces acting on coumarin were analyzed , and the free energy profile was reconstructed by the weighted histogram analysis method ( wham ) using the g_wham program . in this approach , the distance between coms of the drug and lipid bilayer was constrained , and the constraint force was monitored . free energy was then calculated from eq 2(4,16,17,57)2where the mean force applied on the molecule f(z)t in certain bilayer depth z is integrated along the bilayer normal axis beginning in water until the certain bilayer depth z. part of the free energy profile was also calculated from the partitioning displayed in an unbiased simulation using eq 3(15)3where k(z ) is a partition coefficient estimated for a 0.02 nm bin in bilayer depth z , symmetrized for both leaflets . the partition coefficient is calculated from average mass density of coumarin in certain bin and by normalizing this density the reference state is set to have k(z ) = 1 . the minimum free energy ( g = 0 kcal / mol ) of coumarin along the bilayer normal was considered the reference state in all profiles . all free energy profiles were calculated for one lipid leaflet and have been symmetrized for the other one . error estimates of free energy were calculated as integrated standard deviations of the mean calculated either over the bins of 100 bootstraps generated by bayessian bootstrap analysis by the g_wham program in umbrella sampling or over the force distribution in 0.1 nm - spaced positions along the z - axis in constraint simulations . for comparison with experimental data ( see later ) free energy profiles of 7-acetoxy-4-methylcoumarin and 7-acetoxycoumarin along the normal of a dmpc bilayer were calculated ( using a dmpc bilayer structure with 128 dmpc molecules and 3,655 water molecules taken from tieleman s web site , after replacing 20 molecules of water by 10 na and 10 cl ions ) . the lipid bilayer was equilibrated for 200 ns . 7-acetoxy-4-methylcoumarin topology was prepared by the simulation protocol described above for coumarin ( including the use of resp partial charges ) . initial structure was generated from a 20 ns unbiased free simulation . during this simulation , 7-acetoxy-4-methylcoumarin penetrated to 1.8 nm from the middle of the bilayer and was then pulled into the bilayer for 4 ns with a pulling rate of 1 nmns and a pulling force constant of 500 kjmolnm ( 119.5 kcalmolnm ) . the mild force constant was chosen to avoid water artifacts , as the molecule did not penetrate into region 3 freely during the unbiased simulation and was still in touch with water molecules . a 10 ns constraint simulation was performed with the same simulation protocol as applied in the coumarin cf simulation , but the simulation near the equilibrium position ( 0.81.7 nm ) was prolonged to 15 ns per simulation bin . a free energy profile of 7-acetoxycoumarin was obtained by the same protocol as for 7-acetoxy-4-methylcoumarin , except the free simulation lasted 60 ns and during this time 7-acetoxycoumarin penetrated to 0.5 nm from the center of the lipid bilayer , then the molecule was pulled further into the bilayer for 1 ns with a pulling rate of 1 nmns and force constant of 2,000 kjmolnm ( 477.9 kcalmolnm ) . the molecule freely penetrated close to the bilayer center and thus was pulled with a higher force constant than in the previous case . a free energy profile was obtained by z - constraint simulation , which yielded a very large minimum energy zone , with substantial variation in mean forces , so the simulation was prolonged to 15 ns in the interval between 1.0 and 2.0 nm and more frames were added ( so the distance between simulation windows was 0.05 nm in the zone between 1.0 and 2.0 nm ) . five independent unbiased simulations starting from coumarin in water , 3 s long in total ( 2 1 s , 600 ns , 2 200 ns ) , showed a tendency for coumarin to stay at the boundary between regions 2 and 3 , as coumarin was most frequently located 1.4 0.1 nm from the bilayer center ( see supporting information , figure s1 ) . once a coumarin molecule entered the bilayer during the first 10 ns of the simulation , it did not leave the lipid bilayer during the rest of simulation ( figure s1 ) . twelve successful ( and ten unsuccessful ) transitions between leaflets were observed during the 3 s of simulations ( figure s1 ) . the transition between both leaflets took place on a 100 + ns time scale , but the transition process itself was rapid and lasted several nanoseconds . the unbiased simulations also identified a metastable state of coumarin in the bilayer center ( figure 2 ) , where coumarin stayed up to 10 ns . the transition between the bilayer center and one leaflet occurred on time scales of ps up to ns ( figure s1 ) . the bilayer center penetration barrier calculated from the partition coefficient profile ( cf . equation 3 ) was 2.1 kcal / mol , but the water / lipids barrier could not be calculated , as the distribution of coumarin in water was not properly sampled ( figure s1 and figure 2 ) . density profiles of dopc ( blue dash - dotted curves ) and coumarin calculated from unbiased md simulations ( 3 s in total ; red dashed curves ) and from the free energy profile acquired by the cf method ( green dotted curve ) . the density profiles of coumarin obtained from both methods match each other well ( upper panel free energy profiles obtained from biased simulations ( lower panel b ) . both umbrella ( up black curve and uf red curve ) and constraint ( cp dotted blue curve and cf dotted green curve ) simulations provide free energy minima positions for coumarin that overlap well with the maximum density calculated from the free simulation ( cf . upper panel the free energy profiles were calculated for one bilayer leaflet and were symmetrized to the other one ; the density was symmetrized along the middle of the bilayer . up and uf refer to umbrella simulations with initial structures obtained by pulling and free unbiased simulation , respectively ; cp and cf refer to constraint simulation with pulling and free initial structures , respectively . the free energy profiles of coumarin in dopc lipid bilayer reconstructed from four types of biased ( up , cp , uf , and cf ) simulations showed similar trends ( figure 2 ) . typically , the free energy dropped as coumarin entered region 1 ( cf . figure 1 ) . as it moved deeper into the bilayer , the free energy decreased and the global free energy minimum was reached at the border between regions 2 and 3 . when coumarin moved deeper into the bilayer center , the free energy rose . a small local minimum was located in the bilayer center ( region 4 ) . so , one global minimum at 1.351.53 nm ( with a thermally accessible region within 1.051.95 nm at 310 k by thermally accessible region we mean an area with energy barrier of rt ( 0.616 kcal / mol at 310 k ) from the energy minimum ) and one local minimum in the middle of the lipid bilayer were common features of all free energy profiles ( figure 2 ) . the bilayer center penetration barriers ( g ) obtained from the free energy profiles fitted a narrow interval , varying between 2.63.3 kcal / mol ( table 1 , figure 2 ) . the water / lipids barrier ( g ) fitted an interval of 5.76.7 kcal / mol , and the values calculated with pulling initial structures were lower than those calculated in simulations with initial structures from unbiased simulations ( g values derived from up , cp , uf , and cf simulations were 5.7 0.3 , 5.9 0.2 , 6.7 0.1 , and 6.4 0.2 kcal / mol , respectively ; table 1 and figure 2 ) . the up free energy profile also showed a very shallow local minimum at 1.95 nm with an energy barrier of 0.5 kcal / mol ( figure 2 ) . the free energy barrier of this minimum was higher than the free energy error bar estimated by statistical bootstrap analysis ( 0.1 kcal / mol ) , but the error seemed to be underestimated . as the depth of the shallow minimum declined with increasing duration of simulation windows ( see the following paragraph convergence of biased simulations ) and no state corresponding to this minimum was observed in the unbiased simulation ( figure s1 in the supporting information ) , we considered this minimum to be an artifact and called it the artificial minimum . up and uf refer to umbrella simulations with initial structures obtained by pulling and free unbiased simulation , respectively ; cp and cf refer to constraint simulation with pulling and free initial structures , respectively . g and g are water / lipid and bilayer center penetration barriers , respectively . area within reach of a thermal motion is considered to be the area surrounded by an energy barrier of rt ( 0.616 kcal / mol , t = 310 k ) . as such artificial minima may be due to systematic sampling errors , it was of considerable interest to determine the reasons for such behavior . the primary reason lay in the starting structures , which were generated by pulling coumarin along the bilayer normal in the up and cp simulations . the pulling caused deformation of the lipid bilayer , leading to a funnel - shaped bilayer surface depression ( see supporting information , figure s2 ) induced by solvated coumarin ( figure 3a ) . in other words , the pulling procedure produced structures in which coumarin embedded in the bilayer was hydrated by a few water molecules . the defects , namely these which were deeper in lipid bilayer , were eliminated during biased md simulations , as water was expelled from the hydrophobic bilayer interior and bilayer relaxed rapidly on hundreds of picoseconds time scale . the relaxation time grew with increasing distance from the bilayer center . in the area of the artificial minimum ( 1.72.0 nm ) , close to the bilayer surface , the water defects were eliminated on a tens of nanoseconds time scale . it is of considerable interest that relaxation occurred significantly more rapidly in the cp than in up simulations , and thus the membrane deformation was eliminated more rapidly ( figure 3 and figure 4 ) . the lipid bilayer depression was not observed during a spontaneous embedment of coumarin in the lipid bilayer in the unbiased simulations , hence the starting structures for biased simulations based on the snapshots from the unbiased simulation were free of this artifact ( figure 3b ) . initial structures ( at 1.9 nm from the bilayer center ) obtained by pulling ( a ) and free simulation ( b ) show a difference in coumarin hydration . the structure generated by pulling simulations indicates that coumarin is pulled to the lipid bilayer with its solvation shell , which causes funnel - like bilayer deformation . snapshots taken at 10 ns indicate that cp eliminates coumarin hydration more rapidly than up . waters surrounding coumarin are colored as red / white balls.up and uf refer to umbrella simulations with initial structures obtained by pulling and free unbiased simulation , respectively ; cp and cf refer to constraint simulation with pulling and free initial structures , respectively . convergence of free energy profiles , positions of energy minima and energy barriers . the simulation windows within 1.02.5 nm have been simulated for 50 and 100 ns in case of up and uf simulations of cp and cf simulations , respectively ; the rest of each profile is calculated from 30 ns of simulation . free energy profiles calculated from short simulation times ( < 5 ns ) are biased by high error , because of small data set and nonequilibrium starting structures . free energy profiles obtained by up and cp simulations ( left ) show slow elimination of an artificial minimum ( 2.0 nm ) and deepening of the global minimum ( 1.5 nm ) . free energy profiles obtained from uf and cf simulation are consistent in coumarin positioning and have deeper energy minima than those obtained from up and cp simulations . uf refer to umbrella simulations with initial structures obtained by pulling and free unbiased simulation , respectively ; cp and cf refer to constraint simulation with pulling and free initial structures , respectively . the position of the global minimum converged more rapidly in biased simulations starting from free ( unbiased ) simulations ( uf and cf ) than in simulations starting from pulling ( up and cp ) simulations ( figure 4 ) . the free energy profile obtained from up simulation depended strongly on the length of the simulation windows , and two energetically similar minima in region 2 ( one at 1.5 and the other at 2.0 nm ) were observed during the beginning of this simulation ( figure 4 ) . after 10 ns the minimum at 1.5 nm became the global minimum and its position converged to 1.53 nm , while the energy barrier of the artificial minimum decreased to 0.5 kcal / mol . during the first 16 ns of up simulation the area accessible by thermal motion ( gmin+rt ) gradually widened from 0.90 nm after 5 ns to 1.10 nm , and the region accessible by thermal motion thereafter declined to 0.80 nm . g gradually rose throughout the simulation , to a final value ( at 50 ns ) of 5.7 0.3 kcal / mol , while g dropped within the first 16 ns of simulation , slowly rose until 30 ns , and then fluctuated around a final value of 3.2 0.2 kcal / mol ( cf . figure 4 ) . the free energy profile obtained from cp simulation also displayed two minima initially , while the artificial minimum ( at 2.0 nm ) quickly vanished , and after 15 ns there was no sign of this minimum . the area within reach of thermal motion gradually narrowed from 1.38 nm after 5 ns of simulation to 0.51 nm after 40 ns and thereafter remained constant . g gradually rose throughout the simulation , to 5.9 0.2 kcal / mol , while g grew during the first 11 ns of simulation , until 20 ns of simulation it gradually declined to 2.8 0.1 kcal / mol and then fluctuated around this value . in the last 50 ns of the simulation prolonged to 100 ns the position of the energy minimum remained constant ; g fluctuated around 2.8 0.1 kcal / mol and g continued to rise , to 6.2 0.2 kcal / mol . the position of the minimum in the uf free energy profile was almost constant ( within 1.291.35 nm ) during the whole simulation time , with the area accessible by thermal motion slowly widening from 0.44 to 0.66 nm . g slowly decreased during the first 19 ns of simulation , then very slowly increased , and after 30 ns g converged to 6.7 0.1 kcal / mol , while g became convergent after 20 ns , fluctuating within 2.6 0.1 kcal / mol . the cf free energy profile showed a minimum position within 1.291.49 nm , and the area of thermal motion slowly widened from 0.41 to 0.70 nm . g fluctuated around 6.47.0 kcal / mol during the whole simulation time , while g decreased during the first 10 ns of simulation and then fluctuated around 2.93.3 kcal / mol . the prolonged simulation to 100 ns showed similar trends a free energy minimum at 1.29 nm , thermal motion within 0.6 nm , a constant g value of 7.0 kcal / mol after 80 ns , and g already convergent with a final value of 3.1 0.1 kcal / mol . as assignment of partial charges might introduce another systematic sampling error into free energy calculations , we carried out 10 ns long cf simulations with prodrg and mulliken charges ( assigned partial charges are listed in supporting information table s1 ) , to assess the extent to which the partial charges affected the free energy profiles . coumarin with partial charges assigned by prodrg bore a dipole moment of 9.5 d , assignment of mulliken partial charges led to 6.0 d , and resp partial charges resulted in a dipole moment of 4.9 d ( figure 5 ) . the dipole moment based on resp charges was close to that of coumarin in the gas phase calculated by the hybrid dft method ( b3lyp / cc - pvdz ) of 4.6 d , mulliken partial charges represent a compromise between the dipole moment in water ( represented by continuum dielectrics with r = 78.39 ) and heptane ( r = 1.92 ) , which we calculated by the cpcm / b3lyp / cc - pvdz method and that resulted in 6.7 and 5.4 d , respectively . considering these values , the dipole moment stemming from prodrg charges seemed to be unreliably overestimated , which could systematically bias free energy profiles based on prodrg charges . the global minimum of the g profile of coumarin bearing resp partial charges was located at 1.29 nm ( cf with a 10 ns sampling window ) , energy minimum of coumarin bearing mulliken partial charges was localized at 1.20 nm , and the minimum for prodrg - charged coumarin was shifted toward the bilayer / water interface , at 1.62 nm . the global free energy minimum for resp - charged coumarin was also considerably deeper than for mulliken - charged or prodrg - charged coumarin ( g : 7.5 , 5.6 , and 3.3 kcal / mol , respectively ) , and the bilayer center penetration barriers of the systems also differed ( g : 3.1 , 4.6 , and 10.1 kcal / mol , respectively ) ( figure 5 ) . as expected , the energy cost of bilayer center penetration grows with the increasing dipole moment , and reversely the energy barrier between lipid bilayer and water decreases with the growing dipole moment . left panel : free energy profiles calculated for coumarin with prodrg ( red dotted curve ) , mulliken ( blue dashed curve ) , and resp charges ( black curve ) by constraint simulation ( cf ) with initial structures obtained by free simulation using 10 ns windows . coumarin with prodrg partial charges is shifted to the outer part of the lipid bilayer . the bilayer center penetration barriers grow and the water / lipids barriers decrease with increasing dipole moment . the right panel shows that the partial charges ( mapped on the vdw surface ) calculated by resp ( upper part ) and mulliken population analysis ( middle ) are spread along the whole molecule , while partial charges assigned by prodrg ( lower part ) are localized close to coumarin oxygens . to our knowledge , the precise positioning of bare coumarin in a dopc bilayer has not yet been studied experimentally ; therefore , we compared the results of our theoretical calculations to data obtained in experiments with coumarin derivatives . depths of several coumarin derivatives in dimyristoylphosphatidylcholine ( dmpc ) bilayer have been studied in nmr investigations , in which chemical shifts of c - labeled derivatives were used to assess the polarity of the surroundings of c atoms and hence estimate their depth in the lipid bilayer . results of the cited experiments indicate that the mean position of 7-acetoxy-4-methylcoumarin is at the border of regions 2 and 3 in dmpc lipid bilayer ( 0.7 nm from the dmpc choline nitrogens , corresponding to 1.2 nm from the center of the bilayer ) ; c - labeled carbons of the derivative ( c2 and c4 , see figure 6 ) appeared to be located 0.72 and 0.70 nm from the choline nitrogens , corresponding to 1.18 and 1.20 nm from the bilayer center , respectively . another coumarin derivative , 7-acetoxycoumarin , was apparently located closer to the bilayer interface in region 2 , with its c - labeled ( c2 and c4 ) carbons 0.44 and 0.59 nm from the choline nitrogen , corresponding to 1.46 and 1.31 nm from the bilayer center , respectively . we recalculated the experimental positions ( originally expressed as distances from the bilayer surface ) as distances from the bilayer center to facilitate direct comparison with results of this study . in this recalculation , the distance between the dmpc bilayer surface and center was set at 1.9 nm : the mean distance between the bilayer center and maximum density of nitrogens ( regarded as the membrane surface in the cited nmr experiments ) in corresponding md simulations . free energy profiles and structures of coumarin derivatives ( 7-acetoxy-4-methylcoumarin , upper panel ( a ) , and 7-acetoxycoumarin , lower panel ( b ) ) along a dmpc lipid bilayer normal calculated from constraint simulation with initial structures obtained by free simulation ( cf ) . nmr - observed positions of c - labeled carbons ( c2 and c4 ) are displayed as red and green circles , respectively , and the positions of c2 and c4 carbons calculated from simulation are depicted as green and red curves , respectively . the positions of marked carbons of both coumarin derivatives are in good agreement with the positions observed by nmr . for the comparison we employed the most effective simulation protocol of those considered here to calculate the free energy profiles of the coumarin derivatives described above , briefly comprising unbiased simulation followed by constraint simulation with 10 ns simulation bins ( see methods for details ) . the profile obtained for 7-acetoxy-4-methylcoumarin indicated the free energy minimum position of its com to be 1.0 nm from the center of the dmpc bilayer ( figure 6 ) , with a thermally accessible region between 0.8 and 1.3 nm . the thermally accessible region estimated from the simulation ( 0.81.3 nm ) matched that acquired from nmr experiments , where 7-acetoxy-4-methylcoumarin was located 1.2 0.1 nm from the bilayer center . in addition , the positions of its c2 and c4 carbons calculated from simulations ( 1.25 and 1.21 nm , respectively ) agreed well with those estimated from experiments ( 1.18 and 1.20 nm , respectively ) , although the c4 carbon seems to flip - flop between two positions in the bilayer ( the other at 0.76 nm , with ca . the free energy minimum position of the com of 7-acetoxycoumarin in the simulation was located 1.2 nm from the bilayer center , with a thermally accessible region between 0.75 and 1.35 nm . the simulated distances of the c2 and c4 carbons from the bilayer center at this point ( 1.34 and 1.19 nm , respectively ) again matched those obtained from the nmr data reasonably well ( 1.46 and 1.31 nm , respectively , see figure 6 down ) . in summary , the md results for both coumarin derivatives agreed reasonably well with the experimental results , notably 7-acetoxy-4-methylcoumarin was located deeper in the bilayer than 7-acetoxycoumarin , and the carbon atoms positions calculated from simulations matched those deduced from experiments . during the five independent unbiased simulations ( 3 s long in total ) coumarin preferred the lipid bilayer phase rather than the aqueous phase , because it quickly ( within < 10 ns ) entered the lipid bilayer and remained there for the rest of the simulation time ( figure s1 in the supporting information ) . the preferentially occupied position was at 1.4 0.1 nm , at the border of regions 2 and 3 , and the molecule was oriented mainly with its oxygens pointing toward the water phase ( data not shown ) . in addition , coumarin penetrated the lipid bilayer spontaneously , i.e. , moved from one leaflet to the other , remaining at the preferentially occupied positions in both leaflets for several hundreds of nanoseconds between brief ( a few ns ) visits to the lipid bilayer center ( figure s1 in the supporting information ) . similarly , the transition movements were quite rapid , generally occurring within several nanoseconds . some key penetration properties were identified from the unbiased simulations , namely positions of local and global free energy minima and qualitative estimates of the height of energy barriers ( figure 2 ) . the water / lipids barrier , g , seemed to be higher than the bilayer center penetration barrier , g . the number of penetration events allowed us to roughly estimate the absolute value of the bilayer center penetration barrier , at 2.1 kcal / mol ( eq 3 ) . however , the estimated g value should be interpreted with care , due to the limited sampling as only a small number of transitions between the minima were observed , and g could not be calculated as the water phase was not sampled adequately ( figure 2 ) . nevertheless , the spontaneous embedding of coumarin in dopc bilayer strongly indicates that this is a barrierless process and that coumarin prefers the bilayer phase , in accordance with expectations based on coumarin s logpoct / wat value . the final free energy profiles obtained by all simulation protocols ( up , cp , uf , and cf ) were in accord ( figure 2 ) , but those obtained from the unbiased simulations provided more accurate information . the global energy minimum was found at 1.44 0.09 nm , while a local energy minimum was localized in the membrane center . the presence of a local energy minimum in the lipid bilayer center agrees with previous findings presented by bemporad et al . , of such local minima for some other small solutes , e.g. water and acetamide . in our case , the bilayer center penetration barrier ( g ) of coumarin spanned 2.63.3 kcal / mol ( table 1 , figure 2 ) , close to the g estimated roughly from the unbiased simulation ( 2.1 kcal / mol ) . in contrast , the water / lipids barrier ( g ) varied significantly with time and method used ( see below ) . the estimated g from cf simulation ( which is taken as reference , as constraint biasing eliminates possible artificial errors quicker ) was 6.4 0.2 kcal / mol ( figure 2 ) . the free energy profiles therefore confirmed that coumarin more readily penetrates the bilayer than escapes to the water phase . neither accurate unbiased simulation , nor accurate free energy profile calculation , is possible without a careful choice of force field . the berger force field ( using mulliken partial charges calculated at hf/6 - 31 g * level ( in gas phase ) ) used for lipids was tested and shown to provide area per lipid and volume per lipid values that correspond well with experimental values . further , as coumarin parameters were not available in standard data sets for lipid simulations , they had to be acquired separately . generally , atom types and corresponding parameters can be adopted for a nonlipid molecule from the standard data sets quite safely , but the set of partial charges had to be carefully considered , as it may introduce a serious systematic sampling error in lipid bilayer - guest molecule simulations . we addressed this issue by using three sets of partial charges ( figure 5 , table s1 in the supporting information ) : one generated by the prodrg server , one assigned by mulliken population analysis , and the third generated by applying the resp procedure in b3lyp / cc - pvdz calculations of electrostatic potential in gas phase . generally , increasing the dipole moment of a molecule ( by use of prodrg or mulliken partial charges ) resulted in a lower g and higher g , in accordance with expectations , given the higher polarity of coumarin bearing prodrg or mulliken charges in comparison with resp partial charges ( figure 5 ) . with only these profiles it would be difficult to decide which partial charges provided more reliable results . however , prodrg charges led to overestimation of the dipole moment of coumarin and ( as mentioned above ) partial charges assigned by the prodrg server lead to unrealistically strong partitioning in water in cyclohexane / water systems as found by lemkul et al . the latter finding agrees with the trends observed in our lipid bilayer simulations . in summary , resp charges seem to provide more accurate models for simulations of lipid bilayer - guest molecule systems than prodrg charges ( although whether the resp charges should ideally be based on gas phase or solvent - polarized esp , and if they can be robustly combined with the berger force field for lipids , remains to be determined ) . we have shown here that the convergence of free energy profiles was significantly influenced by the generation of initial structures when followed by the biasing method . the biased simulations starting from the pulling simulations ( up and cp ) suffered from bilayer deformation induced by pulling coumarin from the water phase toward the bilayer center ( figure 3 ) . similar bilayer deformations have been repeatedly previously observed and identified as a systematic sampling artifact in biased lipid bilayer simulations . for example , neale et al . observed bilayer deformations when a charged molecule was embedded in the bilayer . we observed a funnel - shape bilayer surface depression ( figure s2 in the supporting information ) , caused by water hydrating the polar parts of coumarin penetrating the lipid bilayer more deeply and thereby exacerbating bilayer deformation during the pulling simulations . the bilayer deformation caused an artificial minimum ( 2.0 nm ) in the free energy profiles in region 2 ( figure 2 ) , whereas in unbiased simulation coumarin never stayed longer in this position , and its behavior showed no sign of reaching a local energy minimum . this artificial minimum was most profound when short simulation times ( < 5 ns ) for each sampling bin were applied , and it slowly disappeared when the simulation time was prolonged ( figure 4 ) . the main reason for the slow convergence and need for longer simulation times was the slow coumarin water shell elimination in region 2 . furthermore , the presence of the artificial minimum led to underestimation of g in simulations using the initial structure set generated by pulling simulation ( cp and up ) . while g values obtained by cf and uf simulations seemed to reach convergence , they did not reach convergence in up and cp simulations during 50 ns of simulation ( or even during 100 ns of cp simulation ) , although both up and cp yielded g values close to those obtained from cf and uf simulations . in this respect , constraint biasing was more effective , as the artificial minimum was eliminated within 15 ns per bin ( in cp ) , while there were signs of the artificial minimum in up simulation even after 50 ns per bin ( figure 4 ) . even longer times may be needed in simulations of polar or charged residues , as previously shown by maccallum et al . and neale et al . , who found that 80 to 205 ns per bin may be required to achieve convergence in umbrella simulations with charged solutes . these water artifacts seem to be present when nonequilibrated initial structures are used for biased simulation . the higher efficiency of constraint over umbrella biasing is also consistent with the recent observation by gunsteren et al . , that constraint - biased simulation using force averaging is the most effective method for calculating potential of mean force with respect to a distance from a given reference point . convergence of the free energy profiles was clearly achieved more rapidly when using starting structures acquired from unbiased ( uf and cf ) simulations in comparison with the pulling simulation ( up and cp ) , since in cases of uf and cf simulation the free energy profiles changed only marginally with increases in the length of the simulation bins ( figure 4 ) . therefore we recommend starting the calculation of free energy profile with an unbiased simulation for all molecules , in a case of more polar molecules a slow pulling simulation ( pulling force constant < 500 kjmolnm ( 119.5 kcalmolnm ) and a pulling rate < 1 nmns ) from the deepest position in the lipid bilayer should follow . thus , this approach was used for comparing the calculated results with experimental data , and the calculated positions of 7-acetoxy-4-methylcoumarin and 7-acetoxycoumarin in dmpc bilayer agreed well with positions derived from nmr experiments ( figure 6 ) . in summary , whenever possible biased simulations should start from geometries acquired from unbiased md simulations , and constraint biasing is the recommended and quickly converging method . the convergence in time of free energy profiles of coumarin along a dopc bilayer normal , calculated by both umbrella sampling and z - constraint techniques , was thoroughly analyzed . two sets of starting structures were also considered : one based on unbiased md simulation and the other on pulling coumarin along the bilayer normal . water defects on the lipid bilayer surface were identified in the structures obtained by pulling simulation but not in structures acquired from unbiased simulation . consequently , the free energy profiles converged more rapidly when starting frames from unbiased simulations were used . the used methods for free energy profile calculation ( umbrella and constraint simulation ) are quite equivalent when applied on an error - free set of starting structures . however , if the membrane defects are present , the z - constraint simulation leads to more rapid convergence than umbrella sampling . in summary , for efficient calculation of convergent free energy profiles of druglike molecules along bilayer normals , we recommend using z - constraint biased md simulations based on as much starting geometries acquired from unbiased md simulations as possible , otherwise when pulling simulation is employed , the biased simulation might need far longer time to reach convergence .

atomistic molecular dynamics ( md ) simulations of druglike molecules embedded in lipid bilayers are of considerable interest as models for drug penetration and positioning in biological membranes . here we analyze partitioning of coumarin in dioleoylphosphatidylcholine ( dopc ) bilayer , based on both multiple , unbiased 3 s md simulations ( total length ) and free energy profiles along the bilayer normal calculated by biased md simulations ( 7 s in total ) . the convergences in time of free energy profiles calculated by both umbrella sampling and z - constraint techniques are thoroughly analyzed . two sets of starting structures are also considered , one from unbiased md simulation and the other from pulling coumarin along the bilayer normal . the structures obtained by pulling simulation contain water defects on the lipid bilayer surface , while those acquired from unbiased simulation have no membrane defects . the free energy profiles converge more rapidly when starting frames from unbiased simulations are used . in addition , z - constraint simulation leads to more rapid convergence than umbrella sampling , due to quicker relaxation of membrane defects . furthermore , we show that the choice of resp , prodrg , or mulliken charges considerably affects the resulting free energy profile of our model drug along the bilayer normal . we recommend using z - constraint biased md simulations based on starting geometries acquired from unbiased md simulations for efficient calculation of convergent free energy profiles of druglike molecules along bilayer normals . the calculation of free energy profile should start with an unbiased simulation , though the polar molecules might need a slow pulling afterward . results obtained with the recommended simulation protocol agree well with available experimental data for two coumarin derivatives .

Introduction Methods Results Discussion Conclusion

PMC4121913

the intrauterine existence of fetus is dependent on one vital organ the placenta . placenta is essential for maintenance of pregnancy and for promoting normal growth and development of fetus . it forms the morphological record of anatomical condition , intrauterine events and intrapartum events of gestation . pregnancy - induced hypertension ( pih ) is the leading cause of maternal mortality and is an important factor in fetal wastage . pregnancy complications like hypertension are reflected in placenta in a significant way both macroscopically and microscopically . several studies have shown that utero - placental blood flow is decreased in pih due to maternal vasospasm . this leads to constriction of fetal stem arteries and has been associated with the changes seen in the placenta of preeclamptic women . maternal vasospasm leads to fetal hypoxia and accordingly it may lead to fetal distress and fetal death . present study has been undertaken to record the data on the morphology , morphometry , and histology of placenta from mothers with pih and correlate the findings with the birth weight of the new born babies . this study was done to find out the morbid changes of the placenta of hypertensive mothers in comparison to normotensive mothers . as placenta is the mirror of maternal and fetal status , it reflects the changes due to maternal hypertension . the study was done in the department of anatomy in collaboration with the department of obstetrics and gynaecology , s.v.s medical college , a tertiary care hospital , mahabubnagar , andhra pradesh , during the period from october 2009 to october 2011 . the study was done in 100 placentas , which were collected from obstetrics and gynecology department . out of the 100 placentas collected , another 50 placentas were collected from preeclamptic and eclamptic cases and served as study group . the cases were divided into three groups , namely , normal , preeclampsia , and eclampsia group . the placenta with attached membranes and umbilical cord was collected soon after delivery , washed in running tap water , labeled , and then fixed with 10% formalin for 4 - 6 weeks . the size , shape , weight , thickness at centre , number of cotyledons , and site of insertion of umbilical cord were noted down . the birth weights of newborn babies were documented and feto - placental weight ratio was calculated . histo - pathological study of placenta was done and the slides were studied under light microscope . the statistical significance between the means of the control group and study groups were analyzed by using students unpaired the placenta with attached membranes and umbilical cord was collected soon after delivery , washed in running tap water , labeled , and then fixed with 10% formalin for 4 - 6 weeks . the size , shape , weight , thickness at centre , number of cotyledons , and site of insertion of umbilical cord were noted down . the birth weights of newborn babies were documented and feto - placental weight ratio was calculated . histo - pathological study of placenta was done and the slides were studied under light microscope . the statistical significance between the means of the control group and study groups were analyzed by using students unpaired from the study , it was observed that the weight of the placenta was less in preeclampsia and eclampsia when compared with normal placenta . the majority of placenta were oval ( 73% ) followed by round placenta ( 27% ) [ table 1 ] . the insertion of the umbilical cord central in 18% of cases and eccentric in 82% of cases . the number of cotyledons varied from 18 to 23 in all groups . comparison of morphological features of placenta in different groups the mean neonatal birth weight was more in normal pregnancy ( 3.14 kg ) . it was less in preeclamptic and eclamptic cases ( < 2.5 kg ) . the feto - placental weight ratio was significantly higher in the hypertensive group than in the control group [ table 1 ] . histological study of placenta showed significant number of syncytial knots , fibrinoid necrosis , areas of calcification and hyalinisation and areas of medial coat proliferation of medium sized blood vessels in the hypertensive group , whereas the control group showed normal histological features . in the present study , the weights of the placenta in study groups were below 500 g. the least weight recorded being 200 g. in the control group , majority of the placenta weighed more than 500 g , the heaviest being 650 g. the observations correlate well with the previous studies done by various workers . reported five cases of toxemia with placental weight less than 300 g. nobis and das , in their study , observed that the placental weight in toxemic cases varies from 279 to 407 g. bhatia et al , in their study , have shown reduced placental weight in severe toxemia , the lowest recorded was 280 g. the change in placental weight observed in the present study between the control group and study groups were statistically significant [ table 1 ] . a significant increase in syncytial knot formation in placental villi indicates the disturbance in the hormonal factors , which may probably lead to altered blood flow . according to robertson , the cause of reduction in blood flow is due to vasculopathies of spiral arteries , which in turn causes reduction in the weight of placenta . it has been recorded that maternal utero - placental blood flow is decreased in preeclampsia because of maternal vasospasm . reduced maternal utero - placental blood flow indirectly leads to constriction of fetal stem arteries . the preeclamptic women will have a lower mean gestation , so the proportion of fetal capillaries will be lower . this relative increase in fetal capillary volume with decrease in proportion of connective tissue will lead to smaller parenchymal volume leading to decrease in placental weight . in the present study , the average diameter of placenta in control group was 18.62 cm , in preeclamptic cases it was 17.33 cm , and in case of eclamptic cases it was 16.24 cm . cibils reported that placenta from pih cases were smaller than normal indicating an underlying pathological process interfering with the normal growth of placenta . the majority of cases showed eccentric insertion ( 82% ) and few showed central insertion ( 18% ) in both the control and study groups . whereas in the earlier studies by nobis and das , the pattern of cord insertion was central in 44.19% , eccentric in 42.17% , and battledore in 1.26% . the neonatal weight was significantly less in study groups when compared with control group ( p < 0.01 ) . the average weight was 3.14 kg in normal pregnancies , 2.44 kg in preeclampsia , and 2.29 kg in eclampsia cases . an earlier study by palaskar had shown the reduced mean weight of the neonates in cases of pih . the arrangement of intracotyledon vasculature is altered in hypertension resulting in low birth weight of the babies . also the reduction in the villous population will interfere with fetal nutrition and growth , leading to decrease in neonatal weight . in the present study , the average feto - placental weight ratio in normal pregnancy was 5.72 0.93 , 6.35 2.05 in preeclamptic cases , and 6.44 2.02 in eclamptic cases . the values corelate with the earlier study by majumdar et al . in the study group , the histology revealed various structural changes such as significant number of syncytial knots , areas of fibrinoid necrosis , areas of medial coat proliferation of medium sized blood vessels , areas of calcification , and areas of hyalinization [ figure 1 ] . a significant increase in syncytial knot formation in placental villi indicates the disturbance in the hormonal factors , which may probably lead to altered morphometry of placenta resulting in pih in the mother and to low birth weight babies . microscopic findings of localized fibrinoid necrosis , medial coat proliferation of arteries , and hyalinization depict the mosaicism of placenta and probably the aftermath of hypertension . again the mosaicism of the placenta probably leads to placental insufficiency and ultimately to fetal growth retardation , thus creating a vicious cycle . pih significantly affects the placenta by reducing its weight and dimensions . however , it does not have any effect on placental shape , umbilical cord insertion , and number of cotyledons on maternal surface . it induces histological changes such as , areas of syncytial knot formation , fibrinoid necrosis , calcified areas , hyalinised areas , and areas of medial coat proliferation of the blood vessels . these changes compromise utero - placental blood flow and significantly reduce the neonatal birth weight .

background : hypertension is one of the most common complication during pregnancy . it contributes significantly to maternal and perinatal morbidity and mortality . this study was designed to investigate the morphological and histopathological changes in placenta from pregnancies complicated with hypertension.objectives:to study the morbid changes in placenta in cases of pregnancy - induced hypertension ( pih ) and to correlate the findings with birth weight of new born babies in comparison with normotensive mothers.materials and methods : the study was done on 100 placentas , out of which 50 were collected from normotensive mothers and the remaining 50 from pih cases . all the placentas were studied morphologically and histologically . the birth weight of neonates was recorded.results:in the present study it was observed that weight and dimensions of placenta was less in study group when compared with control group . the mean neonatal birth weight was more in normal pregnancy and feto - placental weight ratio was significantly high in hypertensive group . histopathological study showed significant number of syncitial knots , areas of fibrinoid necrosis , hyalinization , calcification , and medial coat proliferation of medium sized blood vessels in hypertensive group.conclusion:pih significantly affects the placenta by reducing its weight and dimensions . these changes may cause placental insufficiency as a result of compromised utero - placental blood flow . therefore has an adverse affect on the neonatal birth weight . pih has definite influence on morphology , histology of placenta , and thus affects the growth of the fetus .

INTRODUCTION MATERIALS AND METHODS Collection and examination of placenta RESULTS DISCUSSION CONCLUSION

PMC5096415

six- to 8-week - old male balb / c and c57bl/6 mice ( taconic farms , germantown , ny , usa ) were used in the experiments , and mice were anesthetized using a mixture of ketamine , xylazine , and acepromazine ( 50 mg , 10 mg , and 1 mg / kg body weight , respectively ) for each surgical procedure . all mice were treated according to the arvo statement for the use of animals in ophthalmic and vision research and all protocols were approved by the animal care and use committee , university of california , berkeley . orthotopic corneal transplantation was performed between fully mismatched c57bl/6 ( donors ) and balb / c ( recipients ) , as reported previously . basically , donor central cornea was marked with a 2-mm - diameter microcurette ( katena products , inc . , denville , nj , usa ) and excised with vannas scissors ( storz instruments co. , san dimas , ca , usa ) . the recipient graft bed was similarly prepared with a 1.5-mm - diameter microcurette and the donor button was secured in recipient bed with eight interrupted 11 - 0 nylon sutures ( arosurgical , newport beach , ca , usa ) . the recipient mice were randomized to receive subconjunctival injections of either hamster anti - mouse itga-9 antibody ( 6.4 g ; kindly provided by toshimitsu uede , md , phd , hokkaido university , hokkaido , japan ) or its isotype control hamster igg ( jackson immunoresearch , west grove , pa , usa ) , as reported previously . subconjunctival injection was performed twice a week on the day of transplantation and thereafter up to 8 weeks after the surgery . as described previously , after the transplantation surgery , all eyes were first examined on day 3 and corneal sutures were removed on day 7 . grafts were evaluated by ophthalmic slit - lamp biomicroscopy twice a week for 8 weeks according to the standard scheme . basically , the degree of graft opacification was graded between 0 ( clear and compact graft ) to 5 + ( maximal opacity with total obscuration of the anterior chamber ) . grafts with an opacity score of 2 + or higher after 3 weeks or an opacity score of 3 + or higher at 2 weeks were regarded as rejected . briefly , whole - mount full - thickness corneas were harvested at 8 weeks after transplantation and fixed in acetone for immunofluorescent staining . samples were sequentially incubated with purified rabbit anti - mouse lyve-1 ( abcam , cambridge , ma , usa ) antibody and goat anti - mouse itga-9 antibody ( r&d systems , minneapolis , mn , usa ) , which were visualized by fitc - conjugated donkey anti - rabbit and cy3-conjugated donkey anti - goat secondary antibodies ( jackson immunoresearch laboratories ) , respectively . samples were covered with vector shield mounting medium ( vector laboratories , burlingame , ca , usa ) and examined by an axioimager m1 epifluorescence deconvolution microscope with axiovision 4.8 software ( carl zeiss ag , gttingen , germany ) . briefly , for lg evaluation , lyve-1 vascular structures were analyzed by imagej software ( http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . the lymphatic invasion area was normalized to the total corneal area to obtain a percentage coverage score for each sample . the total corneal area was measured by outlining the innermost lymphatic vessels of the limbal arcade , and lymphatic invasion area was determined by tracing out the contours of the lyve-1 lymphatic network inside the cornea . additionally , the cornea was divided into four equal quadrants in reference to the vertical midline passing through the 6- and 12-o'clock positions , and the nasal and temporal quadrants were used for analysis of polarized lymphatic vessel distribution for each sample . luminal valves also were evaluated and focal itga-9/lyve-1 areas running along the length of the lyve-1 vessels were identified as valves and quantified for each sample . the percentage scores were obtained by normalizing to the means of control condition that were defined as being 100% . mann - whitney u test was used to evaluate the statistical significance of the difference between the groups . the association analysis was performed by the linear mixed model built with the r studio platform ( r studio inc . , all other statistical analysis was performed with prism software ( graphpad , la jolla , ca , usa ) ; six- to 8-week - old male balb / c and c57bl/6 mice ( taconic farms , germantown , ny , usa ) were used in the experiments , and mice were anesthetized using a mixture of ketamine , xylazine , and acepromazine ( 50 mg , 10 mg , and 1 mg / kg body weight , respectively ) for each surgical procedure . all mice were treated according to the arvo statement for the use of animals in ophthalmic and vision research and all protocols were approved by the animal care and use committee , university of california , berkeley . orthotopic corneal transplantation was performed between fully mismatched c57bl/6 ( donors ) and balb / c ( recipients ) , as reported previously . basically , donor central cornea was marked with a 2-mm - diameter microcurette ( katena products , inc . , denville , nj , usa ) and excised with vannas scissors ( storz instruments co. , san dimas , ca , usa ) . the recipient graft bed was similarly prepared with a 1.5-mm - diameter microcurette and the donor button was secured in recipient bed with eight interrupted 11 - 0 nylon sutures ( arosurgical , newport beach , ca , usa ) . the recipient mice were randomized to receive subconjunctival injections of either hamster anti - mouse itga-9 antibody ( 6.4 g ; kindly provided by toshimitsu uede , md , phd , hokkaido university , hokkaido , japan ) or its isotype control hamster igg ( jackson immunoresearch , west grove , pa , usa ) , as reported previously . subconjunctival injection was performed twice a week on the day of transplantation and thereafter up to 8 weeks after the surgery . as described previously , after the transplantation surgery , all eyes were first examined on day 3 and corneal sutures were removed on day 7 . grafts were evaluated by ophthalmic slit - lamp biomicroscopy twice a week for 8 weeks according to the standard scheme . basically , the degree of graft opacification was graded between 0 ( clear and compact graft ) to 5 + ( maximal opacity with total obscuration of the anterior chamber ) . grafts with an opacity score of 2 + or higher after 3 weeks or an opacity score of 3 + or higher at 2 weeks were regarded as rejected . briefly , whole - mount full - thickness corneas were harvested at 8 weeks after transplantation and fixed in acetone for immunofluorescent staining . samples were sequentially incubated with purified rabbit anti - mouse lyve-1 ( abcam , cambridge , ma , usa ) antibody and goat anti - mouse itga-9 antibody ( r&d systems , minneapolis , mn , usa ) , which were visualized by fitc - conjugated donkey anti - rabbit and cy3-conjugated donkey anti - goat secondary antibodies ( jackson immunoresearch laboratories ) , respectively . samples were covered with vector shield mounting medium ( vector laboratories , burlingame , ca , usa ) and examined by an axioimager m1 epifluorescence deconvolution microscope with axiovision 4.8 software ( carl zeiss ag , gttingen , germany ) . briefly , for lg evaluation , lyve-1 vascular structures were analyzed by imagej software ( http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . the lymphatic invasion area was normalized to the total corneal area to obtain a percentage coverage score for each sample . the total corneal area was measured by outlining the innermost lymphatic vessels of the limbal arcade , and lymphatic invasion area was determined by tracing out the contours of the lyve-1 lymphatic network inside the cornea . additionally , the cornea was divided into four equal quadrants in reference to the vertical midline passing through the 6- and 12-o'clock positions , and the nasal and temporal quadrants were used for analysis of polarized lymphatic vessel distribution for each sample . luminal valves also were evaluated and focal itga-9/lyve-1 areas running along the length of the lyve-1 vessels were identified as valves and quantified for each sample . the percentage scores were obtained by normalizing to the means of control condition that were defined as being 100% . mann - whitney u test was used to evaluate the statistical significance of the difference between the groups . the association analysis was performed by the linear mixed model built with the r studio platform ( r studio inc . , all other statistical analysis was performed with prism software ( graphpad , la jolla , ca , usa ) ; p < 0.05 was considered significant . we first studied the effect of itga-9 blockade on corneal lg and vg induced by transplantation . either itga-9 neutralizing body or isotype control was injected subconjunctivally twice a week starting from the surgery date . as demonstrated in figure 1a , following the treatment with the itga-9 blocking antibody , corneal lymphatic vessels contained significantly fewer valves compared with the control condition . summarized data from repetitive experiments are presented in figure 1b ( left ; p < 0.05 ) . however , this treatment had no effect on lg , as shown in figure 1b ( right ) . our further analysis on the ratio of valve quantity to lymphatic invasion area revealed a significant reduction in this parameter in the treated rather than the control condition ( figure 1c ; p < 0.05 ) . ( a ) representative whole - mount immunostaining images demonstrating significantly fewer valves in the itga-9 blocking antibody treated cornea in comparison with isotype control treated cornea . ( b ) comparative quantification on lymphatic valves and lymphatic vessel invasion area in control and treatment conditions . anti the experiment was repeated twice with seven mice in control and eight mice in treatment group . * p < 0.05 ; n.s . , not significant . ( c ) comparative quantification showing significant lower ratio of valves to lymphatic invasion area in response to anti the experiment was repeated twice with seven mice in control and eight mice in treatment group . previously , we reported that corneal lymphatic vessels observe a unique nasal dominant distribution pattern in inflammatory lg . to investigate whether this phenomenon also manifests in transplantation - associated lg and whether it is affected by the itga-9 treatment , we next investigated the effect of itga-9 blockade on the polarity of lg by comparing the nasal and temporal quadrants , as illustrated in figure 2a . our results showed that in both treatment and control groups , lymphatic vessels were more distributed at the nasal side , and itga-9 blockade had no effect on this polarity of corneal lg ( fig . our further association analysis using the linear mixed model also confirmed that the polarized distribution of lg was associated only with corneal regions but not with the anti itga-9 blockade . integrin alpha 9 blockade had no effect on polarized distribution of lymphatic vessels after corneal transplantation . ( b ) comparative quantification showing significantly greater lymphatic invasion area in the nasal rather than the temporal side in both control and treatment conditions . the experiment was repeated twice with seven mice in control and eight mice in treatment group . * p < 0.05 . to further evaluate the effect of itga-9 blockade on corneal graft survival , we examined the grafts in both treatment and control groups and evaluated their survival rate twice a week up to 8 weeks after the surgery . as shown in figure 3 , our results showed a significant promotion of graft survival by this treatment . although graft rejection in both the control and treatment groups started approximately 2.5 weeks after transplantation , a significantly higher percentage of the grafts survived in the treatment group by the end of the 8-week study , as analyzed by the kaplan - meier survival curves ( p < 0.05 ) . ( a ) representative images from slit - lamp examination of rejected and survived grafts in control and treatment conditions , respectively . ( b ) kaplan - meier survival curves showing significantly higher survival rate in the treatment group . * we first studied the effect of itga-9 blockade on corneal lg and vg induced by transplantation . either itga-9 neutralizing body or isotype control was injected subconjunctivally twice a week starting from the surgery date . as demonstrated in figure 1a , following the treatment with the itga-9 blocking antibody , corneal lymphatic vessels contained significantly fewer valves compared with the control condition . summarized data from repetitive experiments are presented in figure 1b ( left ; p < 0.05 ) . however , this treatment had no effect on lg , as shown in figure 1b ( right ) . our further analysis on the ratio of valve quantity to lymphatic invasion area revealed a significant reduction in this parameter in the treated rather than the control condition ( figure 1c ; p < 0.05 ) . ( a ) representative whole - mount immunostaining images demonstrating significantly fewer valves in the itga-9 blocking antibody treated cornea in comparison with isotype control treated cornea . ( b ) comparative quantification on lymphatic valves and lymphatic vessel invasion area in control and treatment conditions . anti the experiment was repeated twice with seven mice in control and eight mice in treatment group . * p < 0.05 ; n.s . , not significant . ( c ) comparative quantification showing significant lower ratio of valves to lymphatic invasion area in response to anti the experiment was repeated twice with seven mice in control and eight mice in treatment group . previously , we reported that corneal lymphatic vessels observe a unique nasal dominant distribution pattern in inflammatory lg . to investigate whether this phenomenon also manifests in transplantation - associated lg and whether it is affected by the itga-9 treatment , we next investigated the effect of itga-9 blockade on the polarity of lg by comparing the nasal and temporal quadrants , as illustrated in figure 2a . our results showed that in both treatment and control groups , lymphatic vessels were more distributed at the nasal side , and itga-9 blockade had no effect on this polarity of corneal lg ( fig . our further association analysis using the linear mixed model also confirmed that the polarized distribution of lg was associated only with corneal regions but not with the anti itga-9 blockade . integrin alpha 9 blockade had no effect on polarized distribution of lymphatic vessels after corneal transplantation . ( b ) comparative quantification showing significantly greater lymphatic invasion area in the nasal rather than the temporal side in both control and treatment conditions . the experiment was repeated twice with seven mice in control and eight mice in treatment group . to further evaluate the effect of itga-9 blockade on corneal graft survival , we examined the grafts in both treatment and control groups and evaluated their survival rate twice a week up to 8 weeks after the surgery . as shown in figure 3 , our results showed a significant promotion of graft survival by this treatment . although graft rejection in both the control and treatment groups started approximately 2.5 weeks after transplantation , a significantly higher percentage of the grafts survived in the treatment group by the end of the 8-week study , as analyzed by the kaplan - meier survival curves ( p < 0.05 ) . ( a ) representative images from slit - lamp examination of rejected and survived grafts in control and treatment conditions , respectively . ( b ) kaplan - meier survival curves showing significantly higher survival rate in the treatment group . in this study , we demonstrated for the first time that itga-9 is critically involved in corneal transplantation induced vg , and its molecular blockade can effectively suppress this process . we have also shown that this treatment strategy does not affect corneal lg or its polarity of nasal distribution . more importantly , we have offered the first evidence showing that by reducing the lymphatic valves but not the vessels themselves , we were able to achieve a higher rate of graft survival . our finding that itga-9 blockade suppressed lymphatic valve formation without disturbing the lymphatic vessels in transplantation is consistent with our previous report on a suture - induced inflammation model . this may be explained by the fact that itga-9 is more highly expressed on the valves than the vessel walls , as shown in supplementary figure 2 with a transplanted cornea . the disparity between lymphatic valves and vessels was also observed during development , in which a reduced number of lymphatic valves , but not vessels , were detected in itga-9 knockout mice . with the treatment regimen used in this study , we did not observe any obvious side effects . for future development of clinical application , it may be possible to achieve the therapeutic effects by using various formats of the antagonists against the itga-9 pathway , such as neutralizing antibodies or small molecules , which merits further investigation and is beyond the scope of this report . this finding indicates a compromise of the immune reflex arc in which the lymphatic pathway serves as the afferent arm . it also aligns well with a previous developmental report that itga-9 knockout mice died shortly after birth from bilateral chylothorax , in which lymphatic vessels were present but displayed compromised integrity . moreover , we have reported that lymphatic vessels are equipped with valves as they become mature and functional . therefore , by targeting on lymphatic valves , we may have interfered with the maturation process of the lymphatic vessels , rendering them dysfunctional . it would be interesting to check if this strategy also affects other indices of the immune responses involved in transplant rejection , such as delayed - type hypersensitivity , which warrants further investigation . in summary it also provides a novel therapeutic strategy to effectively interfere with this pathological process and to improve graft survival . as one of the favorite tools for lymphatic study in general , our results from the cornea may shed some light on the development of new itga-9based therapies to treat broader lymphatic and immune diseases in the body .

purposethe lymphatic pathway mediates transplant rejection . we recently reported that lymphatic vessels develop luminal valves in the cornea during lymphangiogenesis , and these valves express integrin alpha 9 ( itga-9 ) and play a critical role in directing lymph flow . in this study , we used an allogeneic corneal transplantation model to investigate whether itga-9 blockade could suppress valvulogenesis after transplantation , and how this effect would influence the outcomes of the transplants.methodsorthotopic corneal transplantation was performed between fully mismatched c57bl/6 ( donor ) and balb / c ( recipient ) mice . the recipients were randomized to receive subconjunctival injections of either itga-9 blocking antibody or isotype control twice a week for 8 weeks . corneal grafts were assessed in vivo by ophthalmic slit - lamp biomicroscopy and analyzed using kaplan - meier survival curves . additionally , whole - mount full - thickness corneas were evaluated ex vivo by immunofluorescent microscopy on both lymphatic vessels and valves.resultsantiitga-9 treatment suppressed lymphatic valvulogenesis after transplantation . our treatment did not affect lymphatic vessel formation or their nasal polarized distribution in the cornea . more importantly , itga-9 blockade led to a significant promotion of graft survival.conclusionslymphatic valvulogenesis is critically involved in transplant rejection . itga-9 targeting may offer a new and effective strategy to interfere with the immune responses and promote graft survival .

Methods Animals Corneal Transplantation Pharmaceutical Intervention In Vivo Assessment of Grafted Corneas Corneal Immunofluorescent Microscopy Lymphatic Vessel and Valve Quantification Statistical Analysis Results Effect of Itga-9 Blockade on Lymphatic Valvulogenesis After Corneal Transplantation Effect of Itga-9 Blockade on Nasal Dominant Distribution of Lymphatic Vessels Effect of Itga-9 Blockade on Corneal Graft Survival Discussion Supplementary Material

PMC3361252

cancer is a major public health concern in the united states with approximately 25% of total deaths attributed to cancer . the projected number of cancer deaths in 2010 alone was 1500 deaths per day , corresponding to a total of over 560,000 deaths . therefore , investigations into modifiable risk factors that may reduce the rates of cancer mortality continue to be of significant importance . physical activity has been hypothesized to protect against cancer through obesity reduction , improved insulin sensitivity and sex hormone profiles , and lowered inflammation . the world cancer research fund / american institute for cancer research expert panel report concluded that physical activity likely reduces the risk of some cancers and suggested an active lifestyle for protection against cancer progression and mortality . however , concrete recommendations for physical activity to prevent cancer mortality are lacking , in part , due to inconsistencies in the existing evidence , and due to varying methodology and differing definitions of physical activity used . furthermore , at this time associations with physical activity are better established for cancer incidence as compared to mortality [ 4 , 5 ] . a greater understanding of the protective effects of physical activity may enhance existing cancer control strategies and potentially reduce cancer mortality . the present study strengthens the evidence in the current literature by examining longitudinal associations between leisure - time physical activity ( ltpa ) in the third national health and nutrition examination survey ( nhanes iii : 19881994 ) linked to mortality data through 2006 . excess adiposity and aberrations in the insulin - glucose axis are hypothesized to promote carcinogenesis [ 6 , 7 ] and may offset the protective potential of physical activity . it is noteworthy that research in the area of cancer mortality has typically focused on a single risk factor . less attention has been devoted to the combined effects of body weight , insulin resistance ( ir ) , physical activity , and cancer mortality [ 811 ] , and the interrelationships of these risk factors warrant investigation . therefore the current study is designed to expand the physical activity - cancer hypothesis by assessing whether body mass index ( bmi ) and ir modify the relationships of physical activity and cancer mortality . existing data available from this national sample on body weight , laboratory values , self - reported physical activity data , and cancer mortality ( n = 863 ) provides a unique opportunity to conduct these analyses . the results of this study provide data to guide clinical trials that may ultimately contribute to individualized physical activity recommendations for cancer control . the nhanes iii ( 19881994 ) population , a national sample of civilian noninstitutionalized individuals , was selected through a complex , multistage probability design . persons who were 17 + or older were eligible for the mortality follow - up from the date of participation ( 19881994 ) through december 31 , 2006 . this represents the last nhanes iii mortality update . the current analyses included adults 2089 years . per the adult treatment panel ( atp ) definition , individuals were considered to be adults if they were at least 20 years old . nhanes participants 89 + years were assigned an arbitrary age of 90 years for confidentiality purposes and were excluded from the analyses . pregnant women were excluded because their baseline physical activity and bmi may not be an accurate reflection of their usual activity or body weight . additionally , persons with missing values for the pertinent variables were also excluded , resulting in a final sample of 15,535 individuals . the nhanes iii survey consisted of a structured household interview and a standardized physical examination in a mobile examination center at entry into the study . participants self - reported their age , race / ethnicity , level of education , leisure - time physical activity , dietary intakes , alcohol use , current prescription medication , and presence of doctor - diagnosed cancer in a personal interview . race / ethnicity were categorized into ( 1 ) non - hispanic whites , ( 2 ) non - hispanic blacks , ( 3 ) mexican - americans and ( 4 ) other . trained personnel measured height , weight and waist circumference during the in - person examination . the measured height and weight were used to compute bmi . smoking status was assessed during the in - person interview , in which participants reported the use of cigarettes , pipes , and cigars . a fasting blood sample was obtained during the physical examination that was used to measure fasting plasma glucose concentrations . a questionnaire consisting leisure - time physical activity questions including type of activity and frequency of the activity in the past month was queried in the household adult questionnaire administered once at baseline . there was no additional follow - up information on physical activity and other variables after time of entry into the study until the end of mortality follow - up ( figure 1 ) . at baseline , participants were asked for example , if in the past month , did you run or jog ? and in the past month , how often did you jog or run ? similarly , questions were asked on walking , bicycling , swimming , aerobics and/or aerobic dancing , other dancing , calisthenics , yard work , lifting weights , and engagement in up to four other activities that were not mentioned in the survey . frequency of engagement in activities over the past 30 days was reported . these responses were standardized to weekly frequency by using the conversion factors of 4.3 weeks / month and 30.4 days / month per nhanes iii . a validated intensity rating in metabolic equivalents ( met ) was provided by nhanes iii for each activity as defined in the compendium of physical activities . for example , jogging / running was assigned an intensity rating of 8 mets . for this study , low intensity , moderate and vigorous using the standard cut - offs established by pate et al . . low - intensity , activities requiring 36 mets were classified as moderate and > 6 mets were classified as vigorous . participants who did not report engagement in any of the activities , were classified as because individuals engaging in low intensity activities represented < 0.05% ( n = 7 ) of the population , they were combined into the moderate exercises if they reported performing the activity at least 5 times per week and regular vigorous if they reported performing vigorous activities at least 3 times per week . these categories were mutually exclusive ( so that if an individual performed vigorous and low - to - moderate activities , he / she was included in the vigorous activity group and not in both groups ) . additionally , a frequency - weighted ltpa variable was calculated by multiplying the frequency of each activity by its corresponding intensity value and summed . the nhanes iii survey did not collect information for duration of each bout of exercise , which limits more precise estimation of physical activity . the physical activity definitions used in this study are consistent with previously published studies using the nhanes iii population [ 1821 ] . mortality information was obtained from the updated nhanes iii linked mortality files that provide mortality follow - up data from the date of participation in the survey ( 19881994 ) through december 31 , 2006 ( figure 1 ) . mortality was ascertained based upon either death certificates or from a probabilistic match between nhanes iii and the national center for health statistics , national death index ( ndi ) records . cancer mortality was identified using the international classification of diseases , tenth revision ( icd-10 ; nchs 2006 ; icd-10 codes c00c97 ) . persons were considered alive at the end of the follow - up period if they were not matched to a death record . descriptive statistics were generated to assess demographic , lifestyle and dietary attributes related with cancer mortality , and to identify potential confounders . next , unadjusted univariate hazard ratio ( hr ) and 95% confidence intervals ( 95% ci ) of overall cancer mortality and level of ltpa were computed using cox proportional hazard models , using age as the time scale . persons who died from causes other than cancer were censored at the age of death and persons who were considered alive were censored at the end of the study follow - up period . we tested demographic , lifestyle , physiological , and dietary factors as potential confounders of the associations of ltpa and cancer mortality , by entering these additional variables singly into the cox proportional hazards models . if the addition of the variable singly in the model changed the hr for cancer mortality by 10% or more , the variable was added to the final regression model . potential variables tested were age ( continuous and years ) , race or ethnicity ( categorical : white , black , mexican , and other ) , cardiovascular disease ( categorical : yes / no ) , bmi ( categorical : normal weight , overweight and obese ) , smoking ( categorical : never , current , past ) , alcohol consumption ( continuous : number of drinks / day ) , education ( categorical : < 12 and 12 years ) , aspirin use ( yes / no ) total dietary proteins ( continuous : grams / day ) , total dietary fats ( continuous : grams / day ) total dietary carbohydrates ( continuous : grams / day ) , and total calories ( continuous : kilocalories / day ) . we tested for potential effect modification ( considered significant for the purpose of these analyses at alpha value of 0.10 or less ) by race , age , gender , bmi , and ir status for the relationships of physical activity and cancer mortality . a significance level of 0.05 sensitivity analyses were conducted by reanalyzing the associations after excluding persons with a baseline report of doctor - diagnosed cancer ( n = 588 ) . for exploratory purposes , we repeated analyses in adults 40 + years at baseline ( n = 9348 ) . the statistical analyses were performed using sas v.9.1 and sudaan v.10.0.1 sample weights provided by nhanes iii were applied to all the analyses . baseline characteristics of nhanes iii sample are expressed as either weighted frequencies for categorical variables or weighted means with their corresponding standard errors ( ses ) , and minimum and maximum values for continuous variables ( table 1 ) . the population consisted of 76.8% non - hispanic whites , 10.4% non - hispanic blacks , 5.2% mexican - americans , and 7.8% other ethnicities . approximately 85% of the participants reported engaging in any physical activity , with half ( 53.3% ) of the population being moderately and 16.9% being vigorously active on a regular basis . about 15% of the sample was inactive . furthermore , 33% of the participants reported being more active , 21% reported being less active , and 44% estimated about the same level of activity as compared to their peers of the same sex and similar age . over half the population was obese or overweight ( bmi > 25 ) , with a mean waist circumference of 92 cm . twenty - two percent of the population had the insulin resistance syndrome , defined per the atp iii criteria as having at least three of the following five criteria : abdominal obesity ( waist circumference > 102 cm in men or > 88 cm in women ) , insulin resistance ( blood glucose > 110 mg / dl ) , low high density lipoprotein ( hdl ) ( < 40 in men or < 50 in women ) , high serum triglycerides concentration ( > 150 mg / dl ) , and hypertension ( systolic blood pressure > 130 mm hg or diastolic blood pressure > 85 mm hg . ) and 10.67% were insulin resistant ( defined as blood glucose levels > 110 mg / dl ) . in this dataset 5.5% ( n = 863 ) of the population had died from cancer at the end of the follow - up period till 2006 . approximately 15% of the patients who had died of cancer had reported doctor - diagnosed cancer during their in - person baseline interview . overall , nonsignificant inverse associations were observed for overall cancer mortality among persons engaging in any activity , regular low - to - moderate or regular vigorous activity in the whole population ( table 2 ) . individuals who engaged in any activity were 8% and 5% less likely to die of cancer as compared to individuals who were inactive , after adjusting for age ( hr : 0.92 ; 95% ci : 0.711.19 ) and additional variables ( hr : 0.95 ; 95% ci : 0.721.26 ) , respectively . associations were in a similar direction for regular low - to - moderate activity ( hr : 0.92 ; 95% ci : 0.691.21 ) , regular vigorous activity ( hr : 0.66 ; 95% ci : 0.391.13 ) , and for the highest versus lowest quartile of frequency - weighted mets per week ( hr : 0.89 ; 95% ci : 0.681.16 ) after adjusting for age , race , sex , and smoking status , albeit not statistically significant . analyses were repeated after excluding self - reported history of cancer at baseline ( n = 588 ) to assess whether the observed nonsignificant associations persisted . the hrs among individuals with no history of cancer at baseline ranged from 0.82 to 1.09 for all levels of regular ltpa ; however , the hrs were not statistically significant after adjusting for age and additional covariates in the whole population and the conclusions remained unchanged ( table 2 ) . restricting analyses to adults 40 + years at baseline yielded similar results ( data not shown ) . previous research has reported that bmi and ir are associated with cancer mortality [ 6 , 23 , 24 ] . significant effect modification ( a priori considered significant if p < 0.1 ) by ir in the relationships of physical activity and overall cancer mortality were noted ( p = 0.07 ) . therefore , the analyses were stratified by ir status ( yes / no ) as shown in table 3 . among individuals who were not insulin resistant , significant 54% ( hr : 0.46 ; 95% ci : 0.240.87 ) and 49% ( hr : 0.51 ; 95% ci : 0.270.97 ) decreased risk were observed between engagement in regular vigorous activity and overall cancer mortality , after adjusting for age and additional variables , respectively . among individuals who were insulin resistant , an 82% increased risk of cancer mortality was observed ( hr : 1.82 ; 95% ci : 0.714.65 ) , albeit this finding was not statistically significant and therefore can not be considered definitive . these associations persisted when the relationships were reevaluated in a sample after excluding persons with a cancer diagnosis at baseline , in sensitivity analyses ( data not shown ) . although there was no significant effect modification by bmi ( p = 0.355 ) , for exploratory purposes only , the analyses were stratified by bmi < 25 ( normal weight ) and > 25 ( obese or overweight ) . among persons with a lower bmi , the observed inverse associations were more protective for any activity and overall cancer mortality , albeit non - significant after adjusting for age ( hr : 0.82 ; 95% ci : 0.551.22 ) and additional variables ( hr : 0.90 ; 95% ci : 0.611.35 ) as compared to individuals with a bmi > 25 ( ( age - adjusted hr : 1.02 ; 95% ci : 0.731.42 ) and ( adjusted for additional variables hr : 1.03 ; 95% ci : 0.731.46 ) ) . the hr were similarly < 1 but remained non - significant among persons with a normal bmi engaging in regular low - to - moderate or regular vigorous ltpa . the direction of the associations persisted when reevaluated after excluding persons with no cancer diagnosis at baseline ( data not shown ) . next , the frequency - weighted mets / week variable was divided into quartiles in the cox proportional regression models . quartile 1 was the referent group and represented participants with the lowest level of mets expended , and quartile 4 represented the individuals with the highest level of mets expended . although not significant , the hrs for quartiles 2 , 3 , and 4 were < 1 as compared to quartile 1 . the non - significant decreased risk for cancer mortality persisted after excluding persons with a cancer diagnosis at baseline ( data not shown ) . furthermore , hrs were similar when analyses were repeated among individuals who were 40 years or older at baseline . the current study was undertaken to elucidate the relationships between ltpa and cancer mortality in a nationally representative dataset and its linkage to mortality status ( 19882006 ) . the comprehensive data available in the nhanes iii sample on obesity , insulin resistance , and physical activity history provided a unique opportunity to evaluate the combined impact of these factors on longitudinally ascertained cancer mortality , an approach that has typically not been used in the previous studies . although the results of the current study are not statistically significant in the whole population , they are suggestive of protection against overall cancer mortality , specifically for vigorous activity ( hr < 1 ) . this study is similar to previous studies that have evaluated physical activity in relation to overall cancer mortality of site - specific cancer mortality . for example , a recent prospective cohort study among elder japanese adults noted that high levels of physical activity ( 5 or more days per week ) were associated with a 23% decreased risk of overall cancer mortality ( p for trend = 0.02 ) . previous studies have shown that physical activity protects against mortality from some common cancers including lung , prostate [ 10 , 26 ] , breast [ 2729 ] , and colon cancer . we observed a slight attenuation of the non - significant associations in the sensitivity analyses after excluding persons with self - reported cancer at baseline . this could perhaps be due to lifestyle changes among the cancer cases after diagnosis , or due to reverse causality . however , unavailability of follow - up measures of physical activity in the nhanes iii survey limits the complete understanding of this phenomenon . furthermore , the current study suggests that the protection afforded by ltpa may be realized in the absence of aberrations in the insulin - glucose axis . we note that the protective associations of ltpa and cancer mortality were more pronounced among non - insulin - resistant individuals who engaged in vigorous ltpa . several biological mechanisms may explain the potential protective role of physical activity against cancer mortality . insulin , an anabolic hormone , is hypothesized to promote cell proliferation , inhibit apoptosis , and support cancer progression [ 3032 ] . studies have demonstrated that the benefits of physical activity are apparent even in the absence of changes in body weight [ 2 , 35 ] . ir is associated with increased levels of sex hormones and increased levels of inflammation , that may affect cancer risk [ 35 , 37 ] . ltpa may also improve hormonal profiles [ 38 , 39 ] , reduce systemic inflammation , and ultimately delay cancer mortality . despite the potential biological mechanisms of physical activity in cancer biology , associations were not significant for all levels of ltpa in the whole population in this study . some inherent limitations of the nhanes iii survey design and assessment methods for physical activity could have contributed to measurement error and the quantitatively weak findings . it has been hypothesized that the time course of risk factors along the continuum of the cancer process is important and previous studies suggest that physical activity after the diagnosis of cancer influences cancer mortality [ 29 , 41 ] . changes in ltpa patterns over time and engagement in ltpa after cancer diagnosis during the follow - up period were not captured , potentially biasing the results towards the null . next , physical activity was self - reported and was therefore vulnerable to recall bias due to under- or overreporting their level of ltpa . further , the nhanes iii dataset was not well powered to investigate site - specific cancer mortality . lastly , the results of this study might be an underestimation of the associations , because the exposure data were collected in 19881994 and the prevalence of ir as well as the proportion of physically inactive persons have increased in the past two decades [ 4345 ] , emphasizing the importance of physical activity in the current context . in conclusion , this study uniquely utilizes a large , nationally representative sample of us adults and assesses the interrelationship between ltpa , obesity , and ir , two hypothesized risk factors of cancer mortality in the previous literature . the results suggest that the protective effects offered by physical activity against cancer mortality may be realized through the maintenance of normal metabolic function and may thereby serve a potential cancer control tool . if confirmed in additional epidemiologic studies , these findings may have important public health implications in the context of the high rates of insulin resistance and cancer mortality and a large proportion of persons with sedentary lifestyles in the usa . further research is also required to determine the critical periods of exposure to physical activity during the life course with exposure measures before and after cancer diagnosis in relation to cancer outcomes in prospective studies .

longitudinal associations between leisure - time physical activity ( ltpa ) and overall cancer mortality were evaluated within the third national health and nutrition examination survey ( nhanes iii ; 19882006 ; n = 15,535 ) . mortality status was ascertained using the national death index . self - reported ltpa was divided into inactive , regular low - to - moderate and vigorous activity . a frequency - weighted metabolic equivalents ( mets / week ) variable was also computed . hazard ratios ( hrs ) and 95% confidence intervals ( ci ) were calculated for overall cancer mortality in the whole sample , by body mass index categories and insulin resistance ( ir ) status . nonsignificant protective associations were observed for regular low - to - moderate and vigorous activity , and for the highest quartile of mets / week ( hrs range : 0.660.95 ) . individuals without ir engaging in regular vigorous activity had a 48% decreased risk of cancer mortality ( hr : 0.52 ; 95% ci : 0.280.98 ) in multivariate analyses . conversely , nonsignificant positive associations were observed in people with ir . in conclusion , regular vigorous activity may reduce risk of cancer mortality among persons with normal insulin - glucose metabolism in this national sample .

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

PMC4975324

the consequences of atherosclerosis and deaths from atherosclerotic cardiovascular disease in the closest future will replace , in the third world , the frequency of deaths caused by infections ( 1 ) . there are different hypothesis which try to explain the atherogenesis development : lipid hypothesis - proposed the first time by anitschkow in 1913 . the development of atherosclerosis is the result of the gradual accumulation of lipid in the arterial wall , which cause the atheroma characteristics . thrombogenic hypothesis - the atherosclerotic lesions grow with the gradual incorporation of thrombus on arterial wall . this theory is difficult to prove because plaque infiltration by immature blood vessels is common in advanced lesions , consequent hemmorhage and thrombosis also occur frequently and thrombus can appear directly as a result of atherosclerosis . the triggering event in these two hypotheses can be considered the endothelial dysfunction . in the lipid hypothesis because a defective endothelial cell barrier make simpler the lipid accumulation into the arterial intima layer , then the beginning of plaque development . in the thrombogenic theory the dysfunctional endothelium can promote the local platelet aggregation which will be enclosed into the arterial wall . ( 2 ) injury hypothesis - this hypothesis was revised few times leading to the following version , where the endothelial dysfunction from any cause , and not necessary mechanical injury , is very important in the atherosclerosis development . these agents which cause the injury are what today we consider atherosclerosis risk factors : hypertension , hyperlipidemia , cigarette smoking ( 3 , 4 ) . inflammation theory - not only the endothelium play a central role in the phatogenesis of atherosclerosis but also the inflammation ( 2 ) . ross removing the endothelial cells and using a lipid - rich diet , showed that atherosclerosis was developed . today , the endothelial cells are considered as a tissue or organ because of their autocrine , paracrine and endocrine activity . endothelium controls many processes : vascular tone , stimulates the smooth muscle cells ( smc ) , immunity response , monocyte s adhesion , platelet aggregation , nitric oxide ( no ) production . also the no plays the important functions : anti piastrinic activity , reduces the inflammatory cell recruitment into the intima layer preventing the gene expression involved in that process , as gene which encode for intercellular adhesion molecules-1 ( icam-1 ) or vascular cell adhesion molecules-1 ( vcam-1 ) , stimulates the smc ( 5,6,7 ) . in diabetic , hypercholesterolemic and hypertension states these reactive oxygen species interact with no , developing the peroxynitrite ( onoo- ) and subsequently powerfull free radicals , such as hydroxyl ( -oh ) and nitrogen dioxide ( no2 ) . the peroxynitrite can interact with lipoproteins such as ldl ( producing lipoperoxides ) which has different negative effects : cytotoxicity for endothelial cells , promotion of the adhesion of vascular inflammatory cells .ox - ldl are internalized by macrophages with consequent inflammation response and recruitment of lymphocytes in the inflammed area . so there is the production of foam cells , which are the signal for smcs and fibroblasts to realease connective tissue matrix . all these steps lead to plaque development ( 8,9,10 ) . with the growth of plaques and their damaging , the consequent endothelium damaging and thrombus macrophages recognize the ox - ldl by many types of scavenger receptors which are able to bind different kind of ligands . their expression is mediated by inflammation mediators , such as cytokines ( 11 , 12 ) . the inflammation response in the atherosclerotic area , initiates and maintains activation of overlying endothelial cells . the activated cells express different selectines , adhesion molecules ( am ) and chemokines which are proinflammatory cytokines responsible for migration , chemoattraction and activation of leukocytes . ( 9 ) the selectine molecules mediated the inflammatory cell recruitment on the atherosclerotic area . in the contrary to ldl , they are involved in the evolution of chylomicrons , vldl , and they have the ability to block the endothelial cell expression of adhesion molecules and are responsible for reverse cholesterol transport from the periferal tissues to the liver or steroidogenic tissues as adrenal glands or gonads ( 13 , 14 ) . different studies have demonstrated correlation between the levels of c - reactive protein ( crp ) and risk of plaque rupture ( 15,16,17,18 ) . other inflammatory markers as soluble icam-1(19 ) , vcam-1 ( 20 , 21 ) , p - selectin ( 22 ) , interleukin-6 have been shown the primary drivers to crp production ( 23 ) . the lp - pla2 , an enzyme that circulate in the blood can bind the ldl particles . the researchers analyzed data of 32 prospective studies , showing in altogether 79.036 patients that the high concentration of lp - pla2 was associated with an increased risk for coronary artery disease ( the risk was observed to be the same as for hypertension and hypercholesterolemia ) ( 24 ) . the selective inhibition of lp - pla2 has been proposed to reduce the progression of core necrosis and the clinical development of atherosclerosis ( 25 ) . asthma is a chronic inflammatory disease of the airways that involves a very complex cell interaction , mediators , cytokines and chemokines and is associated with variable airflow limitation , superimposed bronchospasm and increased airway responsiveness ( nih guidelines 1997 ) . however , chronic inflammation , associated with persistent symptoms , may contribute to airway remodeling that may not be completely reversible . asthma symptoms are often triggered by : environmental stimuli ( smoke , perfumes , dust mites , animals , fungi / molds , cold air ) and aggravating conditions ( viral upper respiratory infections or uris , rhinitis , sinusitis , gastroesophageal reflux , stress , exercise ) . such triggers may be more important for some asthma phenotypes than others ( 28 , 29 ) . national heart , lung , and blood institute ( nhlbi ) guidelines for the diagnosis and management of asthma , published in 2007(28 ) estimate that in u.s . the prevalence of asthma in children between 0 - 17 years of age was 7.5% in males and 5.5% in females ( 30 ) but , considering other age groups , asthma have higher prevalence in females ( 31 ) . for unknown reasons , asthma has dramatically increased in prevalence over the last several decades along with allergies . although about one half of people with asthma also have allergies , but not all people with allergies have asthma and the association between asthma and allergy , is not clear . multiple reports identified differences among some demographic groups by age , sex , race / ethnicity ( 32 , 33 ) . these reports indicate that population - based asthma prevalence rates , emergency department visit rates , and hospitalization rates were higher for black race patients than whites , higher for females than males , higher for children than adults , and higher for males aged 0 - 17 years age than for females with the same age ( 34 , 33 ) . asthma is a complex disease caused by the interaction of host and environmental factors at some critical period during the development of the immune system . all asthmatic children have a specific contributing factors , so even the therapy may be different ( 35 , 36 ) . if the child s mother has asthma , the child has higher probability to have asthma than if the child s father has asthma . if both parents have asthma , their child has a 50% probability to have asthma . different genes have been identified that may be important in the asthma , including ( adam)33 on chromosome 20p13 , which regulates , metalloproteinase , an enzyme which is involved in the airway smooth muscle cells ( 37 ) . a subgroup of lymphocytes , t helper ( th ) cells type 2 ( th2 ) are the major allergy - asthma controlling cells . th2 cells are responsible for the releasing of immune factors - interleukins , involved in the inflammatory response . some interleukins ( il-4 , il-13 ) are responsible for the first - phase of asthma attack by producing ige , which binding to mast cells allow to release leukotrienes that are responsible for the airway spasms and increased mucus production . other interleukins ( il-5 ) are responsible for the late - phase of asthma attack , where lead the eosinophil accumulation and release other immune factors . the asthma medications have this system as a target , but recently also the t killer cells have been connected with the asthma response and it may be a reason for why some patients does nt respond to the medications ( 28 ) . in contrast to th2 cells which are the asthma and allergy t cells , th1 cells are involved in the infection responses . with the increasing of th1 cell response the th2 cell response decreases . it is known that the th1 response increase with smaller use of antibiotics , increased exposure to other children , and exposure to certain infections , such as the common cold with a subsequent decrease in asthma frequency and a decreased th2 response . this has developed the hygiene hypothesis in which early exposure to allergens and infection may prevent allergic and asthmatic responses ( 38 , 28 ) . it is a non - specific response which the organism activate to tissue damage by exogenous stimuli ( physical : burns , trauma ; chemical : toxic substances ; biological : microorganisms , parasites ) or endogenous stimuli ( metabolic and/or immune disorders ) . it has a defensive purpouse , activates the immune system in the site where there is the biological intrusion and start the tissue reconstruction to repair the damage . interleukin-33 ( also known as il-1f11 ) is a recently found member of the interleukin-1 family ( 39 ) . this expression is upregulated by pro - inflammatory stimulation , thus contributing to the further amplification of inflammatory responses ( 40 ) . il-33 plays an important role in inflammatory diseases as hypersensitive diseases ( asthma ) , autoimmune diseases ( rheumatoid arthritis ) , cardiovascular diseases ( heart failure ) and neurodegenerative diseases ( alzheimer ) , interleukin-33 binds to st2l receptor which is a kind of toll - like receptor superfamily ( 39 ) . interleukin-33 complex consists of st2 receptor and il-1 receptor accessory protein ( il-1rap ) and mediates via tir domain of il-1rap ( 41 ) . interleukin-33 induces the production of many cytokines such as il-4 , il-13 and activates numerous of cells including th2 , basophils , mast cells . consistent with these observations , il-33 is a modulator of inflammation , mediating th2 immune responses . il-33 receptor has a heterodimeric structure which consists of st2 ; il-1 receptor accessory protein ( il-1rap ) mediates the response via tir domain of il-1rap . il-33 binds to this receptor activates the recruitment of myd88 , irak1 and irak4 to the receptor complex activating nf - kb , ikb and other various mapks . modified by authors from adipogen available on axxora.com interleukin-33 has been found to exert a pleiotropic activity atherogenic and cardioprotective in vivo ( 43 ) by inducing th1to - th2 switch and by stimulation the production of il-5 which increases the level of oxildl antibodies . the il-33/st2 complex plays a role similar to the b - type natriuretic peptide ( bnp ) by protecting the heart from harmful cardiomyocyte hypertrophy ( 44 ) . as previously mentioned asthma is a chronic inflammatory disease of the airways that involves a very complex cell interaction , mediators , cytokines and chemokines and is associated with variable airflow limitation , superimposed bronchospasm and increased airway responsiveness . interleukin 33 is a powerful inducer of th2 cells responsible for the releasing of immune factors involved in the inflammatory response . some studies have shown that il-33 concentrations are higher in asthmatics than in healthy subjects ( 45,46,47 ) . recently il-33 expression has been found in endobronchial biopsies with higher levels in asthmatic patients compared to controls , that was more evident in patients with severe asthma ( 46 ) . several studies have been conducted to understand which lung cells were more involved in response to il-33 . it was shown that both , endothelial and epithelial cells are important , but not smooth muscle cells or fibroblasts ( 49 ) . however , experiments conducted on mice by kurokawa et al , revealed that il-33 may contribute to the induction and maintenance of eosinophilic inflammation in the airways probably by action on lung fibroblasts ( 50 ) . the animal model studies have shown a functionally important role for il-33/st2 in asthma and allergic airways inflammation . in a murine ovalbumin - induced airway inflammation model , intranasal il-33 induced antigenspecific il-5 + t cells and promoted allergic airway disease even in the absence of il-4 . intranasal il-33 promoted also an increased airway responsiveness , polarization of macrophages towards an m2 phenotype , globlet cell hyperplasia , eosinophilia , lung accumulation of il-4 , il-5 , il-13(39 ) . more recently zhiguang et al created a transgenic mouse in which the il-33 expression was under the control of cmv ( 51 ) . the histological analysis showed a very high airway inflammation with eosinophils infiltration around bronchi and small blood vessels , hyperplasia of globlet cells and accumulation of mucus on pulmonary tissue . also an increased concentration of il-5 , il-8 , il-13 and ige was detected in bronchoalveolar lavage fluid ( 51 ) . in contrast , the research conduced by liu et al with anti - il-33 treatment ( given intraperitoneally ) , showed a significant decrease of the serum ige , the eosinophils and lymphocytes , il-4 , il-5 and il-13(52 ) . histological examination has shown a significant inhibition of allergen - induced - lung eosinophilic inflammation and mucus hipersecretion ( 52 ) . moreover , blockade with anti - st2 antibodies or st2-lg fusion protein , inhibits th2 cytokine production in vivo , eosinophilic pulmonary inflammation and airways hyper - responsiveness ( 53 ) . to better understand the role of il-33 and his receptor st2 in lung inflammation , different researcher s groups created a mice il-33-deficient . oboki et al demonstrated that 2 sensitizations of il-33 -/- mice with ovalbumin emulsified in alum showed lower lung lymphocyte and eosinophil recruitment , lower airway hyper - responsiveness and inflammation ( 39 ) . based on the model used , the disease can be attenuated by acting on il-33 or st2 , and the data obtained suggest that il-33 is involved in lung inflammation and st2 can be used as an asthma s therapy ( 39 , 52 , 53 , 54 , 55 ) . the mechanism of il-33 action is shown in fig.5 . with the tissue damaging , from infection , contact with allergens or inflammation , il-33 , during asthma , may stimulate the antigen sensitization and th2 cells mediated inflammation due to its ability to activate dendritic cells and to recruit and activate th2 cells . il-33 may induce eosinophilia and higher airway responsiveness by the activation of mast cells and il-13 , activate macrophages and cc - chemokine ligand-11 . nature rev immunol 2010 , 10 , 103 - 10 in a process of atherosclerosis monocytes , mast cells and t cells infiltrate plaques within the intima - media . it was suggested that il-33 may have a protective function during atherosclerosis process by inducing a switch from th1 to th2 . miller et al showed that il-33 administration to apoe-/- mice induced th2 cytokines and protective ox - ldl antibodies , which significantly reduced atherosclerotic plaque development in the aortic sinus ( 56 ) . also mclaren demonstrated on a mice model that il-33 significantly reduces macrophage foam cell formation in vitro in thp-1 macrophages and primary human monocyte derived macrophages ( hmdms ) by decreasing acetylated and oxidized ldl uptake , reducing intracellular total and esterified cholesterol content and by enhancing cholesterol efflux ( 57 ) . they found out that il-33 may have potential action in expression of genes involved in cholesterol esterification and triglyceride storage ( 57 ) . il-33 plays a role in patients with obesity and type 2 diabetes which is linked with atherogenesis ( 58 ) . recent investigations show the expression of il-33 and st 2 in adipocytes and adipose tissue . it was shown that treatment of in vitro cultured adipose tissue cells with il-33 induced production of th2 cytokines ( il-5 , il-13 , il-10 ) and reduced expression of adipogenic and metabolic genes ( c / ebp , srebp-1c , lxr , lxr , and ppar ) ( 39 , 58 ) . furthermore , treatment of genetically obese diabetic ( ob / ob ) mice with il-33 led to protective metabolic effects with reduced adiposity , reduced fasting glucose and improved glucose and insulin tolerance . additionally , mice lacking endogenous st2 and fed a high - fat diet had increased body weight and fat mass and impaired insulin secretion and glucose regulation compared to wt controls fed a high - fat diet ( 39 ) . the protective effects of il-33 on adipocytes appear to be mediated via an increased production of th2 cytokines and a switching of macrophage polarization from an m1 to m2 phenotype ( 44 , 64 ) . moro and yamada reported a new type of innate lymphocytes present in a novel lymphoid structure associated with adipose tissues in the peritoneal cavity ( 59 ) . these cells do not express lineage ( lin ) markers but do express c - kit , sca-1 ( also known as ly6a ) , il7r and il33r . similar lymphoid clusters were found in both , human and mouse mesentery , and this tissue was named falc lin(-)c - kit(+)sca-1(+ ) cells are distinct from lymphoid progenitors and lymphoid tissue inducer cells . these cells proliferate in response to il2 and produce large amounts of t(h)2 cytokines such as il5 , il6 and il13 . il5 and il6 regulate b - cell antibody production and self - renewal of b1 cells ( 59 ) . the consequences of atherosclerosis and deaths from atherosclerotic cardiovascular disease in the closest future will replace , in the third world , the frequency of deaths caused by infections ( 1 ) . there are different hypothesis which try to explain the atherogenesis development : lipid hypothesis - proposed the first time by anitschkow in 1913 . the development of atherosclerosis is the result of the gradual accumulation of lipid in the arterial wall , which cause the atheroma characteristics . thrombogenic hypothesis - the atherosclerotic lesions grow with the gradual incorporation of thrombus on arterial wall . this theory is difficult to prove because plaque infiltration by immature blood vessels is common in advanced lesions , consequent hemmorhage and thrombosis also occur frequently and thrombus can appear directly as a result of atherosclerosis . the triggering event in these two hypotheses can be considered the endothelial dysfunction . in the lipid hypothesis because a defective endothelial cell barrier make simpler the lipid accumulation into the arterial intima layer , then the beginning of plaque development . in the thrombogenic theory the dysfunctional endothelium can promote the local platelet aggregation which will be enclosed into the arterial wall . ( 2 ) injury hypothesis - this hypothesis was revised few times leading to the following version , where the endothelial dysfunction from any cause , and not necessary mechanical injury , is very important in the atherosclerosis development . these agents which cause the injury are what today we consider atherosclerosis risk factors : hypertension , hyperlipidemia , cigarette smoking ( 3 , 4 ) . inflammation theory - not only the endothelium play a central role in the phatogenesis of atherosclerosis but also the inflammation ( 2 ) . ross removing the endothelial cells and using a lipid - rich diet , showed that atherosclerosis was developed . today , the endothelial cells are considered as a tissue or organ because of their autocrine , paracrine and endocrine activity . endothelium controls many processes : vascular tone , stimulates the smooth muscle cells ( smc ) , immunity response , monocyte s adhesion , platelet aggregation , nitric oxide ( no ) production . also the no plays the important functions : anti piastrinic activity , reduces the inflammatory cell recruitment into the intima layer preventing the gene expression involved in that process , as gene which encode for intercellular adhesion molecules-1 ( icam-1 ) or vascular cell adhesion molecules-1 ( vcam-1 ) , stimulates the smc ( 5,6,7 ) . in diabetic , hypercholesterolemic and hypertension states these reactive oxygen species interact with no , developing the peroxynitrite ( onoo- ) and subsequently powerfull free radicals , such as hydroxyl ( -oh ) and nitrogen dioxide ( no2 ) . the peroxynitrite can interact with lipoproteins such as ldl ( producing lipoperoxides ) which has different negative effects : cytotoxicity for endothelial cells , promotion of the adhesion of vascular inflammatory cells .ox - ldl are internalized by macrophages with consequent inflammation response and recruitment of lymphocytes in the inflammed area . so there is the production of foam cells , which are the signal for smcs and fibroblasts to realease connective tissue matrix . all these steps lead to plaque development ( 8,9,10 ) . with the growth of plaques and their damaging , the consequent endothelium damaging and thrombus macrophages recognize the ox - ldl by many types of scavenger receptors which are able to bind different kind of ligands . their expression is mediated by inflammation mediators , such as cytokines ( 11 , 12 ) . the inflammation response in the atherosclerotic area , initiates and maintains activation of overlying endothelial cells . the activated cells express different selectines , adhesion molecules ( am ) and chemokines which are proinflammatory cytokines responsible for migration , chemoattraction and activation of leukocytes . ( 9 ) the selectine molecules mediated the inflammatory cell recruitment on the atherosclerotic area . in the contrary to ldl , they are involved in the evolution of chylomicrons , vldl , and they have the ability to block the endothelial cell expression of adhesion molecules and are responsible for reverse cholesterol transport from the periferal tissues to the liver or steroidogenic tissues as adrenal glands or gonads ( 13 , 14 ) . different studies have demonstrated correlation between the levels of c - reactive protein ( crp ) and risk of plaque rupture ( 15,16,17,18 ) . other inflammatory markers as soluble icam-1(19 ) , vcam-1 ( 20 , 21 ) , p - selectin ( 22 ) , interleukin-6 have been shown the primary drivers to crp production ( 23 ) . the lp - pla2 , an enzyme that circulate in the blood can bind the ldl particles . the researchers analyzed data of 32 prospective studies , showing in altogether 79.036 patients that the high concentration of lp - pla2 was associated with an increased risk for coronary artery disease ( the risk was observed to be the same as for hypertension and hypercholesterolemia ) ( 24 ) . the selective inhibition of lp - pla2 has been proposed to reduce the progression of core necrosis and the clinical development of atherosclerosis ( 25 ) . asthma is a chronic inflammatory disease of the airways that involves a very complex cell interaction , mediators , cytokines and chemokines and is associated with variable airflow limitation , superimposed bronchospasm and increased airway responsiveness ( nih guidelines 1997 ) . however , chronic inflammation , associated with persistent symptoms , may contribute to airway remodeling that may not be completely reversible . asthma symptoms are often triggered by : environmental stimuli ( smoke , perfumes , dust mites , animals , fungi / molds , cold air ) and aggravating conditions ( viral upper respiratory infections or uris , rhinitis , sinusitis , gastroesophageal reflux , stress , exercise ) . such triggers may be more important for some asthma phenotypes than others ( 28 , 29 ) . national heart , lung , and blood institute ( nhlbi ) guidelines for the diagnosis and management of asthma , published in 2007(28 ) estimate that in u.s . the prevalence of asthma in children between 0 - 17 years of age was 7.5% in males and 5.5% in females ( 30 ) but , considering other age groups , asthma have higher prevalence in females ( 31 ) . for unknown reasons , asthma has dramatically increased in prevalence over the last several decades along with allergies . although about one half of people with asthma also have allergies , but not all people with allergies have asthma and the association between asthma and allergy , is not clear . multiple reports identified differences among some demographic groups by age , sex , race / ethnicity ( 32 , 33 ) . these reports indicate that population - based asthma prevalence rates , emergency department visit rates , and hospitalization rates were higher for black race patients than whites , higher for females than males , higher for children than adults , and higher for males aged 0 - 17 years age than for females with the same age ( 34 , 33 ) . asthma is a complex disease caused by the interaction of host and environmental factors at some critical period during the development of the immune system . all asthmatic children have a specific contributing factors , so even the therapy may be different ( 35 , 36 ) . if the child s mother has asthma , the child has higher probability to have asthma than if the child s father has asthma . if both parents have asthma , their child has a 50% probability to have asthma . different genes have been identified that may be important in the asthma , including ( adam)33 on chromosome 20p13 , which regulates , metalloproteinase , an enzyme which is involved in the airway smooth muscle cells ( 37 ) . a subgroup of lymphocytes , t helper ( th ) cells type 2 ( th2 ) are the major allergy - asthma controlling cells . th2 cells are responsible for the releasing of immune factors - interleukins , involved in the inflammatory response . some interleukins ( il-4 , il-13 ) are responsible for the first - phase of asthma attack by producing ige , which binding to mast cells allow to release leukotrienes that are responsible for the airway spasms and increased mucus production . other interleukins ( il-5 ) are responsible for the late - phase of asthma attack , where lead the eosinophil accumulation and release other immune factors . the asthma medications have this system as a target , but recently also the t killer cells have been connected with the asthma response and it may be a reason for why some patients does nt respond to the medications ( 28 ) . in contrast to th2 cells which are the asthma and allergy t cells , th1 cells are involved in the infection responses . with the increasing of th1 cell response the th2 cell response decreases . it is known that the th1 response increase with smaller use of antibiotics , increased exposure to other children , and exposure to certain infections , such as the common cold with a subsequent decrease in asthma frequency and a decreased th2 response . this has developed the hygiene hypothesis in which early exposure to allergens and infection may prevent allergic and asthmatic responses ( 38 , 28 ) . it is a non - specific response which the organism activate to tissue damage by exogenous stimuli ( physical : burns , trauma ; chemical : toxic substances ; biological : microorganisms , parasites ) or endogenous stimuli ( metabolic and/or immune disorders ) . it has a defensive purpouse , activates the immune system in the site where there is the biological intrusion and start the tissue reconstruction to repair the damage . interleukin-33 ( also known as il-1f11 ) is a recently found member of the interleukin-1 family ( 39 ) . this expression is upregulated by pro - inflammatory stimulation , thus contributing to the further amplification of inflammatory responses ( 40 ) . il-33 plays an important role in inflammatory diseases as hypersensitive diseases ( asthma ) , autoimmune diseases ( rheumatoid arthritis ) , cardiovascular diseases ( heart failure ) and neurodegenerative diseases ( alzheimer ) , interleukin-33 binds to st2l receptor which is a kind of toll - like receptor superfamily ( 39 ) . interleukin-33 complex consists of st2 receptor and il-1 receptor accessory protein ( il-1rap ) and mediates via tir domain of il-1rap ( 41 ) . the st2 gene encodes two isoforms : transmembrane st2l and a soluble form sst2 . interleukin-33 induces the production of many cytokines such as il-4 , il-13 and activates numerous of cells including th2 , basophils , mast cells . consistent with these observations , il-33 is a modulator of inflammation , mediating th2 immune responses . il-33 receptor has a heterodimeric structure which consists of st2 ; il-1 receptor accessory protein ( il-1rap ) mediates the response via tir domain of il-1rap . il-33 binds to this receptor activates the recruitment of myd88 , irak1 and irak4 to the receptor complex activating nf - kb , ikb and other various mapks . modified by authors from adipogen available on axxora.com interleukin-33 has been found to exert a pleiotropic activity atherogenic and cardioprotective in vivo ( 43 ) by inducing th1to - th2 switch and by stimulation the production of il-5 which increases the level of oxildl antibodies . the il-33/st2 complex plays a role similar to the b - type natriuretic peptide ( bnp ) by protecting the heart from harmful cardiomyocyte hypertrophy ( 44 ) . as previously mentioned asthma is a chronic inflammatory disease of the airways that involves a very complex cell interaction , mediators , cytokines and chemokines and is associated with variable airflow limitation , superimposed bronchospasm and increased airway responsiveness . interleukin 33 is a powerful inducer of th2 cells responsible for the releasing of immune factors involved in the inflammatory response . some studies have shown that il-33 concentrations are higher in asthmatics than in healthy subjects ( 45,46,47 ) . recently il-33 expression has been found in endobronchial biopsies with higher levels in asthmatic patients compared to controls , that was more evident in patients with severe asthma ( 46 ) . several studies have been conducted to understand which lung cells were more involved in response to il-33 . it was shown that both , endothelial and epithelial cells are important , but not smooth muscle cells or fibroblasts ( 49 ) . however , experiments conducted on mice by kurokawa et al , revealed that il-33 may contribute to the induction and maintenance of eosinophilic inflammation in the airways probably by action on lung fibroblasts ( 50 ) . the animal model studies have shown a functionally important role for il-33/st2 in asthma and allergic airways inflammation . in a murine ovalbumin - induced airway inflammation model , intranasal il-33 induced antigenspecific il-5 + t cells and promoted allergic airway disease even in the absence of il-4 . intranasal il-33 promoted also an increased airway responsiveness , polarization of macrophages towards an m2 phenotype , globlet cell hyperplasia , eosinophilia , lung accumulation of il-4 , il-5 , il-13(39 ) . more recently zhiguang et al created a transgenic mouse in which the il-33 expression was under the control of cmv ( 51 ) . the histological analysis showed a very high airway inflammation with eosinophils infiltration around bronchi and small blood vessels , hyperplasia of globlet cells and accumulation of mucus on pulmonary tissue . also an increased concentration of il-5 , il-8 , il-13 and ige was detected in bronchoalveolar lavage fluid ( 51 ) . in contrast , the research conduced by liu et al with anti - il-33 treatment ( given intraperitoneally ) , showed a significant decrease of the serum ige , the eosinophils and lymphocytes , il-4 , il-5 and il-13(52 ) . histological examination has shown a significant inhibition of allergen - induced - lung eosinophilic inflammation and mucus hipersecretion ( 52 ) . moreover , blockade with anti - st2 antibodies or st2-lg fusion protein , inhibits th2 cytokine production in vivo , eosinophilic pulmonary inflammation and airways hyper - responsiveness ( 53 ) . to better understand the role of il-33 and his receptor st2 in lung inflammation , different researcher s groups created a mice il-33-deficient . oboki et al demonstrated that 2 sensitizations of il-33 -/- mice with ovalbumin emulsified in alum showed lower lung lymphocyte and eosinophil recruitment , lower airway hyper - responsiveness and inflammation ( 39 ) . based on the model used , the disease can be attenuated by acting on il-33 or st2 , and the data obtained suggest that il-33 is involved in lung inflammation and st2 can be used as an asthma s therapy ( 39 , 52 , 53 , 54 , 55 ) . the mechanism of il-33 action is shown in fig.5 . with the tissue damaging , from infection , contact with allergens or inflammation , il-33 , during asthma , may stimulate the antigen sensitization and th2 cells mediated inflammation due to its ability to activate dendritic cells and to recruit and activate th2 cells . il-33 may induce eosinophilia and higher airway responsiveness by the activation of mast cells and il-13 , activate macrophages and cc - chemokine ligand-11 . in a process of atherosclerosis monocytes , mast cells and t cells infiltrate plaques within the intima - media . it was suggested that il-33 may have a protective function during atherosclerosis process by inducing a switch from th1 to th2 . miller et al showed that il-33 administration to apoe-/- mice induced th2 cytokines and protective ox - ldl antibodies , which significantly reduced atherosclerotic plaque development in the aortic sinus ( 56 ) . also mclaren demonstrated on a mice model that il-33 significantly reduces macrophage foam cell formation in vitro in thp-1 macrophages and primary human monocyte derived macrophages ( hmdms ) by decreasing acetylated and oxidized ldl uptake , reducing intracellular total and esterified cholesterol content and by enhancing cholesterol efflux ( 57 ) . they found out that il-33 may have potential action in expression of genes involved in cholesterol esterification and triglyceride storage ( 57 ) . il-33 plays a role in patients with obesity and type 2 diabetes which is linked with atherogenesis ( 58 ) . recent investigations show the expression of il-33 and st 2 in adipocytes and adipose tissue . it was shown that treatment of in vitro cultured adipose tissue cells with il-33 induced production of th2 cytokines ( il-5 , il-13 , il-10 ) and reduced expression of adipogenic and metabolic genes ( c / ebp , srebp-1c , lxr , lxr , and ppar ) ( 39 , 58 ) . furthermore , treatment of genetically obese diabetic ( ob / ob ) mice with il-33 led to protective metabolic effects with reduced adiposity , reduced fasting glucose and improved glucose and insulin tolerance . additionally , mice lacking endogenous st2 and fed a high - fat diet had increased body weight and fat mass and impaired insulin secretion and glucose regulation compared to wt controls fed a high - fat diet ( 39 ) . the protective effects of il-33 on adipocytes appear to be mediated via an increased production of th2 cytokines and a switching of macrophage polarization from an m1 to m2 phenotype ( 44 , 64 ) . moro and yamada reported a new type of innate lymphocytes present in a novel lymphoid structure associated with adipose tissues in the peritoneal cavity ( 59 ) . these cells do not express lineage ( lin ) markers but do express c - kit , sca-1 ( also known as ly6a ) , il7r and il33r . similar lymphoid clusters were found in both , human and mouse mesentery , and this tissue was named falc lin(-)c - kit(+)sca-1(+ ) cells are distinct from lymphoid progenitors and lymphoid tissue inducer cells . these cells proliferate in response to il2 and produce large amounts of t(h)2 cytokines such as il5 , il6 and il13 . il5 and il6 regulate b - cell antibody production and self - renewal of b1 cells ( 59 ) . the studies on il-33 are still running and those already published emphasize the important role of il-33 in th2 cell mediated immunity . il-33 seems to have different protective effects on atherosclerosis , obesity , diabetes and also cardiac fibrosis . and that s not eveything ; st2 seems also to be an important biomarker to predict the mortality in presence of different cardiovascular disorders ( 41 ) . the manipulation of il-33/st2 pathway is a promising new therapeutic strategy in the treatment and prevention of many inflammatory disorders . however , it is important to emphasize that il-33 the exacts role of this cytokine needs to be further explored . in fact many questions about the biology of il-33 still need an answer , including the nuclear effect , the processing and releasing of this cytokine from the cells . the question is how can all the informations derived from in vitro studies and animal models be applied in clinical settings?(60 ) .

interleukin-33 ( il-33 ) is a newly found cytokine of the interleukin-1 ( il-1 ) family . it s mainly expressed by epithelial and endothelial cells . this expression is upregulated by pro - inflammatory stimulation , thus has an important role in inflammatory responses , such as hypersensitive diseases ( asthma ) , autoimmune diseases ( rheumatoid arthritis ) , cardiovascular diseases ( heart failure ) and neurodegenerative diseases ( alzheimer ) . several studies explored the complicated mechanism of il-33 action in asthma and atherosclerosis , as this il is significantly increased in these pathologies , and suggested its potential use in the therapeutic procedures .

INTRODUCTION Atherogenesis Asthma Inflammation Interleukin - 33 Role of IL-33/ST2 in asthma Role of IL-33 in atherosclerosis CONCLUDING REMARKS

PMC2842210

we applied principal components analysis ( pca)16,17 to identify outlier groups ( figure s1 ) . the first pc shows that the siddi have african ancestry , consistent with their origin involving the arab slave trade18 . the second shows that the nyshi and ao naga cluster with the chinese ( chb ) , consistent with them speaking tibeto - burman languages . the third and fourth show that the great andamanese are dispersed , suggesting gene flow from the mainland in the last few generations19 , but that the onge cluster tightly , making them more useful for studying the relationship of the indigenous andamanese to groups worldwide ( note s1 ) . the chenchu are a sixth outlier because of their high minimum fst of 0.052 from all other groups ( table s3 ) . this is much larger than the 0.0033 in a recent study of 23 european groups20 , although a strict comparison is difficult , since european studies have focused on cosmopolitan samples20,21 , which could underestimate differentiation relative to our village - centered sampling . we considered the possibility that the high fst could be an artifact due to marriage between close relatives , which is known to be common in southern india22 and can exaggerate measurements of frequency differentiation . however , when we recalculated fst correcting for consanguinity23 ( appendix ) , the average differentiation decreased only marginally to 0.0100 . we also determined that the high fst was not due to our strategy of sampling diverse groups . restricting to the 9 pairs of groups that were from the same state and traditional caste level , the average inbreeding - corrected fst was 0.0069 ; much higher than the analogous 0.0018 in europe when comparing within regions ( table s3 ) . we propose that the high fst among indian groups could be explained if many groups were founded by a few individuals , followed by limited gene flow8,24 . this hypothesis predicts that within groups , pairs of individuals will tend to have substantial stretches of the genome where they share at least one allele at each snp . we find signals of excess allele sharing in many groups ( figure s2 ) , which as expected tend to occur in the groups that have the highest fst s from all others ( p=0.002 for a correlation ) . to estimate the age of founder events , we measured the genetic distance scale over which allele sharing decays , a procedure that we verified by simulation ( figure s3 ) . six indo - european and dravidian speaking groups have evidence of founder events dating to more than 30 generations ago ( figure s2 ) , including the vysya at more than 100 generations ago . strong endogamy must have applied since then ( average gene flow less than 1 in 30 per generation ) to prevent the genetic signatures of founder events from being erased by gene flow . some historians have argued that caste in modern india is an invention of colonialism25 in the sense that it became more rigid under colonial rule26 . however , our results suggest that many current distinctions among groups are ancient and that strong endogamy must have shaped marriage patterns in india for thousands of years24,27 . the high frequency differentiation among indian groups is medically significant as it shows that population stratification ( systematic ancestry differences between cases and controls that can lead to false - positive disease associations ) may be a confounder in gene mapping studies . this is superficially at odds with a recent report that in indian americans , allele frequency differentiation is lower than among europeans11 . a potential explanation for the discrepancy is that the previous study pooled samples by state of origin , which can mask substructure . for example , when we performed pca on an independent set of 85 gujarati americans28 , we found that they separate into two distinct clusters with high differentiation ( fst = 0.005 ) ( figure s4 ) . similarly , pairs of uttar pradesh and andhra pradesh groups in our data ( excluding the outlying chenchu ) have an average fst of 0.0107 , but their differentiation decreases to 0.0033 when we first pool by state . it was recently suggested that to correct for stratification in india , it may be adequate to adjust for membership in five broad genetic clusters10 . however , our results show that many indian groups have a degree of allele frequency differentiation from their neighbors that is at least as large as that between northern and southern europeans , which is known to be sufficient to cause false positives associations if uncorrected29 . the widespread history of founder events in india is also medically significant because it predicts a high rate of recessive disease . in finland , there is a high rate of recessive diseases that has been shown to be due to a founder event , and that has resulted in a minimum fst of 0.005 with other our data show that many indian groups have a minimum fst with all other groups at least as large ( table 1 ) . haldane wrote 45 years ago that if inter - caste marriages in india become common , various however , it has not been generally appreciated that this applies to groups throughout india , and not only to groups in the south where consanguinity is common22 . we hypothesize that founder effects are responsible for an even higher burden of recessive diseases in india than consanguinity . to test this hypothesis , we used our data to estimate the probability that two alleles from a group share a common ancestor more recently than that group s divergence from other indians , and compared this to the probability that an individual s two alleles share an ancestor in the last few generations due to consanguinity23 . nine of the 15 indian groups for which we could make this assessment had a higher probability of recessive disease due to founder events than to consanguinity , including all the indo - european speaking groups ( table 2 ) . it is important to systematically survey indian groups to identify those with the strongest founder effects , and to prioritize them for studies to identify recessive diseases and map genes . an additional reason why some diseases are expected to occur at elevated frequencies in india is shared descent from a common indian ancestral population9,10 . an example is a 25 base pair deletion in mybpc3 that increases heart failure risk by about 7-fold , and occurs at around 4% throughout india but is nearly absent elsewhere31 . it has recently been shown that power to discover disease risk variants can be increased by modeling indian genetic variation using a reference panel of european and chinese chromosomes32 . however , the example of mybpc3 shows that this is an imperfect solution , since clinically significant alleles that are rare outside of india can not be imputed by studying non - indian genetic variation . it is important to specifically characterize indian variation to permit full powered gene mapping in india , instead of relying on catalogs of variation compiled in distantly related groups . to better understand the genetic ancestry that is only found in india , we carried out a pca of europeans ( ceu ) and chinese ( chb ) along with 22 indian groups ( figure 3 ) . the first pc distinguishes ceu from chb , and the second reflects ancestry that is unique to india31 . the most remarkable feature of the pca is a gradient of proximity to west eurasians ( figure s5 ) ( an analogous pca in europeans did not produce a gradient of proximity to india ; figure s6 ) . indian cline , and hypothesize that it reflects the fact that different indian groups have inherited different proportions of ancestry from ancestral north indians ( ani ) related to west eurasians , and ancestral south indians ( asi ) . to model ani - asi mixture , we selected a subset of 18 groups that formed tight clusters along the indian cline , and included the pathan and sindhi from pakistan14 since they were consistent with the indian cline in the pca but showed greater proximity to west eurasians ( note s2 ) , providing additional information about ani - asi mixture . to test whether any of the 18 indian cline groups is consistent with all ani or all asi ancestry , we applied a novel 3 population test ( methods ) . if group x is related to groups y and w by a simple tree ( through a history of divergence without subsequent mixture ) then if we define the snp allele frequencies as px , py , and pz , the quantity ( px - py)(px - pw ) averaged over snps , should be proportional to the variance in allele frequency since group x split from y and z and thus should be positive . however , this quantity can be negative if x descends from a mixture event ( note s3 and appendix ) . we applied this test to each of the 18 indian cline groups in turn using ceu = y and santhal = w , and obtained significantly negative scores for 16 groups ( table 2 ) as assessed by a jackknife analysis33 ( methods ) . these results do not mean that the indian groups descend from mixtures of european and austro - asiatic speakers , but only that they derive from at least two different groups that are ( distantly ) related to ceu and santhal . for any four groups there are 3 possible simple trees . if ( ( a , b),(c , d ) ) is correct , the allele frequency differences between a and b should be uncorrelated with those between c and d , which we can assess by averaging the quantity ( pa - pb)(pc - pd ) across snps ( appendix ) and testing for consistency with 0 ( methods ) . no indian cline group could be related simply to ceu , onge and west africans ( yri ) after testing all trees ( table s4 ) . we developed a model to study the historical relationship of indian groups to those worldwide , based on the hypothesis that most groups can be approximated as a mixture of two ancestral populations followed by group - specific drift . to fit the model to the data , we computed the squared allele frequency difference between all pairs of groups , and chose parameters by minimizing the difference between observation and expectation ( note s4 ) . the idea of fitting allele frequency differentiation to historical models was first explored by cavalli - sforza and edwards35 and here we extend it to trees with mixture . this approach contrasts with the structure algorithm , which fits data without a tree36 , or a tree in which many groups split simultaneously from an ancestral population followed by mixture37 . while structure is accurate for estimating individual mixture proportions in recently mixed groups , it is not clear whether its estimates of ancient mixture are biased because it does not model hierarchical relationships among groups , leading to inaccurate modeling frequencies in ancestral populations . by contrast , we use a more realistic tree model , and provide a test of fit . applying our model - fitting procedure , we find that the tree ( yri,(ceu , ani),(asi , onge ) ) ) provides an excellent fit to the data from indian groups . in particular , when the pathan , vaish , meghawal and bhil are modeled as mixtures of ani and asi ( figure 4 ) , the observed allele frequency differentiation statistics are all consistent with the theoretical expectation within three standard deviations ( note s4 ) . first , the ani and ceu form a clade , and further analysis shows that the adygei , a caucasian group , are an outgroup ( note s4 ) . many indian and european groups speak indo - european languages , while the adygei speak a northwest caucasian language . it is tempting to hypothesize that the population ancestral to ani and ceu spoke proto - indo - european , which has been reconstructed as ancestral to both sanskrit and european languages38 , although we can not be certain without a date for ani - asi mixture . second , our analysis shows that the onge form a clade with the asi ( note s4 ) , which we verified by running the 4 population test on ( ( yri , papuan)(dai , x ) ) , and finding that it is consistent when x = onge ( z=1.7 ) but inconsistent for all indian cline groups ( z-9 ) ( table s4 ) . previous mtdna analyses suggested that the onge do not share any maternal ancestry with groups outside india within the last ~48,000 years19,39 . while they do share ancestry with some rare haplogroups in some indian tribal populations within the last ~24,000 years 39,40 , this is consistent with our inferred onge - asi clade , as long as the gene flow predated the asi - ani mixture that later occurred on the mainland . while they provide an important framework for testing historical hypotheses , they are oversimplifications . for example , the true ancestral populations of india were probably not homogeneous as we assume in our model but instead were likely to have been formed by clusters of related groups that mixed at different times . however , modeling them as homogeneous fits the data and appears to capture meaningful features of history . estimating the proportions of ani and asi ancestry in india is challenging , since we are unaware of any published methods that produce unbiased estimates of mixture proportion in the absence of accurate ancestral groups . we developed three methods for estimating ancestry , which we verified were accurate even in the face of snp ascertainment bias and some inaccuracies in our phylogenetic model ( note s5 ) , and which we found provided consistent estimates ( table s5 ) . the 18 indian cline groups all have between 39% and 77% ani ancestry based on f3 ancestry estimates ( methods ) , which we quote because it has the smallest standard errors ( table 2 ) . ani ancestry is significantly higher in indo - european than dravidian speakers ( p=0.013 by a 1-sided test)5,6,7,8,41 , suggesting that the ancestral asi may have spoken a dravidian language before mixing with the ani42 . we also find significantly more ani ancestry in traditionally upper than lower or middle caste groups ( p=0.0025)5,6,7,8,41 , and find that traditional caste level is significantly correlated to ani ancestry even after controlling for language ( p=0.0048 ) , suggesting a relationship between the history of caste formation in india and ani - asi mixture . we compared our autosomal estimates of ani ancestry to y chromosome and mtdna haplogroup frequencies . y chromosome analysis has shown that traditionally upper caste and indo - european speaking groups have elevated frequencies of alleles that are also common in west eurasians5,6 . however , mtdna analysis shows elevated frequencies of haplogroups common in west eurasians only in northwest india7,8,43 . comparing the autosomal estimates of ani ancestry to the frequencies of haplogroups characteristic of west eurasians , we find a significant correlation on the y chromosome ( p=0.04 ) and a more marginal correlation in mtdna ( p=0.08 ) ( table s6 and figure s7 ) . the stronger gradient in males , replicating previous reports , could reflect either male gene flow from groups with more ani relatedness into ones with less , or female gene flow in the reverse direction . however , extensive female gene flow in india would be expected to homogenize ani ancestry on the autosomes just as in mtdna , which we do not observe . supporting the view of little female ani ancestry in india , kivisild et al.44 reported that mtdna u2i accounts for 77% of copies in india but ~0% in europe , and u2e accounts for 0% of all copies in india but ~10% in europe . the split is ~50,000 years old , indicating low female gene flow between europe and india since that time . we have documented a high level of population substructure in india , and have shown that the model of mixture between two ancestral populations asi and ani provides an excellent description of genetic variation in many indian groups . a priority for future work should be to estimate a date for the mixture , which may be possible by studying the length of stretches of ani ancestry in indian samples45,46 , and will shed light on the process leading to the present structure of indian groups . a second priority should be to discern the details of the history of the ani and asi before they mixed , including the date of their separation and their history of expansion and contraction ; this may be possible by analyzing allele frequency spectrum47 and linkage disequilibrium data45,48,49 . our findings finally have medical implications . by showing that a large proportion of indian groups descend from strong founder events , these results highlight the importance of identifying recessive diseases in these groups and mapping causal genes . blood samples were collected with informed consent from volunteers . we designate groups by their anthropological name as well as their geographic location , since it has been shown that both are required to specify an effectively endogamous group in india1 . we restricted most analyses to samples that appeared to be unrelated , and to 560,123 autosomal snps for which there was good genotyping completeness and for which there were no signs of problematic genotyping . for some analyses we also intersected our data with illumina 650y genotyping of the human genome diversity panel14 and hapmap13,28 , which produced a merged data set of 119,744 autosomal snps14 . we carried out pca using the eigensoft software17 , assessed allele frequency differentiation among groups using fst , assessed inbreeding in each group using wright s fixation index f23 , and computed standard errors using a block jackknife33 . to detect the signature of founder events in linkage disequilibrium data , we studied all possible pairs of samples for each group , and recorded whether they share 0 , 1 or 2 alleles at each snp ( at snps where both individuals were heterozygous , we recorded 1 allele to be shared to account for the ambiguity in the haplotype phase ) . long stretches of allele sharing can reflect regions that are shared identical by descent from a common founder , and by measuring the exponential decay of allele sharing with distance , we inferred the age of the founder event ( figure s3 ) . to test for a history of mixture , we applied 3 and 4 population tests ( note s3 ) . to infer the proportion of ancestry in each indian cline group in the absence of accurate ancestral populations

india has been underrepresented in genome - wide surveys of human variation . we analyze 25 diverse groups to provide strong evidence for two ancient populations , genetically divergent , that are ancestral to most indians today . one , the ancestral north indians ( ani ) , is genetically close to middle easterners , central asians , and europeans , while the other , the ancestral south indians ( asi ) , is as distinct from ani and east asians as they are from each other . by introducing methods that can estimate ancestry without accurate ancestral populations , we show that ani ancestry ranges from 39 - 71% in india , and is higher in traditionally upper caste and indo - european speakers . groups with only asi ancestry may no longer exist in mainland india . however , the andamanese are an asi - related group without ani ancestry , showing that the peopling of the islands must have occurred before ani - asi gene flow on the mainland . allele frequency differences between groups in india are larger than in europe , reflecting strong founder effects whose signatures have been maintained for thousands of years due to endogamy . we therefore predict that there will be an excess of recessive diseases in india , different in each group , which should be possible to screen and map genetically .

Extensive population structure in India Medical implications Population mixture in Indian history Relationship of Indians to non-Indians Estimates of mixture proportions in India Discussion Methods Summary Supplementary Material

PMC2896846

traditional cancer treatment modalities include surgery , radiation therapy , chemotherapy , and for some cancer types , hormone therapy . although these treatment modalities are life extending for many patients , they are rarely curative for disseminated cancers . the use of cancer vaccines to induce a therapeutic antitumor immune response in the patient has huge potential to complement traditional cancer therapies in a nonoverlapping way . therapeutic cancer vaccines are designed to recalibrate the existing host - tumor interaction , tipping the balance from tumor acceptance towards tumor control to the benefit of the cancer patient . additionally , the highly specific character of the host immune response minimizes the risk for unattractive adverse events associated with most other cancer therapies in use today . such vaccinations have been ongoing since early 1990s based on the long awaited characterization of human tumor antigens recognized by patient t cells . encouraged by sporadic successes , mainly in small phase i trials [ 3 , 4 ] , a high number of trials have been ongoing worldwide , however so far the clinical efficacy as demonstrated in large phase iii trials has in most cases be absent or marginal . recently , however , the us food and drug administration ( fda ) approved an autologous cellular vaccine ( provenge ) for the treatment of prostate cancer . the approval was based a clinical phase iii trial including 512 patients with asymptomatic or minimally symptomatic , metastatic prostate cancer and was shown to increase overall survival ( os ) by 4 months compared to placebo . initial attempts to improve vaccination induced biological and clinical responses focussed on the use of biological modifiers or adjuvants with the aim of increasing the magnitude of the response . to this end , immune stimulatory molecules such as interleukin-2 ( il-2 ) and granulocyte - macrophage colony - stimulating factor ( gm - csf ) have been used as adjuvants in many animal studies and vaccination trials . over the past years it has been realized that the immune response is in fact comprised not only by responder cells and molecules , but also cells and molecules responsible for a counter - response [ 5 , 6 ] . in turn this has led to intensified research into the cells and molecules involved with suppressing immune responses , and ways to inhibit or delete the counter response . in this regard , it has been realized that chemotherapy could be used to specifically target suppressor cells of the immune system or to reboot the immune system for induction of anti - cancer responses . moreover , chemotherapy could lead to immunogenic death of tumor cells , depending on the drug , dose , and administration . concerning the characterization of target epitopes recognized by t cells , the past decades has been characterized by an increased focus on proteins that functionally link to the malignant phenotype , for example , to metastasize and avoid apoptosis [ 1116 ] . importantly , some of the tumor associated antigens that comprise antigenic peptides are functionally related to resistance to conventional therapy , setting the stage for specifically targeting of cells that escape conventional therapy [ 1721 ] . last but not least , the tumor supportive role of cells in the stroma have encouraged the characterization of antigens which are expressed not alone by cancer cells but also or exclusively by cells in the tumor stroma [ 2125 ] . therapeutic strategies based on the above findings are likely to improve the clinical efficacy of therapeutic vaccinations against cancer . nonetheless , combinations of chemotherapy may be even with several drugs in combination with immunological strategies , are complex to evaluate , and demand for careful design of clinical trials as well as development of new ways to evaluate dose and schedule combinations rationally . the perspective , however , is future treatments that combine immunological treatment with conventional therapy leading to increased clinical efficacy to the benefit of the patient . even few years back , the idea of combining chemotherapy with active immune therapy was unheard of . the dogma stated that chemotherapy would jeopardize any immune responses , in part due to the fact that dividing immune cells are vulnerable to chemotherapeutic drugs , and as a consequence concurrent active immune therapy with chemotherapy would be pointless . nonetheless , the concept of combining immune therapy with sequential or even concurrent chemotherapy has lately gained much interest , for several reasons . first , a number of surprising findings suggested that combining immuno- and chemotherapy could lead to better responses in advanced cancer patients . to this end , data from several studies suggest that clinical response to chemotherapy is improved if preceded by immunotherapy [ 2831 ] . a possible explanation for this phenomenon is that cancer cell death by chemotherapy will lead to presentation of antigens that may further activate a treatment - induced t cell response leading to increased killing of cancer cells by t cells . obviously , not only cancer cells are inflicted by systemic chemotherapy but also cells of the tumor stroma and the immune system are influenced and as a consequence any improved reactivity may rely on effects imposed on the stromal cells , immune system , or on cancer cells and probably all may play a role . in general terms , the influence of chemotherapy may have an impact on normal cells of the host ; for example , cells of the immune system as well as an impact on cancer cells . previously , there has been tremendous focus on the use of molecules or cells that would work in direction of a more powerful activation of the immune system in any given immunotherapy , however , the recent acknowledgement that counter - active cells are at play during immune responses has led to a focus on suppressor mechanisms . there seem to be at least two cell types involved as key players in cancer , myeloid derived suppressor cells ( mdsc ) , and regulatory t cells ( treg ) . mdsc are suppressor cells of the innate immune system capable of inhibiting both innate and adaptive immune responses . similarly , regulatory t cells of the adaptive immune system are capable of suppressing immune responses [ 5 , 34 ] . both cell types may be found in high frequencies in cancer patients in blood but also at the tumor site , evidence is now emerging that both cell types are clinically relevant and predictive for patient survival [ 6 , 35 , 36 ] . obviously , these suppressive cells play important roles in controlling and adjusting immune responses in general and are not in any way restricted to cancer patients . hence , the existence of these populations of suppressive cells underscores the self - limiting nature of the immune system , characterized by dual actions , that is , the capacity to kill and destroy upon antigen recognition as well as the capacity to promote repair after antigen clearance , removal of dead cells and microbes , and the subsequent construction of new vessels and down expression of danger signals . this delicate balance is not only governed by presence or absence of antigen , but by a variety of cellular interactions and soluble factors . thus , any immune response is composed by active as well as counter - active cells , and the mechanisms that control the immune response from initiation to full completion and repair are poorly understood . it is now generally accepted that there is a link between inflammation and cancer in turn leading to the notion that a tumor represents a group of cells selected for the capacity to induce immunological repair instead of clearance . moreover , the intimate relation between inflammation and cancer underscores that the interplay between cells of the immune system and cancer cells are ongoing from initiation of oncogenesis and thus that suppressive or repair mechanisms are an intrinsic phenomenon of cancer . although data accumulating over the past few years have revealed much new insight into the counter - active cells of the immune system , we may just have seen the tip of the iceberg . we recently identified regulatory cd8 t cells present in high frequencies in cancer patients , and capable of suppressing cytokine production , proliferation , and cytotoxic activity of other t cells with a hitherto unheard efficacy . t cells are specific for a single peptide from the protein heme oxygenase 1 ( ho-1 ) . interestingly , this protein is expressed by tumor cells as well as during wound healing and is a key molecule in local immune suppression , thus coupling the action of the novel cd8 t - cell population with the overall function of the protein recognized . the characterization of these antigen - specific suppressive cd8 t cells in cancer patients underscores that there are pros and cons of the immune system in relation to the interactions between cells of the immune system and cancer cells . thus , treg , be it cd4 or cd8 cells seem to play a role for suppression of anticancer immune responses , but other cells in the tumor stroma as well as cancer cells themselves may also possess suppressive functions . normal cells in the stroma and a fraction of cancer cells may express indoleamine 2,3-dioxygenase ( ido ) [ 41 , 42 ] . ido may suppress t - cell responses directly by tryptophan deprivation in the microenvironment , and dendritic cells ( dcs ) may also express ido leading to induction of treg . in turn , treg may in fact induce ido expression in dc further linking the suppressive network . the drug 1-mt that inhibit ido activity is in clinical testing , and may represent a suitable conditioning or combination partner to immunotherapy . interestingly , we recently revealed that ido is a target for specific t cells in cancer patients , and that such specific cells are capable of killing ido - expressing tumor cells and also dc provided they express ido . thus , the induction of a response against ido could diminish the suppressive effect of ido and thereby unleash a powerful response against the full spectra of tumor antigens expressed by the cancer cells . strikingly , we have used 1-mt added to peptide stimulation cultures and shown that in some cultures there is a marked increase in inf- secretion measured by elispot , suggesting that pbmc cultures that comprise high frequencies of ido - positive cells can be suppressive to t cells a suppression that can be unleashed by 1-mt . steps to clear treg cells prior to vaccination by a single administration of chemotherapy , for example , cyclophosphamide ( ctx ) have been attempted and have been shown to increase induced immune responses in murine as well as human studies [ 48 , 49 ] . however , a single clearance does not appreciate the continuous development of treg , and other suppressive cells . continued administration of low dose ctx was recently shown to be able to selectively clear treg and would therefore be far more appealing . however , we recently conducted a clinical vaccination trial in melanoma patients using the described low dose ctx without being able to induce treg clearance or even decrease in treg frequency ( manuscript in preparation ) . also taxanes which are widely used in the clinic have been studied for impact on cells of the immune system in particular treg [ 51 , 52 ] . data from such studies suggest that both paclitaxel and docetaxel may impair the frequency of treg either alone or in combination with carboplatin or ctx . it could be speculated that the impact of chemotherapy on treg depends on the subtype of the regulatory cell . to this end , it has been shown that induced treg are rapidly dividing , and such cells could be more vulnerable to chemotherapy . concerning manipulations with mdsc this has been tested in murine models , where gemcitabine were shown to selectively eliminate gr1 positive mdsc . similarly , it has been found that unresponsive tumor infiltrating lymphocytes ( til ) of prostate cancer can gain responsiveness by blockage of arginase 1 ( arg ) known to be expressed by mdsc . cyclooxgenase 2 ( cox-2 ) seems to play a role for induction of mdsc , and administration of cox-2 inhibitors have been shown to decrease mdsc development [ 58 , 59 ] . we are currently running a clinical trial in melanoma patients in which dc vaccination is combined with concurrent administration of the cox-2 inhibitor celebra ( http://www.clinicaltrial.gov/ ) . whereas most attempts to manipulate suppressive cells like mdsc focus on lowering the frequency of the cells , a recent report demonstrated blockage of the suppressive function of mdsc by synthetic triterpenoid whereas the frequency of the cells was unchanged . a potential difficulty concerning selective clearing or blockage of function of mdsc using chemotherapy is that these cells seem to possess different phenotypes in different cancers , and have different ways of actions [ 61 , 62 ] which could be coupled to highly different properties of the cells in response to chemotherapeutic drugs . whether single individual drugs may synergistically work with , for example , therapeutic vaccination by selective clearing or decreasing the activity of cells with suppressive function remains so far elusive , and certainly we are by no means close to having defined optimal dosage , schedule , and so forth , for such combinations . moreover , many cellular approaches are based on the use of allogeneic tumor cell lines transfected with vectors encoding immune stimulatory molecules , which obviously leads to a next level of complexity since the beneficial combination with chemotherapy may depend on the added stimulatory molecule . to this end , two recent studies in which allogeneic , gm - csf - secreting tumor vaccines were administered to cancer patients in combination with ctx ( and doxorubicin ) suggested higher efficacy in the ctx group [ 26 , 63 ] . these highly complex combinations will benefit from designs that are geared to identify the most clinically relevant combination of the interacting drugs . as mentioned , it is still questionable whether selective clearing of immune cells with suppressive function is possible using chemotherapy ; however , chemotherapy could also be beneficially used to reboot the immune system prior to initiation of immune therapy against cancer . for example , the efficacy of act using in vitro expanded til in melanoma patients seems to depend on prior conditioning using chemotherapy and/or whole body irradiation therapy . the biological background for the requirement of conditioning remains unknown but is probably related to creating space thereby enabling homeostatic cell division of transferred cells , and clearing of suppressive cells in the patient . detailed insight into the mechanisms that influence the success of til in act could potentially open the avenue for development of an act regimen that are associated with fewer and less serious side effect . the induction of clinically relevant responses by act using lympho depleting procedures and til transfer could also in part be related to the broad spectra of reactivity comprised in these til cultures . in accordance , we have studied such til cultures from melanoma and head and neck cancer patients for reactivity against a panel of tumor antigens using elispot and tetramer analyses , and in general many of these cultures comprise a large number of t - cell specificities ( paper in preparation ) . t - cell survival and expansion within the host depends on the availability of growth - promoting cytokines and regular encounters with cognate antigen . under ideal conditions small numbers of infused t cells however , this level of in vivo expansion may not occur following the adoptive transfer of tumor antigen - specific t cells owing to the poor immunogenicity of the tumor . hence , an alternative approach is to combine adoptive transfer of t cells with vaccination to facilitate expansion and maintenance of t cells . the combination of act of either in vitro expanded specific t cells or gene - modified t cells with vaccination might provide synergism between the two treatment regimens . chemotherapy may also influence cells of the immune system to more potent activity . to this end , it has been shown that gemcitabine treatment lead to increased efficacy of immune therapy in the absence of any direct effect on cancer cells . as mentioned above ctx may have a role in the clearing of treg and is used together with fludarabin as conditioning prior to act . using ctx alone , it was recently shown in a murine model that the myelosuppressive action may lead to subsequent rebound dc generation with increased capacity to secrete il-12 . with a more direct action on dc , vinblastine has been shown to induce dc maturation , and functionally these dcs were superior to untreated dc in inducing cd8 t - cell responses . using non - cytotoxic concentrations , it was recently shown that several clinically relevant drugs ( paclitaxel , doxorubicin , mitomycin c , and methotrexate ) increase antigen presentation in an autocrine il-12-dependent manner . thus , these data suggest that provided careful examination of the dose and schedule , it might be possible to harness the dual actions of chemotherapy to kill some cells and activate others . chemotherapy may also induce an antitumor immune response by direct influence on the tumor cells . to this end , a panel of chemotherapeutic agents was screened for inducing immunological cell death , each drug was studied functionally for the ability of chemotherapy - killed tumor cells to induce protective immunity upon immunization . the data demonstrated that anthracyclin - treated tumor cells are particularly effective in eliciting an anticancer immune response , and further that the mechanism of the immunogenicity of antracyclin - induced cell death was the rapid preapoptotic translocation of calreticulin to the cell surface . this surface exposure of calreticulin endows cancer cells with an eat me signal to dendritic cells , in turn leading to immunogenic uptake of tumor antigens and activation of tumor - specific t - cell responses . thus , to successfully combine chemotherapy with immune therapy careful selection of the chemotherapeutic agent is required since not all agents induce immunological death . another key denominator of immunogenic cell death is represented by high - mobility group box 1 ( hmgb-1 ) . release of hmgb-1 from dying tumor cells leads to activation of toll - like receptors ( tlrs ) ( 2 and 4 ) and subsequent immune activation . in this respect , hmgb-1 localization in the cytosol is associated with autophagy and cellular escape from apoptosis , in turn conferring resistance to several therapies including immunotherapy . also heat shock proteins ( hsps ) which are upregulated upon specific stress may act as danger signals and be expressed on the cell surface as eat me signals to dc . interestingly , it has been shown that treatment of myeloma cells with the proteasome inhibitor bortezomib leads to surface expression of hsp90 on the cell surface . a vaccination trial in which bortezomib is combined with peptide vaccination targeting the regulators of apoptosis proteins ( raps ) bcl-2 , mcl-1 , and bcl - xl beyond inducing immunogenic tumor cell death , chemotherapy may render cancer cells more susceptible to killing by ctl . thus , 5-fluorouracil ( 5-fu ) , cpt-11 , or cisplatin ( cddp ) were all shown to increase the sensibility of the sw480 colon cancer cell line to killing by t cells , and similar data have been found for renal cell carcinoma cells treated with adriamycin . in both cases the enhanced lytic sensitivity was at least in part due to upregulation of costimulatory molecules on cancer cells , for example , lfa-3 and icam-1 . more recently , ramakrishnan and colleagues showed in a murine model that paclitaxel , cisplatin , and doxorubicin all sensitize tumor cells to more efficient killing by ctl . in this system , however , the responsible mechanism on murine as well and human cancer cells went via upregulation of the mannose-6-phosphate ( m6p ) receptor on cancer cells which is required for granzyme b associated killing . importantly , ctl established upon administration of chemotherapy was capable of off - target killing of neighboring cells at the tumor site even in the absence of antigen expression . the concept of using chemotherapy as an immune adjuvant is rapidly expanding . obviously , future testing in clinical trials will benefit from basic research into the mechanism(s ) of action ; how do the cells die and which pathways are activated for immune activation [ 82 , 83 ] . this will set the stage for initiation of hypothesis driven clinical trials combining chemotherapy and immune therapy and form the basis for more rational biological monitoring . chemotherapeutic agents can induce a series of cellular responses that impact on tumor cell proliferation and survival . perhaps the best studied of these cellular responses is apoptosis , a physiological cell death program that controls normal cell numbers during development and disease . a large number of various drugs in clinical use kill tumor cells by activation of common apoptotic pathways . hence , most cytotoxic anticancer drugs , for example , microtubule binding drugs , dna - damaging agents , and nucleosides induce apoptosis of malignant cells . many drugs are capable of inducing clinical response in the patients with metastatic cancer , however , in most cases the therapy is not curative due to selection of drug - resistant cancer cells . a frustrating property of such acquired resistance of cancer cells is that chemotherapy resistance may lead to cross - resistance to other drugs with different mechanisms of action . drug resistance is a major limiting factor for the effectiveness of chemotherapy in the treatment of disseminated cancer . cancer - associated defects in apoptosis play a vital role in resistance to chemotherapy and radiotherapy . an important reason for this impaired apoptosis is overexpression of rap , that is , the t - cell antigens survivin and proteins of the bcl-2 family . other mechanisms of drug resistance are exemplified by the tumor antigen cyp1b1 which may inactivate cytotoxic or cytostatic drugs thereby influencing the clinical outcome of therapy , and atp transporters which act by transporting drugs out of the cell . the mechanisms of drug resistance mentioned above are associated with expression of proteins that have been shown to be targets for t cell responses [ 1721 , 9095 ] . consequently , the combination of immunotherapy targeting these antigens with conventional chemotherapy appears to be particularly appealing . in such a setting , conventional therapy would kill the majority of the cancer cells , leaving only cells that express high levels of target antigens . such high - expressers would be particularly vulnerable to killing by vaccination - induced t cells . thus , the synergy of these measures could potentially give a more effective treatment than the added effect of either regimen alone , thereby strengthening the already described synergistic effect of anticancer vaccines and chemotherapy . furthermore , it should be noted that chemotherapy only has an effect on dividing cells , whereas , for example , surviving - specific t cells in addition are able to kill resting tumor cells . moreover , also sublethal irradiation may render cancer cells more vulnerable to killing by t cells , implying that even surviving irradiated cancer cells may still facilitate more efficient responses . total body irradiation ( tbi ) is already being used by dudley and colleagues as part of a conditioning regimen prior to act , indicating additive with more intensified tbi . interestingly , a well - known phenomenon related to local radiation therapy is a bystander effect extending to distant untreated metastatic sites an effect potentially mediated by the immune system . one of the inherent properties of cancer cells is genetic instability which in turn allows cancer cells to escape during therapy . obviously , escape is in fact a selection process in which cells carrying an advantageous genetic , epi - genetic , and mirna signature are given a survival and/or growth advantage . as already mentioned , this is a crucial problem concerning chemotherapy , but also for the success of immune therapy this poses a problem . in this regard , antigen loss or hla loss has been described during immune therapy , and the background for this lies in the heterogeneity of cancer cells in turn enabling the presence of cells with escape properties . conversely , stroma cells are genetically stable and possess limited proliferative capacity compared to cancer cells , implying that the risk of hla - loss , antigen - loss , or antigen processing - loss is exceedingly low . some antigens are expressed not only by tumor cells but also by cells in tumor stroma . angiogenesis represents an important step in tumor development , and since antiangiogenic therapy targets the tumor vasculature and prevents tumor growth beyond micro - metastases , combination of antiangiogenic therapy and tumor - specific immunotherapy could lead to a synergistic effect . active immunotherapy targeting endothelial products like vascular endothelial growth factor receptor ( vegfr)-2 protein can delay tumor progression , and it has been shown that vaccination with peptides derived from vegfr-1 inhibits tumor growth in mice . in addition , similar to survivin , bcl-2 and mcl-1 are highly expressed in endothelial cells during tumor angiogenesis . thus , the targeting of survivin , bcl-2 , or mcl-1 in a vaccination setting would beyond tumor cells also target endothelial cells and thus tumor - angiogenesis . indeed this was shown in a mouse model ; vaccination - induced survivin - specific t cells mediated the eradication of lung tumor metastases and the concurrent suppression of angiogenesis at the tumor site . the efficacy of treatment did not inflict on wound healing or fertility of the mice . a primary concern of immunizing against angiogenesis - associated proteins is a potential risk of interference with normal angiogenesis , especially if the effect is sustained . so far no vaccination associated toxicity was observed when late stage melanoma patients were vaccinated with survivin in a compassionate use setting despite the fact that strong ctl responses were introduced in all patients . nevertheless , it is clear that even extensive phase i / ii trials are not geared for analyses of potential side effects presenting several years after termination of the trial , and these issues demand further attention . certainly several targets are available for targeting of tumor stroma in particular for concurrent targeting of tumor cells and stroma cells . as already mentioned , ido are expressed by fractions of cancer cells in vivo , but dcs , mdsc , and other immune cells in the tumor stroma may also be ido positive , and characterized by the same targeting advantages as , for example , endothelial cells . to this end , it has been shown that regulatory t cells can be targeted by immune responses to peptides derived from foxp3 . many current immunological strategies to combat cancer are already focusing on combining different cells and molecules to increase responses , for example , vaccination combined with the addition of cytokines or other immune modulating agents . however , so far most peptide - based vaccination trials have targeted only a single antigen and with the aim to increase efficacy an exciting strategy would be to cotarget biologically connected proteins , for example , rap , in a multiepitope setting . a number of different rap have been described as t - cell antigens in a variety of cancers [ 21 , 90 , 105 , 106 ] . since coexpression of bcl-2 family proteins is a frequent event , simultaneous targeting of bcl-2 proteins may be more efficient than targeting one molecule alone . importantly , drug resistance is casually linked to over expression of rap , for example , survivin and bcl-2 [ 107 , 108 ] . for combination therapies , vaccination against these molecules seems to be ideally suited for targeting of chemoresistant cancer cells upon conventional therapy , and thereby possibly prevents relapse of disease . underscoring this notion , the coexpression of survivin and bcl-2 is associated with poor prognosis in breast cancer , implying that expression of more than one rap is functionally significant at least in some indications . to this end , targeting both the bcl-2 family proteins and survivin would be particularly attractive since they act in different apoptosis pathways , and the targeting of both to kill resistant cells upon chemotherapy would supposedly increase the chances of success . combined with adjuvant chemotherapy after primary surgery , adjuvant vaccination in high risk cancer patients would be suited for a vaccination that specifically targets not only apoptosis pathways but also proteins associated with metastatic behavior , for example , rhoc [ 16 , 111 ] or heparanase . the immune system has a unique capacity to specifically recognize and kill cancer cells while leaving normal cells unharmed . as a consequence , to harness the immune system in therapeutic vaccinations against cancer is a very promising approach for the therapy of disseminated cancer . so far , however , the clinical impact of vaccination has been limited . over the past few years , new insight has been achieved concerning the main suppressive mechanisms that hamper induction of more powerful immune responses , and also revealed new knowledge as to how suppressive cells and molecules could potentially be cleared or inhibited , for example , in combination with chemotherapy . similarly , the immunogenicity of cancer cells has been shown to depend on the death process induced by the specific drug , thus some drugs are more prone to act synergistically with vaccination than others . added to the above , cancer cells treated with chemotherapy may be rendered more vulnerable to killing by t cells , only adding to the potential of improving the efficacy of vaccination when combined with chemotherapy . moreover , antigens may be selected and combined to target various traits of cancer cells and/or target stroma cells in the suppressive tumor environment . the available experimental data should form the basis for initiation of carefully planned hypothesis driven clinical trials that coupled with stringent and robust biological and clinical monitoring will be able to firmly demonstrate the most effective combinations .

the clinical efficacy of most therapeutic vaccines against cancer has not yet met its promise . data are emerging that strongly support the notion that combining immunotherapy with conventional therapies , for example , radiation and chemotherapy may improve efficacy . in particular combination with chemotherapy may lead to improved clinical efficacy by clearing suppressor cells , reboot of the immune system , by rendering tumor cells more susceptible to immune mediated killing , or by activation of cells of the immune system . in addition , a range of tumor antigens have been characterized to allow targeting of proteins coupled to intrinsic properties of cancer cells . for example , proteins associated with drug resistance can be targeted , and form ideal target structures for use in combination with chemotherapy for killing of surviving drug resistant cancer cells . proteins associated with the malignant phenotype can be targeted to specifically target cancer cells , but proteins targeted by immunotherapy may also simultaneously target cancer cells as well as suppressive cells in the tumor stroma .

1. Introduction 2. Combination with Chemotherapy Immunological Reboot, and Immunological Cell Death 3. Chemotherapy Resistance and Immune Targeting 4. Radiation Therapy in Combination with Vaccination 5. Specific or Concurrent Targeting of Stroma Cells 6. Multiepitope Strategies and Additional Immunotherapy 7. Conclusions

PMC4658288

zinc ( zinc is not commercial ) is a public access database and tool set , initially developed to enable ready access to compounds for virtual screening , that has become ever widely used for virtual screening , ligand discovery , pharamcophore screens , benchmarking , and force field development . increasingly , however , investigators have tried to interrogate it for questions that it was not designed to answer . simple questions , such as how many endogenous human metabolites are there , which of these are purchasable , or what natural product or drug does a compound most closely resemble , were surprisingly difficult to answer . with a target in mind , investigators often wanted a focused library biased toward ligands for that target . with new compounds discovered , they often wanted to find the most similar ligands already known for that target . to optimize that ligand , they might look to the availability of starting products for synthesis , asking , for instance , how many boronic acids that contain an indole ring may be purchased in preparative quantities and how soon will they arrive . for these and many related questions , we wondered whether we could make a system that obviated the need for a computer expert s assistance . here , we describe a new version of zinc designed to address these questions , while retaining the ease of use of the original tool . zinc15 is designed to bring together biology and chemoinformatics with a tool that is easy to use for nonexperts , while remaining fully programmable for chemoinformaticians and computational biologists . 1 ) to integrate and curate biological activity , chemical property , and commercial availability data for small molecules from public sources , supplemented by additional calculated properties into a chemistry - aware relational database . 2 ) to build a general query language and report generator that is web url compatible . 3 ) to design a graphical user interface that requires no programming to interrogate the database using this query language . 4 ) to demonstrate and document the use of this tool to answer previously difficult questions . this effort has resulted in zinc 15 , a new research tool for ligand discovery that connects biological activities by gene product , drugs , and natural products with commercial availability . we describe the system and demonstrate its use to answer questions about biologically active and purchasable chemical space that were previously not easy for nonexperts . previously in zinc , compounds stood on their own and were both the subject of queries to zinc and the answers to such queries . an innovation of this version is to identify those molecules that have known biological effects or are of biological origin , such as drugs and natural products , and to link compounds to the proteins and biological processes that they modulate . correspondingly , one can now interrogate zinc regarding the ligands that bind to a particular protein or regarding the proteins that a particular molecule is known to modulate . extending this , one can also ask what biologically active molecules are most like those that bind a particular protein of interest and what proteins such a compound might be predicted to bind , based on chemical similarity to known ligands . in this way , the mission of zinc is expanded from purely compound - centric to one that links molecules to biological targets , processes , and other bioactive small molecules . the biological annotations the identification of molecules as metabolites , drugs , and natural products and the identification of molecules as ligands for particular proteins and processes all derived from other databases and libraries , such as hmdb , chembl , and drugbank , for which zinc is essentially a client and from whose rapid development in the last several years zinc has benefited . what is new here is that zinc cross - references this information with purchasability of reagents ; this much expands its ability to bring readily available reagents to biological questions . doing so has demanded optimization of the mechanics of zinc and its interface . some of these have obvious impacts on the user and the questions they can ask , for instance , expanding the number of molecules they can address by 2 orders of magnitude . for convenience , we divide the results into descriptions of the new associations with which purchasable molecules are freighted and examples of their use . zinc15 draws upon third party databases such as chembl , hmdb , drugbank , and https://clinicaltrials.gov to annotate high information compounds that are active in , or created by , nature . these include biogenic molecules , such as natural products , metabolites , and fda approved drugs , among others ( figure 1a ) . what zinc brings is not only lists of these molecules , which after all are derived from other sources , but their purchasability and , as we will see , their predicted as well as known associations . for instance , there are 70539 metabolites that have been annotated , 15006 of which may be purchased from vendors . restricting this to endogenous human metabolites , there are 46941 molecules , of which 8271 are for sale . properties of catalogs and , in turn , the molecules they contains are ranked in three independent series , thus : biogenic : endogenous human metabolites > nonhuman metabolites > natural products > unknown biogenicity . drug - series : fda approved > world drugs > investigational > in man > in vivo > in cells > in vitro > unknown bioactivity . purchasability : in - stock > via agent > on - demand > boutique > annotated ( not for sale ) . within each series , we classify each compound by the highest level it attains by catalog membership . molecules found in catalogs categorized as containing only endogenous human metabolites such as oxedrine ( zinc733 ) , for instance , inherit that property . compounds are also annotated with molecular properties that speak to their potential behavior and mechanism . for instance , 16485 compounds have appeared in the literature and have been shown to aggregate , a mechanism that is the origin of the greatest number of artifacts in early drug discovery . of these , 15072 are purchasable . interestingly , 53 metabolites have been observed to aggregate as have 23 world drugs , i.e. drugs approved by major national regulatory agencies such as the fda . the zinc15 web interface . a : selected zinc substance subsets showing the number of purchasable bioactive and biogenic subsets . click on the subset name ( 2 ) to browse or download a subset . ( 3 ) estimates of the number of each subset . ( 4 ) inset of dropdown menu providing access to other resources . the page navigation tool ( 1 ) , the get total tool ( 2 ) , the breadcrumbs ( 3 ) , the selection tool ( 4 ) , and the download tool ( 5 ) . click on any molecule s number ( 6 ) to view its detail page . for instance , to answer the following question : how many endogenous human metabolites are there . the user would browse to http://zinc15.docking.org , click on substances in the navigation bar ( top ) , click on subsets ( top , left ) , and select endogenous from the list ( bottom ) ( figure 1a ) . the endogenous human metabolites are displayed , as tiles , but often there are too many to count immediately ( figure 1b ) . to count the number if it is not displayed , the user clicks on the get total button ( top , left ) ( 47319 ) . the user selects now from the selection tool dropdown menu ( top , right ) and clicks on the count button again ( now 7190 ) . to download these , the user would click on the download button ( top , right ) . the same procedure is applicable for drugs , investigational compounds , and biogenic compounds , among others . thus , the user may ask for drugs that are also metabolites or natural products that have been investigated in clinical trials . having drawn on databases such as chembl and drugbank to associate compounds with their individual targets for instance , in zinc15 there are 2737 eukaryotic proteins that have compounds binding at 10 m or better annotated to them . over 100,000 distinct compounds hit at least one eukaryotic target at 10 nm or better , rising to over 1/3 of a million at 10 m . intriguingly , only 361 bacterial proteins have molecules annotated to them amounting to only 4283 compounds at 1 m or better ; the numbers for archael and viral targets are , notwithstanding intense interest , lower still . zinc may be used to acquire focused libraries of ligands annotated to a particular gene . for instance , a user seeking new ligands for the ionotropic glutamate receptor grin1 might want the 59 known ligands as controls in 3d sdf format files of the usual relevant forms expected at physiological ph . in the previous example orthologous genes were combined into gene symbols , but some questions require a particular species be specified . thus , 2025 compounds bind the human beta-2 adrenergic receptor at 10 m or better , while 2050 distinct compounds bind any of its orthologs , which includes the 2025 above plus 25 additional compounds . eight distinct uniprot annotations are available for this gene , and 2021 distinct ligands bind either the rat or human form at 1 m or better . the user may look for molecules either one at a time or in bulk . for a single molecule , the chemical drawing may be seeded with a drug or chemical name , smiles string , smarts pattern , inchi , inchikey , zinc i d , or even original catalog ids such as chembl ids ( figure 2a ) . four buttons below the chemical drawing tool allow for exact , substructure , and two kinds of similarity searches , using either tanimoto or dice coefficients , each based on 512 bit ecfp4 fingerprints . to look up many compounds at once , the user may use the resolver ( figure 2b ) , specifying one molecule per line , again using smiles , name , and i d but not smarts . ( 1 ) the user may seed the drawing with chemical or drug names , zinc ids , inchi , inchikey , or original catalog numbers . the search options may be edited prior to search ( 2 ) , which is run using one of four buttons below drawing ( 3 ) : exact match , substructure , and two kinds of similarity . specify one molecule per line in a file ( 4 ) or by pasting ( 5 ) . additional search options ( 6 ) . format of results may be specified ( 7 , inset 8) prior to running the resolver . buttons to browse subsets or view subsets ( 9 ) as well as general free text search . there is no set limit on the number of molecules that may be returned in a single similarity search calculation . using the api , it should be possible to download 1 million or more compounds , in any format , based on similarity and/or substructure . however , these queries may take a long time and would likely be run in batch mode . zinc is a public resource , and occasionally the most pragmatic solution may be to download a large portion of zinc and run a chemical search locally . we look forward to discovering the practical limits of this new technology . in previous versions of zinc , we supported similarity searches for multiple molecules , apparently in parallel . internally , they were run serially , and the results concatenated . currently , there is no mechanism to run queries with multiple query molecules . 2 ) use the resolver , which is limited to a minimum similarity of 0.7 ( ecfp4 ) . when a hit is found in a screening campaign , a common next step is to identify , possibly model , and then purchase analogs , often called sar - by - catalog . how well explored is the annotated or purchasable chemical space around a compound ? to investigate analogs of olaparib ( zinc 40430143 ) , a recently approved parp inhibitor , the user clicks on substance in the navigation bar and types olaparib in the input line above the drawing tool ( figure 2a ) . at time of writing there were 38885 analogs within a tanimoto of 50% ( ecfp4 ) , 297 of which were in stock for immediate delivery . zinc can answer questions about novelty of a newly discovered ligand for a particular target . for instance , the drug cariprazine is known to hit drd2 , but what are the most similar ligands that hit drd3 or drd4 ? to investigate this , the user would click on substances in the navigation bar and enter cariprazine in the draw / search structure field above the drawing tool ( the molecule appears ) ( figure 2a ) . on the results page , the user selects the relations selector ( the chain link icon ) , selects gene from the popup , types drd3 as the resource name , and clicks on the blue chain link icon to apply this constraint . zinc supports full smarts using rdkit , enabling complex chemical patterns to be matched . the same search tool used for similarity search may be used , in conjunction with the substructure button . smarts pattern searching can be slow , and thus many of these queries will probably end up being run in batch mode . for instance , to find benzylamines , the user would click on substances in the navigation bar , enter the smarts c1ccccc1cn in the draw / search structure bar above the drawing tool , and click on the substructure button below the drawing tool . to select only compounds available in preparative quantities , the users would click on the subsets popup ( the label icon ) and click on bb ( building blocks ) . to only select compounds available for immediate delivery , the user would select now from the same popup . if the user needs to look up more that a few molecules , a bulk facility can simplify this task . to do this , the user selects substances from the navigation bar and using the resolver ( figure 2b ) either selects a file containing molecules to look up or specifies them in the paste smiles field . in either case , there should be one molecule per line , which may be smiles , cas number , name , zinc i d , or an original catalog i d , such as chembl i d . options include allowing close matches , looking for analogs , and whether a single or multiple matches should be returned per input line . the output may be to a web page or a downloadable file in 11 supported formats ( figure 3a ) . when ready , the user clicks resolve file to start the process . if more than 300 molecules are specified , the job is automatically run in batch mode . a. zinc results may be accessed in 11 formats plus the web pages . three line - oriented formats are easy to parse for both people and computers . three machine readable formats provide for rich and flexible data interchange between programs . b. compounds are classified by how they may be purchased based on their current catalog membership . there are five primary levels and three derived levels . c. we classify substances into six levels by the most reactive group they possess , based on smarts patterns.d . 3d representations are associated progressively with the ph range at which they become relevant for docking . these patterns enable new features , such as computing a reactivity attribute for each molecule , accelerating substructure search , and providing a basis for new features . we have calculated patterns for 480 pains patterns using the rdkit version of the guha translation of the original sln format smarts . we also include 40 filtering patterns used in the previous version of zinc for backward compatibility . we have calculated statistics on the prevalence of these functional groups allowing the most popular and the least popular to be easily identified . we compute a reactivity score ( figure 3c ) , which classifies each molecule by the worst functionality it contains , enabling queries as well as subsets that follow community opinion in the tranche browser . for instance , which pains patterns are most common among drugs ? to answer this , the user would select patterns from the navigation bar , click browse , and then click on the drugs column heading twice to sort it in descending order . to find purchasable sulfonyl halides , the user would select patterns from the more menu in the navigation bar and then click browse . in the lookup field , the user would type sulfonyl halide and click on the blue go button . the user can see that there are 85487 sulfonyl halides for sale and clicks on the number to view the substances . the user may further specify building block or now subsets to further narrow the query . the statistics of occurrence documents the popularity of rings by subset . to browse these , the user would select rings from the navigation bar followed by browse . rings may be ordered by their frequency of occurrence in drugs , natural products , or purchasable subsets by clicking on the column heading . the interface allows the user to , for instance , rapidly identify all compounds containing indole rings available in preparative quantities , all investigational compounds containing quinazoline rings , or all compounds containing both pyrimidine and morpholine rings . the latter is interpreted as all substances containing a morpholine ring , and among these , those where any ring in the compound is pyrimidine . interesting questions may be answered from the molecule detail page alone ( figure 4 ) . to look up an individual drug by name or zinc i d , click on substances in the navigation bar and enter its name or code into the text input field above the molecular drawing tool ( we will use the example for isoniazid , zinc1590 ) and click exact ( below , left ) . the molecule detail page contains purchasing information , annotated catalog membership ( figure 4a ) , biological activity data derived from chembl , biological activity predictions from sea and chembl , similar interesting molecules ( figure 4b ) , publications from chembl , chemical patterns , rings , publications from chembl , clinical trial information , and more . molecules may be downloaded in either 2d or 3d in 11 formats ( figure 3a ) . large subsets and slow - to - download ones will be queued and run in batch mode when resources permit . a special class of downloads lead - like and fragment - like subsets , for which we recommend the tranche browser , accessed from the tranches button in the navigation bar ( figure 5 ) . the tranche browser divides physical property space into 121 tranches based on two properties in 11 bins each : the horizontal axis is size ( molecular weight ) and the vertical axis is polarity ( logp ) . the tranche browser allows the user to select the characteristics of the database subset required and then download it , in 2d ( smiles ) for chemoinformatics or 3d formats for docking . the user may select or deselect individual tranches by clicking on them or use the presets selector ( top right ) to choose a popular subset . for 2d databases , the user may also specify purchasability ( figure 3b ) and reactivity ( figure 3c ) and two downloading options , format ( figure 3a ) and download method . in addition to purchasability and reactivity , 3d users may specify restrictions on net molecular charge and ph range ( figure 5 ) . a. showing ( 1 ) zinc i d , name if known , subset membership , ( 2 ) properties and 2d depiction , ( 3 ) 3d representations if available , ( 4 ) purchasing information , and ( 5 ) annotated catalog membership , ( 6 ) breadcrumbs indicating current location , ( 7 ) selection tool for refinement of query , and ( 8) download tool . b. interesting bioactive and biogenic analogs section of molecule detail page : ( 1 ) similar biogenic compounds , ( 2 ) similar bioactive compounds , ( 3 ) compounds with a shared scaffold , ( 4 ) similar aggregators , and ( 5 ) similar purchasable compounds currently too slow to calculate . physical - chemical space has been divided into 11 bins of polarity - hydrophobicity ( calculated logp , vertical ) ( 1 ) and size ( molecular weight , horizontal ) ( 2 ) . subsets of this space may be selected in 2d ( smiles ) or 3d ( figure 3a ) ( 3 ) . purchasability level ( figure 3b ) ( 4 ) and reactivity ( figure 3c ) ( 5 ) may also be specified . for 3d only , net molecular charge ( 6 ) and ph range ( figure 3d ) ( 7 ) may also be specified . presets ( 8) correspond to community practice ( e.g. , lead - like , fragment - like ) , and download ( 9 ) provides for five methods to access the selected molecules . knowing that a molecule resembles a drug or natural product can help generate hypotheses of mechanism of action , while the absence of similar annotated compounds might suggest biological novelty . each substance detail page includes an analogs section , in which the nearest endogenous human metabolite , any metabolite , natural product , drug , in man compounds , and bioactives are shown , if there is one within a tanimoto index of 0.6 ( 512 bit ecfp4 fingerprints ) . a find more button may be used to find more . we have already seen earlier how the most similar compounds annotated for an individual gene target , its major target or subclass , or from a particular catalog may also be found . downloading a 3d screening library for docking subset for docking , the user selects tranches from the navigation bar and then clicks on 3d ( top left ) to switch to the 3d downloading tool ( figure 5 ) . from the preset popup ( top right ) the user chooses lead - like , which sets the molecular weight and logp range of tranches . individual tranches may be selected or deselected by clicking on them . by default , the purchasability selector ( figure 3b ) is set to wait - ok , which includes both in stock and on demand compounds . the reactivity selector ( figure 3c ) is set to mild by default , which includes pains and weakly reactive compounds . to download a script to download the database , the user clicks on the download icon ( top right ) , where two further choices are possible . the user downloads the script , which may then be run to download the database tranches . target classes such as membrane receptors , ion channels , transporters , and enzymes group proteins by function . we have adapted the top two classification tiers in chembl to assign one of 15 major and 42 target subclasses to all genes ( table 2 ) . zinc may be used to select compounds that are known or predicted to hit particular classes of targets . thus , for instance , there are 35080 commercially available ligands for class a gcprs ( purchasable ) , and for epigenetic regulator targets , there are 1286 purchasable annotated ligands . the number of genes total as well as the number of genes for which one or more compounds active at 10 m or better was for sale at the time of publication . the number of distinct compounds that are for sale and not for sale for each target class . zinc encompasses more of the purchasable and annotated chemical space as the catalogs it includes grow in size and number , currently adding around 50 new vendor and annotated catalogs each year . the date each catalog was last updated is available in tabular form and on the detail page of each catalog . two - dimensional smiles have been decoupled from 3d models , allowing us to load molecules for which we do not build noncovalent 3d docking models such as boronic acids , tin , and silicon containing compounds . zinc now includes molecules with molecular weight of up to 1000 da , offering more complete coverage of commercially available chemical space , currently with 220 million molecules , over half of which are for sale . zinc is now a good way to find molecules even if they are too big to be practical for docking , as long as they are available for purchase . building blocks available in preparative quantities are now included and are easier to find . we now use chemaxon s jchem to protonate and prepare biologically relevant tautomers , resulting in an average of 2.2 biologically relevant forms per molecule at physiological ph . zinc now uses the latest version of omega ( openeye scientific software , http://eyesopen.com ) for improved 3d conformations for docking . we have added pdbqt to sdf , mol2 , and our own flexibase formats , for both dock37 and previous versions . zinc15 will now generate dude - style decoys for any molecule directly from the database . zinc now provides iupac inchi and inchikeys for every molecule to allow better interoperability with other resources such as wikipedia , chembl , pubchem , chemspider , and unichem . the first part of an inchikey , which specifies the framework without stereochemistry or protonation , offers a reliable way to find stereoisomers ( e.g. , for praziquantel ) . the canonicalization required by inchis has reduced molecule duplication in zinc so that search results are better estimates of their true values . the anatomical therapeutic chemical classification system , curated by the world health organization , organizes drugs by their therapeutic , pharmacological , and chemical properties . zinc acquires atc codes of drugs via chembl20 and drugbank , allowing drugs to be selected by anatomical , therapeutic , and chemical classes . for instance , the user may find all purchasable drugs for cancer , all dermatologicals of biological origin , and opioid anesthetics such as fentanyl . integration of atc codes also allows for more consistent identification of who - assigned names for substances . we load clinical trials information from https://clinicaltrials.gov , enabling zinc to answer questions about these important compounds . for instance , to see the current clinical trials , the user would click on clinical trials in the navigation bar and select current from the selection tool ( top right ) . it is possible to ask to see which compounds are in clinical trials that hit a eukaryotic target at 10 nm or better and also to ask to see compounds in clinical trials for cancer that are sold by cayman chemicals . publications information from chembl enables the literature to be browsed , active compounds for any paper to be displayed , papers to be found based on which compounds they report on , and many other questions . the user need only enter the pmid of a paper to retrieve all the active structures it reports . suppose the user would like the active compounds from a paper in 2d or 3d form . 2014 , 57 , 9 , the user would click on publication in the navigation bar and enter either the pmid ( here 24684293 ) or select the journal , year , volume , and page number from the selectors . the user clicks search to arrive at the page where the two genes and the first five compounds that bind them at 10 m or better as described in that paper are shown . to download these compounds , use the download selector ( top , center ) . to find out whether any of the compounds reported in this paper can be purchased , the user would click on purchasable in the selection tool ( top , right ) . for example , it turns out that the subnanomolar ligand for protein kinase c , zinc4096162 , is available , in both screening and preparative quantities . this compound , in turn , is reported in 15 papers , which are summarized at the bottom of its detail page or listed at the references relation to zinc4096162 . a new api that is almost identical to the web page url structure allows zinc to be scripted and integrated into third - party applications . documentation for both the web page and the api is available using the help and examples endpoints , and the api ( url ) syntax is described on our wiki . machine - readable formats such as json , xml , sdf , csv , and txt may be read directly into third party client programs such as knime , pipelinepilot , cytoscape , datawarrior , instantjchem , and ipython notebook via pandas . in many cases , the content of the help pages each resource supports up to ten endpoints , including the relation endpoint , which intersects one relation with another . the subsets endpoint allows the curators to define popular subsets of the resource , which can help simplify query syntax . the supported subsets for each resource are available at the respective subsets endpoint . three themes emerge from this work . first , a new research tool zinc15 is now available . it enables chemists and biologists to answer questions that before would have required the assistance of a chemoinformatician . second , zinc has also been improved for experts , enabling them to integrate its features into their applications using the new api . third , zinc has undergone a wide variety of improvements for its original constituency , molecular docking . this required the design of a new database , the use of new software such as rdkit , a new url - compatible query language and report generator , and new web pages designed to simplify complex tasks . the data are structured in 20 resources , which are further divided into subsets . questions may now be asked not only about molecules but also genes and their target classes , catalogs , chemical patterns and rings , publications , and clinical trials as well as individual activity data points . orthologous targets from chembl are now grouped by gene symbol and organized by major and subclasses . the system offers focused libraries of known compounds , purchasable or otherwise , organized by gene , subclass , or major target class . zinc answers questions about chemical novelty and similarity to known drugs , bioactives , and natural products . chemoinformaticsts may now embed zinc and its tools into their own applications . for instance , zinc has been integrated into cytoscape ( zincytoscape ) and r ( spelter ) . the new api offers a modular interface using industry - standard formats like xml and json , and reports are flexible in format and content . resources and their attributes are fully documented and may also be retrieved in a machine - readable format allowing the creation of rich and dynamic tools . the interface accepts molecular queries represented not only as smiles and smarts but also inchi , inchikey , and even binary fingerprints . finally , the virtual screening community can benefit from many innovations and improvements here . among these are new vendors , new annotated catalogs , improved 3d representations , tranches for more efficient physical property subsets , less duplication , faster and more comprehensive searches by similarity and substructure , annotations grouped by gene and organism class , and search results that may be hundreds or thousands of times larger than before . zinc provides a view of biologically precedented and commercially available chemical space organized by genes and the major and subclasses to which they belong . for over one - third of genes that have ligands reported in the literature not a single one of them is for sale , underscoring an urgent need for synthesis to fill gaps in screening libraries . it inherits errors and ambiguity from the catalogs it incorporates , including stereochemical ambiguity , an ongoing challenge with few solutions that are not labor intensive . whereas our goal is to make the interface capable of creating every query without programming expertise , the zinc query and report language ( api ) allow many options for which we have not yet been able to build a point - and - click interface . due to its size and to the generality of questions supported , some queries will take a long time and must be run in batch . batch mode , meant to handle long - running queries , will not be released until december 2015 . , zinc allows compounds to be annotated for bioactivity and biogenicity , adding value to their library . synthetic organic chemists may use zinc to identify neglected metabolites or drugs for synthesis or other bioactives that are not currently purchasable , as well as the building blocks with which to make them . curators of annotated libraries such as chembl and drugbank may use zinc to enhance their offerings with supporting information such as purchasability and biogenicity information . dockers and chemoinformaticians may download commercially available libraries for screening , in 2d or 3d , as well as sets of known actives as controls . medicinal chemists may use zinc to compare their discoveries to what is known publically and then to find purchasable analogs or building blocks to make new libraries . we expect the zinc tools and libraries to have broad utility in the community . new software was written for loading , curating , and querying the database in python using the chemoinformatics software system rdkit 2014_09_01 , the python structured query language toolkit and object relational mapper sqlalchemy version 0.9.8 , and the python web framework flask version 0.10.1 . some sources such as hmdb and drugbank were loaded as several distinct catalogs in zinc allowing us to leverage the curation of metabolite origin such as plant metabolites in hmdb or drug status such as investigational drugs in drugbank . all catalogs in zinc are categorized by their biogenic and bioactivity status , if any . for instance , the approved subset of drugbank was categorized as world drugs since it contains over 100 drugs approved in other countries but not by fda , and the endogenous these values are computed and stored and are accessible in the interface as molecular features . there are four biogenic catalog levels : 1 ) endogenous human metabolites , i.e. compounds that are synthesized in man . interestingly , this may include compounds produced by our bacterial flora ; 2 ) metabolites of any species , i.e. small molecules that are involved in metabolism , development , and reproduction but not metabolites of xenobiotics ; 3 ) biogenic compounds , often called natural products ; 4 ) unknown biogenic status . 1 ) fda approved ; 2 ) world drugs ; 3 ) investigational , compounds reported to be used in clinical trials ; 4 ) in man , which including nutraceuticals , for instance ; 5 ) in vivo , which includes drugbank experimental compounds that have been in animals ; 6 ) in cells , which includes compounds reported active in cell based assays ; 7 ) in vitro , compounds active or assumed active at 10 m or better in a direct binding assay . the categories are ordered to be progressively inclusive within each series , thus all fda approved drugs are also world drugs and all compounds active in cells are also active in vitro . we annotate as building blocks those catalogs of compounds available in preparative quantities , typically 250 mg or more . commercial vendors are categorized by the speed and cost of compound acquisition , allowing the best purchasability of every compound to be computed based on its current catalog membership . catalog categorizations are refined continually by purchasing experience in our lab and reports from colleagues , as follows : 1 ) in stock , delivery in under 2 weeks , 95% typical acquisition success rate ; 2 ) procurement agent , in stock , delivery in 2 weeks , 95% typical acquisition success rate ; 3 ) make - on - demand , delivery typically within 8 to 10 weeks , 70% typical acquisition success rate ; 4 ) boutique , where the cost may be high but still likely cheaper than making it yourself , 70% typical acquisition success rate . source catalogs are processed and loaded into the database ( 2d only ) as follows . name and cas numbers are loaded into a synonyms table , while selected bioactivity and other selected data are stored in a provided_values table . we convert sdf to smiles using rdkit and take the largest organic part of the compound ( desalting ) , enumerating up to four stereoisomers from stereochemically ambiguous smiles using oechem tk version 1.7 ( openeye scientific software , santa fe , nm ) . because of the combinatorial problem of ambiguous stereocenters in sterols , we used smarts filters to prioritize the most probable implied stereoisomers based on biosynthetic pathways ( prof . the smiles are neutralized with mitools ( http://molinspiration.com ) , which also filters out incorrectly coded molecules well . molecules are loaded using python / rdkit scripts by attempting to map them to existing zinc ids or creating new zinc substances as necessary , as well as any additional required datastructures . inchi and inchikeys are calculated on loading , and the inchikey is used as a unique constraint in the database . 512 bit morgan fingerprints with radius 2 ( effectively ecfp4 ) are calculated for each molecule using rdkit . the 3d molecule processing pipeline is now disconnected from the 2d loading process , above . we now use chemaxon s package and the command line tool cxcalc to calculate protonation states and tautomers at or near physiologically relevant ph in three ph tranches . these are physiological , covering roughly ph 6.4 to 8.4 , high , roughly ph 8.4 to 9.0 , and low , roughly ph 5.8 to 6.4 . each protomer is rendered into 3d using jchem s molconvert ( chemaxon , budapest , hungary ) and conformationally sampled using omega ( openeye scientific software , santa fe nm ) . atomic charges and desolvation penalties are calculated using amsol 7.1 and our previously published protocol . files are formatted for docking as flexibase files , mol2 , sdf , and pdbqt . we normalized pki , ic50 , ec50 , ac50 , and pic50 to a single standard pki value , which we rounded to two decimal places . we filtered out data flagged with the data_validity_comment field indicating possible problems in the source document . we associate compounds annotated for protein complexes to each of the genes involved in that complex . two common areas of biology where multigene complexes are observed is for the cell surface receptor integrins and the ligand - gated ion channels such as the nicotinic acetylcholine receptor . for instance , integrin vla-1 is an alpha-1/beta-1 heterodimer of two genes , itga1 and itgb1 , respectively . likewise , nachrs such as ( alpha-3)2(beta-4)3 is a heteropentamer of two genes chrna3 and chrnb4 , respectively . in such cases , for single proteins , we used uniprot gene symbols based on the uniprot accession codes in chembl . orthologs in the trembl part of uniprot often did not have assigned gene symbols , in which case we used the uniprot accession code as a provisional gene name . we assigned target classes and subclasses based on the first two subfields of the protein_class field of the protein_classification table of chembl . in this version of zinc there are 42 subclasses grouped into 15 major target classes : membrane receptor , ion channel , transporter , transcription factor , enzyme , epigenetic , and 9 other catch - all classes for the few cases when none of these fit . we computed 512 bit fingerprints using the morgan algorithm with radius 2 as implemented in rdkit . stereoisomers and some very near neighbors have identical fingerprints , resulting in approximately 50% fewer fingerprints than substances , on average . we grouped fingerprints into three classes , interesting , current , and benched for faster searching . queries that limit their results to annotated compounds need only search fingerprints in the interesting subsets , while benched fingerprints , corresponding to compounds not in any current catalog , are never searched . we have implemented a very general chemical search api that automatically parallelizes chemical search queries using python green threads to search in parallel increments of 1 million molecules at a time . executed on a 64-core computer , we often see full database smarts searches completing in 30 s or less , although smarts can be of almost unlimited complexity , and some queries will certainly take far longer . similarity searches features label molecules with computed properties often derived from catalog membership that would be prohibitive to calculate interactively . there are four biogenic class annotations : biogenic ( natural products ) , metabolites , endogenous human metabolites , and unknown . there are eight bioactivity classes , which includes drugs : fda approved , world drugs , investigational , in man , in vivo , in cells , in vitro , and unknown . there has been considerable interest in pan - assay interference ( pains ) smarts patterns recently . we used the rdkit version of the guha translation of the original 480 pains expressed in sybyl line notation ( sln ) . all molecules in zinc have been annotated and are searchable by pains and other smarts patterns . the reactivity categories are a ) anodyne ; b ) clean ( pains - ok ) ; c ) mild ( weakly reactive , typically as a nucleophile or electrophile ) ; d ) reactive ; e ) unstable or irrelevant for screening . for e , we do not build molecular models for noncovalent docking ( e.g. , boronic acids ) . the web site and api were coded in python using rdkit , sqlalchemy , and flask . a curator s tool ( zincmanage ) is used to load , update , and curate the database . a command line interface ( zinccli ) provides a unix - shell like interface for additional testing and curation . the stereochemical ambiguity problem is particularly acute in sterol rings , but since these are almost always biological in origin and are derived from the sterol biosynthetic pathway , sensible guesses of stereochemistry are reasonable . we have therefore created a special sterol processing pipeline for loading molecules into zinc . leslie kuhn for drawing our attention to this and for providing smarts patterns and advice . we used mitools ( http://molinspiration.com ) to extract ring systems from every zinc molecule and loaded them into the database . we calculate static counts of the number of molecules that have biogenic or bioactivity annotations , allowing rapid reports of approximate counts of numbers of qualifying compounds per ring . we built an interface to the docs table in chembl20 and integrated it into the docs resource on the molecule detail , gene detail , and target detail pages . we attempt to identify names for substances in zinc from who - assigned names via the atc , chembl molecule names , and synonyms extracted from hmdb , drugbank , ttd , and other catalogs . all drug or dietary supplement interventions are then queried using a free text search against substance names to map the indications to the corresponding substances in zinc . there are five classes of endpoints , ten static and an almost unlimited number of dynamic ones . the five endpoint classes are list , detail , relation , subsets , and special . thus , for example , the substances help endpoint provides guidance on how to find substances of interest and a table provides a list of available catalogs in zinc and the time of their last update and shows the available subsets of genes in zinc per chembl20 . the major classes home endpoint provides an overview of target classes in zinc and gives examples of how to query and select individual observations of compound - target affinities from chembl , with or without additional purchasability constraints .

many questions about the biological activity and availability of small molecules remain inaccessible to investigators who could most benefit from their answers . to narrow the gap between chemoinformatics and biology , we have developed a suite of ligand annotation , purchasability , target , and biology association tools , incorporated into zinc and meant for investigators who are not computer specialists . the new version contains over 120 million purchasable drug - like compounds effectively all organic molecules that are for sale a quarter of which are available for immediate delivery . zinc connects purchasable compounds to high - value ones such as metabolites , drugs , natural products , and annotated compounds from the literature . compounds may be accessed by the genes for which they are annotated as well as the major and minor target classes to which those genes belong . it offers new analysis tools that are easy for nonspecialists yet with few limitations for experts . zinc retains its original 3d roots all molecules are available in biologically relevant , ready - to - dock formats . zinc is freely available at http://zinc15.docking.org .

Introduction Results Discussion Methods

PMC4897340

herbal plants are valuable gift of nature for mankind and they are the source of a variety of phytochemicals which are utilized for human and animal diets also . it is capable of synthesizing an overwhelming variety of low molecular weight organic compounds called secondary metabolites , usually with unique and complex structures . the medicinal actions of plants unique to particular plant species or groups are consistent with the concept that the combination of secondary products in a particular plant is taxonomically distinct . it states that around 8590% of the world 's population consumes traditional herbal medicines . in recent decades , studies on phytochemical constituents of medicinal plant and its pharmacological activities have received wide attention [ 36 ] . who has emphasized the need to ensure the quality of medicinal plant products using modern techniques with the application of suitable standards . many modern methods are adapted for identification and quantification of active principle compounds in plant materials . of them , gas chromatography - mass spectrometry ( gc - ms ) has become firmly established as a key technological platform for secondary metabolite profiling in both plant and nonplant species [ 7 , 8 ] . the plant species solena amplexicaulis is commonly called creeping cucumber and belongs to the family cucurbitaceae distributed very seldom in the dry deciduous forest and scrub jungles of tamil nadu . the local healers of tamil nadu and andhra pradesh are prescribing this species for many ailments owing to its effective healing property . the traditional healers are prescribing the tubers , leaves , and seeds of this species for various ailments like spermatorrhoea , thermogenics , diuretics , haemorrhoids , and invigorating and it is a very good appetizer and cardiotonic . the whole plant is a potential source of natural antioxidant [ 12 , 13 ] , antidiabetic , and antibacterial agent also . as the leaves have good anti - inflammatory activity , it is recommended for inflammation , skin lesions , and skin diseases . as the reproductive parts like seeds and tubers are exploited severely for medicinal uses , this species becomes rare sighted in its habitats of tamil nadu . despite these wide medicinal uses , no information on qualitative account of phytochemicals is available for this species . to address this lacuna , gc - ms studies were undertaken to explore the phytochemical constituents present in the leaf , stem , and tuber parts of s. amplexicaulis . the leaf , stem , and tuber parts of s. amplexicaulis were collected separately from the thorny scrub jungles of madukkarai , coimbatore district , tamil nadu , india . the authenticity of the plant was confirmed in botanical survey of india , southern regional centre , coimbatore , by referring to the deposited specimen ( voucher specimen number : cps 313 ) . they were washed thoroughly in tap water , shade - dried , and then homogenized to fine powder and stored in air tight bottles . 50 g of powdered leaf , stem , and tuber parts of s. amplexicaulis was separately extracted with 250 ml methanol at the temperature between 60 and 65c for 24 h by using soxhlet extractor . gc - ms analysis was carried out on a 5975c agilent equipped with a db-5ms agilent fused silica capillary column ( 30 0.25 mm i d ; film thickness : 0.25 m ) , operating in electron impact mode at 70 ev . pure helium ( 99.999% ) was used as carrier gas at a constant flow of 1 ml / min and an injection volume of 1 l was employed ( split ratio is 10 : 1 ) . mass transfer line and injector temperature were set at 230 and 250c , respectively . the oven temperature was programmed from 70 ( isothermal for 3 min ) to 300c ( isothermal for 9 min ) at the rate of 10c / min . total gc running time was 34 min and the ms detection was completed within 35 min . by gc - ms , the compounds were separated and then they were eluted from the column and made enter into the detector which was capable of creating an electronic signal . then the compound entered into the electron ionization ( mass spectroscopy ) detector , where they were bombarded with a stream of electrons causing them to break apart into fragments . the m / z ( mass / charge ) ratio obtained was calibrated from the graph , called the mass spectrum , and is the fingerprint of the molecule . to identify the compounds , the extract was assigned for comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer library and also with the published literature . national institute of standards and technology library sources were also used for matching the identified compounds from the plant materials [ 19 , 20 ] . the gas chromatograms of leaf , stem , and tuber parts of s. amplexicaulis confirmed the presence of various interesting compounds with different retention times as illustrated in figures 1 , 2 , and 3 . the identified compounds and their retention time , molecular formula , molecular weight , peak area ( % ) , structure , category of the compound , and activities related with medicinal uses are given in tables 1 , 2 , and 3 for leaf , stem , and tuber , respectively . six compounds were detected in the methanolic leaf extract of s. amplexicaulis . among them , the most prevailing major compounds were phytol , a diterpene ( peak area : 38.24% ) ( figure 4(a ) ) , carane , a terpene ( peak area : 18.76% ) ( figure 4(b ) ) , and 1-octanamine , an aliphatic amine ( peak area : 16.16% ) . the methanolic stem extract of s. amplexicaulis showed the presence of fifteen different organic compounds . the major phytochemical compounds among them were 4-(4-ethoxyphenyl ) but-3-en-2-one , an aliphatic acid ( peak area : 56.90% ) ( figure 4(c ) ) , trehalose , sucrose ( peak area : 11.49% ) ( figure 4(d ) ) , hexadecanoic acid , methyl ester , a linoleic acid ester ( peak area : 6.52% ) , and 9-octadecenoic acid ( z)- , methyl ester , another linoleic acid ester ( peak area : 6.76% ) . 9,17-octadecadienal ( z)- , an unsaturated aldehyde ( peak area : 21.77% ) ( figure 4(e ) ) , n - hexadecanoic acid , a palmitic acid ( peak area : 21.75% ) ( figure 4(f ) ) , phthalic acid , di(2-propylpentyl ) ester , a dicarboxylic acid ester ( peak area : 9.48% ) , and 9,12-octadecadienoic acid ( z , z)- , a linolenic acid ( peak area : 9.35% ) were the major phytochemicals on the basis of quantity . the gas chromatogram shows that the relative concentrations of various compounds are getting eluted as a function of retention time . the height of the peaks indicates the relative concentrations of the compounds present in the plant . the mass spectrometer analyzes of the compounds eluted at different times to identify the nature and structure of the compounds . the large compound fragments into small compounds give rise to appearance of peaks at different m / z ratios . these mass spectra are fingerprint of that compound which can be identified from the data library . generally , the reliability of medicinal plant for its usage is evaluated by correlating the phytochemical compounds with their biological activities . in the present study , the gc - ms analysis of the methanolic extracts of leaf , stem , and tuber parts of s. amplexicaulis altogether showed the presence of 35 compounds . in this account , the leaf extract contained six compounds among them , phytol ( 38.24% ) is having anticancer , antioxidant , anti - inflammatory , antitumor , antimicrobial , diuretic , and chemopreventive properties and used in vaccine formulations [ 22 , 23 ] . the other compound , carane ( 18.76% ) is having antifeedant and antioxidant properties [ 24 , 25 ] . the methanolic stem and tuber extracts showed the presence of greater number of 14 and 15 compounds , respectively . the six phytoconstituents , namely , undecane , taurolidine , trehalose , hexadecanoic acid methyl ester , 9-octadecenoic acid ( z)- , methyl ester , and benzaldehyde , 2-nitro- , diaminomet hylidenhydrazone in stem extracts have possessed medicinal properties . undecane , an alkane , is an antimicrobial agent , used as carcinogen [ 27 , 28 ] . similarly , the other compound , taurolidine , a taurine amino acid derivative , has antimicrobial , antilipopolysaccharidal , and antitumor properties [ 29 , 30 ] . the linoleic acid esters present in the stem , hexadecanoic acid methyl ester , are reported to have anti - inflammatory , cancer preventive , hepatoprotective , antiarthritic , and anticoronary properties . the other linoleic acid ester , 9-octadecenoic acid ( z)- , methyl ester , is also having anti - inflammatory , antiandrogenic , and anemiagenic properties . the nitrogen compound , benzaldehyde , 2-nitro- , diaminomet hylidenhydrazone , is known to have the property of curing infectious diseases by its antimicrobial activity . in the tuber extracts , the compounds identified , namely , 10,13-octadecadienoic acid methyl ester , trans-13-octadecenoic acid , methyl ester , and 9,12-octadecadienoic acid ( z , z)- , are possessed with anti - inflammatory and cancer preventive characters . the two compounds , namely , tetradecanoic acid and n - hexadecanoic acid , are antioxidants . the phthalic acid , 1,2-benzenedicarboxylic acid , bis(2-methylpropyl ) ester , is used in the preparation of perfumes and cosmetics . the unsaturated alcoholic compound , 9,17-octadecadienal , ( z)- , is reported to have antimicrobial property . the study species s. amplexicaulis is endowed with various medicinal properties maybe due to the presence of all these compounds described . in a similar fashion , certain traditional medicinal plant species of cucurbitaceae have been analyzed phytochemically by using gc - ms and suggested for drug preparation after succeeding in clinical trials [ 34 , 35 ] . the therapeutic properties of the other compounds in all the three parts of s. amplexicaulis were not yet reported . our investigation through the present study revealed that the species s. amplexicaulis is a reliable source of bioactive compounds like fatty acid esters , alcohols , hydrocarbons , alkanes , amines , terpenes , and sugars that justify the traditional usage of this species [ 1618 ] by the local healers in coimbatore and tirupur districts of tamil nadu , india , for various ailments . as gc - ms is the first step towards understanding the nature of active principles [ 36 , 37 ] , further investigation in this species is suggested for the development of novel drugs .

objective . to explore the possible bioactive compounds in the methanolic extracts of leaf , stem , and tuber parts of the medicinal climber , solena amplexicaulis , using gc - ms . methods . gc - ms analysis of the plant extracts were performed by using gc - ms-5975c [ agilent ] and mass spectra of the compounds found in the extract was matched with the data in the library of national institute of standards and technology ( nist ) . results . thirty - five compounds were determined to be present in the parts studied . the active principles with their retention time , molecular formula , molecular weight , peak area , structure , category of the compounds , and activities were predicted . the most prevailing compounds were phytol ( 38.24% ) in leaf , 4-(4-ethoxyphenyl ) but-3-en-2-one ( 56.90% ) in stem , and 9,17-octadecadienal , ( z)- ( 21.77% ) in tuber . conclusion . this study revealed that the species s. amplexicaulis is a good source of many bioactive compounds like terpenes , triazines , esters , alkanes , alcohols , hydrocarbons , aldehydes , amides , and so forth . that justifies the traditional usage of this species .

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

PMC1716729

our aim was to create a tool that would allow the construction of gene lists with homology to any specified protein domain , coupled with the capability to analyze the expression levels of these genes as provided by gene microarray experiments . our motivation to develop such a resource originated from the general lack of such interactivity between existing highly used online resources . we specifically intended to develop a resource that could rapidly provide exhaustive gene - type classification information , concomitantly with expression information of the entire set of members within a specific species type . such a working profile suits numerous areas of investigation , as it is often the case that a priori knowledge of gene family involvement in a given biological process is available , but the individual gene member is unknown . however , if exhaustive gene family lists could be generated with ease , the coupling of such lists to an extensive gene expression base , should help in narrowing down family members playing tissue - specific roles . if such a tool was available , we reasoned it would benefit investigators in a diverse range of disciplines . the core knowledgebase of genespeed is the comprehensive homology categorization of all pfam ( 1 ) protein domain homology within the unigene transcriptomes ( 2 ) of human , mouse , worm and fly , without which its development would have been impossible . this dataset facilitates the generation of gene lists with homology to any specified protein domain by first allowing the user to identify a single protein domain that may characterize a certain protein family of interest and then searching the transcriptome of one of these species for all translations containing homology to this domain . the current version of genespeed only allows queries of single domains ; however , we plan to implement the ability for multiple domain searches in a future version . the resultant list will contain all genes in the transcriptome with homology to the protein domain of interest ( displayed with decreasing similarity ) . in order to assist the user in identifying an initial protein domain of interest the first takes advantage of a custom / manually curated transcription factor characterization of all domains within pfam ( currently 7973 domains ) so that an investigator may browse by transcription factor type . second , we incorporated the gene ontology ( go ) characterization of domains ( 3 ) , which allows browsing by the go organizational tree and the ability to generate a gene list from any specified go node . the third technique allows a more traditional query by gene / domain name , symbol or accession identifier . finally , the capability to search the genespeed database by fold expression level or by tissue specificity . this is made possible through the incorporation of the novartis symatlas2 dataset ( 4 ) , containing expression data from 280 microarrays of 61 mouse and 79 human tissues . these integrated expression data also serve an exploratory role , in that the genes within gene lists generated by the other query techniques mentioned above may be analyzed for fold - change from median , tissue specificity as well as absolute expression level . all resulting datasets and tools were organized into a lamp ( linux / apache / mysql / php ) based dynamic website named genespeed and is available at . the process for populating the genespeed database is illustrated in figure 1 , and is nearly fully automated . ( i ) the first step involves obtaining the full set of pfam domains from the pfam database ( currently representing 7973 domain families ) . ( ii ) a single domain family is selected out of the total of 7973 ( in this example the c2h2-zinc finger domain will be used ) . full pfam alignment file for the c2h2-zinc finger domain ( containing 32 874 distinct c2h2-zinc finger protein sequences from a multitude of different species ) is generated . this process involves using the tblastn algorithm ( 5 ) to search for homology for each of the 38 874 c2h2-zinc finger domains within the unigene est databases for mus musculus , homo sapiens , caenorhabditis elegans and drosophila melanogaster . in order to preserve protein id / aa - aa / e - score value custom perl and python scripts were utilized to perform all tblastn homology searches using the blast pbh cluster maintained at massively parallel technologies inc . ( v ) the tblastn output for this pfam domain is subsequently parsed for expectancy score ( e - score ) , unigene i d , genbank i d , pfam i d , domain range and domain size . these results are sorted first by e - score and then by unigene i d and subsequently all redundancy was eliminated by retaining only the most significant e - score containing record for each unique homologous unigene hit . ( vii ) this process is then repeated for each of the 7973 pfam domains . ( viii ) in addition to the results of the tblastn population process , several other sources of data were incorporated into the genespeed database . information from external databases including unigene ( 2 ) , interpro ( 3 ) , go ( 6 ) and the novartis symatlas2 ( 4 ) were included . from the most recent build of the unigene database we incorporated the unigene i d , gene symbol , gene name , entrez gene , chromosome and cytoband location . in addition , through retired unigene information tables , we implemented a tool that automatically checks for unigene ids that have been retired from the database . when uploading a custom gene list into genespeed , this script scans the list for all retired or changed unigene identifiers . once a retired unigene i d is discovered , the most recent identifier(s ) is displayed for the user . from interpro , we obtained all interpro ids and gene ontology ( go ) information relating domain families to go categories . this information allowed the development of a go browsing tool to search for genes containing domains that have an associated go classification ( 3,6 ) . as a result , these incorporated go classifications may allow for gene - to - gene inference based on an original go - member domain . however , care should be exercised , as protein homology does not always equate functional similarity , especially at the lower go - node levels . as given in the figure : ( 1 ) all domain information and alignments in the pfam database are downloaded from pfam ; ( 2 ) a single domain is selected ( c2h2-zinc finger in this example ) ; ( 3 ) the full alignment ( in this case the c2h2-zinc finger domain full alignment contains 32 874 distinct protein sequences ) is used in a ( 4 ) batch ncbi - based tblastn using the massively parallel pbh blast server ; ( 5 ) tblastn output is parsed and redundancy eliminated ; ( 6 ) non - redundant data are banked into a custom mysql database ; ( 7 ) the process is repeated for all domain families in pfam ( currently 7973 ) ; ( 8) other datasets are integrated with the database . as mentioned above , array data for the 79 human and 61 mouse tissues ( in duplicate ) of the novartis symatlas2 dataset ( 4 ) were included into the genespeed database . these array data were downloaded from novartis ( ) in its mas5 ( affymetrix ) normalized form . in addition to incorporating the absolute expression normalized values for each probeset into the database , we also pre - computed the fold - change differential from median as well as performing an anova statistical test to determine a p - value for tissue specificity . this anova computation compared the mean expression value of a gene for one tissue against the mean expression value of the same gene in all other tissues . such a calculation was repeated for all genes within each tissue type for both human and mouse tissues . all computations were performed within the r statistical environment ( ) running on a linux mandrake 10.2 operating system ( ) . given personal interests in the regulatory dna - binding factors , we also incorporated a custom transcription factor classification of all 7973 pfam protein domains . modeling the outline of a previous classification scheme ( 7 ) , domains within the pfam database were manually evaluated for their relatedness with transcription factors and classified into three hierarchies of superclass , class and subclass . relatedness in this case refers to any domains characteristic of or associated with known transcription factors ( i.e. associated with text queries of relatedness , however , does not imply an absolute transcription factor classification for any particular domain . to clarify this point with an example , the user may browse the genespeed m.musculus division of the database for the transcription factor superclass of helix turn helix ( hth ) and class of fork head / winged helix. this yields a list of domains including the fork head domain as well as 13 other domains and a total of 480 genes . the fork head domain is the signature domain that definitively characterizes the hth and fork head / winged helix transcription factor ; however , 13 other domains have also been found to be associated with these transcription factor proteins . thus , if an interpro - based sub - search of this list is performed for all genes in genespeed that only contain the fork head domain ( pf00250 , ipr001766 ) , the gene list shrinks from 480 to 38 . in translation , 480 genes were identified that contain domains ( albeit some with very low similarity ) that have been associated with fork head / winged helix transcription factors and 38 genes contain a definitive fork head domain . an update consists of running the complete database generation process from the most recent versions of all information / database sources . a general navigation overview for the genespeed database is illustrated in figure 2 . in order to permit users to save custom gene lists while searching the genespeed database , all users of the database , they may browse genespeed , perform queries , create custom gene lists and save these lists into their account . guest users have full access to all database content ; however , to save gene lists , a free account must be established . there is also an upload tool implemented allowing users to upload their own gene lists into the database . this tool receives ( as upload from user ) a unigene gene list , scans the list for all retired or changed unigene identifiers and updates all retired unigene identifiers to the most recent build of the unigene database . any discrepancies , such as retired or divided clusters , will be displayed to the user . once the user 's external list has been uploaded into the database , all the tools of genespeed may be used on the new uploaded list . the primary rationale for including the import tool is to provide rapid domain assignment and other annotation within the genespeed environment for gene lists obtained from external experiments . users may log back into the database at any time to access their previously saved custom gene lists . each user 's gene list information is protected by login name and password and may not be observed by any other user . if using genespeed as a guest user , only a single gene list may be created and manipulated at a given time . ( a. search ) a search is started by first selecting the organism and then the search type . ( b. results ) the results page will display the number of unigene clusters ( genes ) found and the user then has the option to select additional specific information they would like displayed concerning these genes . ( c. sub - searches / tool ) other sub - searches / tools are available to refine the original search including domain sub - search ( finds all domains in a specific gene ) , interpro sub - search ( finds all genes with a specific domain ) , external links ( www links to outside databases ) and novartis symatlas2 ( investigate the expression level of any number of genes with the data contained within the norvartis symatlas2 dataset including 79 human and 61 mouse tissues ) . ( d. user account ) each user is provided with a private account , in which they may store any number of user - specified gene lists to keep for analysis at any later time point . upload tools are also provided , allowing users to analyze any gene list not generated within the genespeed database . a new search ( figure 2 , a. search ) starts with selecting the organism and then the type of search , which includes ( i ) keyword , search the gene name and ( ii ) i d or accession , search by unigene , entrez gene , chromosome or interpro . ( iv ) transcription factor classification , to browse genespeed 's custom transcription factor classification hierarchy . ( v ) microarray expression data , allowing the user to query the database by fold - change from median as well as tissue specificity ( anova p - value ) . once a search has produced a new list ( or a previously saved list is selected from the account page ) the user is prompted to select what information to display for the genes in the resulting list ( figure 2 , b. results ) . output choices include various identifiers ( unigene , pfam , interpro , ensembl , entrez , go ) , gene descriptors ( gene symbol , gene name ) , domain descriptors ( name , size , number ) , mapping information ( chromosome , cytoband ) , transcription factor classification ( superclass , class , subclass ) and blast homology information ( e - score , genbank i d and amino acid range of the original blast query sequence ) . the domain descriptors refer to the name and size of each domain as well as the number of different domains containing homology in each gene sequence . the genbank i d and amino acid range of all original blast queries are also provided so that homology with any domain may be traced back to its original source . the e - score field provides a drop down menu of different e - score values allowing the user to set the e - score cutoff for the resultant output . elaborating on the e - score further , when designing the genespeed database we sought to allow a flexible and open environment allowing the user to have precise control over various selection criteria . because genespeed is a similarity - driven database , the most important criterion for the evaluation of domain sequences in the transcriptome is the expectation - score ( e - score ) . the e - score describes the number of hits that one can expect to encounter by chance when searching by homology in a database . consequently , the score is influenced by the size of the database , and by the size of the query sequence . for any particular hit , as the e - score approaches 0 ( becomes smaller ) , the hit exhibits a more significant degree of similarity with the query . the purpose of an e - score cutoff is to exclude hits generated from the similarity search that do not have sufficient resemblance to the original domain of interest and thus do not represent a homologous domain . unfortunately , due to the dramatic size differences between domain families , and the variations in conserved residues even between similarly sized domains , there is not a clearcut e - score cutoff that may be used for all domains and their corresponding hits in the database . rather , we leave such critical decisions up to the user because both size and content characteristics of each domain must be taken into consideration when evaluating an appropriate e - score cutoff . despite these complications , in most cases , an inverse correlation exists between the domain size and e - score ; therefore , a suitable cutoff may be selected using these criteria . in other less common situations , domain size may not be a suitable tool for selecting the e - score cutoff value . explanations and recommendations for choosing an appropriate e - score cutoff may be found on the genespeed website in the background section . after the results have been displayed , the user has a choice of using other links and tools to further investigate the genes in their list ( figure 2 , c. sub - searches / tools ) : ( i ) domain sub - search identifies all the domains with homology in a specified gene . ( ii ) interpro sub - search identifies all other genes in the genespeed database within the currently selected species displaying homology to the specified domain . ( iv ) novartis symatlas2 , when working in the human or mouse section of the database , the average absolute expression , fold change above median and anova tissue specificity statistical information pre - computed from the symatlas2 dataset may be accessed and visualized for genes contained within a gene list . we have provided several additional resources on the genespeed website to help users navigate and use the database with success . these include a section of real biological scenarios , where we present possible scientific questions and full descriptions on how genespeed may answer these questions . similarly , we have included an faq page as well as a glossary of helpful definitions and descriptions . in addition , we have also included a tutorial section that provides several short tutorials on how to effectively use the tools within the genespeed database . we here describe the construction and use of genespeed , a database providing an extensive homology compilation of all protein domains within the transcriptomes of human , mouse , worm and fly . at present , we have restricted this domain compilation to these four organisms . although unigene databases exist for many species ( at present 76 ) , the transcriptome sequencing effort for most is not approaching the saturated level of the four chosen organisms , and incorporating these additional species , although not limited in a practical sense , was deemed premature . these can later be added , as the sequencing effort and their respective genomics platforms become expanded and further utilized . the homology assemblage employed in the creation of genespeed is based on a computationally demanding batch tblastn population process , which to our knowledge has not been used previously for this purpose . when developing this population process it was necessary to choose an efficient homology detection algorithm . others have shown that a hidden markov model ( hmm ) based approach achieves better performance than gapped blast techniques for homology identification ( 8,9 ) . we , on the other hand , have observed that by using a very large number of domains ( each used as an individual query ) derived from all types of species in the similarity search , a greater number of true hits is possible with the blast algorithm than hmm - based techniques ( results to be published elsewhere ) . by using a large batch of query sequences from a diversity of organisms all representative of a particular protein domain family , it may be possible to more accurately reflect the natural diversity and evolution of amino acid combinations within that family ( details of these experiments and their results are available on the genespeed website ) . included with the domain analysis of these transcriptomes , genespeed provides a toolset for the exploration of these domains , their protein families and the genes in which they reside . relatedness with known transcription factors , go classification , full name , average size , blast domain data and various protein domain identifiers ( pfam , interpro and genbank ) . moreover , with the incorporated blast e - score cutoff tool , the user has control governing the amount of homology displayed when analyzing query results in order to accommodate any domain type or the specific biological question at hand . this is both a unique and indispensable feature of the database , as an appropriate cutoff may significantly fluctuate depending on the size of a domain , type of domain or even the taxonomic limitation / category of a particular protein domain . we have found it highly useful to be able to follow low levels of homology as this often carries certain functional relevance , which may link very distantly related members and often allows bridging between protein superfamilies . such information is normally not available at other domain - based sites as only near - perfect matches are curated as true members ( additional discussion on this topic is available at the genespeed website ) . in essence , genespeed provides users with a very domain - centric online suite for analyzing the transcriptome . it is this domain focus that affords the genespeed database its many unique benefits , such as providing the user the ability to infer protein type classification of a gene based on the composition and homology of its protein domain content . this is particularly noticeable in almost any genespeed query result , where many of the resultant hits are genes that have not been classified in any of the common databases ( genbank , entrez gene , unigene , etc . ) . as an example , a genespeed query for the homeobox domain in human yields 217 genes with significant e - scores . of these , 24 have not been characterized as homeoboxes and 16 have no previous classification associated with them . in such instances the domain content of these genes may be quickly and easily analyzed in the genespeed environment , thereby revealing distinct clues about the possible classification of these unknown genes . to our knowledge , few online biological knowledgebases are dedicated to the task of helping investigators define all genes in a particular proteomic category , which is both species specific and non - redundant , as well as linking this information to searchable gene expression information at this level of detail . only recently have large databases such as ncbi and embl started to interlink genetic , functional and expression datasets . even so , there are still no tools allowing the analysis of large user defined gene lists or the ability to save this information in a private account within the database of interest . furthermore , there is no current resource where an investigator may study protein domain families in a non - redundant manner and within a select organism of choice . we have developed genespeed in an attempt to fulfill these needs within a single database and provide access from a globally accessible website . we have used the genespeed database with significant success in our own research not only to identify particular protein families based on domain content , but also to analyze these gene lists for expression level in our tissue of interest in order to define candidates for follow up in our laboratory . the success by which genespeed has facilitated this process for our research should exemplify its potential value for other investigators .

the genespeed database ( ) is an online database and resource tool facilitating the detailed study of protein domain homology in the transcriptomes of homo sapiens , mus musculus , drosophila melanogaster and caenorhabditis elegans . the population schema for the genespeed database takes advantage of howard parallel cluster technology ( ) and performs exhaustive tblastn searches covering all pre - assigned pfam domain classes in all species ( currently 7973 domain families ) against the respective unigene est databases of the selected four transcriptomes . the resulting database provides a complete annotation of presumed protein domain presence for each unigene cluster . to complement this domain annotation we have also performed a custom transcription factor - family curation of all pfam domains , incorporated the gene ontology classifications for these domains as well as integrated the novartis symatlas2 dataset for both human and mouse which provides rapid and easy access to tissue - based expression analysis . consequently , the genespeed database provides the user with the capability to browse or search the database by any of these specialized criteria as well as more traditional means ( gene identifier , gene symbol , etc . ) , thereby enabling a supervised analysis of gene families through a top - down hierarchical basis defined by domain content , all directly linked to an optimized gene expression dataset .

INTRODUCTION DATABASE CONSTRUCTION NAVIGATION AND USE DISCUSSION

PMC5353418

during last decades , most studies have described this adaptive remodeling also referred to as athletes heart . exercise causes structural , functional , and electrical changes of the heart which are physiological responses to the hemodynamic demands of increased cardiac output during exercise . the main changes are represented by cardiac enlargement with preserved compliance and contractility such that , according to the law laplace , the larger heart can fill and empty larger volumes more efficiently . the type of training and age , sex , ethnicity , genetic factors , and body size can influence cardiac remodeling . actually , in literature , there are many evidences that suggest that the majority of sports induce a variable combination of both endurance and strength exercise , rather than only one of them . while strength training seems to impact minimally on the right ventricle ( rv ) , endurance exercise seems to be associated with the greatest cardiac remodeling . other reports described a disproportionate load on rv , at least during exercise , and this might be the basis for a chronic pro - arrhythmic rv remodeling . indeed , while the reversibility of the changes induced by sport after detraining was considered a typical feature of athletes heart , several studies showed that recovery might be incomplete , in particular for rv changes and this is particularly true in more practiced athletes . moreover , there is some evolving evidence which suggests that some of the exercise - induced changes may be associated with acute and chronic cardiac damage and that in a small number of athletes this may predispose to atrial and ventricular arrhythmias . the observation of acute myocardial injury and chronic myocardial scar in some athletes [ figures 1 and 2 ] , the not completely reversible changes of the rv and an increased prevalence of some arrhythmias among endurance athletes , support the existence of an exercise - induced cardiomyopathy . the aim of this paper is to review current knowledge about physiologic and pathophysiologic changes in the right heart ( rh ) in highly trained athletes . standard doppler echocardiography ( apical 4-chamber view ) showing two possible right ventricle morphologic adaptation to training , even in presence of normal function : dilatation ( a ) and hypertrabeculation ( b ) standard doppler echocardiography outlining increased tricuspidation velocity and inferior vena cava diameter in an endurance athlete , as a result of physiologic volume overload rh clearly participates in the process of enlargement of the athlete 's heart , with an increase in internal diameters and thickness of its free walls , as demonstrated by studies with standard echocardiography . rv shows greater inflow and outflow dimensions comparing with sedentary controls , with no significant difference in the systolic function . in particular , with respect to the type of long - term training , dandrea et al . documented that rh measures were all significantly greater in endurance highly trained athletes , compared to age and sex matched strength - trained athletes . left ventricular ( lv ) stroke volume and pulmonary artery systolic pressure ( pasp ) right sided - valves ( pulmonary and tricuspid ) present a higher prevalence of regurgitation in athletes , possibly as a result of chambers enlargement due to the long - term overload caused by training . these regurgitation are generally considered physiological , because of the absence of structural abnormalities of the valve apparatus and the absence of aliasing at the color - doppler analysis . in close relationship with rh adaptation to long - term training , interesting changes in pulmonary vascular hemodynamics of highly trained athletes can be detected at rest . range of resting pasp values , as estimated by tricuspid regurgitant velocity , have been described by dandrea et al . in both endurance- and strength - trained athletes . the upper limit of normal was 40 mmhg , in the presence of normal pulmonary vascular resistance . endurance - trained athletes showed the highest values , compared with strength - trained athletes , and lv stroke volume was an independent predictor of pasp . resting rv global systolic function as measured by fractional area change and tricuspid annular plain systolic excursion seems to be lower in endurance athletes comparing with nonathletic controls , as reported in recent studies . this finding is in conflict with previous small studies , which reported augmented resting rv systolic function in athletes cohort . a subsequent large cardiovascular magnetic resonance ( cmr ) , found comparable two - dimensional ( 2d ) and 3d rv systolic indexes between endurance athletes and controls . in this setting , a mild reduction in global rv function could be considered a physiological consequence of rv dilation , since an efficient stroke volume will be reached with higher end - diastolic volumes and then at lower ejection fraction . on the other hand , a severe reduction in rv global systolic function should be considered an abnormal finding even among athletes . novel echocardiographic techniques provide data about regional rv systolic function modification among highly trained athletes . in particular , both tissue doppler and 2d - strain derived deformation indexes are reduced at rest in endurance athletes at the rv inlet and mid free wall level [ figures 3 and 4 ] . right ventricle tissue doppler of endurance athlete , showing normal systolic ( sm ) and diastolic ( em and am ) disatolic velocities at a myocardial level right ventricle two - dimensional strain of endurance athlete , showing normal myocardial deformation ( arrow ) inconsistent data have been reported with respect to rv diastolic function in athletes , since several studies reported enhanced rv diastolic function among athletes , while others found no significant differences in comparison with controls . as demonstrated by tissue doppler velocity measurements , the early - diastolic phase of ventricular filling was increased along with a prolonged isometric relaxation time . lv stroke volume was an independent predictor of the early diastolic velocity ( em ) and the time of regional isovolumic release ( rtm ) of the free right ventricular walls . these findings have important implications for the physician involved in the preparticipation screening and follow - up of elite athletes . in particular , further explanation of the resting changes of the right ventricular morphology and function might come from the assessment of the right ventricular performance during exercise , which is an important area of ongoing study . as noted above , changes in dimensions and hemodynamics of rv and pulmonary circulation these make athletes evaluation challenging for the overlap of rv parameters with those detected in pathological conditions . in this context , the evaluation of rv and pulmonary circulation during exercise is of fundamental importance to elucidate the physiological basis of such changes . with respect to pulmonary circulation hemodynamics , a number of studies have reported substantial increases in pulmonary artery pressures during exercise using echocardiographic estimates and direct invasive measures . lewis et al . observed an increase of 1.5 mmhg in mean pulmonary artery pressure for each liter increase in co. thus , an increase in co of 30 l / min would equate to a mean pulmonary artery pressure exceeding 50 mmhg representing an increase of 3-fold or more from the rest . la gerche et al . sought to assess this seemingly disproportionate load using a combination of magnetic resonance and echocardiographic imaging at rest and during exercise to quantify rv systolic wall stress , as compared with that of the lv . using the simple construct of the laplace relationship , we found that during exercise increases in both pressures and volumes were greater for the rv , while increases in wall thickness were relatively less than for the lv . as a result , rv wall stress estimates increased 125% during exercise as compared with a modest 14% increase in lv wall stress . thus , it may be contended that the stress , work , and metabolic demands placed on the rv during exercise are relatively far greater than that of the lv . remembering that the rv is approximately one - quarter the mass of the lv , it would seem better matched to its arterial load at rest than during exercise . the substantial increases in afterload would seem a significant burden for the contractile reserve of the rv and raises the possibility that in the extremes of exercise the rv / pulmonary vascular unit may limit output . however , echocardiographic estimates of contractility seem to increase proportional to increases in pulmonary artery pressures during intense exercise of short duration suggesting that the rv has the contractile reserve to meet exercise demands , at least for a while . while the healthy rv seems capable of meeting the work requirements of short bouts of intense exercise , the aforementioned disparity in ventricular load would suggest that if prolonged exercise can induce cardiac fatigue numerous studies have demonstrated the evidence of cardiac fatigue or injury following intense exercise sustained over many hours , such as following marathon running and ultra - endurance triathlons . biochemical evidence of cardiac injury is common , usually minor and mostly associated with little if any lv dysfunction . in contrast , virtually all studies in which the rv has been assessed following intense endurance exercise have reported rv dysfunction . a recent meta - analysis of multiple postendurance exercise studies confirmed that intense prolonged exercise is associated with a measurable reduction in rv function while lv function is relatively unaffected . the main arguments which are used to support the existence of an exercise - induced cardiomyopathy are : the observation of acute myocardial injury and chronic myocardial scar in some athletes;that exercise - induced remodeling results in structural myocardial changes which are not completely reversible , thereby implying some extracellular expansion , as well as muscular hypertrophy , andan increased prevalence of some arrhythmias among endurance athletes . the observation of acute myocardial injury and chronic myocardial scar in some athletes ; that exercise - induced remodeling results in structural myocardial changes which are not completely reversible , thereby implying some extracellular expansion , as well as muscular hypertrophy , and an increased prevalence of some arrhythmias among endurance athletes . studies of endurance athletes following competitive events in different disciplines and of different durations have shown evidence of acute myocardial injury based on the transient elevation of biomarkers ( troponin and b - type natriuretic peptide ) following these events . a meta - analysis suggested that elevated cardiac troponin levels occur in approximately 50% of endurance exercise event participants . in all studies , cardiac enzyme levels returned to normal within a few days of the exercise prompting a debate regarding the origin , mechanisms , and significance of these biomarkers . however , it would seem that the changes in rv function are both more profound and may provide a better explanation for biomarker elevations . as discussed above , postendurance exercise studies have consistently reported decrements in rv function which are far more substantive than those observed for the lv . also , while multiple studies have documented that there is no relationship between biomarkers of cardiac injury and changes in lv function , two recent studies have documented moderately strong inverse correlations between the decrease in measures of rv function and the increase in release of troponin and b - type natriuretic peptide . achilles heel of the endurance athlete which is disproportionately fatigued or injured following endurance exercise . the question is whether the transient changes in cardiac function and elevations in biomarkers result in any long - term cardiac damage with clinical consequences . a potential deleterious consequence of any cardiac damage is chronic scar or fibrosis that has the potential to affect cardiac function and/or cause arrhythmias . histology provides the only direct evidence of fibrosis and in the case of the heart , this involves an invasive procedure with significant risks . thus , biopsies are performed in those athletes in whom there is a high pretest probability of identifying pathology and hence , it is unsurprising that inflammation and/or fibrosis is identified in a considerable proportion . a multimodality imaging approach to the athlete 's heart aims to differentiate physiological changes due to intensive training in the athlete 's heart from serious cardiac diseases with similar morphological features . perhaps the most promising tool for identifying fibrosis noninvasively is by combining cardiac magnetic resonance imaging following gadolinium contrast . this delayed gadolinium enhancement ( dge ) technique has been investigated in small cohorts of athletes and while it appears that dge tends to be absent in athletes with modest training histories , four recent studies have each reported dge in 12 - 50% of extensively trained veteran athletes la gerche et al . identified dge in 5 of 39 well - trained endurance athletes , and found that those with dge had a more extensive history of training and had greater cardiac dimensions , particularly of the rv . as noted above , increasing interest has been placed in the study of the rv dysfunction noted in high endurance athletes and its relationship with a possible pro - arrhythmic remodeling and an exercise - induced cardiomyopathy . in particular , in some more practiced athletes features of possible chronic rv alteration were seen , that is , dge of the interventricular septum at the site of rv attachment . there is some limitation in the evaluation of rv fibrosis with cmr , such as the difficult visualization of the rv free wall myocardium . then , it is possible that cardiac fibrosis may be underappreciated with current techniques that assess relative enhancement . all the changes in rh morphology and function have important correlates in resting electrocardiography patterns . among these , both complete and incomplete right bundle branch block are common among trained athletes and are important overlap findings with primary conduction disturbance and cardiomyopathies that involve rv , most notably arrhythmogenic right ventricular cardiomyopathy ( arvc ) . there is now reasonably compelling evidence that some cardiac arrhythmias are associated with long - standing endurance training . almost every study conducted in endurance athletes of middle - age or greater has observed an increased prevalence of atrial fibrillation as compared with nonathletic referents . reported that ventricular ectopic beats were common among athletes but was a benign and potentially reversible phenomenon so long as underlying cardiac disease was excluded . in contrast , heidbuchel et al . observed a high incidence of major arrhythmic events ( 39% ) including sudden cardiac death ( 20% ) among 46 athletes who presented with frequent ventricular ectopy or non / sustained ventricular tachycardia that were followed for 5 years . the arrhythmias arose from the rv and were frequently associated with functional and/or structural changes of the rv . the degree to which many years of intense endurance exercise promotes ventricular arrhythmias is yet to be determined . furthermore , it is unknown to what extent these issues are caused by sports practice in its own right as opposed to being an expression of an underlying familial predisposition such as arvc . as noted above , changes in dimensions and hemodynamics of rv and pulmonary circulation can be detected . these make athletes evaluation challenging for the overlap of rv parameters with those detected in pathological conditions . in this context , the evaluation of rv and pulmonary circulation during exercise is of fundamental importance to elucidate the physiological basis of such changes . with respect to pulmonary circulation hemodynamics , a number of studies have reported substantial increases in pulmonary artery pressures during exercise using echocardiographic estimates and direct invasive measures . lewis et al . observed an increase of 1.5 mmhg in mean pulmonary artery pressure for each liter increase in co. thus , an increase in co of 30 l / min would equate to a mean pulmonary artery pressure exceeding 50 mmhg representing an increase of 3-fold or more from the rest . la gerche et al . sought to assess this seemingly disproportionate load using a combination of magnetic resonance and echocardiographic imaging at rest and during exercise to quantify rv systolic wall stress , as compared with that of the lv . using the simple construct of the laplace relationship , we found that during exercise increases in both pressures and volumes were greater for the rv , while increases in wall thickness were relatively less than for the lv . as a result , rv wall stress estimates increased 125% during exercise as compared with a modest 14% increase in lv wall stress . thus , it may be contended that the stress , work , and metabolic demands placed on the rv during exercise are relatively far greater than that of the lv . remembering that the rv is approximately one - quarter the mass of the lv , it would seem better matched to its arterial load at rest than during exercise . the substantial increases in afterload would seem a significant burden for the contractile reserve of the rv and raises the possibility that in the extremes of exercise the rv / pulmonary vascular unit may limit output . however , echocardiographic estimates of contractility seem to increase proportional to increases in pulmonary artery pressures during intense exercise of short duration suggesting that the rv has the contractile reserve to meet exercise demands , at least for a while . while the healthy rv seems capable of meeting the work requirements of short bouts of intense exercise , the aforementioned disparity in ventricular load would suggest that if prolonged exercise can induce cardiac fatigue numerous studies have demonstrated the evidence of cardiac fatigue or injury following intense exercise sustained over many hours , such as following marathon running and ultra - endurance triathlons . biochemical evidence of cardiac injury is common , usually minor and mostly associated with little if any lv dysfunction . in contrast , virtually all studies in which the rv has been assessed following intense endurance exercise have reported rv dysfunction . a recent meta - analysis of multiple postendurance exercise studies confirmed that intense prolonged exercise is associated with a measurable reduction in rv function while lv function is relatively unaffected . the main arguments which are used to support the existence of an exercise - induced cardiomyopathy are : the observation of acute myocardial injury and chronic myocardial scar in some athletes;that exercise - induced remodeling results in structural myocardial changes which are not completely reversible , thereby implying some extracellular expansion , as well as muscular hypertrophy , andan increased prevalence of some arrhythmias among endurance athletes . the observation of acute myocardial injury and chronic myocardial scar in some athletes ; that exercise - induced remodeling results in structural myocardial changes which are not completely reversible , thereby implying some extracellular expansion , as well as muscular hypertrophy , and an increased prevalence of some arrhythmias among endurance athletes . studies of endurance athletes following competitive events in different disciplines and of different durations have shown evidence of acute myocardial injury based on the transient elevation of biomarkers ( troponin and b - type natriuretic peptide ) following these events . a meta - analysis suggested that elevated cardiac troponin levels occur in approximately 50% of endurance exercise event participants . in all studies , cardiac enzyme levels returned to normal within a few days of the exercise prompting a debate regarding the origin , mechanisms , and significance of these biomarkers . however , it would seem that the changes in rv function are both more profound and may provide a better explanation for biomarker elevations . as discussed above , postendurance exercise studies have consistently reported decrements in rv function which are far more substantive than those observed for the lv . also , while multiple studies have documented that there is no relationship between biomarkers of cardiac injury and changes in lv function , two recent studies have documented moderately strong inverse correlations between the decrease in measures of rv function and the increase in release of troponin and b - type natriuretic peptide . achilles heel of the endurance athlete which is disproportionately fatigued or injured following endurance exercise . the question is whether the transient changes in cardiac function and elevations in biomarkers result in any long - term cardiac damage with clinical consequences . a potential deleterious consequence of any cardiac damage is chronic scar or fibrosis that has the potential to affect cardiac function and/or cause arrhythmias . histology provides the only direct evidence of fibrosis and in the case of the heart , this involves an invasive procedure with significant risks . thus , biopsies are performed in those athletes in whom there is a high pretest probability of identifying pathology and hence , it is unsurprising that inflammation and/or fibrosis is identified in a considerable proportion . a multimodality imaging approach to the athlete 's heart aims to differentiate physiological changes due to intensive training in the athlete 's heart from serious cardiac diseases with similar morphological features . perhaps the most promising tool for identifying fibrosis noninvasively is by combining cardiac magnetic resonance imaging following gadolinium contrast . this delayed gadolinium enhancement ( dge ) technique has been investigated in small cohorts of athletes and while it appears that dge tends to be absent in athletes with modest training histories , four recent studies have each reported dge in 12 - 50% of extensively trained veteran athletes la gerche et al . identified dge in 5 of 39 well - trained endurance athletes , and found that those with dge had a more extensive history of training and had greater cardiac dimensions , particularly of the rv . as noted above , increasing interest has been placed in the study of the rv dysfunction noted in high endurance athletes and its relationship with a possible pro - arrhythmic remodeling and an exercise - induced cardiomyopathy . in particular , in some more practiced athletes features of possible chronic rv alteration were seen , that is , dge of the interventricular septum at the site of rv attachment . there is some limitation in the evaluation of rv fibrosis with cmr , such as the difficult visualization of the rv free wall myocardium . then , it is possible that cardiac fibrosis may be underappreciated with current techniques that assess relative enhancement . all the changes in rh morphology and function have important correlates in resting electrocardiography patterns . among these , both complete and incomplete right bundle branch block are common among trained athletes and are important overlap findings with primary conduction disturbance and cardiomyopathies that involve rv , most notably arrhythmogenic right ventricular cardiomyopathy ( arvc ) . there is now reasonably compelling evidence that some cardiac arrhythmias are associated with long - standing endurance training . almost every study conducted in endurance athletes of middle - age or greater has observed an increased prevalence of atrial fibrillation as compared with nonathletic referents . reported that ventricular ectopic beats were common among athletes but was a benign and potentially reversible phenomenon so long as underlying cardiac disease was excluded . in contrast , heidbuchel et al . observed a high incidence of major arrhythmic events ( 39% ) including sudden cardiac death ( 20% ) among 46 athletes who presented with frequent ventricular ectopy or non / sustained ventricular tachycardia that were followed for 5 years . somewhat surprisingly , in 89% of cases the arrhythmias arose from the rv and were frequently associated with functional and/or structural changes of the rv . the degree to which many years of intense endurance exercise promotes ventricular arrhythmias is yet to be determined . furthermore , it is unknown to what extent these issues are caused by sports practice in its own right as opposed to being an expression of an underlying familial predisposition such as arvc . vigorous exercise practice , both for recreational and competitive purpose , is spreading worldwide because of a number of factors , such as documented health benefits , growing numbers of sport events ( i.e. , community - based road running races ) . then , an increase in the number of subjects with features of exercise - induced cardiac remodeling could be expected . it is increasingly important for the cardiologist and sports medicine practitioners to possess at least a basic knowledge of this subject . with respect to the contemplated potential existence of an exercise - induced cardiomyopathy or of any potential excess of arrhythmias in athletes , it is critical to maintain some balance . there are a multitude of health benefits from exercise and these are very unlikely to be outweighed by any small risk of arrhythmias . however , there is much work still to be done on the relative risks and benefits of extreme exercise , but present evidence should not alarm exercise enthusiasts .

several studies have described the adaptive remodeling of the heart during exercise . in some more practiced endurance athletes , there is a disproportionate load on the right ventricle ( rv ) , at least during exercise , and this might be the basis for a chronic pro - arrhythmic rv remodeling . especially , in these kinds of athletes the recovery after detraining might be incomplete , in particular for rv changes . the observation of acute myocardial injury based on transient elevation of biomarkers and chronic myocardial scar , not completely reversible changes of the rv and an increased prevalence of some arrhythmias support the existence of an exercise - induced cardiomyopathy . the aim of this paper is to review current knowledge about changes in the right heart in highly trained athletes and how these change influence cardiac function .

INTRODUCTION THE PHYSIOLOGY Right heart morphological changes and chronic functional adaptation during exercise CONCLUSIONS Financial support and sponsorship Conflicts of interest

PMC4024564

dentin sensitivity after tooth preparation is still one of the major challenges in clinical practice.1 it is determined by short , sharp pain arising from exposed dentin , and is explained with hydrodynamic theory.2,3 according to this theory , blocking dentinal tubules should prevent fluid shifts and prevent dentin sensitivity . desensitizing agents may act by nerve desensitization ( potassium nitrate ) , protein precipitation ( glutaraldehyde , silver nitrate , zinc chloride ) or plugging dentinal tubules ( sodium fluoride , potassium oxalate ) . additionally dentin adhesive sealers , lasers , and homeopathic medication might be used for the same purpose.4 usually hydroxyethylmethacrylate ( hema)/glutaraldehyde,5 oxalate,6 fluoride7 based desensitizing agents are recommended after tooth preparation to reduce dentin hypersensitivity . hema / glutaraldehyde the most commonly used desensitizer , is a dentin adhesive sealer , which also contains benzalkonium chloride and fluoride . hema forms deep resinous tags and occludes the dentinal tubules.8 oxalate , which is another desensitizing agent ; reacts with calcium ions of liberated dentin and forms calcium oxalate crystals . these oxalate crystals block dentinal tubules.9 similarly fluorides decrease the dentin permeability by precipitation of calcium fluoride crystals inside the dentin tubules.9 yet other desensitizing agents nowadays in use are the chlorhexidine and triclosan containing ones . chlorhexidine,10 due to its antibacterial action , has been used as a cavity disinfectant and triclosan11 is an anti - inflammatory agent . another desensitizing treatment for dentin the lasers used for the treatment of dentin hypersensitivity are divided into two groups ; these are either low or middle output power lasers . for the latter ; carbon dioxide laser ( co2 ) , neodymium- or erbium - doped yttrium aluminum garnet lasers ( nd : yag , er : yag lasers ) and erbium , chromium doped : yttrium , scandium , gallium and garnet ( er , cr : ysgg ) lasers are classified.12 nd : yag , er ; cr : ysgg , and co2 lasers can occlude dentinal tubules partially or totally , through their ability to melt peritubular dentin and therefore reduce patients ' hypersensitivity symptoms.13 the mechanism of nd : yag laser effect on dentin hypersensitivity is also by direct nerve analgesia . it has been noted that this type of laser is more effective than the er : yag and co2 lasers.14 self - adhesive resin cements have been recently introduced to simplify the technique sensitive pretreatment steps and to prevent application errors of cementation procedures in fixed prosthodontics.15 these partially hydrophilic cements are used without any pretreatment , and results in literature stating their good bond strength to dentin were reported.16,17,18,19 the adhesion obtained is claimed to rely on micromechanical retention and chemical interaction between monomer acidic groups and hdyroxyapatite.20 these cements ' initial property of hydrophilicity and moisture tolerance provide improved adaptation to the tooth structure.21,22 however in clinical practice , desensitizing agents are frequently used with resin cementation of fixed partial dentures . although desensitizers are helpful by reducing the patient 's discomfort , their effects on bonding performance of adhesive cementation to dentin can not be ignored . there is lack of literature reporting the bond strengths of different kinds of desensitizers when used with the newly developed self - adhesive resin cement . therefore , the present study aimed to compare the effect of five major chemically different groups of desensitizing agents and nd : yag laser application on microtensile bond strength ( mtbs ) between tooth and a recently introduced self - adhesive resin cement . the null - hypothesis was that pretreatment of dentin with different types of desensitizers and nd : yag laser application has no effect on bond strengths . the project was approved by the institutional review board of baskent university , ankara , turkey ( d - da14/02 ) . to determine mtbs , the teeth were stored in distilled water with 0.1% thymol solution at 4 for a maximum of 6 months.23 the root surfaces were cleaned from tissue residue with a scaler . all teeth were then embedded in custom made rectangular molds using autopolymerizing acrylic resin ( steady - resin ; scheu - dental gmbh , iserlohn , germany ) exposing the teeth 3 mm from the acrylic resin surface . the specimens were then grinded until the dentin surfaces were exposed with a coarse grit diamond rotary cutting instrument ( 6856 l-016 gebr . brassler gmbh & co. kg , lemgo , germany ) under water . to standardize the dentin surface texture , the occlusal surface of each tooth was ground off in a mechanical grinder ( metkongripo 2v grinder polisher , metkon instruments ltd , bursa , turkey ) with 600-grit silicon carbide abrasive paper under running water for 30 sec.24 the exposed dentin surfaces were inspected under an optic microscope ( m3 m , wild , heerbrugg , switzerland ) to ensure that no enamel was left . composite resin ( filtek z250 ; 3 m espe ; st paul , mn , usa ) rectangular blocks ( 10 mm in length 3 mm in thickness 5 mm in width ) were prepared in plastic molds and light cured with an light emitting diode divice ( hilux ledmax-550 , benlioglu dental ankara , turkey ) for 60 seconds at 4 different locations with an intensity of 1000 mw / cm . the dentin specimens were secured in a custom - made metallic holding tool to keep the bonding surfaces parallel to the bench . desensitizer agent was applied to dentin surfaces according to the manufacturer 's introductions ( table 1 ) . resin blocks were cemented with relyx u200 self - adhesive resin cement ( 3 m espe ; st . paul , mn , usa ) on the dentin surfaces and loaded by custom - made metallic holding devices , which carried out axial constant load of 2 kg for 60 seconds to standardize the bonding pressure . excess cement was removed , and the bonded specimens were polymerized at 4 different locations of 60 seconds each . all groups were subjected to thermocycling for 1000 cycles between 5 - 55 with a dwelling time of 20 seconds in each bath and transferring time of 10 seconds according to the standards of the international organization for standardization ( iso).25 each bonded specimen was sectioned into microbars ( 6 mm 1 mm 1 mm ) using a microcut ( metkon microcut 175 precision cutter , metkon instruments ltd , bursa , turkey ) . the microbars were inspected under a stereomicroscope ( leica mz 21 , bensheim , germany ) and intact 20 microbars were selected for each group for mtbs tests . each microbar was bonded to the attachment jaw using cyanoacrylate adhesive system ( 502 super glue , evabond group , new taipei city , taiwan ) special care was shown to center the composite - dentin interface at the free space between the jaws of the attachment unit . a crosshead speed of 0.5 mm / min was applied using microtensile tester machine ( bisco , schaumburg , il , usa ) and load at failure was recorded for each specimen . mtbs values were calculated as force to failure ( n)/ bonding area ( mm).26 failure modes were recorded by one operator under an optical microscope ( m3 m , wild , heerbrugg , switzerland ) at a magnification of 25 . these modes were classified as ; adhesive failure , no cement remnants left on dentin surface ; mixed failure , cement remnants partially left on dentin exposed and cohesive failure , dentin surface completely covered with cement or failure in dentin tissue itself . data analysis was performed by using the statistical software ( statistical program for social sciences , ver 11.5 ; spss , inc . , chicago , il , usa ) . whether the distributions of continuous variables were normal or not was determined by using the kolmogorov - smirnov test . kruskal - wallis one - way analysis of variance was used to confirm the difference among the groups at a p=.05 significance level . when the p value of the kruskal - wallis was significant , to determine which group differed from which other , the post hoc conover 's nonparametric statistical analyses was used . a total specimen size of 133 ( n=19 ) was required to detect a microtensile bond strength difference of at least 2.17 mpa between the 2 test material groups , and with a power of 90% at the 5% significance level . the 2.17 mpa value difference was taken from the study of akca et al.27 specimen size estimation was performed by using software ( ncss and pass 2000 ; ncss , inc . , kaysville , ut , usa ) the horizontal lines in the middle of each box indicates the median microtensile bond strength , while the top and bottom borders of the box mark the 25 and 75 percentiles , respectively . the whiskers above and below the box mark indicate the maximum and minimum microtensile bond strength levels . the experimental groups gluma , smart protect , and nd : yag laser showed comparable results with the control group ( p>.05 ) . although there was no statistically significant difference between gluma and the control group , gluma showed higher mtbs values . the experimental test groups treated with aquaprep f ( p=.006 ) , bisblock ( p<.001 ) and cervitec plus ( p=.020 ) showed significanty lower mtbs values in comparison to the control group . the experimental test group treated with 85% of the control group specimens showed adhesive failure , and 15% showed mixed failure . for the gluma group , for the cervitec plus group , 80% of the failures were adhesive , and 20% mixed . 95% of aquaprep f group specimens showed adhesive failure , and cohesive failure was found in 5% of the specimens . in the present study ; gluma , smart protect and nd : yag laser did not affect the mtbs , whereas cervitec plus , aquaprep f and bisblock decreased the mtbs of self - adhesive resin cement to dentin . a limited number of studies have evaluated the impact of gluma on the self - adhesive resin - dentin interface.23,28,29,30 sailer et al.29 showed that glutaraldehyde - containing dentin desensitizer and bonding agent ( gluma and syntac ) tended to positively influence the shear bond strength of the self - adhesive resin cement ( relyx u200 ) to dentin . additionally , sailer et al.28 showed statistically increased shear bond strength values when glutaraldehyde / hema containing desensitizer was applied before self - adhesive cement luting . moreover , stawarczyk et al.23 showed increased shear bond strength values even after aging procedures and also it has been revealed that gluma had a positive effect on the tensile bond strength of self adhesive resin cements even with chewing simulations.30 these results are in agreement with the findings of the current study . although pretreatment of dentin surface with gluma tends to increase mtbs of self - adhesive resin cement to dentin , the difference was not statistically significant in the present study . previously , it has been revealed that the bond strength of resin cement was highly dependent on the hema concentration , with a maximum at 35% , and nearly independent of the glutaraldehyde concentration when greater than 3%.31 additionally qin et al.32 showed that glutaraldehyde in gluma can not cross - link mineralized dentin . actually ; hema depresses the surface tension of water and enhances monomer diffusion into dentin.33 arrais et al.34 showed the presence of a thin layer of resinous structure which was penetrating and occluding dentinal tubules . moreover , they did not show the septa formation in tubule lumen in contrast to another study.35 apart from this , glutaraldehyde / hema products also contain water ; therefore , they may act as rewetting agents . although there is scarce information on the effect of glutaraldehyde / hema on the smear layer and bonding capacity of the self - adhesive resin cement , hema might be responsible for increased bond strength . on the other hand , stawaczyk et al.23 stated that a reaction between glutaraldehyde and phosphate may lead to very strong and stable bonding of gluma desensitizer in combination with self adhesive resin cement . in the current study , pretreatment of dentin surface with aquaprep dndar et al.36 reported increased shear bond strength values with 2 different brands of adhesive cement , when the same desensitizing agent was used . the researchers have attributed the increased bond strength values to hema induced rehydration mechanism allowing time for the penetration of the primer into dentin.36 this finding is contrary to the present study , the differences were in the resin cement types used and the primer used in the mentioned study.36 according to manufacturer 's instruction aquaprep f applied after 15 seconds etching with 32% phosphoric acid , and contains hema - including hydrophilic monomers intend to rehydrate the collapsed collagen matrix caused by air drying . therefore , it is hard to explain the decreased mtbs value for this desensitizing agent . one possible reason may be the precipitates occluding dentinal tubules , and also funnel shaped dentinal tubules which have been showed by arrais et al.34 they determined the function of fluoride introduced to the desensitizer as obstruction of dentinal tubules.34 previous studies document that as a result of the reaction between potassium oxalate and ionized calcium in dentin or dentinal fluid , calcium oxalate crystals form . these crystals , including the tubule orifice , cover the dentin surface thoroughly , therefore ; adhesive resins do not bond well to oxalate - treated dentin.37,38 in the present study , bisblock was applied after 15 seconds of acid etching according to the manufacturer 's instructions . according to pashley et al.39 , potassium oxalate gel application on etched dentin caused the crystal formation inside the tubules rather than on the surface and it is also stated that the crystal formation inside the tubules did not compromise the formation of typical hybrid layer . tay et al.40 showed that when oxalates were used after acid - etching , mtbs values were comparable to the non - treated dentin as well . however in the present study , bisblock decreased the mtbs of self - adhesive resin cement to dentin . the current study confirms the need for cleaning the surface of all calcium oxalate crystal as also stated by aranha et al.41 on the other hand , etching dentin surface after oxalate application to deplete the crystals from the surface as suggested in a previous study,39 might hinder the formation of sufficient hybrid layer for bonding self - adhesive resin cement to dentin . however ; it has been shown that phosphoric acid treatment before self - adhesive cementation may have comparable or increased bond strength results over non treated dentin.16,42 the present study also evaluated the bond strength of desensitizing agents containing antibacterial and anti - inflammatory agents . although triclosan may create low surface free energy and therefore impair the adhesion of resin - based cements due to reduced wettability34 and glutaraldehyde can not crosslink dentin , the application of this luting cement did not compromise bond strength to dentin . on the other hand , in a previous study,23 reaction between glutaraldehyde and phosphate has been proposed to be responsible for the enhanced shear bond strength values of self - adhesive resin cement to dentin . similarly , it has been shown that triclosan containing desensitizing agent did not influence the bonding strength of adhesive cementation ; however the adhesive cement of the previous study was used with the primer of the system.36 in a recent study43 using chlorhexidine digluconate as additional primer with acid - etched resin - bonded dentin has been supported , and also ; it has been declared that chlorhexidine may partially reduce the degradation of the resin - dentin bonds , when incorporated into hydrophilic dental adhesives . additionally santos et al.44 showed that the use of chlorhexidine digluconate as a dentin - cleaning agent has comparable results with non - treated dentin on bond strengths of self - adhesive resin cement , and lin et al.45 showed improvement on the durability of the bond to dentin when chlorhexidine was applied before self - adhesive cementation . on the other hand pretreatment of dentin surface with varying concentrations of chlorhexidine ( 0.2% , 2.0% ) decreased mtbs of self - adhesive resin cement in previous studies.24,26 the current study revealed the negative influence of cervitec plus pretreatment on mtbs of this acidic and hydrophilic , new category of resin cement . the previously mentioned study26 speculated that the precipitates originated from chlorhexidine pretreatment might act as a barrier , limiting the resin cement 's interaction with the surface , diminishing the potential for bonding . in the light of these findings,26 it is possible to explain the decreased mtbs when cervitec plus desensitizing agent was used . when this type of laser irradiation is used , reduction or complete obliteration of the dentinal tubule lumen46 and closure of exposed dentin tubules47 have been shown in many of scanning electron microscope inspections . generally , decreased bond strength values were reported when self - etch , and etch - and - rinse systems were used after nd : yag laser treatment.27,48 reduced bond strength values might be due to tubule obliteration , when self- etching primer was used on the interface of dentin and composite resin,27 because closed tubules may hinder the penetration of resin monomer and resin tag formation may not occur . in the current study nd : yag laser irradiation ( 1 w , 10 hz ) did not influence the mtbs values . a predominance of interfacial debonding between dentin and adhesive cement was noted for both control and dentin desensitized groups in the present study . additionally it has been revealed that self - adhesive resin cements with or without desensitizer presented mainly adhesive failures after water storage and entirely adhesive failure after thermocycling.23 similarly hitz18 et al . showed only adhesive failure of specimens , where self - adhesive resin cement was directly applied on dentin surfaces . however , di hiplito et al.26 showed predominance of cohesive failure when self - adhesive resin cement bonded to smear layer covered dentin , and adhesive failure for chlorhexidine pretreated dentin , on specimens not subjected to thermocycling . in a similar way ; results of sailer et al.29 revealed cohesive failure alone for freshly ground dentin and gluma pretreated dentin when self adhesive resin cement was applied , without the thermocycling process . . showed the negative impact of thermocycling on bond strength of dentin and self - adhesive resin cement interface . contrary to these findings , sailer et al.28 showed predominantly cohesive failure despite the absence of the thermocycling process ; indicating the cement itself was the weakest link and stawarczyk et al.30 also showed cohesive failure before and after aging procedures . adhesive cementation may be more technique sensitive than conventional cementation and the clinical success may be compromised by the technical challenges imposed on the dentist . relyx u200 used in the presented study is newly introduced self - adhesive resin cement with an additional monomer and a new rheology modifier added to the mixture with the processing of the filler particles optimized , which led to a formulation with increased mechanical properties.49 although this study was performed under in vitro conditions , the results provide guidance for clinical trials . the differences among materials and methods used make the results difficult to compare . according to the authors ' opinion effects of 5 different desensitizing agents ( of all kinds of contents in the market today ) and nd : yag laser pretreatment on the bonding compatibility of newly introduced self - adhesive resin cement were compared . hence , this protocol is believed to make a more conclusive decision in which desensitizing material to use when necessary with the new generation self - adhesive resin cements . also , to the authors ' knowledge , there is no study conducted with all kinds of desensitizing agents on this newly developed cement . on the other hand , the present study did not simulate pulpal pressure and dentinal fluid ; however water is formed in dentinal tubules when dentin is exposed . however , self - adhesive resin cement 's initial characteristic of hydrophilicity and moisture tolerance provides improved adaptation to the tooth structure . thus , inherent hydrophilicity of no treatment applied dentin and surface dryness of tubule occlusion succeeded dentin may affect the bonding ability of self - adhesive resin cement . this study investigated mtbs values of a newly introduced self - adhesive resin cement ; the bond strengths might be brand dependent hence , there is need for comparison of mtbs using different kinds of self - adhesive resin cements . in addition , scanning electron microscopy evaluation is needed to determine the adaptation between self - adhesive resin cement and dentin when different desensitizers are used . within the limitations of the presented in vitro study , gluma and smart protect desensitizing agents and nd : yag laser irradiation may be viable options in terms of bond strength when using a self - adhesive resin cement as a luting agent .

purposethe purpose of this study is to evaluate if pre - treatment with desensitizers have a negative effect on microtensile bond strength before cementing a restoration using recently introduced self - adhesive resin cement to dentin.materials and methodsthirty - five human molars ' occlusal surfaces were ground to expose dentin ; and were randomly grouped as ( n=5 ) ; 1 ) gluma-(glutaraldehyde / hema ) 2 ) aqua - prep f-(fluoride ) , 3 ) bisblock-(oxalate ) , 4 ) cervitec plus-(clorhexidine ) , 5 ) smart protect-(triclosan ) , 6 ) nd : yag laser , 7 ) no treatment ( control ) . after applying the selected agent , relyx u200 self - adhesive resin cement was used to bond composite resin blocks to dentin . all groups were subjected to thermocycling for 1000 cycles between 5 - 55. each bonded specimen was sectioned to microbars ( 6 mm 1 mm 1 mm ) ( n=20 ) . specimens were submitted to microtensile bond strength test at a crosshead speed of 0.5 mm / min . kolmogorov - smirnov , levene 's test , kruskal - wallis one - way analysis of variance , and conover 's nonparametric statistical analysis were used ( p<.05).resultsgluma , smart protect and nd : yag laser treatments showed comparable microtensile bond strengths compared with the control group ( p>.05 ) . the microtensile bond strengths of aqua - prep f , and cervitec plus were similar to each other but significantly lower than the control group ( p<.05 ) . bisblock showed the lowest microtensile bond strength among all groups ( p<.001 ) . most groups showed adhesive failure.conclusionwithin the limitation of this study , it is not recommended to use aqua - prep f , cervitec plus and bisblock on dentin when used with a self - adhesive resin cement due to the decrease they cause in bond strength . beside , pre - treatment of dentin with gluma , smart protect , and nd : yag laser do not have a negative effect .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC3204449

polymethyl methacrylate ( pmma ) polymers have been referred as conventional base materials and there is an ongoing effort to improve the properties of denture base materials . the goal is to have longer lasting and more biocompatible denture bases with better mechanical properties and simpler processing techniques that require less time to construct denture.1 mechanical properties of denture base resins are crucial for the clinical success of removable prosthesis . fractures and cracks are the most common types of failures related to base material.2,3 high impact acrylics were developed by the insertion of a rubber compound into the resin to improve impact resistance and strength properties of pmma materials.4,5 on the other hand , the long and rather difficult laboratory procedures of both conventional and high impact resins led to studies about self - cure and light - cure resins.6 self - cure resins offered shorter laboratory procedures but the residual monomer caused an increased risk of tissue reactions and decreased fracture resistance.7 on the other hand , light - curing systems offered simpler laboratory procedures and less risk of allergic reactions as they do not contain methyl metacrylate monomers.8 furthermore , poor fracture resistance of early light - cure systems was improved by the entrance of a new material on the market.9 however , to our knowledge these new self- and light - cure materials have not been comparatively evaluated yet . therefore , the aim of this study was to investigate the 3-point flexural strengths and flexural moduli of these two alternate base materials ( eclipse and weropress ) and compare them to three conventional heat - cure acrylic resins qc 20 , meliodent and paladent and one high impact heat - cure acrylic lucitone 199 . a total of 48 specimens were prepared according to iso 1567 standard with 6 different resins . these patterns were covered with high viscosity polyvinylsiloxane ( silagum putty , dmg , hamburg , germany ) before being invested in conventional flasks with type 3 dental stone ( moldano ; heraeus kulzer , hanau , germany ) . conventional acrylics ( meliodent , qc-20 , paladent 20 ) and high impact lucitone 199 were polymerized at 74 for 9 hours in an automated polymerization unit ( kavo ewl 5501 , kavo electrotechnisches weropress specimens were polymerized in a pressure pot heat cure unit ( ivomat ip3 , ivoclar vivadent ag , schaan , lichtenstein ) under 2 bars of pressure at 45 for 12 minutes . a teflon mold with a transparent plexyglass lid was designed to prepare the eclipse specimens . the eclipse dough was sandwiched in this mold and cured in its specific unit ( enterra vlc curing unit ; degudent gmbh , hanau , germany ) using the recommended 15 minute polymerization cycle . after polymerization the excess materials were removed by trimming with tungsten carbide burs using a handpiece at low speed . both sides of the specimens were polished under running water with # 320- , 400- , and 600-grit silicon carbide papers respectively . before transverse strength test the specimens were immersed in distilled water at room temperature ( 20 2 ) for 15 days . a 3-point bending test device ( mts mini - bionics , model 858 , mts corporation , eden prairie , mn , usa ) was used to determine the flexural strengths and flexural moduli . the device consisted of a loading wedge and a pair of adjustable supporting wedges placed 50 mm apart . the specimens were centered on the supporting wedges and the loading wedge was set to travel at a crosshead speed of 5 mm / min engaged at the center of the upper surface of the specimens . transverse strengths were calculated using the following equation ; where : s = transverse strength ( n / mm ) , p = load at fracture ( n ) , i = distance between the supporting wedges ( mm ) , b = width of the specimen ( mm ) , and d = thickness of the specimen ( mm ) . the mean displacement , maximum load , flexural modulus and flexural strength values and standard deviations were calculated for each group , and the data were analyzed by means of one way analysis of variance ( anova ) ( with mean difference significant at the 0.05 level ) . post hoc analyses ( scheffe test ) were carried out to determine the differences between the groups by using spss statistical software version 11.5 ( spss inc . a total of 48 specimens were prepared according to iso 1567 standard with 6 different resins . these patterns were covered with high viscosity polyvinylsiloxane ( silagum putty , dmg , hamburg , germany ) before being invested in conventional flasks with type 3 dental stone ( moldano ; heraeus kulzer , hanau , germany ) . conventional acrylics ( meliodent , qc-20 , paladent 20 ) and high impact lucitone 199 were polymerized at 74 for 9 hours in an automated polymerization unit ( kavo ewl 5501 , kavo electrotechnisches weropress specimens were polymerized in a pressure pot heat cure unit ( ivomat ip3 , ivoclar vivadent ag , schaan , lichtenstein ) under 2 bars of pressure at 45 for 12 minutes . a teflon mold with a transparent plexyglass lid was designed to prepare the eclipse specimens . the eclipse dough was sandwiched in this mold and cured in its specific unit ( enterra vlc curing unit ; degudent gmbh , hanau , germany ) using the recommended 15 minute polymerization cycle . after polymerization the excess materials were removed by trimming with tungsten carbide burs using a handpiece at low speed . both sides of the specimens were polished under running water with # 320- , 400- , and 600-grit silicon carbide papers respectively . before transverse strength test the specimens were immersed in distilled water at room temperature ( 20 2 ) for 15 days . a 3-point bending test device ( mts mini - bionics , model 858 , mts corporation , eden prairie , mn , usa ) was used to determine the flexural strengths and flexural moduli . the device consisted of a loading wedge and a pair of adjustable supporting wedges placed 50 mm apart . the specimens were centered on the supporting wedges and the loading wedge was set to travel at a crosshead speed of 5 mm / min engaged at the center of the upper surface of the specimens . transverse strengths were calculated using the following equation ; where : s = transverse strength ( n / mm ) , p = load at fracture ( n ) , i = distance between the supporting wedges ( mm ) , b = width of the specimen ( mm ) , and d = thickness of the specimen ( mm ) . the mean displacement , maximum load , flexural modulus and flexural strength values and standard deviations were calculated for each group , and the data were analyzed by means of one way analysis of variance ( anova ) ( with mean difference significant at the 0.05 level ) . post hoc analyses ( scheffe test ) were carried out to determine the differences between the groups by using spss statistical software version 11.5 ( spss inc . the mean flexural strength , flexural modulus , force maximum load and displacement values of eclipse , meliodent , lucitone 199 , weropress , qc 20 and paladent 20 are given in table 2 . one - way anova indicated that there were statistically significant differences in the flexural strengths ( p<.001 ) , flexural moduli ( p<.05 ) , force maximum load ( p<.001 ) and displacement values ( p<.001 ) among the six denture base materials ( table 3 ) . post hoc scheff test indicated that for flexural strength , displacement and force maximum load , the values of eclipse are significantly different from other base materials . only for displacement , besides , there were no significant differences between the values of six denture base materials for flexural modulus ( table 2 ) . the loading characteristics of fracture strength tests imitate clinical situations which a denture base undergoes in the oral environment , we preferred to stick to iso 1567:1999 standard for transverse strength testing.5 even though fatigue fracture is clinically more relevant , as it simulates the clinical failure mechanism more closely , the assessment of flexural strength is easier and thus has been used by other researchers.10 - 12 however , the lack of a thermo - cycling process and loading phase without immersing in artificial saliva could be stated as limitations of this study . it is well known that self - cure systems offer simpler laboratory procedures . however , besides the other mechanical and chemical properties , these materials should have sufficient fracture resistance . to our knowledge , weropress has not been investigated on the basis of flexural properties yet . according to the results of present study , weropress specimens exhibited the lowest transverse strength among all groups but this finding was not statistically significant except for the eclipse group . the use of light - polymerized denture base resins has been supported by several authors , not only for exhibiting acceptable strength and dimensional stability , but also for their relatively complete polymerization without free monomerin addition to their ease of manipulation and elimination of the usual denture processing armamentarium.13 - 17 earlier studies have investigated the mechanical properties of triad ( light - cured resin ) comparing to conventional and high impact resins . the overall results of these studies indicate that the transverse strength of triad is higher than conventional polymethylmethacrylate but lower than high impact resins.18 however , one of the two different base resins we examined , the light - activated urethane dimethacrylate ( udma ) ( eclipse ) , revealed significantly higher flexural strength values where the cold - curing weropress remained within the range of other materials . uzun and hersek19 investigated conventional , hi - impact and strengthened injection mold resins and reported that the transverse strength of six different base materials did not exhibit significant differences which are supported by the findings of similar materials in this study . our results , supporting previous studies , revealed that lucitone 199 has better flexural properties and higher displacement values when compared to conventional resins . however , weropress showed similar displacement and flexural strength values where eclipse specimens exhibited even improved values . therefore , both materials must also be evaluated on the basis of their impact resistance . newly introduced eclipse has been reported to have superior flexural strength and higher flexural modulus when compared to high impact ( lucitone 199 ) , conventional base ( meliodent ) resins and triad resin.6,18 the results of this study are in good agreement with the findings of previous studies . machado et al.18 reported that eclipse exhibited less porosity after polymerization when compared to early udma system ( triad ) . diaz - arnold et al.20 reported that eclipse specimens showed the highest standard deviations in mean flexural strength values ( 15.83 ) in comparison to " so called " high - impact resin groups ( lucitone 199 , diamond d , hi - i , nature - cryl hi - plus other groups ) . they suggested that the high standard deviaton indicated variations in specimen fabrication . according to their comment , as the specimens were not flasked and packed they were not dense enough and this led to the high standard deviation in eclipse group . the eclipse system , as presented by the manufacturer , is not designed for polymerization under pressure . this might be due to the use of semi - transparent white teflon mold instead of the aluminum mold for eclipse group . a possible higher quality of polymerization might have taken place . as the results indicate eclipse resin to have better and weropress to have acceptable flexural properties when compared to well known alternatives , these materials deserve further investigation over their physical properties like impact and fatigue strength , water sorption and solubility and surface characteristics like hardness and staining , to clear out any possible advantages for clinical use . within the limitations of this study following conclusions can be drown ; 1 . regarding the flexural strength properties and simpler processing technique , eclipse system owns a potential as an advantageous alternative to conventional base resins . 2 . weropress system may have similar advantages of easy processing when compared to conventional resins .

purposea new light curing urethane dimethacrylate and a cold curing resin with simpler and faster laboratory procedures may have even improved flexural properties . this study investigated the 3-point flexural strengths and flexural moduli of two alternate base materials.materials and methodsa cold curing resin ( weropress ) and a light curing urethane dimethacrylate base material ( eclipse ) . along with eclipse and weropress , a high impact resin ( lucitone199 ) and three conventional base materials ( qc 20 , meliodent and paladent 20 ) were tested . a 3-point bending test was used to determine the flexural strengths and flexural moduli . the mean displacement , maximum load , flexural modulus and flexural strength values and standard deviations for each group were analyzed by means of one - way analysis of variance ( anova ) ( with mean difference significant at the 0.05 level ) . post hoc analyses ( scheffe test ) were carried out to determine the differences between the groups at a confidence level of 95%.resultsflexural strength , displacement and force maximum load values of eclipse were significantly different from other base materials . displacement values of qc 20 were significantly different from lucitone 199 and weropress.conclusionthe flexural properties and simpler processing technique of eclipse system presents an advantageous alternative to conventional base resins and weropress offers another simple laboratory technique .

INTRODUCTION MATERIALS AND METHODS Specimen Preparation Transverse Strength Testing RESULTS DISCUSSION CONCLUSION

PMC3533635

the main purpose of endodontic therapy is a thorough mechanical and chemical cleansing of the entire pulp cavity and complete obturation with an inert filling material . a clear knowledge of the anatomy of root canal systems is an essential prerequisite to carrying out root canal preparation . many of the problems encountered during root canal treatment occur because of inadequate understanding of the pulp space anatomy . ingle et al have suggested that apical percolation is the main cause of endodontic failures . the main reasons for this failure are incomplete obturation or the presence of an untreated canal . thus , dentists must have a proper knowledge of root canal morphology and the variations of the root canal system before successfully treating a tooth.1 , 4 it is important to be familiar with variations in tooth anatomy and characteristic features among racial groups , as such knowledge can aid in locating , negotiation and management of canals . a number of studies have shown different trends in the shape and number of roots and root canals among different races.4 - 8 these variations appear to be genetically determined,5 , 7 and are important in tracing the racial origins of populations . these have included various methods such as the use of polyester resin impressions , creating transparent samples and the use of radiographs in both in vivo and in vitro studies.,4 - 8 since there are differences in selection of materials and methods used as well as classification of canal configurations , different opinions have arisen about canal morphology . the purpose of this study was to evaluate root canal morphology and the prevalence of second root canal in mandibular permanent canines within an iranian subpopulation . in this descriptive in vitro study , 100 extracted mandibular permanent canines were evaluated . the age and gender of the patients and the reasons for the extraction were not recorded . teeth were immediately placed in 5.25% sodium hypochlorite for 30 minutes after extraction to be disinfected and to remove any organic tissue on the external root surfaces . a box was made for each tooth using rose wax so that it covered from the cej to the apex and there was at least 1 mm space between the root surface and the walls of the box . holding the tooth crown , the box was placed on a vibrator and fast - setting clear acrylic resin with relatively low viscosity was poured to fill it . once resin was set , the boxes were immersed in boiling water for 5 minutes and then rinsed under running cold water for 10 minutes . thus , wax was removed and the teeth were surrounded in acrylic boxes from the cej to the apex . the crowns of the teeth were cut and 2 mm cross sections were made from cej to the apex using diamond disks ( d&z , diamant , germany ) . the sections were examined using a digital stereomicroscope ( olympus , tokyo , japan ) at 40 magnification . the existence of a second canal , canal type and the position of apical foramen according to vertucci were recorded . in a blind manner the samples were re - evaluated if there were any discrepancies and finally the results were recorded in common accord . the data relating to the existence of a second canal and canal type according to vertucci s classification are presented in table 1 . from 100 teeth evaluated , 12 ( 12% ) had 2 canals from which 5 canals were of type ii ( having 2 orifices , 2 separate canals and one apex ) and 7 were of type iii ( having one orifice , 2 separate canals and one apex ) . among the teeth with type iii canal , 4 samples had a bean - shaped orifice dividing into two canals which were separated with a thin wall and joined near the apex . 2 samples ( type iii ) had one orifice in the pulp chamber which was separated into two canals 1 mm further and joined in 4 mm from the apex ( table 2 ) . the last tooth with type iii canal had an oval orifice which changed to a sand watch appearance in sections # 2 and # 3 where two canals were separated by a thin dentinal isthmus . in section # 4 , it had a bean - shaped appearance and 2 canals united in 4 mm from the apex . among the five teeth with type ii canals , three samples had 2 canals with 2 orifices in buccal and lingual which united in 2 mm from the apex . the other two teeth had an orifice with sand watch appearance and 2 canals separated with a thin wall and united near the apex . no lateral canal was found in the specimens and all had one apical foramen . in six teeth , the apical foramina were not located at anatomical apex . in 4 of the latter , in the present study , performed on 100 extracted mandibular canines in an iranian subpopulation , the existence of the second canal was 12% . this is in agreement with the results of hessions ( 11% ) , green(13%)and kaffe et al ( 13.75% ) . it is , however , higher than the results of bellizzi & hartwell ( 4.11% ) and ingle et al ( 6%)while it is lower than the results of vertucci ( 22% ) , calikan et al ( 19.5% ) and sert et al ( 24%).in vertucci s study , 22% of the evaluated mandibular canines had 2 canals , of which 14% were type ii , 2% type iii and 6% type iv.in the present study , 5% were type ii , 7% type iii , and none were of type iv ( 0% ) . the percentage of a second canal was reported to be 24% in the study of sert et al . the existence of types ii , iii and iv in the latter study was estimated 16% , 6.5% and 1.5% , respectively . except for type iii , one - canal mandibular canines ( type i ) was estimated 88% in the present study which is higher than 78% , 80.5% and 76% reported in the similar studies.1 , 4 , 5 in calikan et al s study , 4% of mandibular canines were type iiwhich is similar to the present study . they , however , reported 1.96% type iv canal configuration which was not seen in our study . comparison of the present study with similar previous studies according to the samples size , methods and results is presented in table 2 . lambrianidis et al stated that differences between the results of morphological studies may be due to the differences in method of classification system , sample size and ethnic background of evaluated teeth . in the present study , sample size was chosen similar to previous studies for more precise evaluation.1 , 4 , 5 considering the low prevalence of the studied subject , higher sample size would result in more accurate findings and can be generalized . in a number of studies , clearing technique with demineralization and staining has been used.1 , 4 , 5 others have used only radiography.12 , 14 hession used radiography following infusion of an opaque material to the studied root canals . in the present study transversal sections were made and then evaluated using a stereomicroscope at high magnification . different methods may be a reason for difference in the results of the studies . in almost all of the mentioned studies,4 - 6 , the classification of vertucci was used ; however in certain cases it was accompanied with other classifications . none of the mandibular canines had lateral canal ( 0% ) while the prevalence was reported to be 30% , 33.33% , and 29% in other studies . , one of the most important causes of this controversy seems to be the different techniques used in the studies . the staining technique used in the mentioned studies increases the possibility of finding lateral canals compared to evaluating sections made without staining as we did in our study . in 6% of cases of the present study , apical foramina were not located in anatomical apices ( 4 buccally and 2 lingually ) . in the remaining cases , apical foramina were located in anatomical apices ; however , their lateral or central position could not be evaluated due to the used technique . in vertucci s study apical foramina were located centrally in 15% of the cases and laterally in 85% . sert et al reported 47.5% of apical foramina in anatomical apices and 52.5% out of it . another important cause of differences in results of these studies is the race of populations under study . genetic model is varied among populations and also affects tooth structure.7 , 8 , 13 , 15 various studies have been carried out in different parts of the world on different races and used different methods.1 , 4 - 8 , 10 , 13 , 15 for this purpose , the present study was performed to estimate the existence of the second canal in mandibular canines in an iranian population . the researchers judgment in exploring the second canal can play a major role in the achieved results . this bias can occur in studies using only radiography where one may interpret a radiolucent area as a second canal while others may not have the same judgment . lambrianidis et al also stated that varied results can be due to the differences in genetic model and race in the studied population , sample size , techniques , classification systems and the researchers judgment and diagnosis . the results of the present study revealed that 12% of mandibular canines had two canals of which 5% were type ii and 7% were type iii . these findings emphasize the importance of dentist s knowledge of variations in root canal morphology , since leaving a canal untreated is one of the main reasons of endodontic failures . when treating mandibular canines , the existence of a second canal should be taken into consideration .

background and aims a perfect endodontic treatment necessitates proper understanding of the morphology of canal and pulpal variations . this in vitro study was conducted to demonstrate the in - ternal anatomy of mandibular canine teeth in an iranian population . materials and methods the samples consisted of 100 extracted mandibular permanent canines . the roots of the teeth were molded in acrylic boxes . the crowns of the teeth were cut and 2 mm cross sections were made from cej to the apex . sections were examined using stereomicroscope to reveal the number and location of root canals . results from 100 evaluated teeth , 12 ( 12% ) had 2 canals from which 5 had type ii canal configu - ration and 7 ( 7% ) had type iii . 88% of the specimens had one canal and none were seen to be of type iv . conclusion the findings of this study emphasize the importance of dentist s knowledge of varia - tions in root canal morphology , since leaving a canal untreated is one of the main reasons of endodontic failures . when treating mandibular canines , the existence of a second canal should be taken into consideration .

Introduction Materials and Methods Results Discussion Conclusion

PMC2447745

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

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

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

PMC3687305

during the study period of 15 february 2009 to 15 february 2011 , all danish - speaking pregnant women with pregestational diabetes referred to the center for pregnant women with diabetes , rigshospitalet , before 14 completed gestational weeks with one living intrauterine fetus ( n = 222 ) , were offered participation in the study ( fig . patients were referred from the capital region of denmark and region zealand , covering 2.4 million inhabitants . exclusion criteria were present use of real - time cgm ( n = 7 ) , severe mental or psychiatric barriers ( n = 4 ) , diabetic nephropathy ( n = 3 ) , or severe concurrent comorbidity ( one with severe psoriasis and two with previous gastric bypass surgery ) . if a woman had more than one pregnancy in the study period ( n = 4 ) , the woman was only offered inclusion at first referral . among eligible patients , a total of 123 ( 79% ) women with type 1 diabetes and 31 ( 67% ) women with type 2 diabetes were accepted to take part in the study , of whom 79 ( 51% ) women were randomized to intermittent use of real - time cgm in pregnancy in addition to routine pregnancy care ( see below ) . the major reason for rejecting participation was the possibility of real - time cgm allocation . ( a high - quality color representation of this figure is available in the online issue . ) the research protocol was approved by the danish national committee on biomedical research ethics and the danish data protection agency . they were offered time to think it through , often with their partner , before accepting participation . most women gave written informed consent , were randomized , and had allocated intervention initiated later the same day and at the latest before 14 completed weeks . many women had been informed about the running of the trial beforehand by their local diabetologists , who had all received written information . all women in both study arms followed the routine pregnancy care program for pregestational diabetes with antenatal visits at our clinic at 8 , 12 , 21 , 27 , and 33 gestational weeks . self - monitored plasma glucose measurements were recommended seven times daily ( before and 1.5 h after each main meal and at bedtime ) , and diet and insulin doses were adjusted by the women themselves every third day and in collaboration with an experienced diabetologist every second week . treatment goals for self - monitored plasma glucose values were 4.06.0 mmol / l preprandially , 4.08.0 mmol / l 1.5 h postprandially , and 6.08.0 mmol / l prebedtime . at single - standing preprandial self - monitored plasma glucose values > 8.0 and > 10.0 mmol / l , supplementary rapid - acting insulin was recommended ( 12 and 24 insulin units in the first half of pregnancy and thereafter 24 and 46 insulin units , respectively ) . at self - monitored plasma glucose values < 4.0 mmol / l and/or mild hypoglycemia ( see below ) , supplementary carbohydrate intake of 20 g was recommended . for hba1c , the aim was < 5.6% ( 38 mmol / mol ) after 20 weeks ( 28 ) . hba1c was measured on a dca 2000 analyzer by a latex immunoagglutination inhibition method ( dca 2000 ; bayer , mishawaka , in ) . blood pressure was measured as formerly described ( 25 ) , and if > 135/85 mmhg , treatment was initiated ( 29 ) . elevated urine albumin excretion was defined as albumin - to - creatinine ratio 30 mg / mmol in a random urine sample ( 25 ) . obstetrical ultrasound scanning was performed at all five routine visits and when indicated ( 7 ) . at first pregnancy visit , all women had a dietitian appointment for individual dietary planning following national guidelines for diabetes diet . carbohydrate counting was mainly used in women on insulin pumps , and specific recording of dietary data was not performed . women with bmi < 30 kg / m were advised to gain 1015 kg in pregnancy , whereas women with bmi > 30 kg / m were advised to stay weight - neutral in the first half of pregnancy and thereafter to limit weight gain to 5 kg . all women visited our clinic and/or their local diabetes clinic at 2-week intervals throughout pregnancy , at which time hba1c , blood pressure , weight , and urine examination were registered . twenty - seven ( 22% ) women with type 1 diabetes were on insulin pump therapy ( mainly initiated before pregnancy ) with rapid - acting insulin analogs . most of these women used insulin pumps that could be connected to the prescribed real - time cgm system . the majority were using the bolus calculator , but they did not have the possibility of low glucose suspend . women with type 1 diabetes on multiple daily injections were treated with rapid- and long - acting insulin analogs ( n = 66 ) , rapid - acting insulin analogs with intermediate nph insulin ( n = 19 ) , human short - acting and intermediate nph insulin ( n = 9 ) , or human short - acting insulin and long - acting insulin analog ( n = 2 ) . all but one of 31 women with type 2 diabetes received insulin therapy in pregnancy with insulin aspart mix ( n = 14 ) , intermediate nph insulin combined with rapid - acting insulin analog or human short - acting insulin ( n = 12 ) , or solely intermediate nph insulin ( n = 3 ) or rapid - acting insulin analog ( n = 1 ) . patients were recommended to administer rapid - acting insulin shortly before meals throughout pregnancy . during labor and delivery , self - monitored plasma glucose measurements were performed hourly , aiming for plasma glucose levels of 4.07.0 mmol / l . for study purpose , participants were asked to perform eight daily self - monitored plasma glucose measurements for 6 days , including measurements at 3 a.m. , at study visits at 8 , 12 , 21 , 27 , and 33 weeks . registration of self - monitored plasma glucose values for study purpose was handled as previously described ( 7,25 ) . all women were offered free use of a blood glucose meter with corresponding test strips ( contour ; bayer , wakimachi , japan ) . results on hba1c and insulin doses were obtained at all five study visits and shortly before delivery at median 36 ( range 2939 ) weeks . mild hypoglycemia was defined as events familiar to the patient as hypoglycemia and managed by the patient , whereas severe hypoglycemia was defined as self - reported events with symptoms of hypoglycemia requiring help from another person to actively administer oral carbohydrate or injection of glucose or glucagon in order to restore normal blood glucose level ( 7 ) . information on mild and severe hypoglycemic events was obtained in detailed questionnaires and structured interviews at all study visits and shortly after delivery , as previously described ( 7 ) . during the study period , thyroid dysfunction was treated in 32 women with levothyroxine ( n = 29 ) , thiamazole ( n = 2 ) , or propylthiouracil ( n = 1 ) , resulting in normal thyroid function in all women . a computer - generated randomization program was used and treatment allocation was properly concealed using automated telephone allocation service ( paravox ) provided by an independent organization . trained research personnel ( a.l.s . or project nurse ) provided the women with their arm allocation . participants in the intervention arm were offered intermittent real - time cgm ( guardian real - time continuous glucose monitoring system with the sof - sensor ; medtronic minimed , northridge , ca ) for 6 days at the first pregnancy visit at 8 weeks and at 12 , 21 , 27 and 33 weeks , on top of routine pregnancy care . the women were encouraged to use real - time cgm continuously , especially in cases of hypoglycemia unawareness ( 7,24 ) . a small number of women were only willing to use real - time cgm for 3 days per monitoring period , which was accepted . blinded real - time cgm was not performed in the control arm . at the first pregnancy visit , women were allocated to intervention in which they were educated in sensor insertion and the maintenance of the system on a one - to - one basis for 1 to 2 h by a trained diabetes caregiver ( a.l.s . or one of two project nurses ) ( 25 ) . they were instructed to continue performing self - monitored plasma glucose measurements as recommended and to verify the accuracy of real - time cgm glucose values with self - monitored plasma glucose measurement before making management decisions . at real - time cgm alarms with subsequent plasma glucose < 4.0 real - time cgm alarms for hyperglycemia were tackled on an individual basis , including physical exercise like walking or supplementary rapid - acting insulin . patients were encouraged to contact the clinic at any time and were offered visits on weekdays other than those planned if convenient . the majority of the women preferred to have the sensor inserted in the abdominal skin , but in late pregnancy , during labor or caesarean section , some patients chose to insert the sensor in the upper arm . at enrollment , all women were advised to set the hypoglycemic alarm ( mainly at 3.5 mmol / l ) ( 25 ) . in order to limit excessive alarms , it was in general recommended that patients deactivate the alarm for hyperglycemia , but the alarm was sometimes set at the patient s request ( mainly at 10.0 alarm limits were flexible , and patients were supported in individual alarm settings . shortly after each monitoring period , downloaded real - time cgm data were printed out , with hard copies given to both the participants and health professionals . each real - time cgm reading was discussed with a diabetes caregiver ( mainly a.l.s . using locally developed guidelines on how to interpret real - time cgm readings in pregnancy ( supplementary fig . the primary focus was glycemic trends during nighttime with emphasis on the prevention of hypoglycemia . thereafter , hypoglycemia and pre- and postprandial glucose values during the daytime were evaluated , aiming for glucose values between 4.0 and 8.0 therapeutic adjustments in diet , exercise , and insulin doses were primarily based on self - monitored plasma glucose values , in combination with real - time cgm data . the prescribed device was the only real - time cgm system available at the danish market during study preparation . the prevalence of large - for - gestational - age infants [ i.e. , infant birth weight 90th centile adjusted for sex and gestational age ( 31 ) ] was predefined as the primary outcome of this study . to reflect neonatal morbidity , the prevalence of preterm delivery ( < 37 weeks of gestation ) and/or severe neonatal hypoglycemia ( 2-h plasma glucose < 2.5 mmol / l treated with intravenous glucose infusion ) was selected as the secondary combined end point prior to study onset . other pregnancy outcomes were defined as follows : miscarriage ( before 22 weeks ) , preeclampsia [ blood pressure 140/90 and proteinuria ( 29 ) ] , birth weight sd score ( z - score ) , neonatal hypoglycemia ( 2-h plasma glucose < 2.5 mmol / l ) , and major congenital malformation ( i.e. , abnormality requiring surgery and/or resulting in permanent injury ) . based on the assumption that the prevalence of large - for - gestational - age infants was 50% in our study population ( 7 ) and that the use of real - time cgm could reduce it to 20% , and a type 1 error of 5% and a type 2 error of 20% , the number of patients needed in each arm was 45 . we intended to analyze predefined primary and secondary outcome parameters of women receiving intervention versus control subjects in the entire study population , as well as in the subpopulation of women with type 1 diabetes . the study population of 154 women ( of whom 123 had type 1 diabetes ) was therefore judged to have sufficient power to detect a possible effect of real - time cgm on the prevalence of large - for - gestational - age infants in the entire study population , as well as in women with type 1 diabetes alone . we compared the characteristics of the women using the fisher exact test or test when appropriate for categorical variables and t test or mann whitney test when appropriate for continuous variables . continuous variables were given as median ( range ) , and categorical variables were given as numbers ( percentage ) . analyses were done on an intention - to - treat basis , counting 154 subjects at baseline , and thereafter with the exclusion of women with miscarriages ( n = 5 ) . we primarily analyzed data as planned by comparing women allocated to real - time cgm with control subjects in the entire study population and in women with type 1 diabetes alone . we also analyzed the prevalence of severe hypoglycemia and main outcome parameters in women using real - time cgm per protocol , and despite limited numbers , we analyzed glycemic control and pregnancy outcomes in women with type 2 diabetes and the prevalence of large - for - gestational - age infants in women with type 1 diabetes on insulin pump therapy . six infants were excluded from calculations on neonatal hypoglycemia because of intravenous glucose infusion prior to routine 2-h plasma glucose measurements ( n = 5 ) or perinatal death ( n = 1 ) ( fig . 1 , table 3 , and supplementary table 3 ) . glucose data in table 2 and supplementary table 1 were based on 25,546 self - monitored plasma glucose values , and glucose fractions ( % ) have been calculated for each individual participant after sorting glucose values into intervals ( 3.9 , 4.07.9 , and 8.0 mmol / l ) . statistical analyses were performed using stata 11.1 se software ( stata corp . , college station , tx ) . during the study period of 15 february 2009 to 15 february 2011 , all danish - speaking pregnant women with pregestational diabetes referred to the center for pregnant women with diabetes , rigshospitalet , before 14 completed gestational weeks with one living intrauterine fetus ( n = 222 ) , were offered participation in the study ( fig . patients were referred from the capital region of denmark and region zealand , covering 2.4 million inhabitants . exclusion criteria were present use of real - time cgm ( n = 7 ) , severe mental or psychiatric barriers ( n = 4 ) , diabetic nephropathy ( n = 3 ) , or severe concurrent comorbidity ( one with severe psoriasis and two with previous gastric bypass surgery ) . if a woman had more than one pregnancy in the study period ( n = 4 ) , the woman was only offered inclusion at first referral . among eligible patients , a total of 123 ( 79% ) women with type 1 diabetes and 31 ( 67% ) women with type 2 diabetes were accepted to take part in the study , of whom 79 ( 51% ) women were randomized to intermittent use of real - time cgm in pregnancy in addition to routine pregnancy care ( see below ) . the major reason for rejecting participation was the possibility of real - time cgm allocation . ( a high - quality color representation of this figure is available in the online issue . ) the research protocol was approved by the danish national committee on biomedical research ethics and the danish data protection agency . they were offered time to think it through , often with their partner , before accepting participation . most women gave written informed consent , were randomized , and had allocated intervention initiated later the same day and at the latest before 14 completed weeks . many women had been informed about the running of the trial beforehand by their local diabetologists , who had all received written information . all women in both study arms followed the routine pregnancy care program for pregestational diabetes with antenatal visits at our clinic at 8 , 12 , 21 , 27 , and 33 gestational weeks . self - monitored plasma glucose measurements were recommended seven times daily ( before and 1.5 h after each main meal and at bedtime ) , and diet and insulin doses were adjusted by the women themselves every third day and in collaboration with an experienced diabetologist every second week . treatment goals for self - monitored plasma glucose values were 4.06.0 mmol / l preprandially , 4.08.0 mmol / l 1.5 h postprandially , and 6.08.0 mmol / l prebedtime . at single - standing preprandial self - monitored plasma glucose values > 8.0 and > 10.0 mmol / l , supplementary rapid - acting insulin was recommended ( 12 and 24 insulin units in the first half of pregnancy and thereafter 24 and 46 insulin units , respectively ) . at self - monitored plasma glucose values < 4.0 mmol / l and/or mild hypoglycemia ( see below ) , supplementary carbohydrate intake of 20 g was recommended . for hba1c , the aim was < 5.6% ( 38 mmol / mol ) after 20 weeks ( 28 ) . hba1c was measured on a dca 2000 analyzer by a latex immunoagglutination inhibition method ( dca 2000 ; bayer , mishawaka , in ) . blood pressure was measured as formerly described ( 25 ) , and if > 135/85 mmhg , treatment was initiated ( 29 ) . elevated urine albumin excretion was defined as albumin - to - creatinine ratio 30 mg / mmol in a random urine sample ( 25 ) . obstetrical ultrasound scanning was performed at all five routine visits and when indicated ( 7 ) . at first pregnancy visit , all women had a dietitian appointment for individual dietary planning following national guidelines for diabetes diet . carbohydrate counting was mainly used in women on insulin pumps , and specific recording of dietary data was not performed . women with bmi < 30 kg / m were advised to gain 1015 kg in pregnancy , whereas women with bmi > 30 kg / m were advised to stay weight - neutral in the first half of pregnancy and thereafter to limit weight gain to 5 kg . all women visited our clinic and/or their local diabetes clinic at 2-week intervals throughout pregnancy , at which time hba1c , blood pressure , weight , and urine examination were registered . twenty - seven ( 22% ) women with type 1 diabetes were on insulin pump therapy ( mainly initiated before pregnancy ) with rapid - acting insulin analogs . most of these women used insulin pumps that could be connected to the prescribed real - time cgm system . the majority were using the bolus calculator , but they did not have the possibility of low glucose suspend . women with type 1 diabetes on multiple daily injections were treated with rapid- and long - acting insulin analogs ( n = 66 ) , rapid - acting insulin analogs with intermediate nph insulin ( n = 19 ) , human short - acting and intermediate nph insulin ( n = 9 ) , or human short - acting insulin and long - acting insulin analog ( n = 2 ) . all but one of 31 women with type 2 diabetes received insulin therapy in pregnancy with insulin aspart mix ( n = 14 ) , intermediate nph insulin combined with rapid - acting insulin analog or human short - acting insulin ( n = 12 ) , or solely intermediate nph insulin ( n = 3 ) or rapid - acting insulin analog ( n = 1 ) . patients were recommended to administer rapid - acting insulin shortly before meals throughout pregnancy . during labor and delivery , self - monitored plasma glucose measurements were performed hourly , aiming for plasma glucose levels of 4.07.0 mmol / l . for study purpose , participants were asked to perform eight daily self - monitored plasma glucose measurements for 6 days , including measurements at 3 a.m. , at study visits at 8 , 12 , 21 , 27 , and 33 weeks . registration of self - monitored plasma glucose values for study purpose was handled as previously described ( 7,25 ) . all women were offered free use of a blood glucose meter with corresponding test strips ( contour ; bayer , wakimachi , japan ) . results on hba1c and insulin doses were obtained at all five study visits and shortly before delivery at median 36 ( range 2939 ) weeks . mild hypoglycemia was defined as events familiar to the patient as hypoglycemia and managed by the patient , whereas severe hypoglycemia was defined as self - reported events with symptoms of hypoglycemia requiring help from another person to actively administer oral carbohydrate or injection of glucose or glucagon in order to restore normal blood glucose level ( 7 ) . information on mild and severe hypoglycemic events was obtained in detailed questionnaires and structured interviews at all study visits and shortly after delivery , as previously described ( 7 ) . during the study period , 30 women received antihypertensive treatment , mainly methyldopa ( n = 27 ) . eight women were treated with antidepressive medicine six of them with selective serotonin reuptake inhibitors . thyroid dysfunction was treated in 32 women with levothyroxine ( n = 29 ) , thiamazole ( n = 2 ) , or propylthiouracil ( n = 1 ) , resulting in normal thyroid function in all women . a computer - generated randomization program was used and treatment allocation was properly concealed using automated telephone allocation service ( paravox ) provided by an independent organization . participants were stratified according to type of diabetes . trained research personnel ( a.l.s . or project nurse ) provided the women with their arm allocation . participants in the intervention arm were offered intermittent real - time cgm ( guardian real - time continuous glucose monitoring system with the sof - sensor ; medtronic minimed , northridge , ca ) for 6 days at the first pregnancy visit at 8 weeks and at 12 , 21 , 27 and 33 weeks , on top of routine pregnancy care . the women were encouraged to use real - time cgm continuously , especially in cases of hypoglycemia unawareness ( 7,24 ) . a small number of women were only willing to use real - time cgm for 3 days per monitoring period , which was accepted . blinded real - time cgm was not performed in the control arm . at the first pregnancy visit , women were allocated to intervention in which they were educated in sensor insertion and the maintenance of the system on a one - to - one basis for 1 to 2 h by a trained diabetes caregiver ( a.l.s . or one of two project nurses ) ( 25 ) . they were instructed to continue performing self - monitored plasma glucose measurements as recommended and to verify the accuracy of real - time cgm glucose values with self - monitored plasma glucose measurement before making management decisions . at real - time cgm alarms with subsequent plasma glucose < 4.0 real - time cgm alarms for hyperglycemia were tackled on an individual basis , including physical exercise like walking or supplementary rapid - acting insulin . patients were encouraged to contact the clinic at any time and were offered visits on weekdays other than those planned if convenient . the majority of the women preferred to have the sensor inserted in the abdominal skin , but in late pregnancy , during labor or caesarean section , some patients chose to insert the sensor in the upper arm . at enrollment , all women were advised to set the hypoglycemic alarm ( mainly at 3.5 mmol / l ) ( 25 ) . in order to limit excessive alarms , it was in general recommended that patients deactivate the alarm for hyperglycemia , but the alarm was sometimes set at the patient s request ( mainly at 10.0 shortly after each monitoring period , downloaded real - time cgm data were printed out , with hard copies given to both the participants and health professionals . each real - time cgm reading was discussed with a diabetes caregiver ( mainly a.l.s . using locally developed guidelines on how to interpret real - time cgm readings in pregnancy ( supplementary fig . the primary focus was glycemic trends during nighttime with emphasis on the prevention of hypoglycemia . thereafter , hypoglycemia and pre- and postprandial glucose values during the daytime were evaluated , aiming for glucose values between 4.0 and 8.0 therapeutic adjustments in diet , exercise , and insulin doses were primarily based on self - monitored plasma glucose values , in combination with real - time cgm data . the prescribed device was the only real - time cgm system available at the danish market during study preparation . the prevalence of large - for - gestational - age infants [ i.e. , infant birth weight 90th centile adjusted for sex and gestational age ( 31 ) ] was predefined as the primary outcome of this study . to reflect neonatal morbidity , the prevalence of preterm delivery ( < 37 weeks of gestation ) and/or severe neonatal hypoglycemia ( 2-h plasma glucose < 2.5 mmol / l treated with intravenous glucose infusion ) was selected as the secondary combined end point prior to study onset . other pregnancy outcomes were defined as follows : miscarriage ( before 22 weeks ) , preeclampsia [ blood pressure 140/90 and proteinuria ( 29 ) ] , birth weight sd score ( z - score ) , neonatal hypoglycemia ( 2-h plasma glucose < 2.5 mmol / l ) , and major congenital malformation ( i.e. , abnormality requiring surgery and/or resulting in permanent injury ) . based on the assumption that the prevalence of large - for - gestational - age infants was 50% in our study population ( 7 ) and that the use of real - time cgm could reduce it to 20% , and a type 1 error of 5% and a type 2 error of 20% , the number of patients needed in each arm was 45 . we intended to analyze predefined primary and secondary outcome parameters of women receiving intervention versus control subjects in the entire study population , as well as in the subpopulation of women with type 1 diabetes . the study population of 154 women ( of whom 123 had type 1 diabetes ) was therefore judged to have sufficient power to detect a possible effect of real - time cgm on the prevalence of large - for - gestational - age infants in the entire study population , as well as in women with type 1 diabetes alone . we compared the characteristics of the women using the fisher exact test or test when appropriate for categorical variables and t test or mann whitney test when appropriate for continuous variables . continuous variables were given as median ( range ) , and categorical variables were given as numbers ( percentage ) . analyses were done on an intention - to - treat basis , counting 154 subjects at baseline , and thereafter with the exclusion of women with miscarriages ( n = 5 ) . we primarily analyzed data as planned by comparing women allocated to real - time cgm with control subjects in the entire study population and in women with type 1 diabetes alone . we also analyzed the prevalence of severe hypoglycemia and main outcome parameters in women using real - time cgm per protocol , and despite limited numbers , we analyzed glycemic control and pregnancy outcomes in women with type 2 diabetes and the prevalence of large - for - gestational - age infants in women with type 1 diabetes on insulin pump therapy . six infants were excluded from calculations on neonatal hypoglycemia because of intravenous glucose infusion prior to routine 2-h plasma glucose measurements ( n = 5 ) or perinatal death ( n = 1 ) ( fig . 1 , table 3 , and supplementary table 3 ) . glucose data in table 2 and supplementary table 1 were based on 25,546 self - monitored plasma glucose values , and glucose fractions ( % ) have been calculated for each individual participant after sorting glucose values into intervals ( 3.9 , 4.07.9 , and 8.0 mmol / l ) . statistical analyses were performed using stata 11.1 se software ( stata corp . , college station , tx ) . during the period of 15 february 2009 to 15 february 2011 , 123 women with type 1 diabetes and 31 with type 2 diabetes , with a median age of 32 ( range 1943 ) years , baseline hba1c of 6.7% ( 5.310.7 ) ( 50 [ 3493 ] mmol / mol ) , pregestational bmi of 25.0 ( 18.452.7 ) kg / m , and diabetes duration of 11 ( 138 ) years , were included in the trial . baseline data were comparable between women randomized to real - time cgm ( n = 79 ) and control subjects ( n = 75 ) , both in the entire population ( table 1 ) , and when stratified according to type of diabetes ( data not shown ) . eligible women unwilling to participate ( n = 47 ) were similar to included women with respect to type of diabetes , age , pregestational bmi , baseline hba1c , and parity , but had slightly shorter duration of diabetes ( data not shown ) . the included women were seen in our clinic at all five study visits at 8 ( 513 ) , 12 ( 1114 ) , 21 ( 1925 ) , 27 ( 2129 ) , and 33 ( 3235 ) weeks . self - monitored plasma glucose measurements were documented seven times daily in relation to all study visits in both arms ( table 1 for baseline data ) . real - time cgm was initiated at first pregnancy visit in all 79 women allocated to intervention and was generally well tolerated without severe side effects . forty - nine ( 64% ) women with either type 1 or type 2 diabetes used real - time cgm per protocol ( i.e. , at 8 , 12 , 21 , 27 , and 33 weeks or more ) . near - continuous real - time cgm use ( at least 60% of the time ) was only chosen by five ( 7% ) women . baseline clinical data in 154 included pregnant women with pregestational diabetes hba1c , self - monitored plasma glucose values , and plasma glucose profiles of biochemical hypo- or hyperglycemia were similar throughout pregnancy in women using real - time cgm versus control subjects for the entire study population ( table 2 ) , and for the subpopulation of women with type 1 diabetes alone ( supplementary table 1 ) . the women experienced 4 ( 014 ) mild hypoglycemic events per week , with no difference between the arms . the percentage of women experiencing severe hypoglycemia at least once after inclusion was also similar ( 16 vs. 16% ; p = 0.91 ) . of 38 ( 63% ) women with type 1 diabetes using real - time cgm per protocol , 4 ( 11% ) experienced severe hypoglycemia during the intervention period compared with 11 ( 19% ) among 59 control subjects ( p = 0.28 ) . nineteen ( 16% ) women with type 1 diabetes experienced 59 severe hypoglycemic events , and 5 ( 17% ) women with type 2 diabetes experienced 15 severe hypoglycemic events during study participation , with no difference between the arms ( data not shown ) . five women in the intervention arm experienced in total seven episodes of severe hypoglycemia despite ongoing and technically successful real - time cgm . insulin dosages were similar between women in the intervention versus control arm , both as absolute values and as percentages of pregestational dose at all study visits ( table 1 for baseline data and supplementary table 2 for the entire pregnancy according to type diabetes ) . glycemic control during pregnancy in 149 women with live births the 154 pregnancies resulted in five miscarriages and 149 live births . the predefined primary outcome , large - for - gestational - age infants , occurred to a similar extent in both arms ( 45 vs. 34% ; p = 0.19 ) , and no difference was found between the predefined secondary outcome : the prevalence of preterm delivery and/or severe neonatal hypoglycemia ( 29 vs. 22% ; p = 0.36 ) ( table 3 ) . other maternal and perinatal parameters were also comparable between the arms . even in the subpopulation of women with type 1 diabetes , similar results were found regardless of real - time cgm allocation ( supplementary table 3 ) . maternal and perinatal outcomes in 154 included women with pregestational diabetes for 49 ( 64% ) women with either type 1 or type 2 diabetes using real - time cgm per protocol , the figures for large - for - gestational - age infants and preterm delivery and/or severe neonatal hypoglycemia compared with control subjects ( n = 73 ) were 49 versus 34% ( p = 0.10 ) and 24 versus 22% ( p = 0.75 ) , respectively . for women with type 1 diabetes on insulin pumps ( n = 26 ) , the prevalence of large - for - gestational - age infants was 55 versus 20% ( p = 0.10 ) between the real - time cgm and control arms . major congenital malformations occurred in two infants of women with type 1 diabetes ( one ventricular septal defect combined with coarctation of the aorta and one congenitally corrected transposition of the great arteries ) . one case of perinatal death occurred shortly after delivery in an infant of a woman with type 2 diabetes due to severe shoulder dystocia . hba1c , self - monitored plasma glucose values , and plasma glucose profiles of biochemical hypo- or hyperglycemia were similar throughout pregnancy in women using real - time cgm versus control subjects for the entire study population ( table 2 ) , and for the subpopulation of women with type 1 diabetes alone ( supplementary table 1 ) . the women experienced 4 ( 014 ) mild hypoglycemic events per week , with no difference between the arms . the percentage of women experiencing severe hypoglycemia at least once after inclusion was also similar ( 16 vs. 16% ; p = 0.91 ) . of 38 ( 63% ) women with type 1 diabetes using real - time cgm per protocol , 4 ( 11% ) experienced severe hypoglycemia during the intervention period compared with 11 ( 19% ) among 59 control subjects ( p = 0.28 ) . nineteen ( 16% ) women with type 1 diabetes experienced 59 severe hypoglycemic events , and 5 ( 17% ) women with type 2 diabetes experienced 15 severe hypoglycemic events during study participation , with no difference between the arms ( data not shown ) . five women in the intervention arm experienced in total seven episodes of severe hypoglycemia despite ongoing and technically successful real - time cgm . insulin dosages were similar between women in the intervention versus control arm , both as absolute values and as percentages of pregestational dose at all study visits ( table 1 for baseline data and supplementary table 2 for the entire pregnancy according to type diabetes ) . the predefined primary outcome , large - for - gestational - age infants , occurred to a similar extent in both arms ( 45 vs. 34% ; p = 0.19 ) , and no difference was found between the predefined secondary outcome : the prevalence of preterm delivery and/or severe neonatal hypoglycemia ( 29 vs. 22% ; p = 0.36 ) ( table 3 ) . other maternal and perinatal parameters were also comparable between the arms . even in the subpopulation of women with type 1 diabetes , similar results were found regardless of real - time cgm allocation ( supplementary table 3 ) . maternal and perinatal outcomes in 154 included women with pregestational diabetes for 49 ( 64% ) women with either type 1 or type 2 diabetes using real - time cgm per protocol , the figures for large - for - gestational - age infants and preterm delivery and/or severe neonatal hypoglycemia compared with control subjects ( n = 73 ) were 49 versus 34% ( p = 0.10 ) and 24 versus 22% ( p = 0.75 ) , respectively . for women with type 1 diabetes on insulin pumps ( n = 26 ) , the prevalence of large - for - gestational - age infants was 55 versus 20% ( p = 0.10 ) between the real - time cgm and control arms . major congenital malformations occurred in two infants of women with type 1 diabetes ( one ventricular septal defect combined with coarctation of the aorta and one congenitally corrected transposition of the great arteries ) . one case of perinatal death occurred shortly after delivery in an infant of a woman with type 2 diabetes due to severe shoulder dystocia . in this randomized trial , intermittent use of real - time cgm in pregnancy , in addition to self - monitored plasma glucose measurements seven times daily , did not improve glycemic control or pregnancy outcome in women with pregestational diabetes . these findings are disappointing in the light of real - time cgm efficiency in nonpregnant patients ( 16 ) . however , participants in trials demonstrating an effect on hba1c in both nonpregnant ( 8,11,12,1719 ) and pregnant ( 27 ) populations had higher baseline hba1c compared our cohort of women . the negative findings of this trial are in line with a study on well - controlled nonpregnant patients , in whom hba1c was not further improved by real - time cgm ( 9 ) . recent data indicate the importance of frequent real - time cgm use ( 16 ) . our study was designed with intermittent real - time cgm use in relation to five routine antenatal visits at our clinic , but the women were encouraged to use real - time cgm continuously and offered much support and flexibility in scheduling to attain frequent real - time cgm use . still , only very few women were willing to use real - time cgm continuously . our aim was to improve our routine pregnancy care and therefore to intervene on an unselected cohort of women . ( 27 ) , whereas studies on nonpregnant patients with improved hba1c due to real - time cgm mainly cover selected and highly motivated patients , willing to use real - time cgm continuously ( 10,12,1719 ) . in this trial , sufficient numbers of women were included , and proper allocation with equal distribution of women with type 1 diabetes was secured with computer - generated randomization provided by an independent organization . study arms were comparable at baseline , both in the entire cohort and when analyzed according to type diabetes . we believe that our study design was robust , despite it not being blinded , and broadly comparable to that of murphy et al . real - time cgm had been used for several years in our clinic , and we have previously published case reports suggesting beneficial effects of real - time cgm in pregnancy ( 24,26 ) . therefore , we had expected high compliance to real - time cgm in this trial . however , the relatively low number of per - protocol users has also been observed in the nonpregnant population ( 17,18 ) , and the average use of cgm was comparable to the former study on pregnant women ( 27 ) . we have previously published the results of a questionnaire on patient satisfaction with real - time cgm in early pregnancy based on the present population of women ( 25 ) . this survey demonstrated that real - time cgm inaccuracy compared with self - monitored plasma glucose values , technical challenges , and skin irritation limit compliance ( 25 ) , which is in line with previous studies of nonpregnant ( 12,3235 ) and pregnant ( 27 ) patients . during the first period of real - time cgm , our women experienced up to 12 alarms per 24 h , future development of the real - time cgm systems , leading to improved accuracy in the hypoglycemic range and fewer alarms , may improve compliance . the main barrier in obtaining strict maternal glycemic control is the high risk of severe hypoglycemia , occurring in up to 45% of pregnant women with type 1 diabetes ( 7 ) . a reduction of severe hypoglycemic events due to real - time cgm has been suggested in a study of nonpregnant patients ( 11 ) and in our case study of pregnant women with hypoglycemia unawareness ( 26 ) . therefore , elimination of nocturnal hypoglycemic episodes was emphasized in our guidelines on the interpretation of real - time cgm readings in pregnancy . this may have lead to slightly higher nocturnal glucose levels and could at least partly explain the tendency toward higher birth weight z - score in the intervention arm . the self - monitored plasma glucose levels , which were mainly performed during daytime , and the number of women experiencing at least one severe hypoglycemic event after inclusion were similar in spite of real - time cgm allocation . however , among women with type 1 diabetes using real - time cgm per protocol , there was a tendency toward fewer women with severe hypoglycemia compared with women in the control arm , but numbers were low . surprisingly , we could document severe hypoglycemic events during ongoing real - time cgm without hypoglycemic alarms to warn the patient . this again calls for improved real - time cgm accuracy in the hypoglycemic range and future studies on continuous real - time cgm in pregnant women at high risk of severe hypoglycemia . whether a sum of imbalances including tendencies toward higher pregestational bmi , fewer smokers , and higher weight gain in pregnancy in women in the intervention arm could have the majority of the women with type 1 diabetes in this trial were on basal bolus therapy with multiple daily injections , but patients on insulin pumps may benefit more from real - time cgm ( 18 ) . however , not even in the subset of women treated with insulin pumps in this study was a tendency toward a beneficial effect on large - for - gestational - age infants seen . in conclusion , our data do not support intermittent real - time cgm on a routine basis in an unselected pregnant population of women with pregestational diabetes , already performing self - monitored plasma glucose measurements seven times per day . careful introduction to real - time cgm and interpretation of the real - time cgm readings were offered , but otherwise , the two arms were treated similarly regarding diet , exercise , and insulin therapy . the negative finding of this trial may lead to intensified focus on dietary advice , including carbohydrate counting , stable day - to - day carbohydrate intake , especially in late gestation ( 36 ) , and weight gain within the institute of medicine recommendations ( 37 ) . this trial does not rule out that real - time cgm may be efficient in highly selected pregnant women , and whether introducing continuous real - time cgm prior to pregnancy can reduce the risk of severe hypoglycemia and enhance pregnancy outcome is yet to be examined .

objectiveto assess whether intermittent real - time continuous glucose monitoring ( cgm ) improves glycemic control and pregnancy outcome in unselected women with pregestational diabetes.research design and methodsa total of 123 women with type 1 diabetes and 31 women with type 2 diabetes were randomized to use real - time cgm for 6 days at 8 , 12 , 21 , 27 , and 33 weeks in addition to routine care , including self - monitored plasma glucose seven times daily , or routine care only . to optimize glycemic control , real - time cgm readings were evaluated by a diabetes caregiver . hba1c , self - monitored plasma glucose , severe hypoglycemia , and pregnancy outcomes were recorded , with large - for - gestational - age infants as the primary outcome.resultswomen assigned to real - time cgm ( n = 79 ) had baseline hba1c similar to that of women in the control arm ( n = 75 ) ( median 6.6 [ range 5.310.0 ] vs. 6.8% [ 5.310.7 ] ; p = 0.67 ) ( 49 [ 3486 ] vs. 51 mmol / mol [ 3493 ] ) . forty - nine ( 64% ) women used real - time cgm per protocol . at 33 weeks , hba1c ( 6.1 [ 5.17.8 ] vs. 6.1% [ 4.88.2 ] ; p = 0.39 ) ( 43 [ 3262 ] vs. 43 mmol / mol [ 2966 ] ) and self - monitored plasma glucose ( 6.2 [ 4.77.9 ] vs. 6.2 mmol / l [ 4.97.9 ] ; p = 0.64 ) were comparable regardless of real - time cgm use , and a similar fraction of women had experienced severe hypoglycemia ( 16 vs. 16% ; p = 0.91 ) . the prevalence of large - for - gestational - age infants ( 45 vs. 34% ; p = 0.19 ) and other perinatal outcomes were comparable between the arms.conclusionsin this randomized trial , intermittent use of real - time cgm in pregnancy , in addition to self - monitored plasma glucose seven times daily , did not improve glycemic control or pregnancy outcome in women with pregestational diabetes .

RESEARCH DESIGN AND METHODS Patients Management of diabetes in pregnancy Other medications Randomization Intervention Pregnancy outcome parameters Sample size Statistical analysis RESULTS Glycemic control Pregnancy complications and outcome CONCLUSIONS

PMC4113169

recruitment to the whitehall ii study took place between 1985 and 1988 among all office staff , aged 3555 years , in 20 london - based civil service departments ( 22 ) . informed consent was obtained from all participants , and the university college london medical school committee on the ethics of human research approved the protocol . the target population for the current study was a sample of 5,932 participants ( 4,189 men and 1,743 women , mean age 54.6 years ) for whom data were collected in at least two of the following cycles : from 19911993 to 19971999 , from 19971999 to 20022004 , and from 20022004 to 20072009 . included participants had complete data on type 2 diabetes , psychological distress , frs ( 21 ) , and covariates ( age , sex , socioeconomic status [ ses ] , ethnicity , antidepressant use , smoking , and physical activity ) at baseline . those included in the analyses were free of diabetes at the baseline cycle(s ) and had data on their prediabetes status and frs ( fig . 1 ) . each participant could thus contribute to a minimum of one and a maximum of three cycles . the 5,932 eligible participants produced 13,207 person - observations ; mean follow - up time between baseline and follow - up for each cycle was 5.46 ( sd 0.51 ) years . , venous blood samples were taken from individuals who were fasting 8 h ( those whose clinic visit was in the afternoon had a light fat - free breakfast and they were asked to fast for 5 h ) before undergoing a standard 2-h 75-g oral glucose tolerance test ( ogtt ) ( 1 ) . mmol / l ( 2326 ) , self - reported doctor - diagnosed diabetes , or use of diabetes medication . blood samples were handled at all phases using similar standard protocols , and baseline cases were excluded from the prospective analyses . prediabetes was defined as impaired fasting glucose ( ifg ; a fasting glucose of 5.66.9 mmol / l and 2-h glucose < 7.8 mmol / l ) and/or impaired glucose tolerance ( a fasting glucose < 7 mmol / l and a 2-h postload glucose of 7.811.0 the frs is based on a previously published detailed algorithm ( 21 ) with the following components : ifg , overweight or obesity , low level of hdl , high level of triglycerides , elevated blood pressure or antihypertensive medication , and parental diabetes . , participants with impaired glucose tolerance received the same score as those with ifg ( 10 points in both cases ) . in the framingham offspring study , participants with > 19 points had an 8-year incidence of type 2 diabetes > 15% ( 21 ) ; thus we used this score to determine high risk status . participants with normoglycemia scored 018 in the frs and were further classified into the low - risk group ( 09 points ) and intermediate - risk group ( 1019 ) , the latter range corresponding to the lower ( i.e. , intermediate)-risk group ( 1019 points ) among participants with prediabetes . based on the baseline information on prediabetes status and the frs , participants were classified into four groups : 1 ) no prediabetes , low frs ( 09 ) ; 2 ) no prediabetes , intermediate frs ( 1019 ) ; 3 ) prediabetes , intermediate frs ( 1019 ) ; and 4 ) prediabetes , high frs ( > 19 ) . the 30-item general health questionnaire ( ghq-30 ) was used to assess psychological distress ( 27 ) . the ghq is a screening instrument designed to detect common psychological symptoms , such as depression and anxiety . it is widely used in population - based surveys and trials and has been validated in the whitehall ii study ( 28 ) . each questionnaire item inquires about a specific symptom ; response categories are scored as either 1 or 0 to indicate presence or absence of the symptom . a total score of 5 or more led to individuals being defined as ghq - symptom cases and scores 04 as noncases ( 28 ) . sociodemographic covariates included age , sex , ses ( based on the last known occupational grade and divided into high , intermediate , and low ) , and ethnicity ( white , south asian , or other ) and were all based on survey responses ( 22 ) . antidepressant use ( yes / no ) and smoking ( yes / no ) were based on self - reported information at the baseline survey of each cycle . physical activity was assessed at cycle 1 based on answers to questions about the frequency and duration of participation in mildly energetic ( e.g. , weeding , general housework , bicycle repair ) , moderately energetic ( e.g. , dancing , cycling , leisurely swimming ) , and vigorous physical activity ( e.g. , running , hard swimming , playing squash ) . at cycles 2 and 3 , the questionnaire included 20 items on frequency and duration of participation in different physical activities ( e.g. , walking , cycling , sports ) that were used to compute hours per week of each intensity level . participants were classified as active ( > 2.5 h / week of moderate physical activity or > 1 h / week of vigorous physical activity ) , inactive ( < 1 h / week of moderate physical activity and < 1 h / week of vigorous physical activity ) , or moderately active ( if not active or inactive ) ( 29 ) . after harmonization of data across cycles , we examined associations between psychological distress at baseline and incident type 2 diabetes at follow - up for each cycle . we used generalized estimation equations ( gees ) with a logistic link to control for intraindividual correlation between repeated measurements to estimate odds ratios ( ors ) and their 95% cis . gee analysis was used because repeated measurements were nested within participants ( i.e. , the same individuals could contribute more than one observation to the dataset ) , and the gee method takes into account nonindependence of the within - participant observations when estimating standard errors . we first analyzed the association between psychological distress and the incidence of type 2 diabetes in the total cohort . then we grouped the participants according to their baseline prediabetes status , frs , and psychological distress into eight groups as follows : 1 ) normoglycemia , frs 09 , no distress ( reference group ) ; 2 ) normoglycemia , score 09 , distress ; 3 ) normoglycemia , score 1019 , no distress ; 4 ) normoglycemia , score 1019 , distress ; 5 ) prediabetes , score 1019 , no distress ; 6 ) prediabetes , score 1019 , distress ; 7 ) prediabetes , score > 19 , no distress ; and 8) prediabetes , score > 19 , distress . models were adjusted for age , sex , ses , ethnicity , antidepressant use , smoking , and physical activity . sas version 9.2 ( sas , cary , nc ) was used for all analyses . recruitment to the whitehall ii study took place between 1985 and 1988 among all office staff , aged 3555 years , in 20 london - based civil service departments ( 22 ) . informed consent was obtained from all participants , and the university college london medical school committee on the ethics of human research approved the protocol . the target population for the current study was a sample of 5,932 participants ( 4,189 men and 1,743 women , mean age 54.6 years ) for whom data were collected in at least two of the following cycles : from 19911993 to 19971999 , from 19971999 to 20022004 , and from 20022004 to 20072009 . included participants had complete data on type 2 diabetes , psychological distress , frs ( 21 ) , and covariates ( age , sex , socioeconomic status [ ses ] , ethnicity , antidepressant use , smoking , and physical activity ) at baseline . those included in the analyses were free of diabetes at the baseline cycle(s ) and had data on their prediabetes status and frs ( fig . 1 ) . each participant could thus contribute to a minimum of one and a maximum of three cycles . the 5,932 eligible participants produced 13,207 person - observations ; mean follow - up time between baseline and follow - up for each cycle was 5.46 ( sd 0.51 ) years . at each clinical phase , venous blood samples were taken from individuals who were fasting 8 h ( those whose clinic visit was in the afternoon had a light fat - free breakfast and they were asked to fast for 5 h ) before undergoing a standard 2-h 75-g oral glucose tolerance test ( ogtt ) ( 1 ) . mmol / l ( 2326 ) , self - reported doctor - diagnosed diabetes , or use of diabetes medication . blood samples were handled at all phases using similar standard protocols , and baseline cases were excluded from the prospective analyses . prediabetes was defined as impaired fasting glucose ( ifg ; a fasting glucose of 5.66.9 mmol / l and 2-h glucose < 7.8 mmol / l ) and/or impaired glucose tolerance ( a fasting glucose < 7 mmol / l and a 2-h postload glucose of 7.811.0 mmol / l ) ( 23 ) . the frs is based on a previously published detailed algorithm ( 21 ) with the following components : ifg , overweight or obesity , low level of hdl , high level of triglycerides , elevated blood pressure or antihypertensive medication , and parental diabetes . , participants with impaired glucose tolerance received the same score as those with ifg ( 10 points in both cases ) . in the framingham offspring study , participants with > 19 points had an 8-year incidence of type 2 diabetes > 15% ( 21 ) ; thus we used this score to determine high risk status . participants with normoglycemia scored 018 in the frs and were further classified into the low - risk group ( 09 points ) and intermediate - risk group ( 1019 ) , the latter range corresponding to the lower ( i.e. , intermediate)-risk group ( 1019 points ) among participants with prediabetes . based on the baseline information on prediabetes status and the frs , participants were classified into four groups : 1 ) no prediabetes , low frs ( 09 ) ; 2 ) no prediabetes , intermediate frs ( 1019 ) ; 3 ) prediabetes , intermediate frs ( 1019 ) ; and 4 ) prediabetes , high frs ( > 19 ) . the 30-item general health questionnaire ( ghq-30 ) was used to assess psychological distress ( 27 ) . the ghq is a screening instrument designed to detect common psychological symptoms , such as depression and anxiety . it is widely used in population - based surveys and trials and has been validated in the whitehall ii study ( 28 ) . each questionnaire item inquires about a specific symptom ; response categories are scored as either 1 or 0 to indicate presence or absence of the symptom . a total score of 5 or more led to individuals being defined as ghq - symptom cases and scores 04 as noncases ( 28 ) . sociodemographic covariates included age , sex , ses ( based on the last known occupational grade and divided into high , intermediate , and low ) , and ethnicity ( white , south asian , or other ) and were all based on survey responses ( 22 ) . antidepressant use ( yes / no ) and smoking ( yes / no ) were based on self - reported information at the baseline survey of each cycle . physical activity was assessed at cycle 1 based on answers to questions about the frequency and duration of participation in mildly energetic ( e.g. , weeding , general housework , bicycle repair ) , moderately energetic ( e.g. , dancing , cycling , leisurely swimming ) , and vigorous physical activity ( e.g. , running , hard swimming , playing squash ) . at cycles 2 and 3 , the questionnaire included 20 items on frequency and duration of participation in different physical activities ( e.g. , walking , cycling , sports ) that were used to compute hours per week of each intensity level . participants were classified as active ( > 2.5 h / week of moderate physical activity or > 1 h / week of vigorous physical activity ) , inactive ( < 1 h / week of moderate physical activity and < 1 h / week of vigorous physical activity ) , or moderately active ( if not active or inactive ) ( 29 ) . after harmonization of data across cycles , we examined associations between psychological distress at baseline and incident type 2 diabetes at follow - up for each cycle . we used generalized estimation equations ( gees ) with a logistic link to control for intraindividual correlation between repeated measurements to estimate odds ratios ( ors ) and their 95% cis . gee analysis was used because repeated measurements were nested within participants ( i.e. , the same individuals could contribute more than one observation to the dataset ) , and the gee method takes into account nonindependence of the within - participant observations when estimating standard errors . we first analyzed the association between psychological distress and the incidence of type 2 diabetes in the total cohort . then we grouped the participants according to their baseline prediabetes status , frs , and psychological distress into eight groups as follows : 1 ) normoglycemia , frs 09 , no distress ( reference group ) ; 2 ) normoglycemia , score 09 , distress ; 3 ) normoglycemia , score 1019 , no distress ; 4 ) normoglycemia , score 1019 , distress ; 5 ) prediabetes , score 1019 , no distress ; 6 ) prediabetes , score 1019 , distress ; 7 ) prediabetes , score > 19 , no distress ; and 8) prediabetes , score > 19 , distress . models were adjusted for age , sex , ses , ethnicity , antidepressant use , smoking , and physical activity . sas version 9.2 ( sas , cary , nc ) was used for all analyses . table 1 shows the characteristics of participants at the baseline of each study cycle and in total . the proportion of participants who were male and white , had high ses , were without psychological distress , and had incident type 2 diabetes was greater at the last cycle than at the first . antidepressant use was more prevalent and smoking less prevalent and both high and low physical activity were more prevalent in the latter cycles . characteristics of the participants at the baseline of the three cycles figures are number ( % ) unless otherwise stated . * total n refers to the sum of participants ( n of person - observations ) in total and across the three study cycles ( one participant can contribute to one or more study cycle ) ; n in each study cycle refers to number of participants at that cycle . associations between baseline covariates for participants with normoglycemia and prediabetes by psychological distress are presented in supplementary table 1 . irrespective of the participant s prediabetes status , psychological distress was associated with younger age , female sex , intermediate ses , nonwhite ethnicity , antidepressant use , smoking , and low physical activity . in the total cohort , psychological distress did not predict type 2 diabetes after adjustment for age , sex , ses , and ethnicity ( or 1.16 [ 95% ci 0.941.42 ] ; data not shown ) . we found no interaction between sex and psychological distress ( p = 0.37 ) or between ethnicity and psychological distress ( p = 0.91 ) in the prediction of type 2 diabetes . we then examined whether combinations of prediabetes status , frs , and psychological distress predicted the incidence of type 2 diabetes . type 2 diabetes incidence was low among participants with normoglycemia and a low risk score , irrespective of the presence ( 1.9% ) or absence ( 1.6% ) of psychological distress . among the normoglycemic participants with an intermediate risk score of 1019 , 7.6% of distressed and 6.0% of nondistressed people had type 2 diabetes at follow - up ; the confidence intervals suggesting no association with psychological distress . similarly , among participants with prediabetes and an frs of 1019 , no difference was found in the incidence of type 2 diabetes between those with ( 15.6% ) and without psychological distress ( 15.0% ) . however , among participants with prediabetes and a high frs ( > 19 ) , 40.9% of those with psychological distress developed type 2 diabetes at follow - up compared with only 28.5% of those without psychological distress . unadjusted incidence ( 95% ci ) of type 2 diabetes among participants with normoglycemia and participants with prediabetes ; participants further stratified by the frs and psychological distress . results of the multivariable - adjusted logistic regression models confirm those from the unadjusted analysis by showing a strong dose - response association between prediabetes and frs status , and risk of incident type 2 diabetes ( table 2 ) . moreover , comparisons indicate no difference regarding the association between psychological distress and type 2 diabetes among normoglycemic participants or among those with prediabetes and a lower frs , whereas among participants with prediabetes and a high frs ( > 19 ) , psychological distress was associated with a doubling of the risk of type 2 diabetes . a statistically significant interaction ( p = 0.039 ) high frs group with the other groups combined , as regards the association between psychological distress and incident type 2 diabetes . incidence of type 2 diabetes at follow - up among participants with normoglycemia and participants with prediabetes at baseline ; participants further stratified by frs and psychological distress alternative reference groups are shown in comparisons 18 . models are adjusted for age , sex , ses , ethnicity , antidepressant use , smoking , and physical activity . all comparisons are based on the same data , but they have a different reference group . or , odds ratio . the findings were replicated in sensitivity analysis using an alternative , less stringent cut point of > 18 to define high frs ( supplementary table 2 ) . we examined whether the association between psychological distress and incident type 2 diabetes differed between populations at different baseline risk levels of type 2 diabetes as assessed by the presence of prediabetes and the level of frs . the frs is based on traditional type 2 diabetes risk factors : prediabetes , overweight or obesity , low hdl level , increased level of triglycerides , hypertension , and a history of parental diabetes . in the current study , our main finding was that psychological distress is associated with a doubling of diabetes risk in a high - risk populations . in the overall analysis , psychological distress was not significantly associated with type 2 diabetes . subsequent stratified analysis revealed no association between psychological distress and type 2 diabetes in normoglycemic participants irrespective of the risk score and in participants with prediabetes and a lower frs . however , in the group of participants with prediabetes and high risk score ( > 19 ) , the 5-year incidence of diabetes was 28.5% in those without psychological distress and 40.9% in those with psychological distress at baseline . in the multivariate - adjusted model , the or was twofold increased among those with psychological distress compared with those without . the findings were replicated using a lower cut point ( > 18 ) for defining a high frs . earlier literature suggests that the relationship between psychosocial factors and type 2 diabetes may be complex ( 16,17 ) . earlier findings on the association between psychological factors , such as general stress and work stress , and incident type 2 diabetes have been mixed , including both null and positive findings ( 1017 ) . inconsistencies in earlier research may be due not only to the use of different stress and distress indicators across studies but also , as our present findings suggest , to a failure to recognize that the nondiabetes group might be heterogeneous in terms of vulnerability to distress ; those at an advanced stage of prediabetes may be more affected by the adverse metabolic effects of psychological distress than those at lower risk levels ( 1,17 ) . indeed , the effects of psychological stress factors have been suggested to be synergistic ( 17 ) . rather than a general risk factor in all diabetes - free populations , it might be a stage - specific risk factor that has a much stronger effect among those at an advanced stage of progression toward manifest type 2 diabetes . in line with our results , an earlier report from the whitehall ii study showed that work stress predicted type 2 diabetes among obese , but not nonobese , women ( 14 ) . plausible pathways through which psychological distress may accelerate progression to type 2 diabetes among high - risk individuals include health risk behaviors and direct physiological pathways , such as long - term dysregulation of the hypothalamic - pituitary - adrenal axis , leading to increased levels of glucocorticoids , especially cortisol , and changes in immune system activity , leading to increased concentrations of proinflammatory cytokines ( 1,8,16,17 ) . there is some evidence supporting inflammation and lifestyle factors as mediators between depressive symptoms and incident type 2 diabetes ( 8) . healthcare implications of this study include the notion that psychological distress might hamper the outcomes of intensive lifestyle and other treatment interventions targeted at high - risk groups ( 2,3 ) . psychological distress symptoms , such as stress , anxiety , depression , feelings of hopelessness , and sleep disturbances , have previously been shown to hinder commitment to major , long - term lifestyle changes and reduce adherence to pharmacological treatments ( 30 ) . first , although the study had a high response rate in the successive data collection phases , loss to follow - up accumulated over time , as in most long - term cohort studies . however , large differences in missing data as a function of psychological distress and type 2 diabetes seem unlikely . second , participants of the whitehall ii study are from an occupational cohort that is likely to cover a healthier end of the variation in health status compared with the general population , which limits the generalizability of our findings . as this instrument was not designed to make a psychiatric diagnosis of depression or anxiety , we can not exclude the possibility of confounding by unmeasured depression or anxiety disorders . however , this is unlikely to be a major source of bias because the ghq-30 has been shown to be a valid screening instrument for mental disorders , particularly depression in the whitehall ii study ( 28 ) . the strengths of this study are its large sample size and use of accurate , repeat assessments of all examined variables , use of the frs based on clinical measurements , and ascertainment of diabetes based on the standard 75-g ogtt at each clinical study cycle ( 23,24 ) . however , part of the incident type 2 diabetes identification was based on self - report , although antihyperglycemic medication was confirmed by asking the participants to take their medications or list of medications to the study clinic . of those participants who had self - reported diagnosis of diabetes without evidence on the use of antihyperglycemic medications ( 33.4% of incident cases ) , a substantial proportion was confirmed by a repeat ogtt or by antihyperglycemic medication at the subsequent phase , leaving only 23.6% of all incident diabetes cases unconfirmed . in summary , this observational study suggests that psychological distress may be related to accelerated progression of late - stage prediabetes to clinical diabetes . further investigations are needed to examine mechanisms linking psychological distress to onset of type 2 diabetes in this group . current prevention guidelines do not generally consider psychological factors such as stress or depression ( 31 ) , although some recognize them as contributing factors to be taken into account in efforts to prevent type 2 diabetes ( 32 ) . given the high comorbidity between psychological problems and diabetes and the accumulation of evidence on the role of psychological distress as a predictor of type 2 diabetes , it is important to consider whether more attention should be paid to psychological status among high - risk populations in addition to lifestyle modifications .

objectivewe examined whether psychological distress predicts incident type 2 diabetes and if the association differs between populations at higher or lower risk of type 2 diabetes.research design and methodsthis was a prospective cohort of 5,932 diabetes - free adults ( 4,189 men and 1,743 women , mean age 54.6 years ) with three 5-year data cycles ( 19912009 ) : a total of 13,207 person - observations . participants were classified into four groups according to their prediabetes status and framingham offspring type 2 diabetes risk score : normoglycemia with a risk score of 09 , normoglycemia with a risk score of 1019 , prediabetes with a risk score of 1019 , and prediabetes with a risk score of > 19 . psychological distress was assessed by the general health questionnaire . incident type 2 diabetes was ascertained by 2-h oral glucose tolerance test , doctor diagnosis , or use of antihyperglycemic medication at the 5-year follow - up for each data cycle . adjustments were made for age , sex , ethnicity , socioeconomic status , antidepressant use , smoking , and physical activity.resultsamong participants with normoglycemia and among those with prediabetes combined with a low risk score , psychological distress did not predict type 2 diabetes . diabetes incidence in these groups varied between 1.6 and 15.6% . among participants with prediabetes and a high risk score , 40.9% of those with psychological distress compared with 28.5% of those without distress developed diabetes during the follow - up . the corresponding adjusted odds ratio for psychological distress was 2.07 ( 95% ci 1.193.62).conclusionsthese data suggest that psychological distress is associated with an accelerated progression to manifest diabetes in a subpopulation with advanced prediabetes .

Research Design and Methods Participants and Procedure Ascertainment of Type 2 Diabetes, Prediabetes, and Type 2 Diabetes Risk Status Assessment of Psychological Distress Assessment of Covariates Statistical Analysis Results Conclusions Supplementary Material

PMC4454776

drosophila circadian locomotor rhythms are generated by subsets of clock - containing neurons in the brain . under light - dark ( ld ) cycles , the small ventral lateral neurons ( s - lnvs ; m oscillators ) and a small subset of lateral and dorsal neurons named e oscillators drive morning and evening activity peaks . in constant darkness ( dd ) , the s - lnvs control the synchrony of clock neurons and drive behavioral rhythms , thus serving as the master pacemakers [ 35 ] . nuclear receptors are ligand - dependent transcription factors that regulate diverse biological processes . a number of nuclear receptors are known to play key roles in the molecular clock and its output pathways in mammals . with the exception of unfulfilled ( unf ; dhr51 ) , which is required for the pacemaker function of the s - lnvs , whether nuclear receptors provide important regulatory points in drosophila circadian rhythms remains unclear . to test the requirements of the nuclear receptors in drosophila circadian rhythms , we sought to knock down each of the 18 nuclear receptor genes in the s - lnvs . we chose to use recently generated uas - micrornas ( mirnas ) targeting fly nuclear receptors because each uas line polycistronically expresses two independent mirnas , which permit the efficient silencing of the target with a minimum off - target effect [ 8 , 9 ] . we expressed uas - mirnas with the lnv - specific gal4 driver , pdf - gal4 or gal1118 , and analyzed the locomotor activities of the flies in ld and dd . consistent with our previous results , the knockdown of unf rendered flies arrhythmic in dd . additionally , the knockdown of e75 ( also known as ecdysone - induced protein 75 , eip75b ) in the lnvs led to a similarly high proportion of arrhythmia in dd ( figures s1a and s1b ) . the knockdown of seven - up ( svp ) or estrogen - related receptor ( err ) had a moderate effect on free - running rhythms ( table 1 ) . the e75 gene produces three isoforms of e75 , named e75a , e75b , and e75c , all of which share a large part of the c - terminal region , including the ligand - binding domain , but differ in their n terminus structures . despite these differences , all the isoforms are functionally redundant to some extent in ecdysone - induced developmental processes . e75 isoform redundancy is at least partly explained by the fact that all three isoforms , including e75b ( which lacks dna - binding domain ) , can heterodimerize with the dhr3 nuclear receptor and repress its transcriptional activity [ 12 , 13 ] . our previous rna analysis from isolated lnvs showed that all e75 isoforms are expressed in both larval and adult lnvs [ 14 , 15 ] . the uas - mirnas against e75 targets two sequences in the common c - terminal domain , thereby enabling the knockdown of all the isoforms . expression of uas - e75 mirnas under the control of ubiquitously expressed tubulin - gal4 driver caused near 100% embryonic lethality , which recapitulates the lethal phenotype of the e75 null mutants lacking all isoforms . by contrast , expression of uas - rnai targeting e75 from the vienna drosophila resource center ( vdrc ) collection with tubulin - gal4 had no effect on viability ( figure s2a ) . lnv - targeted expression of e75 mirnas rendered flies arrhythmic , whereas expression of either of the two vdrc uas - rnai lines had no effect on the locomotor rhythms ( figure s2b ) . these results indicate that uas - mirnas efficiently silences the e75 gene and confirm that behavioral arrhythmia is specifically caused by the reduction of the e75 expression in the lnvs . e75 is a homolog of mammalian rev - erb and rev - erb , which are important transcriptional regulators contributing to the molecular clockwork and the clock output [ 1618 ] . intrigued by the high proportion of arrhythmia in the knockdown and the relevance to the circadian rhythms in mammals , we further investigated how e75 contributes to the behavioral rhythm generation in the lnvs . first , we overexpressed e75a , the isoform containing both dna - binding and ligand - binding domains , in the lnvs . these flies showed no differences in locomotor behavior compared with the control ( figures s1a and s1b ) . thus , loss of function , but not overexpression , of e75 in the lnvs impairs the free - running locomotor rhythms . because the knockdown of dhr3 had no effect on behavioral rhythms ( table 1 ) , the e75/dhr3 heterodimer is not a likely candidate that is involved in the behavioral control in the s - lnvs . we next examined the effect of e75 loss of function in developing and adult lnvs separately via stage - specific knockdown using a combination of uas - e75 mirnas , pdf - gal4 , and a temperature - sensitive gal80 expressed under the control of the tubulin promoter ( tub - gal80 ) . to analyze the role of e75 in the developing lnvs , we raised these flies until eclosion at 29c ( the restrictive temperature of gal80 ) , enabling the pdf > e75 mirnas expression . the eclosed flies were transferred to 18c ( the permissive temperature of gal80 ) to stop the expression of e75 mirnas and subsequently tested for locomotor activity under this condition . strikingly , 100% of these flies were arrhythmic in dd . this is consistent with the results of the e75 constitutive knockdown performed at 25c . by staining for pdf and per , we found that all the pdf - positive s - lnvs were undetectable , whereas the l - lnvs and the pdf - negative fifth s - lnv were normal in number and morphology ( figure 1c ) . although this does not exclude the possibility that the s - lnvs are still present , it nevertheless implies that at least multiple genetic programs are severely impaired in the s - lnvs . these results indicate that e75 is critically required for the development of the s - lnvs , which are the master pacemaker neurons for free - running rhythms ; therefore , e75 gene silencing during development renders adult flies arrhythmic in dd . to determine whether e75 in adult lnvs contributes to the generation of locomotor rhythms , we next knocked down e75 only during adulthood by raising the flies carrying uas - e75 mirnas , pdf - gal4 , and tub - gal80 at 18c until eclosion and incubating the adult flies at 29c . the adult - specific lnv - targeted e75 knockdown lengthened the free - running period by over 3 hr ( figures 1d and 1e ) . by staining brains at six time points on the third day in dd ( dd3 ) with anti - pdf / per double staining , we found that both s - lnvs and l - lnvs were present and morphologically normal in the adult - specific e75 knockdown flies ( figure 1f ) , but per levels and oscillations were substantially reduced in the s - lnvs in dd ( figures 2a and 2d ) . these results indicate that e75 is indispensable for robust free - running molecular rhythms in adult s - lnvs . in contrast , per rhythms in the dorsal lateral neurons ( lnds ) were unaffected by this manipulation ( figures 2b and 2d ) . in the dorsal neurons 1 ( dn1s ) , the phase of per rhythms was approximately 12 hr delayed in the knockdown flies compared with the control , indicating that e75 knockdown in adult lnvs non - cell - autonomously slowed down the clocks in the dn1s by approximately 4 hr per day ( figures 2c and 2d ) . the pace of the dn1 clocks matches the behavioral pace in the e75 knockdown flies , suggesting that the dn1s compensate for the dampened rhythms in m oscillators and help drive the behavioral rhythms in these flies . these results are consistent with the previous findings that modulation of the properties of the s - lnvs alters the phase and amplitude of the dn1s [ 7 , 20 , 21 ] , and the dn1s can play a major role in behavioral rhythm generation when s - lnvs are dysfunctional [ 2124 ] . taken together with the results of the e75 developmental knockdown , we conclude that e75 is essential for the development of the s - lnvs and for the molecular clockwork in adult s - lnvs . similar to e75 , the nuclear receptor unf ; dhr51 is expressed in the lnvs throughout development and adulthood and is required for the proper functioning of the s - lnvs as the circadian master pacemakers [ 7 , 15 ] . unf knockdown in developing lnvs does not affect the gross structure of the lnvs but leads to the complete disruption of free - running clocks in adult s - lnvs . unf downregulation in adult lnvs dampens their molecular oscillation in dd , resulting in long - period free - running rhythms . thus , the loss of function of unf in adult lnvs partially phenocopies that of e75 . this further suggests that unf and e75 might function in the same pathway controlling the molecular clocks in the s - lnvs . we next addressed this possibility by testing the genetic interaction between unf and e75 in adult lnvs . interestingly , unf and e75 double knockdown in adult lnvs rendered flies arrhythmic in dd . overexpression of e75 did not alter the lengthened free - running period in the unf knockdown flies ( figures s3a and s3b ) . because behavioral rhythms were normal when double knockdown flies were kept at 18c throughout developmental and adult stages , behavioral arrhythmia in the adult - specific knockdown flies was not caused by leaky mirna expression during development ( figure s3c ) . these results indicate that unf and e75 genetically interact in the pathway controlling the molecular clockwork in the s - lnvs , but e75 is unlikely to be downstream of unf . to understand the mechanisms by which e75 controls molecular clockwork in the s - lnvs and how unf takes part in this process , we sought to identify the e75 downstream target genes . because the adult - specific e75 knockdown in the lnvs not only diminishes the molecular oscillation but also dramatically reduces per levels ( figure 2a and 2d ) , we speculated that e75 controls the transcription of the gene(s ) directly involved in the molecular oscillation . because the dna - binding domains of e75 and mammalian rev - erb and rev - erb are highly conserved , we used the well - characterized rev - erb and rev - erb binding sequences to find potential e75 binding sites in drosophila core clock genes . rev - erb / is known to bind to the ror element ( rore ) ( [ a / t]a[a / t]nt[a / g]ggtca ) as a monomer . interestingly , a consensus rore was found in the first intron of the per gene . moreover , the rore is located near the clk / cyc binding peaks identified in ( figure 3c ) . these bioinformatic analyses predict that e75 could modulate the clk / cyc - mediated transcription of per . to test this possibility , we performed transactivation assays in schneider 2 ( s2 ) cells , which permit the analysis of transcriptional regulation in a non - cycling context . we transiently transfected s2 cells with varying combinations of clk , e75a , and unf expression constructs and analyzed the endogenous per and tim mrna levels by qpcr ( figure 3a ) . in agreement with a previous study , because cyc is expressed in s2 cells , the expression of clk alone significantly upregulated the per and tim rna levels . unf or e75 alone or together but without clk had no effect on the per and tim rna levels . when either unf or e75 was co - expressed with clk , we observed a small but non - significant increase of per mrna levels compared with clk transfection alone . strikingly , the addition of unf and e75 together to clk dose dependently increased per mrna levels by at least 4-fold . neither unf nor e75 affected the tim rna levels when co - expressed with clk . to examine the extent to which unf and e75 transcriptionally control per expression , we generated luciferase reporters to monitor transcription from wild - type per promoter ( per - luc ) and per promoter lacking rore ( delta rore - luc [ drore - luc ] ) in s2 cell co - transcription assay . expression of unf significantly increased clk - dependent transcription from both promoters , whereas e75 caused only a slight and non - significant increase . unf / e75/clk co - expression yielded higher transcriptional activity than unf / clk transfection ; specifically , the increase of the drore - luc activity was statistically significant . intriguingly , drore - luc activity was significantly higher than per - luc in both unf / clk and unf / e75/clk co - transfections ( figure 3b ) . this suggests that transcriptional repression through rore is involved in the overall transcriptional control of per , but other domains within the reporter construct mediate net transcriptional activation . compared to the strong upregulation of per mrna level by unf / e75/clk co - transfection ( figure 3a ) , the difference between unf / clk and unf / e75/clk transfection on per - luc activation was less evident . this probably reflects that regulatory sequences outside the region cloned in the luciferase reporters are also involved in the regulation of per transcription . together with the data indicating that unf / e75 co - transfection synergistically activates transcription of drore - luc , these results suggest that unf upregulates clk / cyc - mediated per transcription and e75 enhances this process , at least via proximal and intronic promoters , except rore . to test whether unf and e75 bind to per regulatory sequences and thereby regulate transcription , we performed chromatin immunoprecipitation ( chip ) in s2 cells transfected with epitope - tagged clk , unf , and e75 and analyzed the binding by qpcr . as expected , clk was bound to the per gene regulatory sequences around circadian regulatory sequence ( crs ) , rore , and a downstream e - box ( e8 ) , reminiscent of prior chip studies [ 26 , 28 ] ( figure s3d ) . strikingly , although e75 binding was unaffected by the co - expression of unf , unf binding was approximately 3- to 4-fold reduced when co - expressed with e75 ( figure 3c ) . these data suggest that both unf and e75 can independently bind to per regulatory sequences , but the presence of e75 increases the turnover of unf binding . this high turnover of unf binding is concomitant with higher transcriptional activity ( figure 3d ) . this model is in concert with the notion that transcriptional activation from highly regulated promoters often requires the turnover of the nuclear receptors and their co - regulators . taken together with the behavioral results ( figure s3a ) , these in vitro data suggest that coordinated action of e75 and unf on per transcription is essential for the molecular clock machinery in the s - lnvs . here , we identified the nuclear receptors e75 and unf as components of the molecular clocks in the s - lnvs . e75 is the closest homolog of mammalian rev - erb and rev - erb , which play important roles in the molecular clock feedback loops . in contrast with rev - erb / , which represses transcription , our results demonstrated that e75 is neither a potent repressor nor a strong activator but potentiates the activation of per transcription by unf . despite these mechanistic divergences , the notion that rev - erb homologs are integral to the molecular oscillators in both drosophila and mammals highlights the significance of transcriptional regulations via nuclear receptors in metazoan circadian clocks . rev - erb and rev - erb are rhythmically transcribed by the clock / bmal1 transcriptional activators , and rev - erbs periodically repress the transcription of bmal1 , thereby forming a feedback loop to ensure robust molecular oscillations of the mammalian clock . a previous study demonstrated that e75 is a cycling target of clk / cyc in the fly head . because e75 has three isoforms , we were unable to determine whether any of the isoforms were rhythmically expressed in the lnvs from the rna profiles of the isolated lnvs . nonetheless , our results indicate that e75 together with unf ( which is not a clk / cyc target ) reinforces the main loop of the core fly clock composed of clk / cyc and per / tim through a feedforward mechanism , showcasing the mechanistic parallels between fly and mammalian clocks . e75 has been demonstrated to covalently bind to heme , and its binding appears to stabilize the e75 and facilitates the binding of nitric oxide ( no ) and carbon monoxide ( co ) . the no / co binding to e75 modulates the transcriptional activity of its known heterodimeric partner dhr3 [ 13 , 30 ] . to test whether similar mechanisms are involved in the action of e75 in the s - lnvs , we attempted to disrupt cellular heme metabolism by knocking down the enzymes in the heme biosynthesis pathway , coproporphyrinogen oxidase ( coprox ) and protoporphyrinogen oxidase ( ppox ) , and the key enzyme in the heme degradation pathway , heme oxygenase ( ho ) . these experiments were inconclusive , as we did not observe any effect on the behavioral rhythms by any knockdown with pdf - gal4 , and knockdown with tim - gal4 was lethal ( data not shown ) . s2 cell experiments showed that unf is a transcriptional activator of per , and concurrent expression of e75 and unf increases the turnover of unf binding to per regulatory sequences . the finding that e75 acts through unf on transcription is consistent with our in vivo data : ( 1 ) depletion of both unf and e75 in adult lnvs abolishes the behavioral rhythms ( figure s3a ) ; ( 2 ) e75 overexpression has no effect on the behavioral rhythms ( figure s1a ) ; and ( 3 ) e75 overexpression does not rescue unf knockdown ( figure s3a ) . although unf mrna levels do not oscillate , unf protein levels cycle in the s - lnvs , peaking at zeitgeber time ( zt)2 and lowest at zt14 . indeed , per is most actively transcribed around zt13 when unf levels are minimum in the s - lnvs . nonetheless , downregulation and arrhythmia of per levels in the s - lnvs is most probably not the sole cause of the altered locomotor rhythms in the unf knockdown , e75 knockdown , and unf / e75 double knockdown . a recent study showed the implication of e75 in the repression of clk transcription , although our results are not in concordance with this observation probably due to the differences in the reagents used for e75 knockdown and the timing of knockdown . deciphering whether e75 and unf heterodimerize or bind to adjacent sequences , how they cooperate with clk / cyc , and whether any ligand is involved in their transcriptional regulation will yield new insights into the diverse mode of nuclear receptor crosstalk and their critical roles in circadian biology .

summarycircadian clocks in large part rely on transcriptional feedback loops . at the core of the clock machinery , the transcriptional activators clock / bmal1 ( in mammals ) and clock / cycle ( clk / cyc ) ( in drosophila ) drive the expression of the period ( per ) family genes . the per - containing complexes inhibit the activity of clock / bmal1 or clk / cyc , thereby forming a negative feedback loop [ 1 ] . in mammals , the ror and rev - erb family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation [ 2 ] . despite the overall similarities between mammalian and drosophila clocks , whether comparable mechanisms via nuclear receptors are required for the drosophila clock remains unknown . we show here that the nuclear receptor e75 , the fly homolog of rev - erb and rev - erb , and the nr2e3 subfamily nuclear receptor unf are components of the molecular clocks in the drosophila pacemaker neurons . in vivo assays in conjunction with the in vitro experiments demonstrate that e75 and unf bind to per regulatory sequences and act together to enhance the clk / cyc - mediated transcription of the per gene , thereby completing the core transcriptional feedback loop necessary for the free - running clockwork . our results identify a missing link in the drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks .

Results Discussion Experimental Procedures

PMC3655566

pediatric glaucoma includes a wide variety of conditions which result in elevated intraocular pressure and optic nerve damage , ranging from primary congenital glaucoma since birth to developmental glaucoma associated with other diseases and acquired glaucoma secondary to multiple causes . depending on the age of the patient , it presents with particular features and circumstances that need to be taken into account and frequently require surgical intervention . traditional surgical procedures are evolving , and the choices are increasing as diagnostic advances , surgical instrumentation , and newer techniques emerge . the prognosis of the disease has significantly improved over the last half century because of the development of angle surgery , trabeculectomy , seton implantation , and use of antimetabolites . the purpose of this paper is to review the literature on the techniques currently available and their results , for the management pediatric glaucoma , and to offer guidelines on what elements to consider when taking a surgical decision for these patients . several issues , pertinent to the visual outcome of pediatric glaucoma , need to be considered before undertaking surgery . unique features of glaucoma in infants are as follows distensibility of the globe from birth until age 2 - 3 which can cause stretching at all levels of the eye : cornea ( increased corneal diameter ) , anterior chamber angle ( shifting of structures ) , sclera ( globe enlargement and axial myopia , generalized scleral thinning , and localized staphylomas ) , optic nerve ( cupping ) , scleral canal ( enlarged disc diameter ) , and lamina cribrosa ( posterior displacement ) [ 27];capability to produce amblyopia through persistent media opacities and uncorrected anisometropia or irregular astigmatism [ 810];corneal opacities initially caused by epithelial corneal edema and later by permanent stromal edema [ 10 , 11 ] and breaks in descemet 's membrane . distensibility of the globe from birth until age 2 - 3 which can cause stretching at all levels of the eye : cornea ( increased corneal diameter ) , anterior chamber angle ( shifting of structures ) , sclera ( globe enlargement and axial myopia , generalized scleral thinning , and localized staphylomas ) , optic nerve ( cupping ) , scleral canal ( enlarged disc diameter ) , and lamina cribrosa ( posterior displacement ) [ 27 ] ; capability to produce amblyopia through persistent media opacities and uncorrected anisometropia or irregular astigmatism [ 810 ] ; corneal opacities initially caused by epithelial corneal edema and later by permanent stromal edema [ 10 , 11 ] and breaks in descemet 's membrane . particular challenges regarding glaucoma surgery in infants are as follows lack of cooperation : requiring examination under sedation or anesthesia before or after surgery;difficulty in protecting consistently and adequately the operated eye , applying medications and monitoring eye for complications and response to surgery . anatomic differences : smaller palpebral fissure and less rigid and thinner sclera , different than expected location of landmarks in buphthalmic eyes.first surgery has a greater chance of success . this is important because it needs to last longer than in adults because of longer life expectancy . therefore , it is not advisable to be performed by the occasional or untrained surgeon on this kind of surgery . it has been recommended by some that it should be done at an ophthalmic referral center that receives at least 20 new cases per year as well as having considerable experience with adult glaucoma surgery . considerable volume and experience of the referral center in dealing not only with skillful glaucoma surgery but also with safe anesthesia is very important . lack of cooperation : requiring examination under sedation or anesthesia before or after surgery;difficulty in protecting consistently and adequately the operated eye , applying medications and monitoring eye for complications and response to surgery . requiring examination under sedation or anesthesia before or after surgery ; difficulty in protecting consistently and adequately the operated eye , applying medications and monitoring eye for complications and response to surgery . anatomic differences : smaller palpebral fissure and less rigid and thinner sclera , different than expected location of landmarks in buphthalmic eyes . this is important because it needs to last longer than in adults because of longer life expectancy . therefore , it is not advisable to be performed by the occasional or untrained surgeon on this kind of surgery . it has been recommended by some that it should be done at an ophthalmic referral center that receives at least 20 new cases per year as well as having considerable experience with adult glaucoma surgery . considerable volume and experience of the referral center in dealing not only with skillful glaucoma surgery but also with safe anesthesia is very important . in order to review in a more systematic way the surgical approaches to the wide variety of glaucomas in children , we will refer to the different glaucomas in children as follows [ 1 , 14 ] : primary congenital glaucoma ( newborn and infantile ) , when an isolated idiopathic developmental abnormality of the anterior chamber angle exists . glaucomas associated with congenital anomalies ; aqueous outflow is reduced due to congenital ocular or systemic disorder.acquired glaucoma ; the outflow impairment is the result of acquired ocular disease or systemic abnormality . primary congenital glaucoma ( newborn and infantile ) , when an isolated idiopathic developmental abnormality of the anterior chamber angle exists . glaucomas associated with congenital anomalies ; aqueous outflow is reduced due to congenital ocular or systemic disorder . acquired glaucoma ; the outflow impairment is the result of acquired ocular disease or systemic abnormality . histopathological correlation has been attempted to support this classification with group i considered mainly affecting the trabecular meshwork ( trabeculodysgenesis ) , while group ii is thought to result from abnormalities in trabecular meshwork , iris , and anterior segment ( iridotrabeculodysgenesis ) [ 15 , 16 ] . this histopathogenic classification has been proposed as the basis to decide whether to perform angle surgery versus a filtering procedure . a problem with this approach is that although a predominance of histopathological abnormalities may exist in some cases , there are also many instances where a combination of elements exists and the decision is still taken based on clinical manifestations , age of appearance , and severity of the disease . the traditional staged approach outlined in most textbooks or review papers on primary congenital glaucoma involves what has been denominated as angle surgery ( goniotomy in cases with relatively clear corneas ) and trabeculotomy ( for those with cloudier cornea ) . if the initial procedure fails then it can be repeated , usually more than twice with goniotomies and twice with trabeculotomies . the next usual procedure is either standard trabeculectomy with the use of antimetabolites or combination of trabeculotomy / trabeculectomy . if this fails , the typical next procedure ( in an eye which still has a reasonable visual potential ) would be a tube implantation . and finally when everything else has failed or the visual potential is so poor , a cyclodestructive procedure which can be repeated several times for iop or pain control is recommended . for patients with the following features : no other ocular or systemic abnormalities , disease noted at least one month after birth but before one year of age , andwith corneal diameters less than 14 mm . no other ocular or systemic abnormalities , disease noted at least one month after birth but before one year of age , and with corneal diameters less than 14 mm . goniotomy ( if the cornea is clear enough for adequate visualization of the goniotomy knife passing across the anterior chamber and incising the trabecular meshwork ) or trabeculotomy ab externo if the cornea is cloudy enough to preclude a safe goniotomy ( or when the surgeon prefers this technique due to prior training or more experience with the procedure even when the cornea is clear ) . both procedures presumably work by allowing a more direct access of aqueous humor into schlemm 's canal and the outflow system . although initially used by de vincentis , in 1893 , for all types of glaucoma , barkan is credited with combining it with gonioscopic view , giving a detailed description of the procedure and reporting its successful use in congenital glaucoma in 1938 . modifications allowed to combine goniotomy with the use of the surgical microscopes , which were introduced into ophthalmology in the early 1950 's [ 20 , 21 ] , but routinely used in large eye institutions dealing with this disease ( such as moorfields eye hospital in london ) until the late 1960 's . the main aims and steps of the procedure have remained unchanged and include entering the anterior chamber through a clear corneal incision and crossing the anterior chamber to the opposite side to incise the trabecular meshwork ( while visualizing the angle with a surgical gonio lens ) and covering an arc of 100110 degrees . the results are excellent when used in patients fulfilling the criteria outlined above . in 1953 , barkan himself described his 17-year results of treating congenital glaucoma with goniotomy and reported an 80% success rate in 188 eyes , with adequate pressure control without medications . shaffer described 287 operated eyes and stated that one or two goniotomies cured 94% of patients diagnosed with glaucoma between 1 month and 24 months of age . broughton and parks also reported their 20-year experience with 50 eyes of patients with primary congenital glaucoma who underwent goniotomy , obtaining an overall 88% success rate ( with a mean followup of five years ) after one or more goniotomies . however , shaffer 's 94% outstanding success rate dropped to 30% when glaucoma was present at birth or after the age of two years . it was suggested that the type of angle abnormality was responsible for the level of success obtained and that this procedure was particularly suited for those cases with incomplete separation of the iris from the trabecular meshwork , which prevented the necessary separation of the corneoscleral trabecular sheets . goniotomy represented an important breakthrough in ophthalmology because for the first time it was possible to treat congenital glaucoma in a successfully and reasonably safe way . the pioneering work of dr . barkan and others , with gonioscopy to perform successful goniotomies , opened new roads in the understanding , diagnosis and treatment of glaucoma in general . useful adjuvants for the procedure such as the use of viscoelastic material [ 2729 ] to allow for a safer pass of the goniotomy knife across the anterior chamber and the use of newer goniolenses that can be utilized with the current surgical microscopes have improved the safety margin and adequate visualization for the goniotomy knife application . a wide variety of direct gonioscopy lenses have been used including the koeppe , barkan , and swan jacob lens . more recently , other useful modifications have been introduced such as the ritch direct panoramic gonioscopy lens , which gives a 160 degree view , obstructing only half the cornea and leaving plenty of working space . ( ritch r , personal communication ; ocular instruments inc . ; bellevue , washington ) ( figure 1 ) . initial results of goniotomy in primary congenital glaucoma have been excellent in western populations and with the characteristics outlined above ( 93.5% of eyes controlled at one year ) , but longer followup studies demonstrate that risk of relapse is a problem , even 30 years after the initial procedure . they noted that patients with symptoms at birth and those who required more than one goniotomy were more likely to relapse . a recent study from east africa reported on the results of goniotomy in 36 children , with advanced disease and late presentation , followed for an average of 1 year . the study suggested that almost 40% of those followed for more than 3 months required repeat goniotomy for adequate control . only 8.5% of corneas were clear preoperatively , while 78% were clear postoperatively . in saudi arabia , however , a study including 254 goniotomies had a success rate of only 52% with the worse outcomes in moderate and severe glaucomas ( only 13% and 0% success rates , resp . ) . these poor results with primary goniotomy ( and also with trabeculotomy ) prompted this institution to start utilizing combined trabeculotomy / trabeculectomy plus mitomycin c as a primary procedure , especially in those more severe cases of primary congenital glaucoma or glaucomas associated with other congenital abnormalities which in that study happened to be the majority ( 70% ) of patients . ideally , the pupil should be constricted , before starting the procedure , to minimize the risk of damaging the lens when crossing the anterior chamber and to facilitate the retraction of the iris from the angle . some preliminary studies with a small number of patients suggest the possibility of using endoscopic goniotomy to treat cases of congenital glaucoma where a cloudy cornea would preclude this type of procedure [ 33 , 34 ] . trabeculotomy was described in 1960 by burian who unroofed schlemm 's canal through an incision radial to the limbus and entered it with a specially made instrument that he called trabeculotome . he called the procedure trabeculotomy ab externo in contrast to goniotomy which was considered a trabeculotomy ab interno . later , in 1966 , harms modified the technique by dissecting a superficial scleral flap similar to the one used in trabeculectomy and then making the radial incision to identify schlemm 's canal and opening it with a modified instrument ( harm 's trabeculotome ) which had two parallel arms , one to open schlemm 's canal and the other one to guide externally the direction of the trabeculotome . pilocarpine drops and/or intracameral miotic agents are advisable to constrict the pupil before the procedure for the same reasons cited for goniotomy . pilocarpine 1% drops three times daily to the operated eye for two to three weeks after the operation have also been recommended to contract the ciliary muscle holding it away from the trabeculum during the healing phase . this technique became fairly popular and preferred by some over goniotomy for the treatment of congenital glaucoma . controversy regarding which was a better initial procedure ( trabeculotomy versus goniotomy ) existed since the introduction of trabeculotomy and remained for a long time , with some authors arguing that trabeculotomy had a better success rate as a single procedure than goniotomy . when the results of both procedures were compared in several studies [ 4042 ] , the conclusion was that they were similar and satisfactory for both techniques , and the argument more or less was settled . quigley reported observations based on 28 trabeculotomies , performed at the wilmer eye institute , with adequate control of iop and stable or improved optic disc status in 80% of eyes followed for one year or longer . main complication was anterior chamber hemorrhage which was mostly self - limited and did not require intervention . less common reported complications are iridodialysis , ruptured descemet 's membrane , and staphyloma formation . there is general consensus that trabeculotomy is the preferred procedure for mild primary congenital glaucoma when corneal opacity would preclude the performance of a goniotomy . there are advantages and disadvantages for each procedure outlined in table i but the final decision to perform goniotomy versus trabeculotomy ( as an initial procedure in primary congenital glaucoma ) rests on the surgeon and includes his / her personal preference and familiarity with the technique ( table 1 ) . trabeculectomy has the advantage of being an operation more familiar to most ophthalmologists , and it has been advocated by some as a primary procedure in congenital glaucoma [ 4446 ] . its mechanism of action is bypassing the aqueous from the anterior chamber to a subconjunctival fistula . the eye is large and the limbal anatomy is frequently distorted , and lack of familiarity with these unusual eyes can lead to complications such as iris and ciliary body incarceration and vitreous loss [ 44 , 4750 ] . another problem is that the superior conjunctival area is utilized , and even after successful surgery , with a normal life expectancy , there is a high likelihood that a significant proportion of these patients will need further surgery within their lifetimes . eyes with a previous failed trabeculectomy have a higher failure rate in the long term . use of antimetabolites has been associated with complications such as flat anterior chambers , hypotony , choroidal detachments , and endophthalmitis in patients with congenital glaucoma and therefore should be reserved for patients with more severe glaucomas or that have failed initial angle surgery [ 47 , 51 ] . milder antimetabolite agents ( 5-fu ) have been also suggested for patients with risk factors for failure . the age at presentation has been identified as a risk factor for failure when performing primary angle surgery . of 50 eyes in which infantile glaucoma was diagnosed at birth , only 26% had pressure controlled by one or two goniotomies . the other 74% ( 37 eyes ) required multiple goniotomies , trabeculotomies , trabeculectomies , and cyclocryotherapy with some of them never reaching control . similar poor results were obtained in those diagnosed as late developing infantile glaucoma after the age of 24 months . severity and duration of glaucoma are other negative predictive factors for success with either surgery . in such cases , with anticipated worse prognosis , a primary trabeculectomy with antimetabolites has been performed by some . a study at wills eye hospital showed a clear preference for trabeculectomy in primary congenital glaucoma as a secondary procedure when angle surgery failed , while it was used mostly as a primary procedure in aphakic glaucoma patients . results at one year were abysmal in aphakic patients ( 0% success ) compared with 76.9% in phakic patients , even when combined with mitomycin c. other studies in patients with primary congenital glaucoma and secondary phakic glaucomas also reported reasonable results with mitomycin c trabeculectomy [ 51 , 55 ] . concern about long - term complications in pediatric patients with a long life ahead of them has been raised because the thin , avascular bleb observed sometimes with this kind of surgery can produce late bleb leaks , bleb - related endophthalmitis , and long - term hypotony [ 5661 ] . there is also certain reluctance to utilize mitomycin and other antimetabolites in children because of the possibility , at least in theory , of secondary neoplasms as it occurs in other areas of pediatrics . because trabeculectomy without mitomycin c in children , who have failed other glaucoma procedures , has a much lower and unacceptable success rate ( less than 50% at 18 months in a retrospective study ) , enhancement with these agents is widely utilized . mitomycin c trabeculectomy in pediatric patients results in reasonable success rates of 6787% at one year ; however , a significant drop to 58 - 59% at two years has been reported by two different authors [ 61 , 63 ] , reflecting the higher tendency in children compared to adults to scar and occlude the filtering site . besides the use of antimetabolites , other modifications that have been suggested to improve the outcome of this surgery in pediatric patients have been the use of a fornix base flap because of its lower rates of bleb - related infections , use of releasable sutures , and use of healon gv left into the anterior chamber at the end of the procedure to prevent early postoperative hypotony and shallow anterior chamber . in these very elongated eyes , luntz and others have recommended to perform the sclera - corneal incision at the most anterior location possible , since a corneal site entry reduces the risk of blocking the opening by the ciliary body and iris adherences as well as vitreous loss [ 49 , 67 ] . unlike adults , where paracentesis tends to be self - sealing , in young children it can leak profusely due to increased tissue elasticity . in our institution , we routinely close the paracentesis tract at the end of the procedure with an absorbable suture ( 100 polyglycolic acid suture with spatulated needle ) and bury the knot . if this suture becomes loose before it reabsorbs , it is important to remove it to avoid the risk of suture - related microbial keratitis . cycloplegic agents are useful in the immediate postoperative period to relax the ciliary body and facilitate deepening of the anterior chamber . maul et al . first reported the use of this procedure in 1980 , for a child with severe bilateral primary congenital glaucoma who had not been controlled with initial goniotomy . after that , the results of this procedure have been mostly reported in middle eastern and indian populations , where it was used preferentially because it was felt that congenital glaucoma did not respond as well to angle surgery as previously described in western populations [ 31 , 69 , 70 ] . the intended mechanism of action for combining these two procedures is to gain access to dual outflow , through schlemm 's canal and/or the trabeculectomy fistula . one study in saudi arabia which prompted an institution to search for other options , such as this combined procedure , reported a disappointing 67% success rate with trabeculotomy alone at one year follow - up . . they hypothesized that the poor results compared with those from the western literature were because these patients had more severe degree of disease due to a higher rate of consanguinity and poor prognostic indicators such as larger corneal diameters , presentation since birth , and higher intraocular pressures . a small study of nine arab children , with primary congenital glaucoma , who underwent primary trabeculotomy / trabeculectomy before one year of age , demonstrated a much better success rate of 93.5% in contrast with 72% of those that underwent trabeculectomy alone . a theoretical advantage of the combined procedure is that it provides two major outflow pathways , the incised trabecular meshwork by the trabeculotome and the excised trabecular meshwork block and filtering bleb by the trabeculectomy [ 32 , 69 ] . a practical advantage was that in those cases where schlemm 's canal could not be identified , something that has been reported in up to 1115% of trabeculotomies [ 37 , 69 ] , the procedure would not result in failure , since the trabeculectomy pathway could still function . mandal et al . reported a 94.4% success rate of primary trabeculotomy - trabeculectomy in 122 eyes with primary congenital glaucoma operated in india , with a mean follow - up of approximately a year . complications included one case each of premature penetration into anterior chamber , vitreous prolapse and descemet detachment , plus 10 cases of shallow anterior chamber from which only two required surgical reformation . , at king khaled eye specialist hospital a tertiary level referral center in saudi arabia , studied retrospectively a very large sample of 532 patients ( 820 eyes ) , who underwent goniotomy , trabeculotomy , or combined trabeculotomy - trabeculectomy with mitomycin c as an initial glaucoma procedure . almost 70% of eyes had either moderate or severe glaucoma as per corneal enlargement ( > 13 mm ) corneal haze and higher levels of iop . all procedures resulted in high success rates ( > 80% ) for the mild form of primary congenital glaucoma . however , combined trabeculotomy - trabeculectomy with mitomycin c gave the best results for moderate ( 80% ) and severe ( 70% ) cases of primary congenital glaucoma in contrast with only 40% and 10% , respectively , for trabeculotomy in those cases with moderate and severe glaucoma . an important point to make regarding the combined procedure is that although it has been described by several authors as making the trabeculectomy ( incision and scleral block excision ) an extension of the initial incision for the trabeculotomy [ 6870 ] , it can be done using an alternate technique . our preference is to make a separate incision , limbal and more anterior , right under the hinge of the scleral flap , when entering the anterior chamber and then to perform a sclerotomy using controlled bites with the kelly descemet punch . this maneuver minimizes the chances of iris and ciliary body prolapse and incarceration mentioned by some authors [ 47 , 49 ] . glaucoma drainage implant surgery has a definitive role in managing infants and other children with glaucoma refractory to angle surgery and trabeculectomy . a tube is placed in the anterior chamber of the eye and aqueous flows through the tube and into the subconjunctival space to a plate which is placed at least 8 - 9 mm posterior to the limbus . the first glaucoma drainage implant used in the pediatric population was the molteno implant ( iop inc . , costa mesa , ca , usa ) in 1973 , followed by the baerveldt implant ( pharmacia and upjohn inc . , kalamazoo , mi , usa ) [ 72 , 73 ] and ahmed valve implant ( new world medical inc . , rancho cucamonga , ca , usa ) . the ahmed valve implant has a unidirectional valve restriction flow mechanism , designed to open when the aqueous pressure is higher than 8 mmhg . this is highly effective in reducing the risk of early postoperative hypotony compared to nonvalved implants ( molteno , baerveldt ) , which require special surgical maneuvers to reduce the flow or a two - stage procedure to avoid this problem . molteno , baerveldt , and ahmed implants have been the most common devices used in children and at the present time , judging from the current literature , baerveldt and ahmed implants are those mostly used ( the choice depending mostly on surgeon preference and individual case circumstances ) . comparison of gdis ( ahmed and baerveldt ) versus mmc trabeculectomy in children younger than 2 years of age in a retrospective , age - matched , comparative study resulted in better iop control with the gdis than the mmc trabeculectomy group with cumulative success rates of 87% versus 36% , respectively , at one year and even a larger difference of 53% versus 19% at 6 years . complication rates requiring reoperation though were more frequent among the gdis patients than among those with mitomycin - c ( mmc ) trabeculectomy ( 45.7% versus 12.5% , resp . ) . a prospective , randomized study comparing ahmed implant versus mmc trabeculectomy in pediatric aphakic glaucoma seemed to show higher qualified success in the ahmed ( 67% ) versus the mmc trabeculectomy group ( 40% ) and complication rates higher in the mmc trabeculectomy ( 40% ) than ahmed implant ( 26.7% ) although the differences were not statistically significant . success figures for individual aqueous shunt devices vary widely ( 3193% ) , but it is difficult to compare the success rates because different studies include very different populations , lengths of follow - up , surgical techniques , and types of devices [ 72 , 79 ] . for instance , after two years of follow - up , one study found a very low success rate of 31% while another one had 86% success at the same interval . however , the first study evaluated much younger patients , operated within the first two years of life , while the latter looked at older patients ( average age 6 years ) . molteno , baerveldt , shocket , krupin , ahmed , and optimed implants have been all used in children . currently , baerveldt and ahmed implants are the most commonly used in adults and children ( the choice depending mostly on surgeon preference and individual case circumstances ) . several studies with ahmed implants in the pediatric population have suggested that congenital glaucoma may be associated with higher failure rate than other pediatric glaucomas [ 8385 ] . other studies , however , did not find a correlation between surgical failure and glaucoma type in the pediatric population [ 81 , 86 , 87 ] . one of the largest studies and with longer follow - ups of gdis ( ahmed and baerveldt ) in pediatric glaucoma included 38 eyes with congenital glaucoma and 32 eyes with aphakic glaucoma . one - year success rates were 92% and 90% in the congenital and aphakic groups , respectively , but decreased to 42% and 55% after 10 years . there was a preference to implant ahmed valve in congenital glaucoma while baerveldt was preferred for aphakic patients . another study of ahmed implant in children reported a high cumulative success rate of 89% at 6 months just after two years , and they explained that their lower longer - term success than other studies might have been caused because of their use of smaller plate sizes in a number of patients . age of the patient did not clearly affect success rates of gdis [ 81 , 88 ] , but complications and rate of re - operations seem to be higher than in adults . some authors utilizing avg did not find a correlation between failure and prior glaucoma surgery [ 81 , 87 ] , while others [ 84 , 89 , 90 ] noted that eyes with previous glaucoma surgeries showed significantly worse results . there is a limited and contradictory information with respect to the effect of intraoperative adjunctive mmc use and gdis in pediatric glaucoma . several studies of aqueous shunt implantation with and without adjunctive mmc in adults did not show a benefit to intraoperative mmc use [ 9193 ] , and it had been assumed that the same was true for pediatric patients . several authors [ 82 , 94 ] confirmed no post operative difference , in iop measurement or complications , with or without mmc in studies including children . but then , al - mobarak and khan surprisingly found a shorter survival time ( 22 months versus 16 months ) and a much lower cumulative probability of success ( 31% versus 80% ) , respectively , when comparing the patients treated with ahmed and mitomycin c and those without it . it has been argued that because of the retrospective nature of this study , a selection bias could have occurred with the mitomycin c patients having worse type of glaucomas [ 79 , 95 , 96 ] . because of the lack of evidence that mitomycin c improves the outcomes with gdis and because of the potential complications , it is generally agreed that it is better to avoid its use [ 79 , 97 ] . the size of the implant is another important consideration because it has been demonstrated that the degree of iop reduction achieved postoperatively is directly proportional to the end plate size . this correlation is observed up to a certain degree , since a study comparing baerveldt 500 mm versus 350 mm did not show lower intraocular pressures with the larger implant . currently the ahmed implant comes in a smaller pediatric version denominated fp8 model with 96 mm surface ( 9.6 mm wide/10 mm long ) and adult version or fp 7 model with a larger surface of 184 mm ( 13 mm wide/16 mm long ) . in our experience , it is almost always possible ( except for nanophthalmic and premature baby eyes ) to place the larger adult size ahmed implant in children , and this should increase the chances of obtaining lower intraocular pressures than with the smaller implant size . studies in adults have shown encouraging results after the use of a second implant when the first one had failed [ 99101 ] . surgical management of a child with an already failed aqueous shunt with a second shunt implantation in a different quadrant , without removing the first one , is possible . a couple of studies in children which utilized the agv reported reasonable rates of success after the second implant [ 87 , 88 ] . ou et al , treating primary congenital glaucoma , reported a cumulative probability of success of 86% at 1 and two years and 69% at five years after the second agv implant ( figure 2 ) . postoperative complications after gdis in children are numerous , some of them occurring more frequently than in adults and requiring postoperative intervention more often . this higher incidence of complications in children compared to adults is probably related to different factors such as buphthalmic eyes with a thinner sclera , a growing orbit and eye , and more frequent eye rubbing in children . use of ahmed implant has been described in special glaucomas that require minimizing hypotony as much as possible , such as sturge - weber syndrome , and the results have been satisfactory . with nonvalved implants such as baerveldt or molteno , temporary occlusion of the tube with different modalities ( internal occlusion with a removable suture , external ligation with reabsorbable sutures or , a combination of both ) have been utilized to minimize immediate postoperative hypotony . those complications that are either more commonly cited or more serious , endangering the eye or vision , that need to be taken into consideration are as follows.early postoperative complications ( within one week after surgery ) : shallow [ 77 , 84 , 104 ] and flat anterior chamber [ 78 , 88 ] , hypotony , hyphema , choroidal detachment [ 82 , 84 ] and suprachoroidal hemorrhage [ 78 , 83 ] , corneal tube contact [ 77 , 86 , 88 ] , cataract formation [ 77 , 84 ] , secondary pupil and iris abnormalities [ 73 , 76 ] , and retinal detachment . intermediate postoperative period ( after one week to three months ) hypertensive phase [ 85 , 87 , 105 ] , hypotony after suture removal , bleb encapsulation . late complications ( three months to years ) : tube exposure ( figure 3 ) , endophthalmitis which is often associated with tube extrusion [ 81 , 108 ] , fibrous ingrowth , cyclitic membrane and persistent hypotony , and ocular motility abnormalities [ 74 , 84 , 109111 ] . adult studies have attempted to determine whether either one of the most utilized gdis ( ahmed and baerveldt ) is more successful and/or safer than the other one , and the results so far have been mixed without giving clear superiority to one device over the other , except for perhaps a slightly lower iop reduction for baerveldt and fewer and less serious complications for ahmed . for children , we do not have prospective , randomized studies to evaluate this issue , and therefore the choice of the specific device is determined by surgeon personal experience , preference , and availability of the shunt and special circumstances of the case . either one offers advantages or disadvantages that need to be taken into account , especially for these more complicated patients . preferences around the baerveldt implant cite a lower incidence of encapsulation and lower intraocular pressures with less medications , while the ahmed implant is preferred because of less immediate postoperative hypotony complications prevented by the valve mechanism . early postoperative complications ( within one week after surgery ) : shallow [ 77 , 84 , 104 ] and flat anterior chamber [ 78 , 88 ] , hypotony , hyphema , choroidal detachment [ 82 , 84 ] and suprachoroidal hemorrhage [ 78 , 83 ] , corneal tube contact [ 77 , 86 , 88 ] , cataract formation [ 77 , 84 ] , secondary pupil and iris abnormalities [ 73 , 76 ] , and retinal detachment . intermediate postoperative period ( after one week to three months ) hypertensive phase [ 85 , 87 , 105 ] , hypotony after suture removal , bleb encapsulation . late complications ( three months to years ) : tube exposure ( figure 3 ) , endophthalmitis which is often associated with tube extrusion [ 81 , 108 ] , fibrous ingrowth , cyclitic membrane and persistent hypotony , and ocular motility abnormalities [ 74 , 84 , 109111 ] . adult studies have attempted to determine whether either one of the most utilized gdis ( ahmed and baerveldt ) is more successful and/or safer than the other one , and the results so far have been mixed without giving clear superiority to one device over the other , except for perhaps a slightly lower iop reduction for baerveldt and fewer and less serious complications for ahmed . for children , we do not have prospective , randomized studies to evaluate this issue , and therefore the choice of the specific device is determined by surgeon personal experience , preference , and availability of the shunt and special circumstances of the case . either one offers advantages or disadvantages that need to be taken into account , especially for these more complicated patients . preferences around the baerveldt implant cite a lower incidence of encapsulation and lower intraocular pressures with less medications , while the ahmed implant is preferred because of less immediate postoperative hypotony complications prevented by the valve mechanism . cyclodestructive procedures in pediatric glaucoma are usually reserved for those challenging cases that have failed multiple more conservative treatments and for those patients with anatomic abnormalities that preclude traditional surgeries . . their mechanism of action is through ablation of the ciliary body and resultant reduction of aqueous production . this procedure was introduced since 1950 and decreased the intraocular pressure by freezing and destroying the ciliary body epithelium . other devastating complications including phthisis , retinal detachment , and sympathetic ophthalmia have been reported [ 117119 ] . a long - term evaluation study in pediatric patients reported a higher incidence of phthisis bulbi in aniridic patients . although cyclocryotherapy was utilized for a while for refractory and poor visual potential pediatric patients , it is not a preferred cyclodestructive procedure any longer because it has been gradually replaced by less aggressive and more targeted procedures such as laser cyclophotocoagulation either transclerally [ 122 , 123 ] or endoscopically [ 124 , 125 ] , which result in less inflammation and complications . transcleral diode laser cyclophotocoagulation . introduced in the early 1990 's , it rapidly replaced other laser methods of transcleral cyclophotocoagulation [ 128 , 129 ] used before . transcleral yag laser cyclophotocoagulation in children resulted in very disappointing results , with one study reporting that after ten patients were treated , only half had controlled iop , and there was loss of vision in four patients and phthisis bulbi in one . transcleral diode laser cyclophotocoagulation , on the other hand , had a convenient compact design and less side effects , in particular avoiding the occurrence of sympathetic ophthalmia , a dreaded complication of yag transcleral cyclophotocoagulation [ 131 , 132 ] . diode laser cyclophotocoagulation was utilized in a variety of pediatric glaucomas since its introduction , but the reports suggested that the response in children was less than in adults [ 133 , 134 ] . it was hypothesized that younger eyes may recover faster from the treatment than older patients . an overall success rate of 50% in pediatric refractory glaucomas has been cited , including retreated patients ( average 2.2 procedures per eye ) and a high retreatment rate of 70% and most failures occurring during the first 6 months after treatment . a problem with performing adequate transcleral cyclophotocoagulation in congenital glaucoma eyes with aberrant anatomy features is to get accurate localization of the ciliary body . transillumination , which we routinely use at our institution with a fiber optic probe , is recommended [ 113 , 135 ] . , there is no significant decrease in the number of glaucoma medications and it is considered mostly as an adjunctive therapy [ 113 , 134 ] . a relatively large study of 77 pediatric glaucoma eyes noted an initial adequate iop reduction in 62% , but this fell to 37% by one year . repeat treatments in 72% accomplished useful iop reduction for a year or more , but 13% of patients did not respond at all . noted complications were retinal detachment in three eyes and significant inflammation in 10% of eyes . diode laser transcleral cyclophotocoagulation still should be reserved for patients with limited visual potential ( 20/100 or worse ) , while other incisional procedures should be attempted for patients with better visual acuity . reasonable indications for this procedure are ( 1 ) advanced glaucoma with previous failed multiple procedures ; ( 2 ) markedly elevated iop on acute presentation , where at least temporary iop control is required before undertaking more definitive surgery ; ( 3 ) treatment of a blind painful eye with an elevated iop ; ( 4 ) markedly elevated iop , where the fellow eye has undergone surgery and it is desirable to defer surgery until the fellow eye is more stable ; ( 5 ) moderately elevated iop with maximum medical therapy where the risks of drainage surgery are high ( severe complications in the fellow eye ) or where incisional surgery was declined by parents . some authors suggest caution when cycloablation surgery fails and tube surgery is undertaken ( to consider two - stage tube procedure ) , since it has been noted that some cases are associated with chronic postoperative hypotony [ 86 , 106 ] . a complication observed with diode laser cyclophotocoagulation , predominantly in younger patients , is scleral thinning ( figure 5 ) . it is probably an overlooked complication because in many cases it tends to be mild and without clinical implications . although probably very rare , actual scleral perforation requiring suturing of the sclera has been reported [ 137 , 138 ] , and this highlights the need to lower the energy levels utilized for pediatric glaucoma cases with thinner sclera . in our institution , we tend to start at roughly half the level of initial energy utilized for adults and then increase gradually until a mild popping sound is heard , which is the attempted threshold . endolaser cyclophotocoagulation ( ecp ) . described first in 1992 , this procedure accomplishes cycloablation through direct visualization ( in contrast with other transcleral cycloablation procedures which just estimate the location of the ciliary body ) . it uses a 20 gauge instrument , with endoscopic view through a monitor and a diode laser treating each individual ciliary process until whitening and shrinkage is observed . initial use in children was tentative , due to the concern of possible serious complications or phthisis bulbi from this new procedure . it was utilized first in a few eyes for a pilot study , with poor visual prognosis and with limited amount of cyclophotocoagulation ( 180 degrees ) . after it was felt that it was relatively safe , it was used in patients with better visual potential , with higher levels of energy , and a wider extent of treatment ( 270 degrees ) . the results were encouraging with no sight - threatening complications , severe hypotony , or significant pain or inflammation . at 3 years of follow - up , 50% ( five eyes ) were considered success and 50% ( five eyes ) failures . a larger study by the same group , including 36 eyes that were followed for an average period of a year and a half , described their wider experience . their success rate was 34% with one quarter of eyes needed retreatment at least once . postoperative complications included retinal detachment in 2 patients , prephthisis in 1 patient , and progression of vision loss from hand motion to no light perception in 1 patient . they concluded that this procedure is moderately effective for the management of difficult pediatric glaucomas and that an aphakic patient may have an increased risk of complications . it is worth noting that a number of phakic patients ( 14 ) were treated in this study and they did not find any new development of cataracts after the procedure . endolaser cyclophotocoagulation offers the advantage of more targeted , visualized end point of treatment and ability to titrate the amount and extent of treatment as advantages , while adding the potential possible complications associated with intraocular procedures ( infection , suprachoroidal serous and hemorrhagic detachments , and intraocular pressure spikes related to viscoelastic retention ) . in our opinion , it is the most useful resource in pseudophakic and aphakic patients who are not candidates for some reason for tubes or that have thin and abnormal scleras that would prevent the use of transcleral cyclodestructive procedures . although no significant complications have been reported from its limited use in phakic patients , we do not recommend its use in these patients because of the high risk of damage to the crystalline lens during the procedure . the rational for performing this kind of surgery is that a larger extent of exposed schlemm 's canal will yield a lower intraocular pressure than partial opening of the lumen . 360-degree trabeculotomy surgery was initiated by smith in 1960 in cadaver eyes using suture material . he described using two separate radial incisions to thread a piece of nylon into schlemm 's canal and tensioning the suture from both ends opening into the anterior chamber . beck and lynch in 1995 refined the technique by using 60 polypropylene suture , which was threaded all around the 360 circumference of schlemm 's canal and reported success of 87% of treated eyes with congenital glaucoma . in 2011 , beck et al . reported a 77% success of same procedure in cases of primary congenital glaucoma considered to have a poor prognosis ( onset at birth , presentation after 1 year of age , failure of initial goniotomy ) . visual outcomes and intraocular pressure ( iop ) control have been shown to be better with 360-degree trabeculotomy than with multiple goniotomy procedures . however , when threading schlemm 's canal with a suture , there is a risk of misdirection into the suprachoroidal space [ 144 , 145 ] . use of an illuminated microcatheter avoids this potential complication as the tip with the illuminated ( flashing or steady light ) continuously indicates its position within the canal or whether it starts to go astray . recently reviewed the results of 11 eyes with primary or secondary congenital or juvenile glaucoma that underwent circumferential trabeculotomy performed with an illuminated microcatheter and reported a 91.6% qualified and 83.3% unqualified success rate with short - term ( 8 to 12 months ) followup . transient hyphema was common , but no major complications were seen in this series . in another retrospective consecutive chart review of 16 eyes , there was a 47.0% reduction in iop at 6 months , although average antiglaucoma medications use was not significantly reduced from baseline . the superiority of canaloplasty over other more traditional techniques in adults still needs to be demonstrated by randomized , controlled studies that utilize only one intervention ( instead of several interventions added to the basic procedure of schlemm 's canal catheterization such as deep sclerectomy , circumferential vasodilatation of the canal , tensioning of the canal with a nylon ligature ) . still we believe that 360-degree trabeculotomy with a lighted probe offers significant advantages over the traditional trabeculotomy technique in children , and it deserves further consideration . in contrast with traditional trabeculotomy , this procedure adds certainty with regards to adequate identification and probing of schlemm 's canal for the whole360-degree circumference in a single session . potential risks are that while retrieving the more rigid ( than the prolene suture ) catheter throughout the anterior chamber damage to the lens or other structures , a more extensive descemet detachment than that produced by a traditional trabeculotome could occur . we have utilized this procedure for a limited number of congenital glaucoma cases , with both the 250 micron catheter ( iscience interventional , menlopark , ca ) or a battery operated smaller caliber catheter with apparently reasonable preliminary results ( unpublished data ) ( figure 6 ) . we believe that a prospective , randomized , controlled study comparing this procedure with either trabeculotomy or goniotomy in congenital glaucoma patients with similar level of pathology is necessary to make a more definitive conclusion about its place in the treatment of this kind of glaucoma . nonpenetrating surgery has attracted more interest during the last decade [ 148150 ] for its potential to decrease intraocular pressure without some of the immediate postoperative hypotony and long - term bleb complications of traditional filtering surgery . its use in pediatric glaucoma has been fairly circumscribed , and there are only a handful of studies describing its results in the english ophthalmic literature [ 149 , 151 ] . deep sclerectomy involves the dissection of a deep scleral flap , deroofing of schlemm 's canal , and preserving the structural integrity of the trabecular meshwork . its mechanism of action is not entirely clear , but a combination of a more diffuse filtering bleb formation and uveoscleral and transcleral flow have been cited . it has been proposed by some [ 154 , 155 ] as an alternative to other procedures in high risk pediatric glaucoma cases such as sturge - weber syndrome , where it is desirable to minimize sudden hypotony and the resultant possibility of massive choroidal serous or hemorrhagic detachments , which can lead to catastrophic outcomes . its use has been reported in a few studies for the treatment of primary congenital glaucoma and congenital glaucoma refractory to treatment . it has been proposed as an alternative to nonangle surgery , because it potentially reduces the complications of immediate postoperative hypotony and overfiltration and the side effects of performing a peripheral iridectomy and avoids the complications of long - term filtering blebs , including serious intraocular infections . although its risk profile appears better than penetrating procedures such as trabeculectomy , its utilization has not become so generalized partly because it is technically more demanding and because it is technically more demanding and also because of surgeon wide variability in fashioning the deep scleral flap [ 152 , 157 ] . prospective , randomized , comparative studies with other traditional procedures are still lacking and are difficult to perform , because pediatric glaucoma is an uncommon disease in most places and matching of study samples is more difficult with the variety and levels of disease on these patients . because in saudi arabia congenital glaucoma is more common and a more severe disease than in other countries [ 31 , 50 ] , dealing with the severe problems and complications of traditional penetrating surgery has prompted trying alternatives , such as deep sclerectomy . preliminary results from an ongoing study reviewing the results of deep sclerectomy , as a primary procedure , in 74 eyes with primary congenital glaucoma and at least three years of follow - up suggest an overall success rate of 82.4% . difficulties with this procedure in children are that they have a thinner and more elastic sclera and variable anatomical features , and it is not always possible to identify schlemm 's canal , all of which make a procedure already technically demanding , even more challenging and more likely to be performed at highly specialized centers in treating this condition . one study reported abysmal results ( 100% failure ) of the procedure in eight patients who had already failed other glaucoma surgeries and a high rate of failure to successfully perform the procedure as well as serious complications including a case of vitreous hemorrhage and other with vitreous loss and retinal detachment . other authors have reported much better results when they used deep sclerectomy as an initial procedure in congenital glaucoma , with a success rate of 75% at last follow - up and therefore advocate this technique as a primary intervention . , one of the main advocates of deep sclerectomy , published the results of performing combined deep sclerectomy and trabeculectomy in 35 eyes of patients with a variety of pediatric glaucomas with a mean follow - up of almost four years . they reported a complete and qualified success rates , based on cumulative survival curves , after 9 years of 52.3% and 70.6% . useful pearls when performing deep sclerectomy in pediatric glaucoma are ( 1 ) more careful dissection of the deep flap ; ( 2 ) if antimetabolites are used , to apply them before dissecting the superficial scleral flap ; ( 3 ) not to attempt deroofing of schlemm 's canal as this tissue is difficult to identify and peel and may result in perforation . in our experience , deep sclerectomy , when done properly , is another helpful and relatively safe procedure in the armamentarium to manage congenital glaucoma especially in its mild form . further data with a relatively larger number of patients should be available to the ophthalmic community in the near future . we do not advocate the use of this procedure for cases with other congenital secondary glaucomas , where the disease process may be more complicated and the angle may be closed or abnormal . we hope that its use may also contribute to a better understanding of congenital glaucoma mechanisms . ab interno trabeculectomy using a mechanical device such as the trabectome has been mainly used for adult forms of glaucoma . its aim and presumed mechanism of action is to enhance outflow via increased access to schlemm 's canal , allowing aqueous to escape the anterior chamber without the impedance of the strip of trabecular meshwork and inner wall of schlemm 's canal that are removed . in order to perform this procedure in an efficient manner , the cornea needs to be relatively clear in order to clearly visualize the anterior chamber angle , and trabecular meshwork landmarks must be clearly visible . when the anterior chamber is deep and the angle structures clearly visualized in pediatric glaucoma with a large cornea , this procedure is suitable . however , corneal clouding and/or presence of haab 's striae may preclude clear visualization of the anterior chamber angle . in addition , aberrant or incomplete development of the meshwork and schlemm 's canal complex in pediatric glaucoma may prevent electrocautery stripping of the meshwork . therefore , one could envision the trabectome being potentially useful in milder cases of primary congenital glaucoma and other forms of pediatric glaucoma , where the angle structures are well developed and presence of schlemm 's canal identified . it may also be useful in other secondary forms of glaucoma where the angle remains relatively open such as what may be seen in pediatric glaucoma associated with certain forms of uveitis . there are no case series that specifically address the use of trabectome in pediatric glaucoma . the use of the trabectome in pediatric glaucoma was described by minckler and colleagues in a large case series presented at the american ophthalmological society along with the published discussion . however , specifics of the technique , types of patients , and outcomes were not clearly described in this paper . future studies focusing on this specific minimally invasive surgical technique will hopefully provide new information on the benefit of the trabectome in pediatric glaucoma . although for every glaucoma procedure the most immediate objective is the reduction of harmful elevated intraocular pressure , in children other aims need to be kept in mind when deciding which procedure to perform and when to do it . with children we are running against time because the sooner the child develops clear media , improved visual acuity , and binocularity the better . it is well known that in spite of adequate normalization of iop , many elements hamper the development of a normal vision in these children ; however , aiming at preserving or restoring the best possible visual function is an important final goal . while choosing a procedure it is important to keep in mind that the immediate goals of the surgery are normalization of the intraocular pressure and clearing of the cornea as soon as possible . in the long term , the aims of the procedure are prevention of ( further ) optic nerve damage and peripheral vision integrity and preservation or restoration of the capability to develop as close as normal binocular visual function . the management of pediatric glaucoma in its different forms is still quite challenging and the visual and long - term results variable depending on the severity and type of disease . the number and type of newer surgical procedures and modifications to traditional ones have improved our choices and capability to treat this condition . although it is generally agreed that angle surgery is the best initial approach for milder cases of primary congenital glaucoma , the surgical procedure to use for more severe cases , secondary glaucomas , or failed angle surgery cases is less clear cut . prospective , randomized , comparative studies are scarce because of the infrequent and variable nature of the condition , but the surgeon facing such patients still needs to make the best informed choice regarding which procedure to use and what are the chances of success . some procedures , like goniotomy , except for some minor modifications , are almost in their eighties but have stood the test of time and remained as useful and strong as when they revolutionized the field of congenital glaucoma surgery at first . others like trabeculotomy are in their fifties and also have maintained their status in the initial management of congenital glaucoma , keeping the same indications . others , like trabeculectomy , also in their fifties have undergone a number of important modifications , such as the use of antimetabolites to improve the outcome and the shift to fornix base conjunctival opening to encourage more diffuse , posterior , thicker blebs , but even then still have a significant rate of failure and complications especially in pediatric patients . there are some procedures in their 20 's like the baerveldt and ahmed implants which have made a big difference in the management of difficult or refractory cases that before would have been candidates only for cyclodestructive procedures , which also have become more refined and targeted allowing using them at earlier stages . and finally , the newest procedures ( 360 trabeculotomy , trabectome , and deep sclerectomy ) barely getting to their first decade still need to prove themselves in a disease that requires long - term , life - long control . in the end , the continued efforts of many researchers , surgeons , and clinicians on this field have improved the outlook and chances for the life of our young patients with such difficult disease which has life - lasting consequences .

currently , there are numerous choices for the treatment of pediatric glaucoma depending on the type of glaucoma , the age of the patient , and other particularities of the condition discussed in this review . traditionally , goniotomy and trabeculotomy ab externo have been the preferred choices of treatment for congenital glaucoma , and a variety of adult procedures adapted to children have been utilized for other types of pediatric glaucoma with variable results and complications . more recently , seton implantations of different types have become more popular to use in children , and newer techniques have become available including visualized cannulation and opening of schlemm 's canal , deep sclerectomy , trabectome , and milder more directed cyclodestructive procedures such as endolaser and transcleral diode laser cyclophotocoagulation . this paper reviews the different surgical techniques currently available , their indications , results , and most common complications to allow the surgeon treating these conditions to make a more informed choice in each particular case . although the outcome of surgical treatment in pediatric glaucoma has improved significantly , its treatment remains challenging .

1. Introduction 2. Primary Congenital Glaucoma 3. Conclusions

PMC155674

dynamic steady state aerobic exercise lasting between 5 and 30 minutes is associated with an increase in cardiac output secondary to an increase in heart rate and stroke volume . the heightened stroke volume is due , in part , to a starling - mediated increase in end - diastolic volume ( i.e. preload reserve ) and to enhanced systolic emptying secondary to an increase in myocardial contractility ( i.e. contractile reserve ) . during dynamic steady state exercise lasting > 30 minutes , stroke volume has been shown to gradually decrease while cardiac output remains unaltered due to a compensatory elevation in heart rate . a widely held belief in exercise physiology is that the prolonged strenuous exercise ( pse ) mediated decline in stroke volume may be due , in part , to a reduction in preload reserve secondary to the diminished plasma volume associated with dehydration and thermoregulatory peripheral vasodilatation . although alterations in preload reserve may contribute to the decreased stroke volume , several investigators have found that the decline in left ventricular ( lv ) systolic performance associated with pse was independent of changes in lv preload or afterload . these findings suggest that the alteration in lv systolic function after pse may result in " cardiac fatigue " due , in part , to an impairment in myocardial contractility ( reviewed in ) . a limitation of a majority of investigations that have examined the effect of pse on lv systolic performance was that echocardiographic assessment of lv function was obtained prior to and approximately 20 to 45 minutes after completing prolonged exercise . however , a previous investigation has found that echocardiographic images obtained after cessation of exercise are not representative of the acute lv changes that occur during exertion . therefore , the time - course of alteration in lv systolic performance during prolonged intense exercise ( similar to that observed during a competitive endurance event ) is not well known . finally , there is a paucity of investigations that have examined the effect of pse on lv systolic function in female athletes . the purpose of this investigation was to assess lv systolic function prior to , during and immediately after performing pse in female triathletes . we hypothesized that pse would result in a decline in lv systolic function secondary to a decline in myocardial contractility . the participants for this investigation consisted of 13 healthy trained female triathletes with normal lv systolic function ( table 1 ) . the subjects were in the early phase of their annual training regimen and had not participated in a competitive endurance event for at least three months prior to testing . written informed consent was obtained from each subject in accordance with guidelines established by the health research ethics board at our institution . ( hrmax = maximal heart rate ) the subjects performed an incremental exercise treadmill test to exhaustion during which time oxygen uptake , carbon dioxide production , minute ventilation and heart rate were continuously measured . expired gases were collected , sampled and averaged every 15 seconds using a parvomedics metabolic cart ( parvomedics , salt lake city , ut ) and heart rate data was measured using a polar vantage xl heart rate monitor ( polar electro oy , finland ) . the graded exercise test began at a running velocity of 161 mminand increased by 13.4 mminevery two minutes until the breakaway point in the ventilatory equivalent for carbon dioxide , after which stages were reduced to one minute . in an attempt to simulate the bicycle and running portions of an olympic - distance triathlon event , participants performed a 40 kilometre simulated cycling time trial using self - selected gears on their own bicycles mounted to a computrainer ( racermate , seattle wa ) , followed by a 10 kilometre run on a treadmill ( star trac , irvine ca ) . incorporation of the two modes of exercise reduced the boredom associated with stationary bicycle exercise and enabled participants to compare the effort with past racing experiences . at 30-minute intervals , the subjects stopped exercising , dismounted from the apparatus and were placed in the left lateral supine position during which time echocardiographic images , heart rate and blood pressure were obtained . the total time to dismount the exercise apparatus , acquire a satisfactory echocardiographic image and resume exercising was 2.8 0.2 minutes . the subjects were weighed prior to and following the race and fluid consumption was recorded for each participant . body temperature was not monitored , however participants were cooled with fans throughout the exercise session . lv imaging was performed with a commercially available ultrasound instrument ( hewlett packard , sonos 5500 ) with a 3.5 mhz transducer . two - dimensional transthoracic images were obtained from the parasternal short - axis view at the level of the mid - papillary muscles according to american society of echocardiography guidelines . lv images were obtained at rest , at 30-minute intervals during exercise and immediately ( < 45 seconds ) after cessation of pse . the images were analysed offline at a later date and the following measures were obtained and averaged over four cardiac cycles : end - diastolic cavity area ( largest endocardial area ) and end - systolic cavity area ( smallest endocardial cavity area ) . lv fractional area change and lv end - systolic meridional wall stress were calculated by standard methods . in addition , the systolic blood pressure end - systolic cavity area relation was used as an estimate of myocardial contractility . if a significant time effect was found then a post - hoc neuman keuls test was performed . the participants for this investigation consisted of 13 healthy trained female triathletes with normal lv systolic function ( table 1 ) . the subjects were in the early phase of their annual training regimen and had not participated in a competitive endurance event for at least three months prior to testing . written informed consent was obtained from each subject in accordance with guidelines established by the health research ethics board at our institution . the subjects performed an incremental exercise treadmill test to exhaustion during which time oxygen uptake , carbon dioxide production , minute ventilation and heart rate were continuously measured . expired gases were collected , sampled and averaged every 15 seconds using a parvomedics metabolic cart ( parvomedics , salt lake city , ut ) and heart rate data was measured using a polar vantage xl heart rate monitor ( polar electro oy , finland ) . the graded exercise test began at a running velocity of 161 mminand increased by 13.4 mminevery two minutes until the breakaway point in the ventilatory equivalent for carbon dioxide , after which stages were reduced to one minute . in an attempt to simulate the bicycle and running portions of an olympic - distance triathlon event , participants performed a 40 kilometre simulated cycling time trial using self - selected gears on their own bicycles mounted to a computrainer ( racermate , seattle wa ) , followed by a 10 kilometre run on a treadmill ( star trac , irvine ca ) . incorporation of the two modes of exercise reduced the boredom associated with stationary bicycle exercise and enabled participants to compare the effort with past racing experiences . at 30-minute intervals , the subjects stopped exercising , dismounted from the apparatus and were placed in the left lateral supine position during which time echocardiographic images , heart rate and blood pressure were obtained . the total time to dismount the exercise apparatus , the subjects were weighed prior to and following the race and fluid consumption was recorded for each participant . body temperature was not monitored , however participants were cooled with fans throughout the exercise session . lv imaging was performed with a commercially available ultrasound instrument ( hewlett packard , sonos 5500 ) with a 3.5 mhz transducer . two - dimensional transthoracic images were obtained from the parasternal short - axis view at the level of the mid - papillary muscles according to american society of echocardiography guidelines . lv images were obtained at rest , at 30-minute intervals during exercise and immediately ( < 45 seconds ) after cessation of pse . the images were analysed offline at a later date and the following measures were obtained and averaged over four cardiac cycles : end - diastolic cavity area ( largest endocardial area ) and end - systolic cavity area ( smallest endocardial cavity area ) . lv fractional area change and lv end - systolic meridional wall stress were calculated by standard methods . in addition , the systolic blood pressure end - systolic cavity area relation was used as an estimate of myocardial contractility . if a significant time effect was found then a post - hoc neuman keuls test was performed . subjects exercised at an average intensity equivalent to 90 3% of their maximal heart rate . heart rate gradually increased from an average of 162 15 beats / min for the initial 15 minutes of exercise to 175 8 beats / min during the final 15 minutes of exercise ( figure 1 ) . average weight loss following the exercise bout was 0.8 0.6 kg despite an average fluid intake of 1090 455 ml . . heart rate , systolic blood pressure and the systolic blood pressure end - systolic cavity area relation were significantly higher at 30 , 60 and 90 minutes of exertion compared to pre - exercise baseline values ( table 3 ) . the systolic blood pressure end - systolic cavity area relation was significantly lower at the completion of pse compared to the 60-minute time - period . the end - systolic cavity area was significantly lower than resting values after 30 , 60 and 90 minutes of exercise . a similar trend was found for end - diastolic cavity area with the 90-minute measure being significantly lower than rest . fractional area change was significantly higher after one - hour of exercise and returned to resting values upon cessation of exercise . left ventricular end - systolic wall stress was significantly higher at 90 minutes and immediately after completing pse compared to resting values ( table 3 ) . the fractional area change end systolic wall stress relationship increased with exercise and remained above resting values upon completion of pse ( figure 2 ) . left ventricular systolic performance in relation to wall stress prior to , one hour into and immediately following prolonged strenuous exercise . ( * = p < 0.05 vs pre ; = p < 0.05 vs 60 minutes ; edca , end - diastolic cavity area ; esca , end - systolic cavity area ; esws , end - systolic wall stress ; fac , fractional area change ; hr , heart rate ; sbp , systolic blood pressure ; sbp - esca relation , surrogate for myocardial contractility ) subjects exercised at an average intensity equivalent to 90 3% of their maximal heart rate . heart rate gradually increased from an average of 162 15 beats / min for the initial 15 minutes of exercise to 175 8 beats / min during the final 15 minutes of exercise ( figure 1 ) . average weight loss following the exercise bout was 0.8 0.6 kg despite an average fluid intake of 1090 455 ml . heart rate , systolic blood pressure and the systolic blood pressure end - systolic cavity area relation were significantly higher at 30 , 60 and 90 minutes of exertion compared to pre - exercise baseline values ( table 3 ) . the systolic blood pressure end - systolic cavity area relation was significantly lower at the completion of pse compared to the 60-minute time - period . the end - systolic cavity area was significantly lower than resting values after 30 , 60 and 90 minutes of exercise . a similar trend was found for end - diastolic cavity area with the 90-minute measure being significantly lower than rest . fractional area change was significantly higher after one - hour of exercise and returned to resting values upon cessation of exercise . left ventricular end - systolic wall stress was significantly higher at 90 minutes and immediately after completing pse compared to resting values ( table 3 ) . the fractional area change end systolic wall stress relationship increased with exercise and remained above resting values upon completion of pse ( figure 2 ) . left ventricular systolic performance in relation to wall stress prior to , one hour into and immediately following prolonged strenuous exercise . ( * = p < 0.05 vs pre ; = p < 0.05 vs 60 minutes ; edca , end - diastolic cavity area ; esca , end - systolic cavity area ; esws , end - systolic wall stress ; fac , fractional area change ; hr , heart rate ; sbp , systolic blood pressure ; sbp - esca relation , surrogate for myocardial contractility ) the major new finding of this investigation was that pse exercise was associated with an enhanced lv systolic function secondary to the heightened myocardial contractility . this finding is contrary to our " a priori " hypothesis and discordant with previous investigations that found a decline in lv systolic function after performing pse . however , our findings support the findings of others who demonstrated that lv systolic function remains above resting levels throughout prolonged exercise . previous investigators have found that pse of varying exercise durations ( i.e. , 1.6 to 24 hours ) was associated with a decline in lv fractional shortening that returned towards pre - exercise baseline values one to two days after cessation of exercise . the mechanism(s ) responsible for the reduced lv systolic performance appears to be due , in part , to a decline in end - diastolic volume and subsequent attenuated use of the starling mechanism and to a reduction in myocardial contractility . a limitation of a majority of previous investigations was that lv systolic function was examined after the cessation of pse , therefore , the time - course of the decline in lv systolic function and the underlying mechanisms responsible for this change during prolonged exertion are not well known . in the present investigation , fractional area change increased during exercise and did not decline below pre - exercise baseline values . the heightened systolic performance appears to be secondary to an increase in myocardial contractility ( i.e. , increased systolic blood pressure end - systolic cavity area relation ) as end - diastolic cavity area ( i.e. , preload ) decreased while lv wall stress ( i.e. , afterload ) increased during exertion . in addition , the fractional area change - end systolic wall stress relationship increased with exercise and remained elevated throughout the 2 hours of pse ( figure 2 ) . this finding is in contrast to other investigations which demonstrated that the fractional area change - end systolic wall stress relationship declined following pse lasting three and greater than 12 hours . although these authors report a decline in lv systolic function following prolonged exercise , they did not measure lv function during exercise . to date only two other investigations have examined lv systolic function throughout ( i.e. , 1 to 2.5 hours ) pse and in both cases exercise was performed by younger healthy male athletes . consistent with our results , both investigations found that pse was not associated with lv systolic dysfunction as ejection fraction was always greater than pre - exercise baseline values . moreover , palatini and colleagues found that the increased lv ejection fraction was due , in part , to increased myocardial contractility . the mechanism responsible for the heightened myocardial contractility during pse has not been well studied , however , it may be related to the increased beta - adrenergic stimulation of the myocardium and/or to the force - frequency relation . regardless of the underlying mechanisms , our findings confirm and extend previous exercise echocardiographic or radionuclide angiographic investigations by revealing that pse does not appear to negatively alter lv systolic function in younger female triathletes . our finding that lv end - diastolic cavity area ( i.e. , preload ) decreased during pse despite our athletes consuming > 250 ml of fluid every 30 minutes during exertion is similar to the findings of goodman and associates . the attenuated preload reserve has been linked to the increased heart rate associated with performing prolonged exercise . however , in the current investigation , the reduced preload reserve does not appear to be related to an exercise mediated rise in heart rate since the 13% decline in end - diastolic cavity area that occurred during the first 90 minutes of exercise and its subsequent return towards baseline values at the cessation of pse occurred at similar heart rates . furthermore , an exercise - mediated increase in sympathetic stimulation and tachycardia have been shown to decrease the time constant of lv pressure fall during isovolumic relaxation and minimal lv pressure while increasing the peak mitral valve pressure gradient , early diastolic filling rate and end - diastolic volume , despite a marked reduction in the duration of diastole . it is possible that our attenuated preload reserve may be secondary to ventricular interaction or to a pericardial constraint to lv filling . douglas and colleagues found disparate left and right ventricular end - diastolic cardiac area responses after performing pse . more specifically , upon cessation of pse the right ventricular end - diastolic cavity area increased compared to pre - exercise baseline values while the lv end - diastolic cavity area was smaller than basal values . therefore , it is possible that sudden postural changes ( i.e. , a change form the upright exercising posture to the supine left lateral position ) that occurred in current investigation may have resulted in increased right ventricular end - diastolic cavity area that via diastolic ventricular interaction ( with or without pericardial constraint ) may have reduced lv compliance and filling . this hypothesis does not seem likely , however , as the lv end - diastolic cavity area decreased during the first 90 minutes of exercise and returned to baseline values at the cessation of exercise despite similar postural perturbations during all 2-d image acquisitions . finally , it is possible that heightened lv wall stress associated with pse may have resulted in reduced lv relaxation and subsequent decrease in lv preload during exertion . currently , there has only been one laboratory investigation that has examined the stroke volume response during pse in younger ( 31 years ) female triathletes . in that investigation , the athletes performed five hours of cycling exercise followed by three hours of treadmill running during which time cardiac output was measured ( co2-rebreathing method ) . the main finding of this investigation was that stroke volume decreased during the first 30 minutes of cycling exercise and remained lower than pre - exercise values during most of the 8-hour exercise session . the heart rate gradually increased during the prolonged exercise session , however , it did not fully compensate for the reduced stroke volume and as a consequence cardiac output decreased during exertion . a limitation of this examination was that the underlying mechanisms ( i.e. , loading conditions and myocardial contractility ) responsible for the reduced stroke volume were not measured . however , our finding that the pse mediated decline in preload reserve was offset by an increase in contractile reserve that resulted in no alteration in stroke area during exercise is divergent from the above findings . the disparity of findings between these investigations is not related to the participant 's age , mode of exercise , or fluid replenishment , which were similar in both examinations . the only major difference between these investigations was that the subjects in our investigation exercised at a substantially higher exercise intensity and approximately one - quarter of the duration compared to the subjects in the above investigation . a series of limitations that may have affected the results of this investigation must be addressed . first , in order to compare our findings with previous investigations that performed supine echocardiograms post pse , we elected to have our subjects momentarily stop exercising and quickly lie in the left lateral supine position while the echocardiographic images were acquired . a limitation with our method is that it does not fully represent the loading conditions and heart rate that occur during exercise . in addition , the supine posture should enhance venous return and as a consequence may result in divergent right and left ventricular interactions that may not occur to the same extent during upright exercise . in spite of the above limitations , our results are clearly consistent with recent exercise echocardiographic or radionuclide angiographic examinations that found that pse did not result in a decline in lv systolic dysfunction . although our images were obtained in the supine position we did observe heightened contractile function at all time points throughout the exercise session . heart rate values did decline significantly during imaging ( figure 1 ) , however the phenomenon of " cardiac fatigue " was initially described in a resting state in similar supine positions following pse . if lv contractile dysfunction was evident at rest in the supine position with heart < 100 bpm following pse in these previous investigations , one would expect to observe changes in contractile function not only during exercise but at heart rates between exercise and resting values , similar to those we observed in our investigation ( 100113 bpm ) . moreover , our findings are identical to those of palatini and associates who revealed that the heightened ejection fraction during exercise was due , in part , to an increase in myocardial contractility . although palatini 's group measured cardiac output during exercise , the lv contractile response was identical to what we observed at lower heart rates . these data also parallel the findings of goodman and colleagues , despite the fact that our athletes exercised at significantly higher exercise intensities . therefore , it is likely that the imaging methods we used in this investigation reflect the contractile status of the lv during exercise despite the alteration in posture and the lower heart rate values during image acquisition . a second limitation of this investigation is that the systolic blood pressure end - systolic cavity relation is an indirect measure of end - systolic elastance . however , it is not feasible to perform invasive lv pressure - volume assessments in healthy younger athletes while they perform two hours of pse . despite this limitation , our finding that fractional area change increased despite a decline in end - diastolic cavity area ( i.e. , preload ) with a concomitant increase in lv wall stress ( i.e. , afterload ) at a constant exercise heart rate suggests that the heightened systolic function was related to an increase in myocardial contractility . as previously mentioned , there has been a paucity of investigations that have investigated the effects of pse in female athletes . therefore , our purpose was to investigate the effects of pse on lv systolic function in female triathletes while a secondary purpose was to determine the mechanism(s ) responsible for the changes in lv systolic performance . as a result , we were not interested in examining the gender effects of pse on lv systolic function and as such we did not have a comparison group of male triathletes . however , our results confirm and extend previous findings ( in younger male athletes ) by revealing that pse does not appear to result in lv systolic dysfunction in female endurance athletes . finally , we chose two divergent modes of exercise ( i.e. , cycling and treadmill running ) since these types of exercise were previously shown to result in a decline in stroke volume , cardiac output and lv systolic function in younger athletic females . therefore , it is possible that we may have observed a decline in lv systolic function if the subjects performed the same mode of exercise over our allotted time period . a significant number of the investigations that have reported altered lv systolic performance after pse have obtained lv images immediately following multisport events such as the hawaii ironman world championships ( reviewed in ) . athletes in these events exercise in three different body positions and echocardiographic data obtained from these studies consistently report altered lv systolic performance following pse . we therefore chose to mimic such an environment within a laboratory setting to quantify lv systolic performance throughout exercise , rather than following exercise . in addition , although postural changes may alter loading conditions of the lv during exercise , our images were consistent as they were all obtained with the athletes lying supine . therefore it is our contention that lv function during image acquisition was not affected by changes in postural position during pse . alternatively , it may have been possible that a decline in lv systolic function could have occurred if our subjects had exercised for a longer time period ( i.e. , > 2 hours ) . however , this does not seem realistic based on the findings of o'toole and associates who revealed that a decline in stroke volume and cardiac output occurred within the first 3060 minutes of low - intensity cycle ergometer exercise in younger female triathletes . a series of limitations that may have affected the results of this investigation must be addressed . first , in order to compare our findings with previous investigations that performed supine echocardiograms post pse , we elected to have our subjects momentarily stop exercising and quickly lie in the left lateral supine position while the echocardiographic images were acquired . a limitation with our method is that it does not fully represent the loading conditions and heart rate that occur during exercise . in addition , the supine posture should enhance venous return and as a consequence may result in divergent right and left ventricular interactions that may not occur to the same extent during upright exercise . in spite of the above limitations , our results are clearly consistent with recent exercise echocardiographic or radionuclide angiographic examinations that found that pse did not result in a decline in lv systolic dysfunction . although our images were obtained in the supine position we did observe heightened contractile function at all time points throughout the exercise session . heart rate values did decline significantly during imaging ( figure 1 ) , however the phenomenon of " cardiac fatigue " was initially described in a resting state in similar supine positions following pse . if lv contractile dysfunction was evident at rest in the supine position with heart < 100 bpm following pse in these previous investigations , one would expect to observe changes in contractile function not only during exercise but at heart rates between exercise and resting values , similar to those we observed in our investigation ( 100113 bpm ) . moreover , our findings are identical to those of palatini and associates who revealed that the heightened ejection fraction during exercise was due , in part , to an increase in myocardial contractility . although palatini 's group measured cardiac output during exercise , the lv contractile response was identical to what we observed at lower heart rates . these data also parallel the findings of goodman and colleagues , despite the fact that our athletes exercised at significantly higher exercise intensities . therefore , it is likely that the imaging methods we used in this investigation reflect the contractile status of the lv during exercise despite the alteration in posture and the lower heart rate values during image acquisition . a second limitation of this investigation is that the systolic blood pressure end - systolic cavity relation is an indirect measure of end - systolic elastance . however , it is not feasible to perform invasive lv pressure - volume assessments in healthy younger athletes while they perform two hours of pse . despite this limitation , our finding that fractional area change increased despite a decline in end - diastolic cavity area ( i.e. , preload ) with a concomitant increase in lv wall stress ( i.e. , afterload ) at a constant exercise heart rate suggests that the heightened systolic function was related to an increase in myocardial contractility . as previously mentioned , there has been a paucity of investigations that have investigated the effects of pse in female athletes . therefore , our purpose was to investigate the effects of pse on lv systolic function in female triathletes while a secondary purpose was to determine the mechanism(s ) responsible for the changes in lv systolic performance . as a result , we were not interested in examining the gender effects of pse on lv systolic function and as such we did not have a comparison group of male triathletes . however , our results confirm and extend previous findings ( in younger male athletes ) by revealing that pse does not appear to result in lv systolic dysfunction in female endurance athletes . finally , we chose two divergent modes of exercise ( i.e. , cycling and treadmill running ) since these types of exercise were previously shown to result in a decline in stroke volume , cardiac output and lv systolic function in younger athletic females . therefore , it is possible that we may have observed a decline in lv systolic function if the subjects performed the same mode of exercise over our allotted time period . a significant number of the investigations that have reported altered lv systolic performance after pse have obtained lv images immediately following multisport events such as the hawaii ironman world championships ( reviewed in ) . athletes in these events exercise in three different body positions and echocardiographic data obtained from these studies consistently report altered lv systolic performance following pse . we therefore chose to mimic such an environment within a laboratory setting to quantify lv systolic performance throughout exercise , rather than following exercise . in addition , although postural changes may alter loading conditions of the lv during exercise , our images were consistent as they were all obtained with the athletes lying supine . therefore it is our contention that lv function during image acquisition was not affected by changes in postural position during pse . alternatively , it may have been possible that a decline in lv systolic function could have occurred if our subjects had exercised for a longer time period ( i.e. , > 2 hours ) . however , this does not seem realistic based on the findings of o'toole and associates who revealed that a decline in stroke volume and cardiac output occurred within the first 3060 minutes of low - intensity cycle ergometer exercise in younger female triathletes . in summary , pse as performed by female triathletes was associated with an increase in lv systolic function . the heightened fractional area change during pse appears to be secondary to the increase in myocardial contractility as this form of exercise was associated with a decline in lv preload and a concomitant increase in lv afterload . these findings confirm a series of recent investigations , in younger males , that found that pse did not result in lv systolic dysfunction . moreover , they extend these findings and show that pse does not appear to result in lv systolic dysfunction in younger female triathletes . jm conceived the study , and participated in its design , coordination and data collection . we thank dave buchaski and the hewlett - packard corporation ( canada ) for providing the ultrasound machine for this investigation .

backgroundthe effect of prolonged strenuous exercise ( pse ) on left ventricular ( lv ) systolic function has not been well studied in younger female triathletes . this study examined lv systolic function prior to , during and immediately following pse ( i.e. , 40 km bicycle time trial followed by a 10 km run ) in 13 younger ( 29 6 years ) female triathletes.methodstwo-dimensional echocardiographic images were obtained prior to , at 30-minute intervals during and immediately following pse . heart rate , systolic blood pressure , end - diastolic and end - systolic cavity areas were measured at each time point . echocardiographic and hemodynamic measures were also combined to obtain lv end - systolic wall stress and myocardial contractility ( i.e. , systolic blood pressure end - systolic cavity area relation).resultssubjects exercised at an intensity equivalent to 90 3% of maximal heart rate . heart rate , systolic blood pressure , systolic blood pressure end - systolic cavity area relation and fractional area change increased while end - diastolic and end - systolic cavity areas decreased during exertion.conclusionspse is associated with enhanced lv systolic function secondary to an increase in myocardial contractility in younger female triathletes .

Background Methods Study Population Baseline Testing Race Simulation Left ventricular imaging Statistical Analysis Results Race simulation Echocardiographic and hemodynamic measurements Discussion Limitations Conclusions Author's Contributions Acknowledgements

PMC2808899

a toxic substance is a small molecule , peptide , or protein that is capable of causing injury or death . toxins , both natural and man - made , represent an important class of poisonous compounds that are ubiquitous in nature , in homes and in the workplace . common toxins include pollutants , pesticides , preservatives , drugs , venoms , food toxins , cosmetic toxins , dyes and cleaning compounds . indeed , common toxins are so ubiquitous that most adults will have been exposed to or will have while the concentration of most toxins in adults is generally below hazardous levels , their ubiquity essentially makes them an integral part of the human metabolome ( 1 ) . this fact , combined with the recent advances in chemical detection technologies for monitoring low abundance compounds , has greatly increased the need to identify , quantify , classify and understand these common toxins ( 2 ) . though a great deal of information has been acquired about toxic substances and their mechanism of action over the past 100 years , much of this information tends to be highly dispersed and of limited accessibility often being confined to specialized textbooks , obscure government documents or subscription - requiring scientific journals . a number of excellent , web - accessible toxic substance databases have started to appear , such as the animal toxin database ( atdb ) ( 3 ) , supertoxic ( 4 ) , aggregated computational toxicology resource ( actor ) ( 5 ) and the comparative toxicogenomics database ( ctd ) ( 6 ) . in particular , actor and supertoxic provide bioassy data and chemical structure information for a very large number of industrial or pharmaceutically interesting chemicals ( > 60 000 for supertoxic , > 500 000 for actor ) . atdb with 3844 peptide toxins and ctd with 5056 chemicals are somewhat more specialized , offering data on channel targets ( atdb ) or chemical - gene interactions ( ctd ) for a much smaller number of better - understood toxins . each of these databases addresses the needs of certain communities such as animal physiologists ( atdb ) , toxicogenomics specialists ( ctd ) , environmental or industrial regulators ( actor ) or medicinal chemists interested in toxicity prediction ( supertoxic ) . however , compared to a number of other widely used biochemical or metabolomic databases such as human metabolome database ( hmdb ) ( 1 ) , drugbank ( 7 ) or pharmgkb ( 8) most of today s online toxin or toxic compound databases are relatively lightly annotated , with fewer than 10 data fields per compound . furthermore , the data model adopted by many databases seems to be focused on providing a little information about everything ( including somewhat exotic or extremely rare compounds ) at the expense of including a lot of information about those toxins that nearly everyone encounters . consequently it is difficult to find answers to common questions such as : how does this poison work ? what enzymes , receptors or organs does this toxin target ? where is this chemical found ? how does this toxin get into the body ? how can i treat someone who has been exposed to this substance ? where can i find more information about this chemical ? in other words , the information contained in these resources does not fully address the needs of physicians , toxicologists , clinical chemists , metabolomics researchers , educators and the general public . indeed , members of these communities typically need in - depth , biological , chemical and mechanistic information about clinically relevant or commonly encountered toxins . in an effort to address these needs t3db is a multi - purpose , freely available , online toxicology resource that provides in - depth , quantitative and molecular - scale information about toxins and their associated targets . in terms of its layout , content and depth of coverage , the t3db is modeled closely after two other cheminformatic or metabolomics resources previously developed in our laboratory : the hmdb and drugbank . as the name indicates , the t3db is primarily intended to be a database that links toxins with their biological ( usually protein ) targets . this molecular interaction information is further supplemented with detailed descriptions of the toxin 's ; mechanism of action , its metabolism in the human body , its lethal or toxic dose levels , its potential carcinogenicity , exposure sources , symptoms or health effects and suggested treatment options . in total each toxin listed in t3db contains more than 80 data fields covering the structure , chemistry , physico - chemical properties , toxicology , route of delivery , treatment and target protein / macromolecule properties . t3db currently contains over 2900 toxin entries corresponding to more than 34 000 different synonyms . these toxins are further connected to some 1300 protein targets through almost 33 500 toxin and toxin - target bonds which , in turn , are supported by more than 3100 references . the entire database , including text , sequence , structure and image data , occupies nearly 16 gigabytes of data most of which can be freely downloaded . table 1.summary of the data fields or data types found in each toxcardtoxin and medical informationtoxin target protein/ enzyme informationcommon namenamecompound typemechanism of action ( in humans)descriptiondescriptionsynonyms / iupac namesynonymschemical formulagene namechemical structureprotein sequencecas registry numbernumber of residuesinchi identifiermolecular weightpubchem compound idtheoretical pikegg compound idgo classificationuniprot idgeneral functionomim idpathwayschebi idreactionsbiocyc idsignalssupertoxic idtransmembrane regionsctd idessentialitystitch iddomain functiondrugbank idgenbank i d proteinpdb iduniprotkb idactor idcellular locationwikipedia linkgene sequencemonoisotopic massgenbank gene idmol filegenecard idpdb filegenatlas idsmileshgnc idappearancechromosome locationmelting pointlocusboiling pointsnpsdensitygeneral referencessolubilitypredicted logpspecific gravityflash pointvapour pressureroute of exposuremechanism of actionmetabolismtoxicity values ( experimental ld50s and lc50s)lethal dose ( for humans)carcinogenicity ( iarc classification)uses / sourcesminimum risk levelhealth effectssymptomstreatmentgeneral referencesprotein targets summary of the data fields or data types found in each toxcard the diversity of data types , required breadth of knowledge and widespread nature of the required data made the assembly of the t3db both difficult and time - consuming . the t3db represents the accumulation of both qualitative and quantitative information derived from a variety of sources , including scientific journals , government records , chemical safety documents ( i.e. msds or material safety data sheets ) , textbooks , encyclopedias , online protein and protein structure databases , on - line metabolomic databases , electronic drug and pharmaceutical databases as well as existing toxin and toxicology databases . to compile , confirm and validate this comprehensive collection of data , more than a dozen toxicology , pharmacology and biochemistry textbooks , several thousands journal articles , nearly 20 different electronic databases , and at least 20 in - house or web - based programs were individually searched , accessed , compared , written , or run over the course of the past year . the team of t3db contributors and annotators consisted of eight bioinformaticians with dual training in computing science and molecular biology / chemistry . the t3db is designed to be a fully searchable web resource with many built - in tools and features for viewing , sorting and extracting toxin and toxin - target annotation , including structures and gene and protein sequences . a screenshot montage illustrating the types of viewing and searching options available is shown in figure 1 . detailed instructions on where to locate and how to use these browsing / search tools are provided on the t3db homepage . as with any web - enabled database , the t3db supports standard text queries through the text search box located on the home page . it also offers general database browsing using the browse button located in the t3db navigation bar . to facilitate browsing , the t3db is divided into synoptic summary tables which , in turn , are linked to more detailed all of the t3db s summary tables can be rapidly browsed , sorted or reformatted in a manner similar to the way pubmed abstracts may be viewed . clicking on the toxcard button found in the leftmost column of any given t3db summary table opens a webpage describing the toxin of interest in much greater detail . each toxcard entry contains over 80 data fields , with 50 data fields devoted to chemical and toxicological / medical data and 30 data fields ( each ) devoted to describing the toxin target(s ) . figure 1.a screenshot montage of the toxin , toxin - target database ( t3db ) showing several of t3db s search and display tools describing the toxin toxaphene . left : toxin browse view ; toxcard for toxaphene ; chemical structure similarity search for tozaphene ; t3db chemquery window ; t3db data extractor for toxin and toxin - target data fields . a screenshot montage of the toxin , toxin - target database ( t3db ) showing several of t3db s search and display tools describing the toxin toxaphene . clockwise from top left : toxin browse view ; toxcard for toxaphene ; chemical structure similarity search for tozaphene ; t3db chemquery window ; t3db data extractor for toxin and toxin - target data fields . a toxcard begins with various identifiers and descriptors ( names , synonyms , compound description , structure image , related database links and i d numbers ) , followed by additional structure and physico - chemical property information . the remainder of data on the toxin is devoted to providing detailed toxicity and toxicological data , including route of delivery , mechanism of action , medical information and toxicity measurements . each of these targets are described by some 30 data fields that include both chemical and biological information , as well as details on their role in the mechanism of action of the toxin . in addition to providing comprehensive numeric , sequence and textual data , each toxcard also contains hyperlinks to other databases , abstracts , digital images and interactive applets for viewing molecular structures . a key feature that distinguishes the t3db from other on - line toxin or toxicology resources is its extensive support for higher - level database searching and selecting functions . in addition to the data viewing and sorting features already described , the t3db also offers a local blast ( 9 ) search that supports both single and multiple sequence queries , a boolean text search based on kinosearch ( http://www.rectangular.com/kinosearch/ ) , a chemical structure search utility based on chemaxon s marvinview , and a relational data extraction tool similar to that found in drugbank and the hmdb ( 1 ) . these can all be accessed via the database navigation bar located at the top of every t3db page . t3db s simple text search box ( located at the top of most t3db pages ) supports text matching , text match rankings , mis - spellings ( offering suggestions for incorrectly spelled words ) and highlights text where the word is found . t3db s textquery function uses the same kinosearch engine , but also supports more sophisticated text querying functions ( boolean logic , multi - word matching and parenthetical groupings ) as well as data - field - specific queries such as finding the query word only in the compound source field . t3db s sequence searching utility ( seqsearch ) allows users to search through t3db s collection of 1300 known ( human ) toxin targets . this service potentially allows users to identify both orthologous and paralogous targets for known toxins or toxin targets . it also facilitates the identification of potential targets from other animal species . with seqsearch , gene or protein sequences may be searched against the t3db s sequence database of identified toxin - target sequences by pasting the fasta formatted sequence ( or sequences ) into the seqsearch query box and pressing the submit button . t3db s structure similarity search tool ( chemquery ) can be used in a similar manner as its seqsearch tool . users may sketch ( through chemaxon s freely available chemical sketching applet ) or paste a smiles string ( 10 ) of a query compound into the chemquery window . submitting the query launches a structure similarity search that looks for common substructures from the query compound that matches the t3db s database of known toxic compounds . users can also select the type of search ( exact or tanimoto score ) to be performed . high - scoring hits are presented in a tabular format with hyperlinks to the corresponding toxcards ( which in turn links to the targets ) . the chemquery tool allows users to quickly determine whether their compound of interest is a known toxin or chemically related to a known toxin and which target(s ) it may act upon . in addition to these structure similarity searches , the chemquery utility also supports compound searches on the basis of chemical formula and molecular weight ranges . the t3db s data extraction utility ( data extractor ) employs a simple relational database system that allows users to select one or more data fields and to search for ranges , occurrences or partial occurrences of words , strings , or numbers . the data extractor uses clickable web forms so that users may intuitively construct sql - like queries . using a few mouse clicks , it is relatively simple to construct complex queries ( find all toxins that target acetylcholinesterase and are pesticides ) or to build a series of highly customized tables . the output from these queries t3db has a perl - based web - friendly front - end attached to a sophisticated mysql relational database ( version : 5.0.51a ) . the database is maintained on an apple xserve located on the university of alberta campus . all data is entered directly onto the database s ruby on rails web - based laboratory information management system ( t3-lims ) . each toxcard and targetcard has an edit page , which allows administrators to manually make changes to the database entries . the public user interface and the lims both read from the same database , and there is no intermediate database for rendered html pages . this hub is a restful web resource that automatically stores and updates chemical properties such as molecular weight , solubility and log p. additionally , the hub renders the structure images and thumbnails visible on the public site . t3db s structure hub uses a combination of jchem base ( from chemaxon ) and ruby on rails running in a jruby environment on a glassfish server . the centralized nature of this hub helps to maintain consistency for all structures stored in t3db . whenever a structure is changed or fixed , all properties are automatically re - calculated and made available on the public site . unlike the situation for drugs where formal approval processes and tight regulations allows one to easily identify whether a compound is a drug or not , the situation for toxins is not so simple . first , there is the issue of defining exactly what a toxin is . normally , the word given that many man - made toxic substances are also produced by living organisms or are the byproducts of living organisms ( albeit at low concentrations ) , this distinction seems a little arbitrary . therefore we have chosen to use the term toxin to refer to both man - made and naturally produced substances that exhibit some form of toxicity ( i.e. an acute reaction , injury or death ) . in constructing the t3db a distinction between all posssible toxins and technically almost all of the 38-million known chemical substances ( 11 ) could be toxic if consumed , injected or inhaled in sufficient quantities . yet only a tiny fraction of known chemicals ever make it out of the laboratory and into everyday use ( perhaps < 20 000 compounds ) . therefore common toxins would have to be defined as those compounds that can be found in the home , the environment or the workplace and which of have had a recorded medical consequence ( i.e. acute reaction , injury or death ) . while this definition may seem reasonable for instance , essential compounds such as vitamins , sugar , oxygen and even water can be prove to be toxic if consumed / inhaled in extreme excess . yet their inclusion in a toxin database like t3db would be certainly stretch the limits of believability . therefore we chose to further refine the inclusion criteria to limit ourselves to those compounds that have been routinely identified as hazardous in relatively low concentrations ( < 1 mm for some , < 1 m for others ) and which appear on multiple toxin / poison lists provided by government agencies such as toxnet ( 12 ) or the toxicological and medical literature . in each case , the toxicity of each compound was assessed by examining the available toxicity measurements and health effects , such as minimum lethal dose , ld50 , lc50 values and carcinogenicity . furthermore , because many common toxic substances , such as cleaning agents , gasoline , kerosene , glue , paint thinners are complex mixtures of many compounds , we also limited t3db to include only those substances where specific toxic components could be identified and where the chemical structures to those components were known . as a result , common or name - brand household cleaning products are not included in t3db but their toxic components are . obviously these criteria may lead to the exclusion of some rare or interesting toxic compounds or toxic mixtures , but our focus for t3db was on creating a chemically - oriented database that could be broadly useful , comprehensively annotated and easily managed . as has been previously noted , t3db toxins were identified using a number of methods , including literature surveys , data mining of other databases , using toxicology textbooks and examining lists of controlled or banned substances . the toxic compounds found using these sources were also used to derive additional substances that were toxic by relation ( such as metal salts or structurally similar compounds ) and which can be found in the household , workplace or environment . once identified the toxins were further assessed using the common toxin inclusion criteria described above . in order to ensure both completeness and correctness , each toxin record entered into t3db was reviewed and validated by a member of the curation team after being annotated by another member . additional spot checks were routinely performed on each entry by other members of the curation group . several software packages including text - mining tools , chemical parameter calculators and protein annotation tools were developed , modified and used to aid in data entry and data validation . this includes search tools such as biospider ( 13 ) and polysearch ( 14 ) , which collate and display text ( as well as images ) from multiple sources allowing t3db s curators to compare , assess , enter and correct toxin and toxin - target information . much of the annotation in t3db was done manually , especially in areas regarding toxicology and biology , such as mechanisms of action , route of delivery , health effects and target identification . this qualitative and quantitative information was derived from a variety of sources , including scientific journals , government records , material safety data sheets ( msds ) , textbooks , encyclopedias and existing toxicology databases . for example , we frequently used existing chemical - protein or chemical gene interaction databases such as the ctd and stitch ( 15 ) to identify potential toxin targets . once these potential targets were identified , the corresponding references were manually reviewed by multiple curators to confirm which of the genes / proteins were true targets . in annotating the toxic action of drugs , we often used the drug mechanism information found in t3db s sister database drugbank . to facilitate and monitor the data entry process , all of t3db s data is entered into a centralized laboratory information management system ( lims ) , allowing all changes and edits to the t3db to be monitored , time - stamped and automatically transferred . relative to other recently published toxic substance databases , t3db probably has the smallest number of toxins or poisons in its collection ( 2900 in t3db versus 500 000 compounds in actor ) . this situation was quite deliberate as t3db was designed to be a database for common toxins as opposed to a database for all known toxic substances . while t3db s breadth of coverage is limited , it depth of coverage is not . with more than 80 data fields for each toxin and with more than 16 gb of data , we believe t3db offers a considerable amount of useful chemical , biological and toxicological data . as with most toxic substance databases t3db this usually indicates that the data has yet to be measured or the information has yet to be reported in the literature . in some cases , the data probably exists , but our annotation team has yet to locate or validate it . in addition to the existence of empty data fields , t3db is also missing some compounds . currently more than 500 compounds are still on the t3db to do list and will be added ( at a rate of 50 compounds a week ) to the database over the coming months . furthermore , new toxins are always being identified and these will need to be added as they are reported ( if sufficient information exists ) . because t3db strives for in - depth coverage , not all toxic compounds make it to the database . those that lack any significant biological or toxicological data ( i.e. the claim for toxicity is not supported by any study or biomedical report ) or those that do not have a known chemical structure will not be included in the database . despite these caveats and limitations , we believe t3db represents a new and important model for toxic substance databases . with a focus on depth as opposed to breadth , we are hopeful that the rich content in t3db will allow researchers , educators and members of the general public to uncover answers to many common questions about toxins and poisons . likewise , with its unique emphasis on common substances ( i.e. those that can normally be detected with modern analytical methods ) we believe that the t3db should also prove to be a valuable resource in toxico - metabolomics and clinical toxicology research . the fact that t3db provides a wide range of chemical and biological data as well as a broad collection of search and display tools also represents a significant new development for toxic substance databases . we are certainly hoping that some of these ideas may find there way into other toxic substance databases . over the coming two years we also plan to bring in additional data ( spectroscopic , tissue / biofluid concentration data ) and to add new features ( spectral matching , pathway images , target prediction ) to this database , with the intent of making t3db a comprehensive , one - stop - shop for toxicological and toxico - metabolomics research . alberta advanced education and technology ( aaet ) ; the canadian institutes of health research ( cihr ) ; genome alberta , a division of genome canada . funding for open access charge : canadian institutes of health research .

in an effort to capture meaningful biological , chemical and mechanistic information about clinically relevant , commonly encountered or important toxins , we have developed the toxin and toxin - target database ( t3db ) . the t3db is a unique bioinformatics resource that compiles comprehensive information about common or ubiquitous toxins and their toxin - targets into a single electronic repository . the database currently contains over 2900 small molecule and peptide toxins , 1300 toxin - targets and more than 33 000 toxin - target associations . each t3db record ( toxcard ) contains over 80 data fields providing detailed information on chemical properties and descriptors , toxicity values , protein and gene sequences ( for both targets and toxins ) , molecular and cellular interaction data , toxicological data , mechanistic information and references . this information has been manually extracted and manually verified from numerous sources , including other electronic databases , government documents , textbooks and scientific journals . a key focus of the t3db is on providing depth over breadth with detailed descriptions , mechanisms of action , and information on toxins and toxin - targets . t3db is fully searchable and supports extensive text , sequence , chemical structure and relational query searches , similar to those found in the human metabolome database ( hmdb ) and drugbank . potential applications of the t3db include clinical metabolomics , toxin target prediction , toxicity prediction and toxicology education . the t3db is available online at http://www.t3db.org .

INTRODUCTION DATABASE DESCRIPTION DATABASE IMPLEMENTATION CRITERIA FOR INCLUSION IN T3DB QUALITY ASSURANCE, COMPLETENESS AND CURATION LIMITATIONS AND CONCLUSION FUNDING

PMC4403610

musa acuminata ( banana ) is one of the most important fruit crops of world as well as of india . banana could be considered poor man 's apple , and it is the cheapest among all other fruits in the country . the pathogen is soil - borne and remains viable up to several years and cause 20%80% loss of banana . several disease management strategies can be used such as crop rotation , burning infected plants or plant parts , and application of carbendazim . methods mentioned have limited success , and the application of synthetic fungicides may result in undesirable effects on the environment . an alternative to above strategies for managing fusarium wilt is the use of biological control . biocontrol agent can be a beneficial organism ( live or dead ) or its part such as cell wall , protein , and oligosaccharides . while using live organisms as a biocontrol agent , appropriate conditions for maintaining it should be strictly followed . nevertheless , if part of the organism such as cell wall , protein , oligosaccharide , or attenuated / killed organism is used then strict conditions are not required . plants , humans , and animals give instantaneous response to the pathogen or its part . animals and humans produce antibodies against pathogen or vaccine , similarly plants response to pathogen attack by producing pr - proteins , defense - related enzymes , plantibodies , and phytoalexins . study of pr - proteins or defense - related enzymes are key to any plant disease resistance mechanism . farmers in gujarat , india purchase tissue culture plants every year for sowing in the fields with the expectations of getting high production and high profit . foc being soil borne may enter the plant and cause disease anytime after sowing and affects fruit production . if plants are immunized , that is , accumulation of defense - related enzymes occurs before the attack of pathogen then the pathogen can be successfully warded off , and loss can be minimized . elicitor used here is acting as a vaccine ( derived from the dead fungus ) to protect the plant . the aim of the present study was to compare the interaction of dead and live pathogen with banana plants ( grand naine variety ) . grand naine is a large fruit yielding dwarf cavendish variety with height of 6.5 to 7.5 ft introduced to india from israel remains choice of farmers as the bunches of banana fruits can be harvested within twelve to thirteen months from the date of planting . the plant response was documented using several marker enzymes to analyze whether the plant could differentiate the signals from dead and live pathogen . another important aim was to check if the response generated using dead pathogen was sufficient to ward off the forced inoculated live pathogen . cubense ( foc ) from fusarial wilt infected banana plants was maintained on potato dextrose agar ( pda ) at 27c . for liquid culture of fungus , 8-mm agar plug of 3 - 4 w k old culture was inoculated in potato dextrose broth ( pdb ) and incubated at 27c for 21 day on pdb . the media with mycelium was autoclaved at 121c for 20 minutes , crushed intensely in grinder , and was used as dead fungi for treatment . two months old banana ( grand naine variety ) plantlets were procured from gujarat state fertilizers and chemicals ( gsfc ) , baroda , gujarat . plant roots of grand naine variety were exposed without damaging them by carefully removing the surrounding soil . dead and live fungus suspensions were prepared by mixing 1 g of dead fungus and live fungus per liter of distilled water . per plant , 1 ml of dead and live pathogen suspension was administered against control plants treated with 1 ml of distilled water in exposed root region . after treatment , changes in levels of defense - related enzymes in leaves were assayed after each successive day till seventh day [ 4 , 5 ] . leaves were excised up to seven days at regular interval of 24 h for estimation of defense related enzymes , namely , pox , ppo , -1,3 glucanase , chitinase , and total phenolics from both control and treated plants . experiments were repeated twice under similar conditions with different sets of plants , and all the analyses were performed in triplicates . ppo , -1,3 glucanase , chitinase , and total phenolics assays were done as described by meena et al . . to ensure the induction of resistance , plant roots treated with dead pathogen was exposed to spore suspension of foc ( 10 spores / ml ) where plants treated with distil water was used as control and exposed to same quantity of spore suspension as experimental . the aim of this study was to investigate interaction of dead and live pathogen , fusarium oxysporum f.sp . cubense a causative agent of panama disease , with banana plants ( grand naine ) . interaction was determined using accumulation of several defense related enzymes , namely , pox , ppo , -1,3 glucanase , chitinase , and total phenolics as markers for induction of defense . hypothesis was that plants could differentiate signals from dead and live pathogen and in response to signals from dead pathogen , it can induce defense enzymes , which could protect plants upon subsequent exposure to pathogen . pox activity induced earlier in dead and live pathogen treated plants and retained elevated levels compared to control plants . pox activity increased 4- and 2.5-fold in dead and live fungus treated plants , respectively . highest pox activity in plant treated with dead and live pathogen was observed on 6th and 7th day , respectively ( figure 1 ) . comprehensible difference in ppo activity was observed on treatment of dead and live pathogen . with live fungus interaction , plants showed to induce 3-fold ppo activity from first day onward , reached highest level on 4th day , and thereafter reached near basal . however , dead fungus treatment failed to mount significant induction in ppo activity for initial five days of interaction followed by 2- and 3-fold induction in ppo activity on 6th and 7th days , respectively compared to control plants ( figure 2 ) . interaction of dead and live fungus with banana plants resulted in induction of -1,3 glucanase activities from 1st day onward . up to five days , pattern of -1,3 glucanase activity was very similar in both treatments . from the 5th day onward , -1,3 glucanase activities stabilized at 2- to 2.5- fold higher levels in live fungus interaction as compared to control . in case of dead fungus interaction , gradual increase in -1,3 glucanase activity was observed from 5th day onward with highest increase 3.5-fold on 7th day as compared to control ( figure 3 ) . chitinase activity induced from 1st day in banana plants treated with dead and live pathogenic fungus . figure 4 showed stiff rise ( 10-fold ) in chitinase activity on 2nd and 3rd day followed by rapid fall and retention of about 1.5 fold chitinase activity in banana treated with live fungus compared to control plants . whereas , in dead fungus treated plants , chitinase activity increased twofold on 1st day followed by gradual increase up to 4-fold on 7th day . in dead fungus treated plants , total phenolics content gradually increased and reached to maximum on 7th day ( 2-fold ) . whereas in live fungus treated plants , total phenolics content increased on 3rd day followed by sharp decline to reach basal level as compared to control plants ( figure 5 ) . control plants and plants treated with dead pathogen were forced inoculated with the live fungal spores and observed for the development of symptoms . in control plants , the characteristic symptoms of fusarial wilt were observed in the first week after forced inoculation followed by aggravation of disease condition after 15 days . however , no symptoms of fusarium wilt were observed even after two months after forced inoculation in plants treated with dead pathogen . in the present study , analysis of plants response towards dead and live pathogenic strain was carried out using induction of several key marker enzymes associated with plant defense mechanism . development of effective , durable , economic , and environmentally sound strategies for the control of crop diseases could be possible through an improved understanding of the interactions between plants and pathogenic agents . the ability of a pathogen to produce a disease in a host plant is usually the exception , not the rule . this is because plants have an innate ability to recognize potential invading pathogens and to set up successful defenses . on the other hand , successful pathogens produce diseases because they are capable to evade detection or suppress host defense mechanisms , or both . when plants are challenged by a pathogen , early local defense reactions and delayed , systemic responses get activated in order to counteract the pathogen attack . among the early local responses , the hypersensitive response ( hr ) leads to a local programmed cell death in order to deprive the pathogens of their nutrition base . later on , the plant can develop systemic acquired resistance ( sar ) leading to resistance throughout the whole plant in an unspecific manner towards a broad spectrum of pathogens . in case of sar , signal is transmitted from infected tissue in the whole plant for induction of overall defense gene expression . this demonstrates that signal perception in initial pathogen recognition and signal transduction to initiate further defense responses is essential for plants to counteract phytopathogens . some defenses are constitutive , such as various pre - formed antimicrobial compounds , whereas others activated by pathogen recognition . recognition process includes product of a dominant or semidominant resistance r gene present in the plant and the corresponding dominant avirulence ( avr ) factor encoded by or derived from the pathogen . recognition of avr factor by host plant starts one or more signal transduction pathways that activate several of plant 's defenses , thus compromising ability of pathogen to colonize plant [ 11 , 12 ] . in the present study , interaction of banana plants with dead and live pathogen resulted in induction of pox activity . as dead pathogen was capable to generate initial recognition signals ; however , it can not counter the plant response , which leads to induction of pox activity more than live fungus . peroxidases are a well - known class of pr proteins and induced in host plant tissues by pathogen infection . they belong to pr - protein 9 subfamily and are expressed to limit cellular spreading of infection through establishment of structural barriers or generation of highly toxic environments by massively producing ros and rns . peroxidase activity or peroxidase gene expression in higher plants is , indeed , induced by fungi , bacteria , viruses , and viroids . cross - linking of the phenolic monomers in oxidative coupling of lignin subunits has been associated with peroxidase using h2o2 as oxidant . one significant event in plant defense reactions is oxidative burst , a common early response of host plant cells to pathogen attack and elicitor treatment . our results showed increase in pox activity in dead fungus treated as well as fungus treated plants as compared to control . this result indicates that elicitor slowly increases level of lignin formation , suberization , and hypersensitive response . other enzymes , ppos are a group of copper containing enzymes that catalyze oxidation of hydroxy phenols to their quinone derivatives , which have antimicrobial activity . because of its reaction products and wound inducibility , ppo plays a role in defense against plant pathogens . plant immediately respond to pathogen so there is immediate rise in ppo indicating immediate synthesis of antimicrobials to ward off pathogen . in elicitor treated plant ppo activity increases slowly day by day indicating that plant has got stimuli to increase ppo . in case of live pathogen interaction , there was immediate response by plants increasing ppo 5-fold on the first day , which starts decreasing from the 6th day indicating multiplication of fungus in the plant system . however , dead pathogen completely fails to mount this response , which strongly suggests that plants are capable to differentiate signals from live and dead pathogen . increase in ppo activity was reported in banana roots treated with foc - derived elicitors by thakker et al . . marked increase in ppo activity was observed in banana roots treated with psuedomonas fluorescens against fusarial wilt . -1,3 glucan and chitin , polymer of n - acetylglucosamine ( nag ) are major cell wall components of many fungi . since -1,3 glucanase and chitinases have been shown to be capable of attacking cell wall of fungal pathogens , these enzymes have been proposed as direct defense enzymes of plants . we observed an increase in chitinase and -1,3 glucanase activity indicating plants ready mechanism to ward off pathogen by directly degrading the pathogen cell wall and in turn protecting the plant . in our previous study , we found antifusaric activity of elicitor induced -1,3 glucanase that showed swelling of mycelia after one hour incubation of pathogen with purified elicitor . endochitinase purified from barley was capable of inhibiting the growth of trichoderma rescei , alternaria alternate and neurospora crassa . in addition , mauch et al . reported that in combination , chitinase and -1,3 glucanase act synergistically to inhibit fungal growth . phenolic acids are involved in phytoalexin accumulation , biosynthesis of lignin , and formation of structural barrier , and play a main role in the resistance against pathogen . marked accumulation of phenolics was observed on the 3rd third day in fungus treated plants indicating the plants sensitivity to pathogen , and it attempt to protect itself by the formation of structural barriers . further , from the 4th day activity decreases showing successful multiplication and establishment of pathogen by overcoming structural barriers formed by plants . in dead pathogen - treated plants , we found slow increase in phenolics activity indicating ability to recognize dead pathogen as some foreign body , but it is not multiplying ; therefore , it will not break structural barrier . marked accumulation of phenols leading to suppression of fusarial wilt was observed in tomato plants . similar results were observed in banana plants in vitro condition . when tomato plants were treated with catechol , marked accumulation of phenols anna and dubery investigated that subfraction of cell - wall bound phenolics , ester - bound phenolics , glycoside bound phenolics , and free phenolics increased 6.3- , 4.2- , 3.0- , and 2.3-folds , respectively , upon induction . in plants treated with dead pathogen , a forced inoculation of live pathogen was performed to assess induced plant 's ability to ward off pathogen . interestingly no symptoms of fusarial wilt were observed in these plants even though pathogen was in close proximity . the present study strongly supports the view of preparation of plant vaccines for combating devastating disease like fusarium wilt of banana . dead pathogen preparation was not only successful in mounting defense response but also in protecting plants upon subsequent infections . therefore , it could be potential candidate for plant vaccine preparation to combat panama disease .

the aim of the present study was to scrutinize the response of banana ( grand naine variety ) plants when interacting with dead or live pathogen , fusarium oxysporum f.sp . cubense , a causative agent of panama disease . response of plants was evaluated in terms of induction of defense - related marker enzyme activity , namely , peroxidase ( pox ) , polyphenol oxidase ( ppo ) , -1,3 glucanase , chitinase , and phenolics . plant 's interaction with live pathogen resulted in early induction of defense to restrain penetration as well as antimicrobial productions . however , pathogen overcame the defense of plant and caused disease . interaction with dead pathogen resulted in escalating defense response in plants . later on plants inoculated with dead pathogen showed resistance to even forced inoculation of live pathogen . results obtained in the present study suggest that dead pathogen was able to mount defense response in plants and provide resistance to panama disease upon subsequent exposure . therefore , preparation from dead pathogen could be a potential candidate as a biocontrol agent or plant vaccine to combat panama disease .

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

PMC3000473

parathyroid carcinoma is a rare disorder , accounting for 0.1% to 5.0% of all cases of primary hyperparathyroidism [ 15 ] . the disease presents at a mean age of 50 years and is equally likely to occur in males as in females [ 26 ] ( table 1 ) . clinical and biochemical manifestations are those of severe primary hyperparathyroidism often with renal and skeletal complications [ 25 , 7 ] . a palpable neck mass can be felt in 3076% of patients [ 3 , 5 , 8 , 9 ] . parathyroid carcinoma is associated with germline and somatic mutations in the hrpt2 gene [ 3 , 10 ] and with irradiation of the neck for other pathologies [ 3 , 11 ] . table 1overview of cases of parathyroid carcinoma published between 2001 and 2010authorage / sexnumber of operationsradiotherapychemotherapyembolisationradiofrequency ablationpth immunisationcalcitonincinacalcetbisphosphonatesdevelopment of local recurrence or distant metastasisoutcomefollow - up ( years)savli 47/f1noa / fod1tyler 44/m2yesyesa / fod5.5dotzenrath 43/m4yesyesdod1243/f12yesyesa / wd862/f2yesd / other9.5yamashita 43/m2yesa / wd12eurelings 45/f1yesyesyesdod4brown 51/m2noa / fod2bhansali 40/m2yesyesyesnodod0.5hundley 33/f5yesa / fod20schmidt 76/f1noa / fod1schoretsanitis 55/f1noa / fod2dionisi 35/m2yesyesdod4rufener 67/m3yesa / fod4munson 41/f1yesnoa / fod5.546/f1yesnoa / fod4.549/f1yesnoa / fod5.557/f1yesnoa / fod4.5dogan 50/f1noa / fod1.5betea 50/f2yesyesyesyesa / wd11kern 54/f2yesyesdod2.5kirkby - bott 44/m2yesyesyesdod3.570/m1yesnoa / fod736/f1yesnoa / fod566/m2yesyesdod846/m1yesyesyesa / wd125/f1noa / fod144/m1noa / fod1lin 38/m1noa / fod0.5ahmad 42/f1yesyesa / fod1chiofalo 66/m1yesnoa / fod1cheah 32/m2noa / fod0.542/f1noa / fod0.552/f2yesa / fod5.5yoshida 61/f2yesyesa / wd18.5ikeda 48/m4yesyesa / wd6pahlavan 21/m2yesnoa / fod2agarwal 22/f2yesa / fod9mezhir 64/f7yesyesyesyesa / fod8placzkowski 51/f4yesyesyesyesyesa / fod12sahasranam 53/m1noa / fod2tkaczyk 55/m2yesnoa / fod1tan 55/f1noa / fod0.5iwata 61/m1noa / fod3montenegro 50/m2yesyesyesdod5rathi 45/f1noa / fod0.5artinyan 71/m2yesyesyesyesyesa / fod0.5cetani 53/m3yesyesa / fod2temmim 63/f1noa / fod2tamura 70/m1yesa / wd0.5schoretsanitis 72/m1noa / fod478/f1noa / fod455/f1noa / fod671/m1yesdod275/m1noa / fod4kung 72/f1noa / fod2marcy 42/f1noa / fod1yuan 38/f2noa / fod4rock 60/m1yesnoa / fod0.5yong 23/m1yesyesnoa / fod2chaychi 79/f1noa / fod0.5tochio 42/m2yesyesyesa / wd8.551 2/30m:32f1.9 0.218/62 ( 29%)4/62 ( 6%)1/62 ( 2%)3/62 ( 5%)1/62 ( 2%)3/62 ( 5%)5/62 ( 8%)14/62 ( 23%)27/62 ( 44%)4.3 0.5results are expressed as mean sedod dead of disease , d / other dead of other cause , a / wd alive with disease , a / fod alive free of disease overview of cases of parathyroid carcinoma published between 2001 and 2010 results are expressed as mean se dod dead of disease , d / other dead of other cause , a / wd alive with disease , a / fod alive free of disease a diagnosis of parathyroid carcinoma is suggested by the presence of intra - operative features of local invasion and the diagnosis is confirmed by the world health organization histopathological criteria for parathyroid carcinoma . these include the presence of vascular invasion , perineural space invasion , capsular penetration with growth into adjacent tissues and/or metastasis . although parathyroid carcinoma is a slow - growing tumour , it inevitably metastasises and is invariably fatal due to the eventual inability to control hypercalcaemia . survival is estimated to range from 50% to 86% at 5 years and 3570% at 10 years [ 16 , 13 , 14 ] . the most effective treatment for parathyroid carcinoma is radical surgery with en bloc removal of the lesion together with the ipsilateral thyroid , thyroid isthmus and lymph nodes [ 2 , 3 , 5 , 13 , 15 ] . surgery is also the treatment of choice in case of local recurrence or development of metastases [ 5 , 7 , 16 ] , although radiotherapy [ 25 , 8 , 9 ] , chemotherapy [ 3 , 5 , 9 , 17 , 18 ] , embolisation or radio frequency ( rf ) ablation [ 1921 ] may also be attempted . we report here the nearly two decades odyssey of a male patient with multiple metastases from parathyroid carcinoma , describing in the process the challenges and pitfalls we encountered in the management of this difficult malignancy . we also review all original case reports and case series on parathyroid carcinoma , fulfilling the who criteria and with a follow - up of at least 6 months after initial parathyroidectomy , which were published in the english literature since the last large review on this topic in 2001 . the literature search was conducted in pubmed using the keywords ( parathyroid neoplasms or parathyroid carcinoma or ( case reports or case report or case or cases ) . patients with changes suggestive of carcinoma in the context of autonomous ( tertiary ) hyperparathyroidism were excluded because of the controversy in diagnosing malignancy in this case . of an initial 872 publications , only 45 fulfilled the who criteria for parathyroid carcinoma and follow - up as per our search criteria , and were selected for further analysis . a 29-year - old man presented to his primary care physician with a 2-month history of severe weight loss ( 20 kg ) associated with polyuria , polydipsia , nausea , muscle weakness , difficulty in concentrating and debilitating tiredness . on physical examination , he was clinically dehydrated and had a palpable hard , fixed swelling in the left side of the neck . laboratory investigations revealed a severe hypercalcaemia of 4.9 mmol / l ( normal range 2.152.55 mmol / l ) , a decreased creatinine clearance of 45 ml / min ( normal > 120 ml / min ) and a 10-fold increased serum intact parathyroid hormone ( pth ) concentration : 88 pmol / l ( normal no further investigations were requested ; the patient was intensively rehydrated and referred for parathyroidectomy . at neck exploration , an enlarged parathyroid gland ( 3.5 3 3.5 cm ) invading the ipsilateral thyroid lobe was found and removed from the left side of the neck ( fig . 1 ) . the diagnosis of parathyroid carcinoma was confirmed histologically by the finding of capsular and vascular invasion . serum calcium and pth concentrations normalised within a few days of surgery associated with a significant improvement in renal function . a second surgery was performed within 1 month of the first surgery to ensure complete removal of all malignant tissue and provide a negative surgical margin . the patient was started on a course of radiotherapy , which had to be discontinued before completion because of severe pharyngitis and oesophagitis . 1disease course of our patient spanning over 17 years disease course of our patient spanning over 17 years a recurrence of hyperparathyroidism was documented on routine laboratory control 3 years after parathyroidectomy . mmol / l , a pth concentration of 8.4 pmol / l and a stable creatinine clearance of 75 ml / min . tc99m - mibi - spect and ultrasonography identified a lesion in the left side of the neck , which was found to be a local recurrence of the primary tumour at extensive bilateral neck exploration . the recurrent tumour had invaded the oesophagus , part of which was excised and reconstructed using tissue from the sternocleidomastoid muscle . the edges of the excised specimen were tumour free and resected draining lymph nodes were clear of tumour tissue . dna analysis of the resected recurrent tumour demonstrated a somatic hrpt2 mutation , c.165delc located on exon 2 . despite the radical nature of the surgery , mild hyperparathyroidism persisted post - operatively , suggesting the presence of residual probably metastatic tumour tissue . no further localisation studies or surgery were planned principally because of the patient s reluctance to undergo further surgery but also because of the mild nature of the hyperparathyroidism . closer clinical monitoring was arranged . over the following 4 years , serum calcium and pth concentrations and bone turnover markers slowly increased , and periods of dehydration were associated with transient worsening of renal function , which remained , however , on the whole stable despite persistent hypercalciuria and development of nephrocalcinosis . progressive bone loss , predominantly cortical , was also documented and treatment with the non - nitrogen - containing bisphosphonate clodronate was started , resulting in normalisation of bone turnover and stabilisation of bmd measurements . ultrasound ( us ) , selective venous sampling for pth ( svs ) and ct scan of the neck succeeded in localising a small dense lesion in the left supraclavicular region . ct scan of the thorax also revealed a small solitary lesion of 1-cm radius in the inferior lobe of the right lung ( fig . the patient refused to undergo a thoracotomy , but did agree to a fourth neck exploration at which a lymph node metastasis was excised from the left supraclavicular region ( fig . 1 ) . although less severe , hyperparathyroidism persisted post - operatively , as expected due to the unresected lung metastasis . in contrast , radiofrequency ablation ( rfa ) of the lesion , although complicated by an episode of severe pleuritis , was very successful in achieving biochemical remission , with serum pth concentrations decreasing from 265 pmol / l to < 1 pmol / l ( fig . 2 ) , suggesting that the metastasis targeted with rfa was the only source of pth secretion . however , remission was not permanent and pth levels started to rise within 9 months of rfa . 3 ) , which was complicated by multiple small cerebral infarcts resulting in right haemianopia , dysphasia , acalculia and transient epilepsy . disappointedly , there was also only partial improvement in serum calcium and pth concentrations , which continued to increase within 2 months of lobectomy ( fig . 3 ) . 2radiological ( a pre - rf ablation , b after rf ablation ) and biochemical changes after rf ablation of the lung metastasis , demonstrating a severe drop in serum pth concentrations , which persisted for almost a yearfig . 3radiological and biochemical changes following lobectomy of the lung metastasis , which resulted in only partial improvement in serum calcium and pth concentrations radiological ( a pre - rf ablation , b after rf ablation ) and biochemical changes after rf ablation of the lung metastasis , demonstrating a severe drop in serum pth concentrations , which persisted for almost a year radiological and biochemical changes following lobectomy of the lung metastasis , which resulted in only partial improvement in serum calcium and pth concentrations further localisation studies , in the form of tc99m - mibi - spect and a ct scan of the neck and thorax , were unable to localise any residual pathological parathyroid tissue suggesting the presence of ( micro)metastasis . the now available calcimimetic cinacalcet was prescribed eventually at a maximum dose of 90 mg twice daily , which decreased pth concentrations from 120 pmol / l to 85 pmol / l , stabilised calcium concentrations to about 2.80 in the course of the following year , serum calcium and pth concentrations further increased , however , despite maximal doses of cinacalcet and intermittent intravenous bisphosphonates . repeat tc99m - mibi - spect and ct scan of the thorax , now demonstrated a new large ( 4 3 2 cm ) subcarinal mediastinal metastasis , which was successfully excised using a transpericardial approach ( fig . the elimination of the source of pth secretion was associated with a very severe hungry bone syndrome for which the patient needed intensive care for over 2 months , requiring very large doses of iv administered calcium ( up to 14 g / day ) and maximal doses of active vitamin d metabolites . remission lasted for more than 1 year , after which pth levels started to rise again and a new metastatic lesion was localised pretracheally in the upper mediastinum on ct scan of the thorax . there was also suggestion of a second subcarinal lesion at the site of the previously excised metastasis . 4ct scan of the thorax demonstrating a large ( 4 3 2 cm ) subcarinal mediastinal lymph node metastasis ( white arrow ) ( a , b ) , which was successfully excised using a transpericardial approach ( c ) ct scan of the thorax demonstrating a large ( 4 3 2 cm ) subcarinal mediastinal lymph node metastasis ( white arrow ) ( a , b ) , which was successfully excised using a transpericardial approach ( c ) in view of the complications associated with his previous surgeries , the patient was at that stage again reluctant to undergo any further surgery . within the next few months , pth steadily increased associated with uncontrollable hypercalcaemia and deteriorating renal function eventually requiring haemodialysis using a very - low - dialysate calcium concentration . the pretracheal lesion was accessible from the neck and was radically removed by a cervical approach ( fig . 1 ) . there were no post - operative complications and serum pth concentration decreased from 590 pmol / l to 155 pmol / l , as expected due to the remaining subcarinal mediastinal metastasis . surgical removal of this metastasis would have required a third thoracotomy , which would be associated with too high an operative risk , especially in view of the patient s poor clinical condition . excision of the metastasis by mediastinoscopy was also not considered to be an option because of multiple scarring due to previous surgeries . the patient remains on haemodialysis and is still treated with cinacalcet , with intermittent courses of intravenous bisphosphonates . the patient was not considered to be a candidate for pth immunisation [ 23 , 24 ] because of surgical clearance of cervical and mediastinal lymph nodes , poor general immune status and recurring infections . sixty - two new cases have been reported in the literature since the last published review in 2001 , and data of these cases are summarised in table 1 . the median age of the reported patients was 50 years ( 2179 ) and there was an equal number of men and women reported . data on initial presentation were available in 43 of the 62 patients ( 69% ) , 37 of which were symptomatic ( 86% ) . polyuria and polydipsia were reported in nine patients ( 21% ) , constipation in seven ( 16% ) , tiredness in 13 ( 30% ) , bone and joint pain in 17 ( 40% ) , muscle weakness in seven ( 16% ) , nausea and vomiting in 10 ( 23% ) and weight loss in seven patients ( 16% ) . seventeen patients ( 40% ) had renal stones and six patients ( 14% ) had sustained a documented fracture . a palpable neck mass was reported in 22 of 27 patients in whom this was checked ( 81% ) . similar to the review of 2001 , surgery is still the most frequently chosen therapy for the initial treatment of parathyroid carcinoma but also for the treatment of recurrent or metastatic parathyroid carcinoma ( table 1 ) . it is of note that the use of both chemotherapy and radiotherapy has decreased over the last 10 years , likely due to several reports and reviews which showed disappointing results [ 25 ] . the last case report on the use of chemotherapy and radiotherapy dates from 2005 and 2008 , respectively ( table 1 ) . in contrast to the review of 2001 , reports on the use of the newer techniques embolisation ( n = 1 ) , radiofrequency ablation ( n = 3 ) and pth immunisation ( n = 1 ) have been recently published , although the number of cases reported is still very limited . use of the calcimimetic , cinacalcet , has increased over the last few years after the first report in 2007 , while the use of bisphosphonates has remained stable . the median follow - up reported in the 62 cases is relatively short at 3 years ( range 0.520 years ) , considering that the average time to a first recurrence is also approximately 3 years [ 1 , 3 ] . this could explain why only 27 of the 62 patients ( 44% ) developed a local recurrence and/or distant metastasis and nine patients ( 15% ) died as a result of parathyroid carcinoma during the reported follow - up period . to our knowledge , we report here one of the longest survival and follow - up in one centre of a patient with recurrent metastatic parathyroid carcinoma . although parathyroid carcinoma is a slow - growing tumour , it is associated with a relatively high incidence of local recurrence ( 4082% within 5 years ) [ 13 , 5 , 6 , 13 ] . metastases tend to occur later in the course of the disease with spread to cervical nodes ( 30% ) , lung ( 40% ) and liver ( 10% ) , via both lymphatic and haematogenous routes . survival is estimated to range from 35% to 70% at 10 years [ 16 , 13 , 14 ] , with an average survival of 6 years after local recurrence , and of 4.5 years after the development of distant metastases . in patients with parathyroid carcinoma , determinants of survival include delay in diagnosis , the presence of locally invasive features , distant metastasis at diagnosis and the radical nature of initial surgery [ 3 , 6 , 13 , 14 ] . a further important determinant of survival and disease - free survival is the due care taken at initial surgery to avoid rupturing the capsule of the gland to prevent local seeding of tumour tissue . initial surgery has been reported not to be radical in up to 48% of patients because of failure to establish the diagnosis of parathyroid carcinoma pre- or intra - operatively [ 6 , 14 ] . this was also the case in our patient at initial surgery , despite very suggestive clinical features of parathyroid carcinoma , in the form of severe hyperparathyroidism in a young male patient , palpable neck swelling and intra - operative features of local invasion . a second surgery had to be performed within 1 month of the first to ensure complete removal of all malignant tissue and to provide a negative surgical margin . the main determinant of survival in all patients remains , however , the eventual inability to control hypercalcaemia [ 2 , 5 ] and its association with fatal renal and cardiovascular complications [ 1 , 2 , 5 , 7 ] . end - stage renal failure has been reported in up to 84% of patients due the deleterious effects of severe persistent hypercalcaemia on kidney function [ 2 , 5 , 7 ] . a persistently elevated circulating pth level is also associated with increased bone turnover and bone loss predominantly at cortical sites and with increased risk of fractures . in parathyroid carcinoma , the main goal of treatment is control of hyperparathyroidism by eradicating the source of pth secretion [ 7 , 16 ] . although the morbidity associated with re - exploration of the neck is estimated to be 617% , that of re - exploration of the mediastinum or lungs is clearly much higher . in our patient , the recurrent laryngeal nerve had to be unavoidably sacrificed during the second surgical intervention due to invasive growth of remnants of the primary tumour , resulting in transient loss of voice and permanent hoarseness . the increasing morbidity attached to repeated surgical interventions has led to the search for other treatment options , such as radiotherapy , chemotherapy , rf ablation , embolisation , use of the calcimimetic cinacalcet and pth immunisation . although parathyroid tumours are relatively resistant to radiotherapy [ 25 ] , this management approach showed some promise as adjuvant treatment for microscopic residual disease [ 8 , 9 ] . in our patient , the prescribed course of radiotherapy could not be completed due to the development of severe side effects in the form of pharyngitis and oesophagitis . embolisation of localised and/or ectopic parathyroid adenomas has been reported to have only limited success [ 19 , 2527 ] , and in metastatic parathyroid carcinoma , embolisation has only been described in combination with rf ablation in one patient . in our patient , the outcome of twice attempted embolisation of a lung metastasis was disappointing , largely because it was not technically possible to selectively embolise the arterial branch that fed the lung metastasis . the outcome of combined embolisation and rf ablation in a patient with liver metastasis due to parathyroid carcinoma , and the effectiveness of rf ablation in various types of cancer has led us to consider rf ablation in our patient . this approach was indeed very successful in eradicating pth production by the metastasis , as has also been subsequently reported in two patients with parathyroid carcinoma and lung metastases [ 20 , 21 ] . the beneficial effect was unfortunately transient , lasting only 9 months after the procedure , when pth production was documented to resume from the same site . in parathyroid carcinoma , attempts at reducing tumour load are not always successful , in which case control of serum calcium by other means becomes central to prevent the deleterious effects of hypercalcaemia on various organ systems . intensive rehydration , use of medications such as bisphosphonates and calcimimetics and use of dialysis represent diverse means to control hypercalcaemia and are often used in combination . bisphosphonates decrease the skeletal efflux of calcium from bone by suppressing osteoclast - mediated bone resorption and overall bone turnover . these agents , however , have no effect on renal tubular reabsorption of calcium so that they improve hypercalcaemia but do not normalise serum calcium [ 3 , 29 ] . cinacalcet is a calcimimetic , which reduces parathyroid hormone secretion by binding to the calcium - sensing receptor on parathyroid cells , increasing the sensitivity of these cells to extracellular calcium concentrations [ 15 , 30 , 31 ] . these agents are widely used in the management of secondary and tertiary hyperparathyroidism in renal failure and have been also recently registered for the management of primary hyperparathyroidism , including that due to parathyroid carcinoma . in our patient , combined treatment with bisphosphonates and cinacalcet resulted in reasonable control of the hypercalcaemia initially , but eventually failed to do so , even when used at maximal doses . dialysing against low - dialysate calcium became necessary in order to better control the hypercalcaemia . the very high operative risks associated with a third thoracotomy , the patient s poor lung function and current metabolic status despite regular dialysis , preclude the option of further surgery to remove the identified source of pth secretion . pth immunisation is being explored as a potential non - invasive option , although experience with this approach is still limited and the patient s general condition may not permit its use [ 23 , 24 , 32 ] . although we are rapidly running out of options in the management of our patient , we believe that we have nonetheless managed to secure for him a longer albeit not disease - free survival . this clinical case with a follow - up spanning over 17 years illustrates that the long - term management of patients with metastatic parathyroid carcinoma remains indeed a daunting task , despite all recent imaging , surgical and medical advances .

a 29-year - old man presented to his primary care physician with nausea , severe weight loss and muscle weakness . he had a hard , fixed neck swelling . he was severely hypercalcaemic with 10-fold increased parathyroid hormone ( pth ) concentrations . a diagnosis of primary hyperparathyroidism was established and the patient was referred for parathyroidectomy . at neck exploration , an enlarged parathyroid gland with invasive growth into the thyroid gland was found and removed , lymph nodes were cleared and hemithyroidectomy was performed . a suspected diagnosis of parathyroid carcinoma was confirmed histologically . serum calcium and pth levels normalised post - operatively , but hyperparathyroidism recurred within 3 years of surgery . over the following 17 years , control of hypercalcaemia represented the most difficult challenge despite variable success achieved with repeated surgical interventions , embolisations , radiofrequency ablation of metastases and treatment with calcimimetics , bisphosphonates and haemodialysis using low - dialysate calcium . in this paper , we report the challenges and pitfalls we encountered in the management of our patient over nearly two decades of follow - up and review recent literature on the topic .

Introduction Case Report Review of the Literature 20012010 Discussion

PMC3926413

the word metabolism comes from greek metabol which means change or transformation , and in fact the levels of most cellular metabolites change with half times of minutes , seconds , or even faster . during the past years the complexity and dynamics nature of the metabolites have become increasingly apparent [ 16 ] . the comprehensive analysis of a large set of metabolites , now referred to as metabolomics , present in a biological sample , has emerged as an important tool in functional genomics and systems biology [ 7 , 8 ] . since it is downstream of central dogma ( not like transcriptome and proteome at higher cascade ) , the metabolome should show greater effects of genetic or physiological changes [ 9 , 10 ] and is therefore much closer related to the phenotype expressed by an organism . metabolic fingerprinting ( an analysis of intracellular metabolites profiling ) and metabolic footprinting ( an analysis of extracellular metabolites profiling ) have succeeded in experimental characterization of genetic mutants on the basis of combined intracellular and extracellular metabolite data measured by mass spectrometry ( ms ) [ 5 , 8 , 11 , 12 ] . however , the measurement of extracellular metabolites , often referred to as footprinting [ 4 , 11 ] , represents several advantages over the analysis of intracellular compounds , often referred to as metabolic fingerprinting , for microbial cultures . although the functional analysis using metabolic fingerprinting can classify the different phenotypes of several mutants , it is difficult to scale up for high - throughput screening of many mutants as it is quite time - consuming and subject to technical difficulties caused by the rapid turnover and the need of quenching and extracting of metabolites from the extracellular space [ 5 , 11 ] . analysis of metabolite profiles can be done using different analytical techniques , but recent technical advances in ms have brought this technology to the forefront among methods for metabolome analysis due to the high sensitivity and separation efficiency [ 13 , 14 ] . gas chromatography coupled to ms ( gc - ms ) is the most extensively used technique in metabolome analysis , as it is effective for resolving complex biological mixtures and hereby enable reliably identification of the analyzed compounds . however , gc - ms can only analyze metabolites which are stable at the high temperatures present in gc and they have to be volatile or can be made volatile upon chemical derivatization . most metabolites are not volatile and many of them are not stable at high temperature or can not be derivatized . recently , direct infusion - mass spectrometry ( di - ms ) has been reported as an alternative to gc - ms and it seems to be an ideal analytical tool for high - throughput metabolome analysis [ 1620 ] . the most significant feature of esi mode for di - ms is that it is a very soft ionization technique which , in many cases , will produce protonated molecular species ( in positive esi ) for a broad range of different compounds with very high sensitivity . with high sensitivity and atmospheric pressure ionization , di - ms can analyze the majority of metabolites in the sample in a few seconds without any chromatographic separation [ 16 , 17 , 19 , 20 ] . an obvious extension of a di - ms approach is to store the spectra in a database using the database software included with most instruments . this will give a sample identification database rather than compound identification as anticipated by the manufactures [ 15 , 17 , 19 , 21 , 22 ] . here we present the use of dynamic metabolic footprinting analyzed by di - ms for phenotypic information of yeast . using novel data analysis methods we show that it is possible to extract the key information about the metabolism from footprinting data when yeast is grown on different carbon sources . the data analysis is based on identification of significantly correlated metabolites over time , which corresponds to flux - ratios for different metabolites . the advantage of this approach is that it is not sensitive to a single time point and , therefore , better allows for analysis of mutants having different growth rates . all reagents used for metabolite analysis and the medium for yeast cultures were prepared using analytical grade ingredients . methanol , acetonitrile , and formic acid used for mass spectrometry were obtained from sigma - aldrich ( st . louis , mo , usa ) . milliq - purified water was used during the sample preparation for hplc and di - ms analyses . the s. cerevisiae strain by4709 ( mat ura30 ) obtained from the european s. cerevisiae archive for functional analysis ( euroscarf , frankfurt , germany ) was used in this study . the strain was grown in a minimal synthetic medium supplemented with a metabolite cocktail according to allen et al . the metabolite cocktail contained a selection of amino acids , organic acids , and organic bases . the medium was based on yeast nitrogen base ( ynb ) without amino acids ( bd difco , franklin lakes , nj , usa ) and the metabolite cocktail was composed of l - arginine , l - aspartate , l - glutamate , l - histidine , l - leucine , l - lysine , l - methionine , l - serine , l - threonine , l - tryptophan , l - valine , citrate , fumarate , malate , pyruvate , succinate , cytosine , and uracil . all compounds from the metabolite cocktail had a concentration of 1 mm in the final medium . cultivations were performed with 3 different carbon sources , that is , glucose , glycerol , and ethanol . each carbon source is dissolved in water , autoclaved separately , and finally added to give a final concentration 0.67 c - mole / l corresponding to ( 20.00 g / l glucose , 15.34 g / l ethanol , or 20.45 cultivations were performed in 5 replicates in 500 ml baffled shake flasks ( 150 rpm ) , at 30c . each flask contained 100 ml of footprinting media prepared according to allen et al . and closed with cotton plugs . during cultivation , the culture purity was monitored on a regular basis by phase contrast microscopy and they east growth was monitored by od measurements at 600 nm . biomass dry weight was determined by filtering a known volume of fermentation broth , approximately 5 ml , through a dried , preweighed nitrocellulose filter ( sartorius stedim biotech s.a . ) with a pore size 0.45 m . the filter was dried to constant weight in a microwave oven at 150 w for 10 min , cooled in a desiccator , and the weight was measured . besides , the optical density was determined at 600 nm using a genesis 20 ( thermo spectronic ) , spectrophotometer . samples were diluted with water to obtain od measurements in the linear range of 0.10.5 od units . samples harvested from the cultivation broth were immediately filtered through a 0.45 m pore - size cellulose acetate filter ( vwr ) and stored at 20c until analysis . glucose , glycerol , ethanol , succinate , and acetate concentrations were determined by hplc analysis using an aminex hpx-87h column ( biorad , hercules , ca ) and all the conditions were set following zaldivar et al . . briefly , the separation was performed at 45c , with sulfuric acid ( 5 mm ) at a flow rate of 0.5 ml / min as the mobile phase . subsequent determinations of yield coefficients for extracellular metabolites as well as biomass were based on linear regressions of their concentration as a function of the residual glucose concentration in the exponential growth phase . the yeast strain by4709 was grown on three different carbon sources : glucose , ethanol , and glycerol . for each carbon source 5 replicates were carried out and the samples were taken every 3 hours from 3 hr until 30 hr such that they represented different growth phases for growth on the different carbon sources . two - milliliter samples were centrifuged ( 8,000 g , 10 min ) and the supernatant was stored at 18c until further di - ms analysis . the supernatants were diluted fivefold with acetonitrile right before they were analyzed by di - ms . the sample preparation for the dynamic metabolic footprinting analysis was performed as illustrated in the metabolic footprinting pipeline in figure 1 . the di - ms analysis was performed on a system setup with an agilent 1100 microflow hlpc pump , lc - packings autosampler coupled to a micromass ( waters , manchester ) q - tof system with an electrospray ionization interface . the instrument was tuned for maximal sensitivity at low flow rate and minimal fragmentation using leucine - enkephalin , followed by external calibration using a mixture of peg200 and 400 in acetonitrile - water . the samples were diluted fivefold in acetonitrile ( with 1 g/l leucine - enkephalin as an internal standard mass reference ) and centrifuged at 10,000 g for 1 min . the supernatants were transferred into 200 l hplc vial inserts and the vials were placed in the autosampler . the sequence of samples was randomly injected in order to decrease the effects from instrumental bias . the samples were analyzed by infusion of 5 l sample into the esi source of q - tof ms at a flow rate of 20 l / min . a carrier flow of methanol was used at a rate 15 l / min from the lc - pump through the autosampler ; just before the ion source a flow of 2% formic acid in water was fed into the solvent stream from a syringe pump at a rate of 5 l / min using a t - piece giving a combined flow of 20 l / min of 75% methanol - water with 0.5% formic acid going into the source . mass spectra were acquired in both positive and negative mode and data were collected for 3 min / sample between 50 and 1000 da / e at a rate of one continuum scan / second . the q - tof conditions were the following : capillary voltage 3,000 v in positive mode and 2,600 v in negative mode , desolvation temperature 150c , dry gas , desolvation gas at 300 l / hr , nebulizer flow 20 l / hr , source temperature 90c , and cone voltage optimized to minimal fragmentation approximately 40 v in positive mode and 30 v in negative mode . initially , the raw data were processed according to the method described by hansen and smedsgaard . the ms data ( as shown in figure s1a ) were preprocessed in the following way : for each sample , the elution profile were detected , 40 continuum scans ( 1 s / scan ) were summarized to a single continuum spectrum to reduce noise , followed by background subtraction and calculating the centroid spectrum ( figure s1b ) . then , all data were normalized based on the ion count of leucine - enkephalin ( m / z 556.2771 ) internal standard to reduce technical variability from the instrumental bias . the result was a matrix where each row corresponded to a sample and each of the columns to a metabolite . the width of each column roughly corresponded to 0.5 m / z . in the following , two criteria were applied for selecting ( cherry - picking ) the metabolites.the first criteria dealt with screening for the single metabolites in each injected sample showing proper changes over time ( either decreasing or increasing or a combination of both ) . since the changing pattern can either be linear or nonlinear ( e.g. , exponential or sigmoidal pattern ) , the data smoothness step was required for the nonlinear function by fitting to the ( standardized ) time profiles . in the later step , those selected metabolites which have the actual trend in the profiles ( noise excluded ) will further be analyzed.for the next criteria , we identified metabolite pairs which show the covariance across time . each metabolite was paired up and plotted against each other to investigate the correlation pattern of individual pair and the regression line was fitted to the data . finally , the correlation ( r of the regression line ) and the p value were calculated . the first criteria dealt with screening for the single metabolites in each injected sample showing proper changes over time ( either decreasing or increasing or a combination of both ) . since the changing pattern can either be linear or nonlinear ( e.g. , exponential or sigmoidal pattern ) , the data smoothness step was required for the nonlinear function by fitting to the ( standardized ) time profiles . in the later step , those selected metabolites which have the actual trend in the profiles ( noise excluded ) for the next criteria , we identified metabolite pairs which show the covariance across time . each metabolite was paired up and plotted against each other to investigate the correlation pattern of individual pair and the regression line was fitted to the data . finally , the correlation ( r of the regression line ) and the p value were calculated . the intercept , slope ( which is equivalent to what is often referred to as yield coefficients ) , r , and the p value of the regression line for each metabolite were stored in a matrix of dimension as the number of metabolites . since many of the identified metabolites are related and covary we used only the selected metabolites to analyze the variance present in data . whereas ordinary principal component analysis ( pca ) is widely used in data processing and dimensionality reduction , pca in general suffer from the fact that each component is a linear combination of all the original variables . even if some variables contain random noise , those will be assigned weights ( loadings ) in the linear combination . thus , it is often difficult to interpret the results . as for ordinary pca , sparse pca finds sets of sparse vectors ( having a relatively small number of nonzero elements ) for use as weights ( loadings ) in the linear combinations while still explaining most of the variance present in the data . for all samples , based on the p value calculated for each pair of ions , we could generate a symmetric matrix of correlations with the ions along both axes . the upper triangular part of the matrix was extracted and unwrapped ( figure s2 ) . based on the matrix wt ( 1 ) esi - mode ( 2 ) source ( 3 ) rep ( 5 ) rows and ( n 1)n/2 columns , where n is the number of ions present , we investigated the variation ( informational ) content by sparse principal component analysis and further identified metabolites that additionally correlated in response to time for growth on the different carbon sources . therefore , the analysis becomes very dependent on the growth rate of the organism and also on factors such as inoculum size . in order to circumvent this problem we developed a pipeline for dynamic metabolic footprinting ( figure 1 ) . based on hplc measurements of the carbon sources and metabolic products the overall kinetics of substrate uptake could be determined . for the glucose cultures , the cells took approximately 12 hours to finish their exponential phase with a glucose consumption rate of 0.304 g glucose / gdw / h ( see table 2 and figure 3 ) . in contrast , the cells growing on a nonfermentative carbon source took 21 hours to finish their exponential phase with a consumption rate of 0.027 g ethanol / gdw h and 0.048 g glycerol / gdw / h , respectively . even though they were grown in different initial concentrations in terms of g substrate / l the c - mole amounts were exactly the same ( 0.67 c - mole / l ) for all three carbon sources . comparing the specific consumption rate in terms of c - mole ( see figure 3 and also table 2 ) , the cells could consume glucose approximately 10 times faster than ethanol and glycerol ( i.e. , 10.133 , 1.117 , and 1.564 c - mmole of carbon / gdw h for glucose , ethanol , and glycerol , resp . ) . not surprisingly , yeast cells preferred glucose which is a fermentable carbon source more than the 2 nonfermentable carbon sources . there is as mentioned in the introduction an interest in using metabolic footprinting as the turnover rate for extracellular metabolites is much longer , than that for intra - cellular metabolites ( fingerprinting ) and footprinting does not require any complicated procedure for the quenching and extraction steps . however , using a single data point can cause problems with capturing the phenotype of different mutants , and the approach proposed here may therefore allow for wider use of metabolic footprinting . here we demonstrate high - throughput metabolic footprinting using di - ms , which allow us to cover more than 2,000 ion peaks ( both from positive and negative esi mode ) , which cover many metabolites in different pathways to be analyzed at the same time . from the di - ms analysis results and its integrated signal spectra ( figures s1a and b ) , we pointed out how the technique was so consistent giving such a high reproducibility in all 5 biological replicates . even though it is complicated to interpret biological meaning from thousands of ion peaks , we here demonstrate the cherry - picking criteria to identify a list of single metabolites that showed significant change over time . the di - ms analysis showed that amino acids were best detected in positive mode , probably due to the amino group . the typical base peak ion was [ m + h ] and [ m + ch3cn + h ] . after conversion and alignment of the data according to hansen and smedsgaard , each of the metabolites was first analyzed for trends over time . the resulting matrix had 300 rows ( one for each sample ) and approximately 1500 columns . after binning the data , as part of the initial procedure , columns containing no data were removed . the first cherry - picking criteria identified a list of single metabolites that showed the proper change over time . after this step , the number of columns in the matrix was reduced to 145 metabolites . two examples of identified metabolites can be seen in the top two figures in figure 2 . as the next criteria , the degree of covariance over time was calculated . figure 2 ( bottom picture ) illustrates the process , where the two metabolites were picked during the first selection step , and shows covariance across time . for all metabolite pairs , the intercept , slope ( yields ) , r , and the p value of the regression line for each metabolite were stored in a matrix of dimension as the number of metabolites . in order to evaluate the information present in the matrix of metabolite pairs , we initially analyzed the variation in yields by pca to identify the key metabolites which are significantly different between the sample groups and to how well they can separate growth on the different carbon source from each other . based on the matrix columns as shown with the cherry - picking method ( figure 2 ) , we calculated the pca loading for pca analysis . figure 4 illustrates the scores and loadings for the first two principal components using ordinary pca . as can be seen from the plots , the yields of the selected metabolite pairs show a nice separation of the carbon sources . still , when looking at the loadings we see that all metabolite pairs are assigned weights . sparse pca was used to obtain sets of sparse vectors for weights ( loadings ) in the linear combinations while explaining most of the variance present in the data . figure 5 illustrates the effect of using of sparse pca rather than ordinary pca on the loadings . we see that most of the metabolite pairs have been assigned zero weight , whereas only a few pairs have been assigned a weight . although most of the weights in the loadings are zero , we still see the same grouping into carbon sources as when using ordinary pca . as can be seen from the sparse pca , a few pairs of metabolites can separate the data into distinct carbon sources . next , we wish to investigate those metabolites in further detail . from the reconstruction of the correlation networks it was found that pairs from pc1 are most important as they generate the largest network . pc1 segregates the signal from glucose from the other two carbon sources ( ethanol and glycerol ) . the size of key metabolites network represents the level of differences in dynamic pattern among the sample groups [ 6 , 21 ] . pairs of ms ions that correlated over time were identified as those the sparse pca were assigned weights . only the metabolite pairs from the loadings corresponding to the sparse pc1 and sparse pc2 were extracted . there were a total of 48 pairs , which corresponds to correlation between 44 ions as key metabolites : 24 pairs between 17 metabolites for pc1 and 3 pairs between 3 metabolites for pc2 in positive esi mode ( see table 3 ) , and 17 pairs between 19 metabolites for pc1 and 4 pairs between 5 metabolites for pc2 in negative esi mode ( see table 4 ) . a few metabolites were identified both in positive and negative esi mode , and the number of metabolites identified was 45 . for each ion the corresponding metabolite was identified using the database developed by hjer - pedersen , and a list of the identified metabolites for the two modes are given in tables 5 and 6 . moreover , the fragmentation patterns from esi - ms of each metabolite were also carefully considered and compared according to the golm metabolome databases to confirm the possible calculated mass per charge . for most of the ions there is a unique identification of the corresponding metabolite , but for some ions more than one metabolite has the corresponding mass , for example , for the ion 179.0353 m / z there are three potential candidates for the corresponding metabolite , namely , 3-(4-hydroxyphenyl ) pyruvate , glucose , and myo - inositol . in this particular case glucose is the most likely metabolite and it was chosen , but cases where there could not be made a clear assignment both metabolite names were used . pc1 ions primarily separate the glucose samples from the ethanol and glycerol samples and pc2 mainly separates the ethanol samples from the glycerol samples . the metabolite pairs from pc1 represent a network as illustrated in figure 6 , and the metabolite pairs from pc2 represent two networks as illustrated in figure 7 . in these networks it is observed that most of the key metabolites are amino acids that are linked to cyclic amp or cyclic damp , but glucose is also clearly correlated to several metabolites . the color code indicates the slope with green indicating decreasing concentration with time and red increasing concentration with time . a few metabolites do not change much in concentration profile or do not have a monotonous concentration profile during the whole fermentation , for example , their concentration are increasing in one part of the fermentation and decreasing in another part of the fermentation , and these are marked blue in the networks . it is interesting to note that there is a positive correlation between camp and most amino acids , whereas the glucose concentration is negatively correlated with the concentration of several metabolites , for example , glycerol . the pca results , both ordinary pca , and sparse pca showed that the main variance in the data is caused by the 3 different c - sources : glucose , ethanol , and glycerol . the sparse pca finds sets of sparse vectors for use as weights in the linear combinations while still explaining most of the variance present in the data . it is built on the fact that pca can be written as a regression - type optimization problem , with a quadratic penalty ; the lasso penalty ( via the elastic net ) can then be directly integrated into the regression criterion , leading to a modified pca with sparse loadings . according to the results we obtained from both ordinary pca ( figure 3 ) and sparse pca ( figure 4 ) , it is clearly shown that the sparse pca gives a better clustering among the groups compared to the normal pca results . focusing on the consistency of this analysis , the results from pca indicated that the main variance in the data is caused by the different c - sources with high reproducibility . the 5 replicates are separated into the same cluster based on the dynamic metabolic profile which is promising . the key metabolites network on pc1 which contain more metabolite nodes compared to that on pc2 show the higher difference between glucose and ethanol or glucose and glycerol cultures while there are less differences between glycerol and ethanol cultures . in other words , the dynamic pattern of the glucose culture is more unique than the rest . so , we can obviously see that there are largest dynamic changes in metabolite concentrations during growth on glucose , which can be explained by the fact that glucose is a fermentative carbon source whereas there is purely respiratory growth on ethanol and glycerol . the 4 main hubs of the network are glucose , cyclic amp , cyclic damp , and nicotinate - d - ribonucleotide ( namn ) as illustrated in the key metabolites network on pc1 ( figure 6 ) . the levels of these metabolites are linked to the biosynthesis of many amino acids and also affected by the concentrations of glucose and also amino acids . since fermentable sugars are specific stimulators for camp synthesis in yeast cells , the ras / camp pathway is activated by both growth signal ( e.g. , glucose ) and stress signals ( e.g. , uv radiation and starvation ) . our finding from the correlation analyses is consistent with this as it shows that glucose and camp have the same dynamic pattern at all conditions . since low camp concentration stimulates the uptake of and l - leucine , we can see that the uptake rate of leucine was increasing ( the extracellular leucine level was decreasing ) , while the extracellular camp level was decreasing . from the key metabolites network on pc2 ( figure 7 ) , we also found the link between methionine , glycine , 4-phospho - hydroxy - l - threonine , and pyridoxine . as the methionine is required for synthesis of phosphatidylcholine ( pc ) via methylation of phosphatidylethanolamine ( pe ) , these two metabolites have high correlation ( sharing the same pattern ) in correlation analysis result . in a similar case , 4-phospho - hydroxy - l - threonine ( 4hlt , the precursor to pyridoxal 5-phosphate biosynthesis ) was also linked to glycine , serine , and threonine metabolism and also pyridoxal phosphate synthesis pathway via cyclic amp and cyclic damp . in conclusion we here show that through dynamic metabolic footprinting it is possible to identify correlations between metabolites that can be used to provide new insight into possible regulatory structures . furthermore , our correlation analysis may be used for identification of key biomarkers for specific phenotypes during dynamic growth conditions .

metabolic footprinting offers a relatively easy approach to exploit the potentials of metabolomics for phenotypic characterization of microbial cells . to capture the highly dynamic nature of metabolites , we propose the use of dynamic metabolic footprinting instead of the traditional method which relies on analysis at a single time point . using direct infusion - mass spectrometry ( di - ms ) , we could observe the dynamic metabolic footprinting in yeast s. cerevisiae by4709 ( wild type ) cultured on 3 different c - sources ( glucose , glycerol , and ethanol ) and sampled along 10 time points with 5 biological replicates . in order to analyze the dynamic mass spectrometry data , we developed the novel analysis methods that allow us to perform correlation analysis to identify metabolites that significantly correlate over time during growth on the different carbon sources . both positive and negative electrospray ionization ( esi ) modes were performed to obtain the complete information about the metabolite content . using sparse principal component analysis ( sparse pca ) , we further identified those pairs of metabolites that significantly contribute to the separation . from the list of significant metabolite pairs , we reconstructed an interaction map that provides information of how different metabolic pathways have correlated patterns during growth on the different carbon sources .

1. Introduction 2. Materials and Methods 3. Results and Discussions

PMC428583

crohn 's disease is a chronic intestinal disorder of unknown etiology characterized by weight loss , abdominal pain , diarrhea , arthritis and the development of fistulae and abscesses . it causes significant morbidity and affects approximately 1 in 1000 individuals in the developed world . crohn 's disease is believed to ensue from the action of an environmental trigger(s ) including alteration in host intestinal flora on a genetically susceptible host mucosal immune system and intestinal epithelial barrier . a variety of crohn 's disease susceptibility loci have been identified by genetic mapping studies . the first crohn 's disease susceptibility gene , nod2 , was identified definitively in 2002 by positional cloning and linkage disequilibrium mapping as well as candidate gene approaches . nod2 encodes an intracellular receptor for muramyl dipeptide , a component of the peptidoglycan moiety of bacterial cell walls , and triggers a cascade of signaling events resulting in the activation of nf - kappa b and the host innate immune system . crohn 's disease - associated mutations in nod2 result in defective nf kappa b activation , suggesting that crohn 's disease may represent , in part , a defect in innate immunity . a second gene that has recently been linked to crohn 's disease and ulcerative colitis susceptibility is the multidrug resistance transporter 1 ( mdr1 ) . a single nucleotide polymorphism ( snp ) in the coding region of the gene ( ala893ser / thr ) has been found to occur more frequently in patients with crohn 's disease and a second snp ( c3435 t ) has been associated with ulcerative colitis susceptibility . the mdr1 gene encodes an atp - binding cassette ( abc ) family member that pumps neutral and cationic hydrophobic molecules out of the cell and plays a role in resistance to chemotherapy . intriguingly , mdr1-/- mice develop spontaneous colitis in the presence of normal intestinal bacteria . the mdr1 protein may also play a role in host defense against intracellular bacteria by extruding them from the cell , explaining its protective role in intestinal inflammation , but this hypothesis identification of additional crohn 's disease susceptibility genes is important to complete the puzzle of crohn 's disease pathogenesis and to develop specific , targeted immunotherapies . a region of broad susceptibility to inflammatory bowel disease has been identified on chromosome 5q31 - 5q33 and is known as ibd5 . within this region , a crohn 's disease susceptibility haplotype comprising a cytokine cluster on 5q31 has been identified . interestingly , a missense substitution in slc22a4 , a gene in this region that is a downstream target of the transcription factor , runx1 , is associated with susceptibility to crohn 's disease . moreover , an intronic snp in a runx1 binding site of slc22a4 has been found in rheumatoid arthritis , an autoimmune disease that sometime occurs in individuals and families affected by crohn 's disease . polymorphisms in the promoter of the cd14 gene , which plays a critical role in lipopolysaccharide signaling and is located downstream from the cytokine cluster , have been linked to crohn 's disease susceptibility in a case - control study . to our knowledge , no crohn 's disease - related polymorphisms in genes located in the 5q32 or 5q33 region have been reported . using microarray analysis to examine gene expression in endoscopic colonic biopsies from patients with newly diagnosed , untreated crohn 's disease , we identified an overexpressed gene on 5q33 , csf1r ( unpublished data ) . the csf1r is a tyrosine kinase receptor proto - oncogene involved in monocyte to macrophage differentiation . although expression of csf1r has been detected in epithelial cells of other organs , the expression of csf1r in the intestine has not been well documented [ 15 - 18 ] . here we report the results of a case - control study of louisiana patients with crohn 's disease and ethnically similar controls showing increased prevalence of the t allele of a snp ( a2033 t ) * near an intronic runx1 binding site in the csf1r gene in patients with crohn 's disease . we also show , using immunohistochemistry , that the csf1r protein is expressed in the superficial epithelium of the ileum and colon . * this snp occurs 2033 base pairs from the 3 ' end of the eleventh exon of the csf1r gene . patients ( n = 111 ) and controls ( n = 108 ) were recruited in the study from children 's hospital of new orleans and private practices in southeastern louisiana and western mississippi after louisiana health sciences center institutional review board ( irb ) approval and informed consent and assent . patient and control dna were also obtained from archival colonic tissue blocks after irb approval . genomic dna was obtained from one of 3 sources for all subjects : peripheral blood buffy coat , buccal swab or paraffin - embedded archival tissue blocks . for blood , ten ml of whole blood was collected in purple top , edta tubes and buffy coats prepared using red blood cell lysis buffer ( nh4cl , nh4hco3 , h2o ) , pellet buffer ( 1 m trix hcl ph 8.0 , 0.5 m edta , nacl , h2o ) , 10% sds and proteinase k. buffy coats were heated in a water bath overnight at 56c and stored at -20c . dna was extracted using phenol : chloroform : isoamyl alcohol , followed by chloroform , and precipitated in 100% ethanol . after air drying , the pellet was resuspended in te buffer and its quantity and integrity were verified by 1% agarose gel and spectrophotometry ( beckman coulter , du640b ) . dna was extracted from buccal swabs ( epicentre technologies , madison , wisconsin ) following the manufacturer 's instructions . briefly , swabs were placed in dna extraction solution , mixed for ten seconds and incubated at 60c for 30 min , then a total of 16 min at 98c . after centrifugation at 10,000 g at 4c , the supernatant was transferred to a clean tube and stored at -20c . for archival tissue blocks , 3 sections of 10 m were cut and incubated twice with 1 ml of n - octane ( sigma , st . after centrifugation at 10,000 g at room temperature ( rt ) , the pellet was resuspended in 1 ml 100% etoh and then in 1 ml 75% etoh . after the last centrifugation , the pellet was resuspended in 85 l of pellet buffer ( 10 mm tris - hcl , ph 8.0 , 10 mm edta , ph 8.0 , 150 mm nacl ) followed by 5 l of proteinase k ( 20 mg / ml ) ( invitrogen , grand island , ny ) and 10 l 10% sds ( invitrogen ) . one hundred l of phenol chloroform : iso - amylalcohol ( 50:1 ) ( sigma ) was added and the sample was centrifuged at 10,000 g for 5 min at rt . the aqueous phase was transferred to a clean tube and 100 ul of chloroform were added ( sigma ) . the sample was centrifuged at 10,000 g for 5 min and the aqueous phase transferred to a clean tube and mixed with 200 l 100% ethanol ( aldrich ) and incubated at -70c for at least 1 hr . the dna was precipitated by centrifugation and resuspended in te buffer . reverse primers to amplify dna in the vicinity of the snp of interest were designed using the primer quest(integrated dna technologies ( idt ) , coralville , ia ) program and ordered from idt . the primer sequences are : ( f ) 5'ttc tct gag cag ctc caa tg3 ' and ( r ) 3'cca cag aca ggc cac ttc tt5 ' . master mix for pcr was prepared using taq polymerase , dntps and other reagents from invitrogen ( carlsbad , ca ) . after optimization of conditions , pcr reactions were carried out in a bio - rad i - cycler . the pcr product was resolved on a 1% agarose gel and purified using qiaquick dna purification kit ( qiagen , valencia , ca ) . forward and reverse dna sequencing to detect the a2033 t snp in intron 11 of the csf1r gene was performed in the lsu sequencing core . briefly , in a 0.2 ml pcr tube , dna template , primer , bigdye terminator ready reaction mix ( pe applied biosystems , foster city , ca ) , 5x sequencing mix , and hplc water were combined to 20 l . tubes were placed in a thermal cycler ( geneamp pcr 9700 ) set to the following program . 30 cycles 96c 10 seconds extension products were purified by adding 3 m naoac , ph 4.6 and 95% etoh to reaction tubes for 20 m and spinning tubes upright at 3600 rpm for 30 m. tubes were then inverted and spun at 700 rpm for 1 m. after washing the pellet in 70% etoh , tubes were spun at 3600 for 10 m. the procedure beginning with inversion of tubes was repeated , tubes centrifuged at 700 rpm for 1 m and air dried . to analyze the sequencing reaction , formamide was added to each tube and denatured for 3 m at 95c , followed by wet ice . the sequencing gel was prepared using urea , hplc water , long ranger 50% ( pe applied biosystems ) and 10x tbe buffer , stirring for 1 h. 10% aps and temed were added to the filtered gel solutions and gel was loaded into a cassette with glass plates in an abi 3100 automated sequencer equipped with abi prism data collection software . four different dyes were used to identify the a , c , g , and t extension reactions using an argon laser . slides cut from paraffin - embedded tissue blocks were deparaffinized , hydrated , and blocked with 3% hydrogen peroxide at rt for 15 min . after rinsing in distilled water , they were placed in pbs for 2 min and then blocked with biocare 's background sniper ( biocare medical , walnut creek , ca ) for 10 min at rt . slides were incubated with primary antibody ( rabbit polyclonal antibody to human c - fms , cymbus biotechnology , ltd . , chandlers ford , hants , uk ) at a dilution of 1:100 for 60 min at rt and , after rinsing with pbs , incubated with secondary antibody ( mach 2 rabbit - hrp polymer , biocare medical ) for 30 min at rt . after rinsing with pbs , slides were placed in diaminobenzamide for 7 min at rt , rinsed in 2 changes of distilled water , counterstained with hematoxylin , dehydrated and mounted with resinous medium . numbers of patients with the t allele of the a2033 t snp were compared to numbers of controls using a chi - square statistic . an odds ratio with 95% confidence interval was calculated using sas software ( sas , cary , nc ) . patients ( n = 111 ) and controls ( n = 108 ) were recruited in the study from children 's hospital of new orleans and private practices in southeastern louisiana and western mississippi after louisiana health sciences center institutional review board ( irb ) approval and informed consent and assent . patient and control dna were also obtained from archival colonic tissue blocks after irb approval . genomic dna was obtained from one of 3 sources for all subjects : peripheral blood buffy coat , buccal swab or paraffin - embedded archival tissue blocks . for blood , ten ml of whole blood was collected in purple top , edta tubes and buffy coats prepared using red blood cell lysis buffer ( nh4cl , nh4hco3 , h2o ) , pellet buffer ( 1 m trix hcl ph 8.0 , 0.5 m edta , nacl , h2o ) , 10% sds and proteinase k. buffy coats were heated in a water bath overnight at 56c and stored at -20c . dna was extracted using phenol : chloroform : isoamyl alcohol , followed by chloroform , and precipitated in 100% ethanol . after air drying , the pellet was resuspended in te buffer and its quantity and integrity were verified by 1% agarose gel and spectrophotometry ( beckman coulter , du640b ) . dna was extracted from buccal swabs ( epicentre technologies , madison , wisconsin ) following the manufacturer 's instructions . briefly , swabs were placed in dna extraction solution , mixed for ten seconds and incubated at 60c for 30 min , then a total of 16 min at 98c . after centrifugation at 10,000 g at 4c , the supernatant was transferred to a clean tube and stored at -20c . for archival tissue blocks , 3 sections of 10 m were cut and incubated twice with 1 ml of n - octane ( sigma , st . after centrifugation at 10,000 g at room temperature ( rt ) , the pellet was resuspended in 1 ml 100% etoh and then in 1 ml 75% etoh . after the last centrifugation , the pellet was resuspended in 85 l of pellet buffer ( 10 mm tris - hcl , ph 8.0 , 10 mm edta , ph 8.0 , 150 mm nacl ) followed by 5 l of proteinase k ( 20 mg / ml ) ( invitrogen , grand island , ny ) and 10 l 10% sds ( invitrogen ) . one hundred l of phenol chloroform : iso - amylalcohol ( 50:1 ) ( sigma ) was added and the sample was centrifuged at 10,000 g for 5 min at rt . the aqueous phase was transferred to a clean tube and 100 ul of chloroform were added ( sigma ) . the sample was centrifuged at 10,000 g for 5 min and the aqueous phase transferred to a clean tube and mixed with 200 l 100% ethanol ( aldrich ) and incubated at -70c for at least 1 hr . the dna was precipitated by centrifugation and resuspended in te buffer . forward and reverse primers to amplify dna in the vicinity of the snp of interest were designed using the primer quest(integrated dna technologies ( idt ) , coralville , ia ) program and ordered from idt . the primer sequences are : ( f ) 5'ttc tct gag cag ctc caa tg3 ' and ( r ) 3'cca cag aca ggc cac ttc tt5 ' . master mix for pcr was prepared using taq polymerase , dntps and other reagents from invitrogen ( carlsbad , ca ) . after optimization of conditions , pcr reactions were carried out in a bio - rad i - cycler . the pcr product was resolved on a 1% agarose gel and purified using qiaquick dna purification kit ( qiagen , valencia , ca ) . forward and reverse dna sequencing to detect the a2033 t snp in intron 11 of the csf1r gene was performed in the lsu sequencing core . briefly , in a 0.2 ml pcr tube , dna template , primer , bigdye terminator ready reaction mix ( pe applied biosystems , foster city , ca ) , 5x sequencing mix , and hplc water were combined to 20 l . tubes were placed in a thermal cycler ( geneamp pcr 9700 ) set to the following program . 30 cycles 96c 10 seconds extension products were purified by adding 3 m naoac , ph 4.6 and 95% etoh to reaction tubes for 20 m and spinning tubes upright at 3600 rpm for 30 m. tubes were then inverted and spun at 700 rpm for 1 m. after washing the pellet in 70% etoh , tubes were spun at 3600 for 10 m. the procedure beginning with inversion of tubes was repeated , tubes centrifuged at 700 rpm for 1 m and air dried . to analyze the sequencing reaction , formamide was added to each tube and denatured for 3 m at 95c , followed by wet ice . the sequencing gel was prepared using urea , hplc water , long ranger 50% ( pe applied biosystems ) and 10x tbe buffer , stirring for 1 h. 10% aps and temed were added to the filtered gel solutions and gel was loaded into a cassette with glass plates in an abi 3100 automated sequencer equipped with abi prism data collection software . four different dyes were used to identify the a , c , g , and t extension reactions using an argon laser . slides cut from paraffin - embedded tissue blocks were deparaffinized , hydrated , and blocked with 3% hydrogen peroxide at rt for 15 min . after rinsing in distilled water , they were placed in pbs for 2 min and then blocked with biocare 's background sniper ( biocare medical , walnut creek , ca ) for 10 min at rt . slides were incubated with primary antibody ( rabbit polyclonal antibody to human c - fms , cymbus biotechnology , ltd . , chandlers ford , hants , uk ) at a dilution of 1:100 for 60 min at rt and , after rinsing with pbs , incubated with secondary antibody ( mach 2 rabbit - hrp polymer , biocare medical ) for 30 min at rt . after rinsing with pbs , slides were placed in diaminobenzamide for 7 min at rt , rinsed in 2 changes of distilled water , counterstained with hematoxylin , dehydrated and mounted with resinous medium . numbers of patients with the t allele of the a2033 t snp were compared to numbers of controls using a chi - square statistic . an odds ratio with 95% confidence interval was calculated using sas software ( sas , cary , nc ) . a snp ( a2033 t ) was detected in the eleventh intron of the csf1r gene that was located 77 base pairs downstream from a runx1 binding site ( tgtggt ) . forward and reverse sequencing of this snp was performed in 111 patients with crohn 's disease and 108 controls ( table 1 ) . thirty patients with crohn 's disease ( 27% ) but only fourteen controls ( 13% ) had the t allele of the snp ( = 6.74 , p < 0.01 , o.r . data from the stratification of crohn 's patients and control patients by ethnicity ( table 2 ) suggest that the differential expression of the snp was more pronounced in some ethnic groups ( acadian ) than in others ( african american ) . in the case of crohn 's patients of acadian descent , the rate of the t allele ( 47% vs. 23% ) was significantly higher than the rate of the t allele in all other ethnicities combined ( table 3 ; = 4.01 , p < 0.05 , o.r . since there were more controls of acadian descent than patients of acadian descent ( table 2 ) , it is unlikely that the higher rate of the t allele of the a2033 t snp noted in crohn 's patients in general ( table 1 ) can be attributed to ethnicity . however , to exclude the possibility that acadian ethnicity was a confounding variable , we compared rates of the t allele in non - acadian patients with crohn 's disease to those of non - acadian controls ( table 4 ) . in this analysis , patients with crohn 's disease still had significantly higher rates of the t allele than controls . crohn 's disease status vs csf1r a2033 t snp : all patients = 6.74 ( p < 0.01 ) o.r.:2.49 ( 1.23 < o.r . < 5.01 ) a2033 t snp allele by ethnicity ethnicity vs csf1r a2003 t snp : crohn 's patients = 4.81 ( p < 0.05 ) o.r.:3.04 ( 1.09 < o.r . < 8.47 ) crohn 's disease status vs csf1r a2033 t snp : non - acadian patients fisher exact test ( p = 0.025 ) o.r.:2.45 ( 0.97 < o.r . < 6.17 ) paraffin - embedded , formalin - fixed tissue sections from control patients were stained with a rabbit anti - human polyclonal antibody to csf1r and read by a pathologist . positive cytoplasmic staining was noted in the superficial epithelium of the ileum and colon with differentiated cells being sloughed off into intestinal lumen staining most vividly ( figure 1a,1b,1c ) . examination of the intracellular staining pattern revealed a characteristic staining pattern with the terminal web and the lateral junctions of intestinal epithelial cells ( figure 1a ) . sections from paraffin - embedded tissue blocks of normal human ileum and colon were stained with polyclonal rabbit anti - human antisera to csf1r and a horseradish peroxidase - conjugated goat anti - rabbit secondary antibody with diaminobenzamide used as chromagen . a. superficial epithelium of the terminal ileum with cytoplasmic staining of the terminal web and lateral junctions of the epithelial cells ( green arrow ) . b. staining of the superficial epithelium of the colon . c. staining of the superficial epithelium of the ileum with an epithelial cell being sloughed off into the lumen ( green arrow ) a snp ( a2033 t ) was detected in the eleventh intron of the csf1r gene that was located 77 base pairs downstream from a runx1 binding site ( tgtggt ) . forward and reverse sequencing of this snp was performed in 111 patients with crohn 's disease and 108 controls ( table 1 ) . thirty patients with crohn 's disease ( 27% ) but only fourteen controls ( 13% ) had the t allele of the snp ( = 6.74 , p < 0.01 , o.r . data from the stratification of crohn 's patients and control patients by ethnicity ( table 2 ) suggest that the differential expression of the snp was more pronounced in some ethnic groups ( acadian ) than in others ( african american ) . in the case of crohn 's patients of acadian descent , the rate of the t allele ( 47% vs. 23% ) was significantly higher than the rate of the t allele in all other ethnicities combined ( table 3 ; = 4.01 , p < 0.05 , o.r . since there were more controls of acadian descent than patients of acadian descent ( table 2 ) , it is unlikely that the higher rate of the t allele of the a2033 t snp noted in crohn 's patients in general ( table 1 ) can be attributed to ethnicity . however , to exclude the possibility that acadian ethnicity was a confounding variable , we compared rates of the t allele in non - acadian patients with crohn 's disease to those of non - acadian controls ( table 4 ) . in this analysis , patients with crohn 's disease still had significantly higher rates of the t allele than controls . crohn 's disease status vs csf1r a2033 t snp : all patients = 6.74 ( p < 0.01 ) o.r.:2.49 ( 1.23 < o.r . < 5.01 ) a2033 t snp allele by ethnicity ethnicity vs csf1r a2003 t snp : crohn 's patients = 4.81 ( p < 0.05 ) o.r.:3.04 ( 1.09 < o.r . < 8.47 ) crohn 's disease status vs csf1r a2033 t snp : non - acadian patients fisher exact test ( p = 0.025 ) o.r.:2.45 ( 0.97 < o.r . paraffin - embedded , formalin - fixed tissue sections from control patients were stained with a rabbit anti - human polyclonal antibody to csf1r and read by a pathologist . positive cytoplasmic staining was noted in the superficial epithelium of the ileum and colon with differentiated cells being sloughed off into intestinal lumen staining most vividly ( figure 1a,1b,1c ) . examination of the intracellular staining pattern revealed a characteristic staining pattern with the terminal web and the lateral junctions of intestinal epithelial cells ( figure 1a ) . sections from paraffin - embedded tissue blocks of normal human ileum and colon were stained with polyclonal rabbit anti - human antisera to csf1r and a horseradish peroxidase - conjugated goat anti - rabbit secondary antibody with diaminobenzamide used as chromagen . a. superficial epithelium of the terminal ileum with cytoplasmic staining of the terminal web and lateral junctions of the epithelial cells ( green arrow ) . b. staining of the superficial epithelium of the colon . c. staining of the superficial epithelium of the ileum with an epithelial cell being sloughed off into the lumen ( green arrow ) . based on gene expression data , chromosomal location and biological function , we have evidence that the colony stimulating factor 1 receptor gene may contribute to crohn 's disease susceptibility . using a case - control study , we have shown that a snp in an intron of this gene is associated with crohn 's disease . whether this snp is in close proximity to another disease - causing snp in the same ( or a neighboring ) gene or is itself disruptive of gene functioning in a way that increases susceptibility to crohn 's disease will be the focus of future studies . the csf1r gene is an intriguing candidate gene for crohn 's disease susceptibility for several reasons . first , it is involved in innate immunity and host defense against fungi and certain bacteria such as listeria that have been postulated to play a role in crohn 's disease pathogenesis . second , csf1r is involved in an intracellular signal transduction cascade linking the g alpha i2 receptor to the transcription factor stat3 . in nih3t3 cells expressing a dominant negative g alpha i2 , stat3 phosphorylation by v - fms ( oncogenic csf1r ) was inhibited . this is significant because targeted disruption of either the g alpha i2 gene or the stat3 transcription factor ( in monocytes ) results in inflammatory bowel disease in rodents . these data suggest that a signaling pathway involving g alpha i2 , stat3 and csf1r is critical for protection against intestinal inflammation . one possible mechanism is the regulation of il-10 , a cytokine known to be essential for normal intestinal homeostasis . third , acute myelogenous leukemia and myelodysplasia , two conditions that may occur with increased frequency in the context of crohn 's disease , are associated with polymorphisms or deletions in the csf1r gene [ 25 - 27 ] . there is a paucity of literature regarding the expression of the csf1r protein in the intestine despite documentation of its presence in the epithelium of a variety of other tissues including breast , ovary , endometrium , lung and prostate [ 15 - 18,28 ] . therefore , we examined its expression by immunostaining and found it to be expressed in the cytoplasm of certain epithelial cells of the superficial epithelium and villous tips of the ileum and colon , including cells that were being sloughed into the lumen . because of this superficial location of staining , it is tempting to hypothesize that the csf1r protein plays a role in differentiation of intestinal epithelial cells as it does in macrophages . the most intense cytoplasmic staining occurred in the terminal web of the epithelial cell and in the lateral junctions of the cells . the localization of csf1r in actin - rich areas of the cell is not surprising in view of data from in vitro studies demonstrating that the csf1r protein mediates morphological changes in macrophages through the regulation of paxillin and focal adhesions . what role the csf1r protein might play in cytoskeletal regulation in either mononuclear cells or in intestinal epithelial cells the intestine as well as its expression pattern and pathogenic role in inflammatory bowel disease remain to be investigated further . studies of the prevalence of nod2 polymorphisms in patients with crohn 's disease point to a subset of patients with ileal and fibrostenotic disease who are more likely to have the nod2 genotype . moreover , specific nod2 polymorphisms are more prevalent in some ethnic groups . the numbers of patients enrolled in the current study do not permit conclusive analysis of disease subtype or ethnicity . however , we did find that patients of acadian descent ( descendants of migrs from french canada ) have a higher prevalence of the disease - associated snp in csf1r . this is interesting because the population in which the ibd5 susceptibility locus was originally identified was , in part , french canadian . we do not know what the significance , if any , is of the location of the a2033 t snp near a binding site for the transcription factor , runx1 . it is intriguing to note , however , that snps in runx1 binding sites and in the runx1 gene itself have been associated with a variety of autoimmune conditions , including psoriasis , systemic lupus erythematosus , type i diabetes mellitus and rheumatoid arthritis [ 11,33 - 35 ] . since the csf1r gene is a target of runx1 and has multiple runx1 binding sites in several introns , complete sequencing of each of these sites will be performed to investigate the hypothesis that defective runx1 binding is related to crohn 's disease susceptibility . in a case - control study of louisiana patients with crohn 's disease , we have detected a snp ( a2033 t ) in the eleventh intron of the csf1r gene that is significantly associated with the disease . azv performed the molecular genetic studies and assisted in the recruitment of patients , the data analysis and the drafting of the paper . em conceived of the study , recruited patients , performed the data analysis and drafted the manuscript . this work was sponsored in part by grants from the louisiana board of regents , the toler foundation , the louisiana digestive health foundation and the solomon family to e.m .

backgroundpolymorphisms in several genes ( nod2 , mdr1 , slc22a4 ) have been associated with susceptibility to crohn 's disease . identification of the remaining crohn 's susceptibility genes is essential for the development of disease - specific targets for immunotherapy . using gene expression analysis , we identified a differentially expressed gene on 5q33 , the colony stimulating factor 1 receptor ( csf1r ) gene , and hypothesized that it is a crohn 's susceptibility gene . the csf1r gene is involved in monocyte to macrophage differentiation and in innate immunity.methodspatients provided informed consent prior to entry into the study as approved by the institutional review board at lsu health sciences center . we performed forward and reverse sequencing of genomic dna from 111 unrelated patients with crohn 's disease and 108 controls . we also stained paraffin - embedded , ileal and colonic tissue sections from patients with crohn 's disease and controls with a polyclonal antibody raised against the human csf1r protein.resultsa single nucleotide polymorphism ( a2033 t ) near a runx1 binding site in the eleventh intron of the colony stimulating factor 1 receptor was identified . the t allele of this single nucleotide polymorphism occurred in 27% of patients with crohn 's disease but in only 13% of controls ( x2 = 6.74 , p < 0.01 , odds ratio ( o.r . ) = 2.49 , 1.23 < o.r . < 5.01 ) . using immunohistochemistry , positive staining with a polyclonal antibody to csf1r was observed in the superficial epithelium of ileal and colonic tissue sections.conclusionswe conclude that the colony stimulating factor receptor 1 gene may be a susceptibility gene for crohn 's disease .

Introduction Methods Patients DNA extraction and purification PCR DNA Sequencing Immunohistochemistry Data analysis Results Association of the T allele of the A2033T SNP with Crohn's disease especially in patients of Acadian descent The CSF1R protein is expressed in the superficial epithelium of the ileum and colon Discussion Conclusions Competing interests Authors' contributions Acknowledgments

PMC5357695

frontotemporal lobar degeneration ( ftld ) is a common cause of early onset dementia ( rabinovici and miller , 2010 ) . its prevalence and incidence is similar to alzheimer 's disease in individuals under the age of 65 years . behavioral variant frontotemporal dementia ( bvftd ) , its most common subtype , is characterized by deep alterations in behavior and personality ( neary et al . , 1998 ) . in 2011 , new diagnostic criteria were suggested that divide bvftd into three different diagnostic categories : probable bvftd , if clinical criteria are complemented with fitting imaging data ( frontal and/or anterior temporal atrophy , hypometabolism or hypoperfusion ) , and bvftd with , when either a known pathogenic mutation or histopathological evidence is present ( rascovsky et al . , 2011 ) . recent meta - analyses on the neural correlates of bvftd validated these imaging criteria and showed a decreased gray matter density ( gmd ) mainly in the frontal cortex , suggesting that bvftd is mainly a frontal brain disease in contrast to other neurodegenerative diseases ( pan et al . , 2012 , schroeter , 2012 , schroeter and neumann , 2011 , schroeter et al . , 2007 , schroeter et al . , 2008 , schroeter et al . incorporating imaging criteria into diagnostic algorithms for bvftd is a decisive step in improving these criteria . the aim of our study was to further validate imaging criteria for bvftd by measuring brain atrophy with magnetic resonance imaging ( mri ) in a multi - centric cohort , and by using this data to predict diagnosis in each single patient . note that usage of multi - center data is a precondition for future application in clinical routine . accordingly , we calculated structural brain differences between bvftd patients and healthy controls with voxel based morphometry ( vbm ) ( ashburner and friston , 2000 ) using the multi - center cohort of the german ftld consortium . recently , machine learning techniques have been applied to neuroimaging data in order to distinguish between patients and control subjects or different diseases on an individual level ( dukart et al . , 2011 , dukart et al . we used support vector machine ( svm ) algorithms to differentiate between bvftd patients and healthy controls on an individual level . we hypothesized that bvftd is characterized mainly by frontotemporal lobe atrophy and that svm algorithms identify bvftd patient and control subjects with a high accuracy that might even be increased by focusing on disease - specific brain areas , namely fronto(temporal ) regions , the insula and basal ganglia in comparison with whole brain approaches , based on recent meta - analyses ( pan et al . ( 2011 ) . of those fifty - two bvftd patients twenty met the criteria for possible bvftd , twenty - eight the criteria for probable bvftd , and four met the criteria for genetically confirmed bvftd . twenty - four were female , and duration since initial symptoms averaged 3.7 3.7 years ( results are generally reported as mean standard deviation ) . disease severity was 5.5 3.5 or 7.7 4.2 as measured with the clinical dementia rating scale ( cdr ) and the ftld - modified clinical dementia rating scale ( ftld - cdr ) . the data was retrieved from the german ftld consortium , a prospective multicenter study on ftld ( http://www.ftld.de ; otto et al . , 2011 ) . fifty - two ( twenty - four female ) control subjects were recruited overall , thirty subjects from the different centers in the german ftld consortium 's study and , additionally , twenty - two from the max - planck - institute leipzig database . all subjects were thoroughly examined by taking a case history , neurological examination , neuropsychological testing and mri ( see below ) . overall the bvftd and the healthy control subject group were exactly gender matched and did n't differ in mean age ( bvftd 61.5 10.0 years ; healthy control subjects 63.1 9.8 years ; student 's t - test p = 0.76 ) . the study was approved by the ethics committees of the university of ulm , and of the other universities involved in the ftld consortium , and was in accordance with the latest version of the declaration of helsinki . high resolution magnetization - prepared rapid gradient echo ( mprage ) t1-images were acquired by seven different centers throughout germany . mris were performed on five different 3 tesla siemens scanners ( allegra ulm , biograph munich , triotim erlangen & goettingen , verio leipzig & rostock , skyra bonn ) with three different sequences ( erlangen / bonn / goettingen / leipzig / munich : tr = 2300 ms , ti = 900 ms , te = 2.98 ms ; rostock : tr = 2500 ms , ti = 1100 ms , te = 2.98 ms ; ulm : tr = 2200 ms , ti = 1200 ms , te = 4.38 ms ) . in total , the 52 bvftd patients were compared to 52 healthy control subjects . the first one consisted of 19 , exactly center - matched , patients and control subjects , i.e. patients were compared with control subjects from exactly the same center , to enable a maximally controlled pilot analysis . the second analysis included all available subjects , and could therefore not be exactly matched according to center . both groups had no significant differences in age and gender distribution ( see above ) . gmd differences between bvftd patients and the control cohort were analyzed with vbm by applying the vbm8 toolbox ( http://dbm.neuro.uni-jena.de/vbm/vbm8-for-spm8/ ) , implemented in spm8 ( wellcome trust centre for neuroimaging , london , uk ) , running on matlab 7.1 . firstly , high resolution images were normalized to the same stereotactic space by registering each individual image to the same template image , the montreal neurological institute ( mni ) 152 standard template , using non - linear transformations . the images were segmented into gray matter , white matter , and cerebrospinal fluid volumes . the gray matter volumes were then smoothed by convolving an isotropic gaussian kernel ( 12 12 12 mm ) . the result of the preprocessing steps is referred to as gmd , which was compared in the next step . the statistical analysis was performed voxel - wise by using the general linear model and implementing a two - sample t - test . results were controlled for potentially confounding factors , namely age , gender , total intracranial volume , and the center where the mri was conducted . a significance threshold of p < 0.05 , family wise error ( fwe ) corrected on the cluster level , was used for the 19 vs. 19 pilot analysis . for the final 52 vs. 52 analysis a threshold of p < 0.05 fwe corrected on the voxel level was used due to higher statistical power by the larger number of participants . note that center effects were controlled for in both group comparisons ( see below ) . these analyses were performed to control for potential center effects in the aforementioned group comparisons . to investigate the influence of inter - center variability , group comparisons ( patients vs. control subjects ) were conducted repeatedly with the leave one center out approach , i.e. excluding systematically subjects from one center from the analysis . after these processing steps , a conjunction analysis was performed by overlaying the results of the six , respectively seven different vbm analyses , in order to identify those brain networks that were affected by bvftd in all analyses , and accordingly , independent from center . the threshold was set to p < 0.05 fwe on the cluster level here . the classification was obtained by svm , using the libsvm software package ( software available at http://www.csie.ntu.edu.tw/~cjlin/libsvm ) ( chang and lin , 2011 ) . svms are supervised learning models , used to analyze data , build classifiers and regression analyses . they work in two phases . in the training phase , a subset of the available data points , in this study all the voxels of the brain or all the voxels of the region of interest are used to find a linear hyperplane to separate the two classes ( e.g. patients vs. controls ) optimally . in the testing phase , new , previously unseen data is classified depending on their relative position to the hyperplane . for classification leave one ( subject ) out approach . in this approach one subject of each group ( e.g. patients and control ) is used for testing and the remaining ones are used for training the classifier , until every subject has been left out . the masks were created using the wake forest university school of medicine ( wfu ) pickatlas ( maldjian et al . , 2003 ) . classifications were run for both of the above mentioned analyses , and with different regions of interest for each analysis ( see table 1 ) . ( 2011 ) . of those fifty - two bvftd patients twenty met the criteria for possible bvftd , twenty - eight the criteria for probable bvftd , and four met the criteria for genetically confirmed bvftd . twenty - four were female , and duration since initial symptoms averaged 3.7 3.7 years ( results are generally reported as mean standard deviation ) . disease severity was 5.5 3.5 or 7.7 4.2 as measured with the clinical dementia rating scale ( cdr ) and the ftld - modified clinical dementia rating scale ( ftld - cdr ) . the data was retrieved from the german ftld consortium , a prospective multicenter study on ftld ( http://www.ftld.de ; otto et al . , 2011 ) . fifty - two ( twenty - four female ) control subjects were recruited overall , thirty subjects from the different centers in the german ftld consortium 's study and , additionally , twenty - two from the max - planck - institute leipzig database . all subjects were thoroughly examined by taking a case history , neurological examination , neuropsychological testing and mri ( see below ) . overall the bvftd and the healthy control subject group were exactly gender matched and did n't differ in mean age ( bvftd 61.5 10.0 years ; healthy control subjects 63.1 9.8 years ; student 's t - test p = 0.76 ) . the study was approved by the ethics committees of the university of ulm , and of the other universities involved in the ftld consortium , and was in accordance with the latest version of the declaration of helsinki . high resolution magnetization - prepared rapid gradient echo ( mprage ) t1-images were acquired by seven different centers throughout germany . mris were performed on five different 3 tesla siemens scanners ( allegra ulm , biograph munich , triotim erlangen & goettingen , verio leipzig & rostock , skyra bonn ) with three different sequences ( erlangen / bonn / goettingen / leipzig / munich : tr = 2300 ms , ti = 900 ms , te = 2.98 ms ; rostock : tr = 2500 ms , ti = 1100 ms , te = 2.98 ms ; ulm : tr = 2200 ms , ti = 1200 ms , te = 4.38 ms ) . in total , the 52 bvftd patients were compared to 52 healthy control subjects . the first one consisted of 19 , exactly center - matched , patients and control subjects , i.e. patients were compared with control subjects from exactly the same center , to enable a maximally controlled pilot analysis . the second analysis included all available subjects , and could therefore not be exactly matched according to center . both groups had no significant differences in age and gender distribution ( see above ) . gmd differences between bvftd patients and the control cohort were analyzed with vbm by applying the vbm8 toolbox ( http://dbm.neuro.uni-jena.de/vbm/vbm8-for-spm8/ ) , implemented in spm8 ( wellcome trust centre for neuroimaging , london , uk ) , running on matlab 7.1 . firstly , high resolution images were normalized to the same stereotactic space by registering each individual image to the same template image , the montreal neurological institute ( mni ) 152 standard template , using non - linear transformations . the images were segmented into gray matter , white matter , and cerebrospinal fluid volumes . the gray matter volumes were then smoothed by convolving an isotropic gaussian kernel ( 12 12 12 mm ) . the result of the preprocessing steps is referred to as gmd , which was compared in the next step . the statistical analysis was performed voxel - wise by using the general linear model and implementing a two - sample t - test . results were controlled for potentially confounding factors , namely age , gender , total intracranial volume , and the center where the mri was conducted . a significance threshold of p < 0.05 , family wise error ( fwe ) corrected on the cluster level , was used for the 19 vs. 19 pilot analysis . for the final 52 vs. 52 analysis a threshold of p < 0.05 fwe corrected on the voxel level was used due to higher statistical power by the larger number of participants . note that center effects were controlled for in both group comparisons ( see below ) . these analyses were performed to control for potential center effects in the aforementioned group comparisons . to investigate the influence of inter - center variability , group comparisons ( patients vs. control subjects ) were conducted repeatedly with the leave one center out approach , i.e. excluding systematically subjects from one center from the analysis . after these processing steps , a conjunction analysis was performed by overlaying the results of the six , respectively seven different vbm analyses , in order to identify those brain networks that were affected by bvftd in all analyses , and accordingly , independent from center . the threshold was set to p < 0.05 fwe on the cluster level here . the classification was obtained by svm , using the libsvm software package ( software available at http://www.csie.ntu.edu.tw/~cjlin/libsvm ) ( chang and lin , 2011 ) . svms are supervised learning models , used to analyze data , build classifiers and regression analyses . a subset of the available data points , in this study all the voxels of the brain or all the voxels of the region of interest are used to find a linear hyperplane to separate the two classes ( e.g. patients vs. controls ) optimally . in the testing phase , new , previously unseen data is classified depending on their relative position to the hyperplane . for classification leave one ( subject ) out approach . in this approach one subject of each group ( e.g. patients and control ) is used for testing and the remaining ones are used for training the classifier , until every subject has been left out . the masks were created using the wake forest university school of medicine ( wfu ) pickatlas ( maldjian et al . , 2003 ) . classifications were run for both of the above mentioned analyses , and with different regions of interest for each analysis ( see table 1 ) . 1 , the vbm - analysis revealed reductions in gmd mainly in the frontal and temporal lobe , insulae and basal ganglia in bvftd . for the first center - matched analysis with 19 bvftd patients vs. 19 control subjects , brain regions included bilaterally the globus pallidus , frontal pole , insular cortex , temporal pole , left precentral gyrus , postcentral gyrus , amygdala , heschl 's gyrus , and central opercular gyrus . the second analysis including 52 bvftd patients vs. 52 control subjects revealed changes bilaterally in the anterior cingulate , frontomedian , paracingulate cortices , superior , middle and inferior frontal gyri , frontal pole , orbital gyrus , insular cortex , temporal pole , middle temporal gyrus , amygdala , putamen , nucleus accumbens , and right pre- and postcentral gyrus . note that neither disease duration nor disease severity as measured with the ftld - cdr correlated with gmd in our study , preventing a potential impact of these two clinical factors on our results . as opposed to this , the 52 bvftd vs. 52 control subjects analysis showed a high concordance in both , the frontal and temporal lobes , insula and the basal ganglia . more specifically it identified the superior frontal gyrus , frontal pole , frontoorbital and frontomedian cortex , paracingulate and anterior cingulate / midcingulate cortex , middle and superior temporal gyrus , planum polare , insular cortex , putamen , nucleus accumbens , left angular gyrus , and right supramarginal gyrus ( see fig . results of the final analyses , investigating the possibility to individually classify patients solely from mri data , are illustrated in detail ( accuracy , sensitivity , specificity , positive and negative predictive value ) in table 1 . classification accuracy ranged from 71.1% to 78.9% in the 19 bvftd patients vs. 19 control subjects analyses , and from 78.8% to 84.6% in the 52 bvftd patients vs. 52 control subjects analyses . obviously , classification accuracy was generally strong with higher values in the larger cohort , presumably due to a higher statistical power . accordingly , description of results will be focused on the larger cohort including 52 patients and 52 control subjects in the following . the frontal lobe mask revealed better results for accuracy than the temporal lobe mask ( 80.7% vs. 78.8% ) . both masks reached slightly lower accuracy than using all the voxels of the brain ( 81.7% ) . adding the insula and basal ganglia to the frontal mask increased accuracy to 82.7% . the highest accuracy with 84.6% could be obtained by using a mask including the frontal and temporal lobe alone , or combined with the insula and basal ganglia . 3 illustrates weights of voxels most relevant for svm classification between bvftd patients and healthy controls across the whole brain . relevant voxels for bvftd classification were located in the frontal and temporal cortex , the insula , basal ganglia , and the cerebellum . 1 , the vbm - analysis revealed reductions in gmd mainly in the frontal and temporal lobe , insulae and basal ganglia in bvftd . for the first center - matched analysis with 19 bvftd patients vs. 19 control subjects , brain regions included bilaterally the globus pallidus , frontal pole , insular cortex , temporal pole , left precentral gyrus , postcentral gyrus , amygdala , heschl 's gyrus , and central opercular gyrus . the second analysis including 52 bvftd patients vs. 52 control subjects revealed changes bilaterally in the anterior cingulate , frontomedian , paracingulate cortices , superior , middle and inferior frontal gyri , frontal pole , orbital gyrus , insular cortex , temporal pole , middle temporal gyrus , amygdala , putamen , nucleus accumbens , and right pre- and postcentral gyrus . note that neither disease duration nor disease severity as measured with the ftld - cdr correlated with gmd in our study , preventing a potential impact of these two clinical factors on our results . as opposed to this , the 52 bvftd vs. 52 control subjects analysis showed a high concordance in both , the frontal and temporal lobes , insula and the basal ganglia . more specifically it identified the superior frontal gyrus , frontal pole , frontoorbital and frontomedian cortex , paracingulate and anterior cingulate / midcingulate cortex , middle and superior temporal gyrus , planum polare , insular cortex , putamen , nucleus accumbens , left angular gyrus , and right supramarginal gyrus ( see fig . results of the final analyses , investigating the possibility to individually classify patients solely from mri data , are illustrated in detail ( accuracy , sensitivity , specificity , positive and negative predictive value ) in table 1 . classification accuracy ranged from 71.1% to 78.9% in the 19 bvftd patients vs. 19 control subjects analyses , and from 78.8% to 84.6% in the 52 bvftd patients vs. 52 control subjects analyses . obviously , classification accuracy was generally strong with higher values in the larger cohort , presumably due to a higher statistical power . accordingly , description of results will be focused on the larger cohort including 52 patients and 52 control subjects in the following . the frontal lobe mask revealed better results for accuracy than the temporal lobe mask ( 80.7% vs. 78.8% ) . both masks reached slightly lower accuracy than using all the voxels of the brain ( 81.7% ) . adding the insula and basal ganglia to the frontal mask increased accuracy to 82.7% . the highest accuracy with 84.6% could be obtained by using a mask including the frontal and temporal lobe alone , or combined with the insula and basal ganglia . 3 illustrates weights of voxels most relevant for svm classification between bvftd patients and healthy controls across the whole brain . relevant voxels for bvftd classification were located in the frontal and temporal cortex , the insula , basal ganglia , and the cerebellum . this study aimed at further validating imaging criteria for bvftd by measuring brain atrophy with mri in a multi - centric cohort , and by predicting diagnosis in each patient individually based on pattern recognition algorithms ( svm classification ) . in line with our hypothesis the group comparison between bvftd and control subjects revealed most pronounced and most consistent gmd loss in frontal brain regions in both of the conducted group comparison analyses . remarkably , the conjunction analysis of the smaller of the two cohorts revealed the greatest overlap between the centers in the bilateral frontal pole . this is of particular note since this cohort was characterized by a lower average of duration since initial symptoms occurred ( 2.4 2.6 vs. 3.7 3.7 years ) , and , accordingly , lower disease severity ( cdr 5.0 3.5 vs. 5.5 3.5 ; ftld - cdr 5.8 4.1 vs. 7.7 4.2 ; student 's t - test p = 0.12 , 0.62 , 0.52 ) as compared to the larger cohort , suggesting that the frontal lobe may be the first affected brain region in bvftd patients , as has been postulated before ( schroeter et al . , the results support the assumption that bvftd might be regarded as a mainly frontal disease as suggested by powerful meta - analyses across imaging studies , histopathological studies , and conceptual theories for this neurodegenerative disease ( kim et al . , 2012 , pan et al . , 2012 , schroeter , 2012 , schroeter et al . furthermore , we found gmd differences in the insulae and basal ganglia in agreement with the aforementioned meta - analytic and histopathological findings , and , additionally , in the temporal lobe . such regional alterations have been previously described for different subtypes of bvftd ( whitwell et al . , 2009 ) and in autopsy proven ftld patients ( rabinovici et al . , 2007 ) . in line with the major aim of this study , the diagnosis bvftd could be predicted in individual patients by applying pattern recognition algorithms to mri data as shown for the first time to our knowledge . the svm analyses supplied very reasonable results , varying in accuracy between 71.1% and 78.9% in the 19 vs. 19 comparison and between 78.8% and 84.6% in the 52 vs. 52 comparison , depending on the selected region of interest . using all voxels of the frontal lobe led to better classification accuracy than using all voxels of the temporal lobe . this again supports the assumption that the primarily affected or core network lies within the frontal lobe . the highest accuracy was reached by using all the voxels of the frontal and temporal lobe alone or together with the insula and basal ganglia in order to distinguish between patients and control subjects ( table 1 ) . these results underline the importance of the insula and basal ganglia in bvftd ( kim et al . , 2012 , schroeter et al . , 2014 ) the relevance of the four aforementioned brain regions for pattern classification was confirmed by the analysis investigating the importance of the several brain regions contributing to the correct classification in the whole brain analysis ( see fig . one might conclude that svm classification enables individual diagnosis of bvftd with structural mri a finding supporting the inclusion of imaging criteria into diagnostic algorithms . for clinical applications , we suggest conducting analyses across the frontal , temporal lobes , insula and basal ganglia in order to distinguish between patients and healthy control subjects with highest reliability . incorporating additional biomarkers into the classifier , such as from cerebrospinal fluid , or other imaging modalities , such as positron emission tomography , , 2011 , dukart et al . , 2013a , steinacker et al . , 2017 , tahmasian et al . , 2016 ) for clinical purposes in the framework of personalized medicine future studies shall focus on detecting neurodegenerative diseases with such approaches as early as possible , i.e. in prestages such as mild cognitive or mild behavioral impairment or mild neurocognitive disorder , where treatment might be easier to conduct , and allow differential diagnosis with other neurodegenerative disease subtypes . , previous studies have shown that bvftd / ftld can be differentiated with high accuracy from alzheimer 's disease with the same approaches ( dukart et al . , 2011 , klppel et al . , 2008 , mller et al . , 2016 ) . note that regions - of - interest and analysis techniques might differ in diagnostic vs. differential diagnostic procedures as shown for imaging approaches recently ( dukart et al . , 2010 , dukart et al . , 2011 , dukart et al . , 2013a , dukart et al . , 2013b ) inter - center variability , as revealed in the conjunction analysis of the group comparisons , was more prominent in the smaller cohort ( fig . firstly , the disease stages might vary between centers , and secondly , the centers used different mri scanners or even scanning parameters . the overlap between detected gmd differences across the different centers in the larger cohort shows a greater similarity between the centers in the expected areas of the frontal and temporal lobe , and the basal ganglia , presumably due to higher numbers of subjects included and , consequently , higher statistical power . in sum , the conjunction analyses prove that disease effects are greater than the effect of inter - center variability . we suggest applying such conjunction analyses with the leave one center out approach , i.e. excluding repeatedly subjects from each center from the analysis , also to other multi - centric data to control for inter - center differences / bias . one might discuss as a limitation of this study the different scanning conditions between the centers , varying from different types of scanners to different scanning sequences . more subtle effects of the disease might therefore be overlooked or some over - interpreted because of inter - center variability . we tried to approach this problem with the center conjunction analyses ( see above ) , which pointed out the main areas showing gmd loss across all centers . note that this approach can be transferred to other multi - center studies to validate their findings . here on the other hand the multi - center design might be discussed as an important advantage of the study . by conducting analyses across multi - centric data we guaranteed a real - life scenario ensuring that our approach can be transferred to and applied in clinical settings . another shortcoming of this study is the fact that most of the patients do not have a definite diagnosis yet , i.e. no detected pathogenic mutation or histopathological evidence . in order to be as precise as possible only patients diagnosed according to the newest diagnostic criteria recently , new diagnostic criteria were suggested that incorporate imaging criteria into diagnostic algorithms for behavioral variant frontotemporal dementia ( bvftd ) . the study aimed at validating these imaging criteria for individual diagnosis by predicting diagnosis from imaging data with machine learning algorithms . brain atrophy was measured with structural magnetic resonance imaging ( mri ) at 3 tesla in a multi - centric cohort of 52 bvftd patients and 52 healthy control subjects from the german ftld consortium 's study . note that usage of multi - center data is a precondition for future application in clinical routine . support vector machine classification predicted diagnosis in single patients with a high accuracy of up to 84.6% , where accuracy was higher in a region - of - interest approach focusing on a disease - specific network including frontotemporal , insular regions and basal ganglia in comparison with the whole brain approach . our study demonstrates that mri , a widespread imaging technology , can individually identify bvftd with high accuracy , paving the road to personalized diagnostic approaches in the future .

purposefrontotemporal lobar degeneration ( ftld ) is a common cause of early onset dementia . behavioral variant frontotemporal dementia ( bvftd ) , its most common subtype , is characterized by deep alterations in behavior and personality . in 2011 , new diagnostic criteria were suggested that incorporate imaging criteria into diagnostic algorithms . the study aimed at validating the potential of imaging criteria to individually predict diagnosis with machine learning algorithms.materials & methodsbrain atrophy was measured with structural magnetic resonance imaging ( mri ) at 3 tesla in a multi - centric cohort of 52 bvftd patients and 52 healthy control subjects from the german ftld consortium 's study . beside group comparisons , diagnosis bvftd vs. controls was individually predicted in each subject with support vector machine classification in mri data across the whole brain or in frontotemporal , insular regions , and basal ganglia known to be mainly affected based on recent meta - analyses . multi - center effects were controlled for with a new method , leave one center out conjunction analyses , i.e. repeatedly excluding subjects from each center from the analysis.resultsgroup comparisons revealed atrophy in , most consistently , the frontal lobe in bvftd beside alterations in the insula , basal ganglia and temporal lobe . most remarkably , support vector machine classification enabled predicting diagnosis in single patients with a high accuracy of up to 84.6% , where accuracy was highest in a region - of - interest approach focusing on frontotemporal , insular regions , and basal ganglia in comparison with the whole brain approach.conclusionour study demonstrates that mri , a widespread imaging technology , can individually identify bvftd with high accuracy in multi - center imaging data , paving the road to personalized diagnostic approaches in the future .

Introduction Methods Patients and control subjects Acquisition and analysis of imaging data Group comparison patients vs. control subjects VBM analyses Group comparison patients vs. control subjects conjunction analyses Group comparison patients vs. control subjects SVM classification analyses Results Group comparison patients vs. control subjects VBM analyses Group comparison patients vs. control subjects conjunction analyses Group comparison patients vs. controls subjects SVM classification analyses Discussion Conclusion Conflict of interest

PMC4392485

inguinal herniorrhaphy remains one of the most common general surgical operations , with approximately 15% performed for recurrence and there is little available evidence on the optimal management of recurrent inguinal hernia . the repair of the resulting recurrent hernia is a daunting task because of already weakened tissues and obscured and distorted anatomy and therefore failure rate of these repairs using an open anterior approach may reach as high as 36% . in 1988 , nyhus stated that the evolution of the posterior preperitoneal approach for recurrent inguinal hernia repair made it the procedure of choice for the management of all recurrent groin hernias . in previous published data , the author reported that posterior preperitoneal approach gives results far superior to those of the commonly used anterior approach . the laparoscopic transabdominal preperitoneal ( tapp ) inguinal hernia repair is an evolving technique associated with the well - known advantages of a minimally invasive approach . a recent study stated that for the treatment of recurrent inguinal hernias , the first choice should be given to the laparoscopic method , especially for young , physically active , non - obese patients . some researchers advised that greater efforts should be undertaken to make laparoscopic repair easier , safer , and less expensive . the present work aimed at comparing three approaches in repair of recurrent inguinal hernia : anterior inguinal approach , open properitoneal repair and transabdominal pro - peritoneal repair regarding complications and early recurrence . patients in this study were divided randomly into three main groups : a , b , and c. group a patients were subjected to open posterior preperitoneal approach , those of group b were subjected to transinguinal anterior tension - free repair and group c patients were subjected to tapp . all of our patients were gentlemen with total number was 180 patients ; 60 for each group , their ages ranged between 42 and 65 years . the study started from january 2007 to april 2011 and included all patients having unilateral recurrent inguinal scrotal and irreducible hernias . patients with primary inguinal hernia , patients with marked obesity ( bmi > 35 ) and asa grade 3 and beyond were excluded . calculation of the sample size included the number of participants to be recruited for the study using the mathematical equation . the authors used two equations to calculate the minimum number required to reliably answer the research question . randomization was performed prior to the study using a computer - generated table of random numbers for group assignment . operations were performed in port - fouad general hospital , port - fouad , port - said , egypt and in the university hospital , department of surgery , faculty of medicine , suez canal university , egypt . the open preperitoneal approach to the inguinal region was performed under general or regional anesthesia , as originally described by nyhus . through a lower abdominal transverse incision , a 15 15 cm polypropylene mesh with a slit was inserted in the preperitoneal space and fixed with non - absorbable sutures to pubic tubercle and cooper 's ligament . the mesh was passed behind the cord and manipulated to lay flat against the posterior inguinal floor overlapping the entire myopectineal orifice.the anterior tension - free repair , as defined by lichtenstein et al . large pore - sized ( 1.6 mm ) , monofilament heavy - weight polypropylene meshes were used ( prolene ; ethicon , egypt ) . really our patients were oriented to the type of repair and the other observers were unaware to operative techniques of the study groups.the laparoscopic transabdominal preperitoneal repair ( tapp ) : the open preperitoneal approach to the inguinal region was performed under general or regional anesthesia , as originally described by nyhus . through a lower abdominal transverse incision , a 15 15 cm polypropylene mesh with a slit was inserted in the preperitoneal space and fixed with non - absorbable sutures to pubic tubercle and cooper 's ligament . the mesh was passed behind the cord and manipulated to lay flat against the posterior inguinal floor overlapping the entire myopectineal orifice . the anterior tension - free repair , as defined by lichtenstein et al . , large pore - sized ( 1.6 mm ) , monofilament heavy - weight polypropylene meshes were used ( prolene ; ethicon , egypt ) . really our patients were oriented to the type of repair and the other observers were unaware to operative techniques of the study groups . the properitoneal space was dissected from lateral to medial at the level of the retroinguinal ( bogros ' ) space , with parietalization of the spermatic cord posteriorly and outwards . the dissection was continued medially toward the retropubic space , extending behind the symphysis pubis and iliopubic tract , exposing the pectineal ligament . the peritoneum forming the hernia sac was pulled in , separating it from the cord structures . a 15 10 cm sheet of polypropylene mesh was placed so as to cover the hesselbach 's triangle , the indirect space , and the femoral ring areas . the mesh was fixed using an endoscopic multifire hernia stapler ( ethicon , johnson and johnson ) , beginning at the pubic tubercle and proceeding laterally . the peritoneum is tightly closed using 3/0 running vicryl suture . the fascia at the two 10/12 mm port site is closed using 2 - 0 prolene sutures . the primary end point of the study was the recurrence of the hernia , defined as a clinically detectable characteristic swelling in the groin and diagnosed by two authors . the secondary end points were time off from work , defined as the number of days between the day of surgery and the first day a patient returned to work , postoperative pain , scrotal swelling , and wound infections . regarding the postoperative pain , we considered the visual analog scale pain score , prosthesis awareness and return to normal physical activity . chronic pain was defined as pain lasting more than 3 months and was studied in relation to age , body mass index and operative procedure . this system is a 3-scale system ; with maximum score as 7 points and minimum as 2 points . analog scale pain score ( 1 - 10 ) : mild ( 1 - 4 ) = 1 point , moderate ( 5 - 7 ) = 2 points , severe ( 8 - 10 ) = 3 points.prosthesis awareness : yes = 1 point , no = 0 point.physical activity : pain only on exertion = 1 pain limits some daily activity = 2 , disabling pain = 3 . analog scale pain score ( 1 - 10 ) : mild ( 1 - 4 ) = 1 point , moderate ( 5 - 7 ) = 2 points , severe ( 8 - 10 ) = 3 points . physical activity : pain only on exertion = 1 pain limits some daily activity = 2 , disabling pain = 3 . ethical approval for this study was granted by the ethical review committee under supervision of the general director of port- fouad general hospital , port - fouad , port - said , egypt . the statistical tests were run on a compatible personal computer using the statistical package for social scientists ( spss ) for windows 15 . chi - square distribution was used for studying the frequencies of recurrence , pain , hospital stay , and postoperative complications . the mean values of the groups were compared by one - way analysis of variance ( anova ) and paired comparisons of the groups were done using the paired student t - test . patients in this study were divided randomly into three main groups : a , b , and c. group a patients were subjected to open posterior preperitoneal approach , those of group b were subjected to transinguinal anterior tension - free repair and group c patients were subjected to tapp . all of our patients were gentlemen with total number was 180 patients ; 60 for each group , their ages ranged between 42 and 65 years . the study started from january 2007 to april 2011 and included all patients having unilateral recurrent inguinal scrotal and irreducible hernias . patients with primary inguinal hernia , patients with marked obesity ( bmi > 35 ) and asa grade 3 and beyond were excluded . calculation of the sample size included the number of participants to be recruited for the study using the mathematical equation . the authors used two equations to calculate the minimum number required to reliably answer the research question . randomization was performed prior to the study using a computer - generated table of random numbers for group assignment . operations were performed in port - fouad general hospital , port - fouad , port - said , egypt and in the university hospital , department of surgery , faculty of medicine , suez canal university , egypt . the open preperitoneal approach to the inguinal region was performed under general or regional anesthesia , as originally described by nyhus . through a lower abdominal transverse incision , a 15 15 cm polypropylene mesh with a slit was inserted in the preperitoneal space and fixed with non - absorbable sutures to pubic tubercle and cooper 's ligament . the mesh was passed behind the cord and manipulated to lay flat against the posterior inguinal floor overlapping the entire myopectineal orifice.the anterior tension - free repair , as defined by lichtenstein et al . large pore - sized ( 1.6 mm ) , monofilament heavy - weight polypropylene meshes were used ( prolene ; ethicon , egypt ) . really our patients were oriented to the type of repair and the other observers were unaware to operative techniques of the study groups.the laparoscopic transabdominal preperitoneal repair ( tapp ) : the open preperitoneal approach to the inguinal region was performed under general or regional anesthesia , as originally described by nyhus . through a lower abdominal transverse incision , the anterior rectus sheath was incised and the rectus muscle reflected medially . a 15 15 cm polypropylene mesh with a slit was inserted in the preperitoneal space and fixed with non - absorbable sutures to pubic tubercle and cooper 's ligament . the mesh was passed behind the cord and manipulated to lay flat against the posterior inguinal floor overlapping the entire myopectineal orifice . the anterior tension - free repair , as defined by lichtenstein et al . , large pore - sized ( 1.6 mm ) , monofilament heavy - weight polypropylene meshes were used ( prolene ; ethicon , egypt ) . really our patients were oriented to the type of repair and the other observers were unaware to operative techniques of the study groups . the properitoneal space was dissected from lateral to medial at the level of the retroinguinal ( bogros ' ) space , with parietalization of the spermatic cord posteriorly and outwards . the dissection was continued medially toward the retropubic space , extending behind the symphysis pubis and iliopubic tract , exposing the pectineal ligament . the peritoneum forming the hernia sac was pulled in , separating it from the cord structures . a 15 10 cm sheet of polypropylene mesh was placed so as to cover the hesselbach 's triangle , the indirect space , and the femoral ring areas . the mesh was fixed using an endoscopic multifire hernia stapler ( ethicon , johnson and johnson ) , beginning at the pubic tubercle and proceeding laterally . the peritoneum is tightly closed using 3/0 running vicryl suture . the trocars are removed under direct vision , and the peritoneum is deflated . the fascia at the two 10/12 mm port site is closed using 2 - 0 prolene sutures . the primary end point of the study was the recurrence of the hernia , defined as a clinically detectable characteristic swelling in the groin and diagnosed by two authors . the secondary end points were time off from work , defined as the number of days between the day of surgery and the first day a patient returned to work , postoperative pain , scrotal swelling , and wound infections . regarding the postoperative pain , we considered the visual analog scale pain score , prosthesis awareness and return to normal physical activity . chronic pain was defined as pain lasting more than 3 months and was studied in relation to age , body mass index and operative procedure . this system is a 3-scale system ; with maximum score as 7 points and minimum as 2 points . analog scale pain score ( 1 - 10 ) : mild ( 1 - 4 ) = 1 point , moderate ( 5 - 7 ) = 2 points , severe ( 8 - 10 ) = 3 points.prosthesis awareness : yes = 1 point , no = 0 point.physical activity : pain only on exertion = 1 pain limits some daily activity = 2 , disabling pain = 3 . analog scale pain score ( 1 - 10 ) : mild ( 1 - 4 ) = 1 point , moderate ( 5 - 7 ) = 2 points , severe ( 8 - 10 ) = 3 points . physical activity : pain only on exertion = 1 pain limits some daily activity = 2 , disabling pain = 3 . written consents were obtained from all patients before the study . the steps of operative interferences was granted by the ethical review committee under supervision of the general director of port- fouad general hospital , port - fouad , port - said , egypt . the statistical tests were run on a compatible personal computer using the statistical package for social scientists ( spss ) for windows 15 . chi - square distribution was used for studying the frequencies of recurrence , pain , hospital stay , and postoperative complications . the mean values of the groups were compared by one - way analysis of variance ( anova ) and paired comparisons of the groups were done using the paired student t - test . there was no statistical difference between the three groups regarding patients ' age and body mass index [ table 1 ] . showing subdivision of the three groups regarding age and body mass index age ranged between 42 and 65 years with a mean age as 53.5 years . follow - up assessment was at the 1st week after discharge then at 1st month and through regular visit of 6 months duration or by a telephone call thereafter . follow up included patients ' complaint , if any , clinical examination and ultrasonography if needed . the maximum follow - up period was 60 months and the minimum was 22 months with a mean value as 41 26.87 months . a complete follow - up was obtained in 56/60 ( 93.3% ) of patients in group a , 54/60 ( 90% ) of patients in group b , and 58/60 ( 96.2% ) in group c. the mean operative time in group a was 71.6 min 25.47 ( 40 - 120 ) . in group b , the mean value was 94.7 min 28.5 ( 60 - 150 ) . in group c , the operative time was calculated just after the induction of anesthesia and including the time required for the setup of the laparoscopy . the mean operative time in group c was 122.5 min 31 ( 80 - 170 ) . the mean hospital stay was 1- 3 days ( 2.1 0.8 ) in group a , 2 - 6 days ( 3.7 1.5 ) in group b and 1 - 2 days ( 1.3 0.5 ) in group c. in the other hand , the mean time to return work was 8.2 1.15 ( 7 - 10 ) days in group a while in group b was 11.2 2.3 ( 7 - 15 ) and in group c was 7.6 1.4 ( 6 - 9 ) days . therefore , the mean time off from work in group a was 10.3 1.95 days and in group b was 14.9 3.8 while in group c was 8.9 1.9 [ p < 0.05 ] . chronic postoperative pain was observed in 8 patients in group a ( 13.33% ) , in 18 patients in group b ( 30% ) , and 6 patients in group c ( 10% ) . table 2 shows the detailed descriptions of pain in the three groups as well as the final pain score per patient [ p < 0.003 ] . the authors found that 6/8 patients in group a were in the age group < 50 years and the other two were > 50 year while regarding the body mass index , all 8 patients were < 30 . in group b , bmi < 25 kg / m and the remaining 6 were in the age group > 50 and bmi < 30 kg / m2 . however , 5/6 patients in group c were in the age group < 50 years while regarding the body mass index , all 6 patients were < 30 . all patients who were aware of the presence of prosthesis and pain on exertion belonged to the smaller age group < 50 and the less bmi < 25 kg / m . showing the detailed descriptions of pain in both groups as well as the final pain score per patient regarding the early postoperative complications , 6 patients ( 10% ) in group a developed mild scrotal swelling due to tissue edema not for hematoma formation and wound seroma formation while in group b , the figure was higher as 12 patients ( 20% ) experienced mild to moderate scrotal swelling and seroma . in group c , wound infection was seen in three patients ( 5% ) in groups a and b necessitating only dressing in the outpatient clinic under cover of systemic antibiotics with no need to remove the mesh but those patients developed re - recurrence thereafter . in group c , four patients ( 6.7% ) showed superficial port site infection necessitating only dressing in the outpatient clinic . testicular atrophy was not seen and only two recurrences ( 3.3% ) in group a all over the period of follow up . five cases of testicular atrophy ( 8.3% ) and four cases of hernia recurrence ( 6.25% ) were seen in group b. however , in group c three patients came with recurrence with no testicular atrophy reported [ p 0.05 ] . moreover , the overall postoperative complication rates were 18.3% and 40% in group a and group b , respectively with significant distribution { p < 0.01 } [ table 3 ] . showing the detailed descriptions of results in the three groups with corresponding p - values the ideal method for repair of inguinal hernia would cause minimal discomfort to the patient , both during the surgical procedure and in the postoperative course . it would be technically simple to perform and easy to learn , would have a low rate of complications and recurrence , and would require only a short period of convalescence . however , the most effective method in any given patient is not clearly defined and consequently surgery for recurrent inguinal hernia after mesh repair is usually a difficult operation due to the disadvantage of re - operating through dense fibrotic scar tissue around the mesh with the risk of testicular damage and a large number of local hematoma . to avoid the disadvantage of re - operating through scar tissue and dense fibrotic scar tissue around the mesh , the open posterior preperitoneal mesh repair was popularized by nyhus as a good alternative for recurrent inguinal hernias . in previous study , saber and co - workers 1 reported that open preperitoneal hernia repair offers many advantages over the transinguinal repair for recurrent hernia . laparoscopic transabdominal preperitoneal ( tapp ) procedure generally is accepted for the repair of primary and recurrent hernias that follow conventional transinguinal repair . in the present study , we found that the time of hospital stay and sick leaves and accordingly the time off from work all were reduced in patients with open posterior preperitoneal approach and tapp compared with the anterior approach and the difference was statistically significant . many studies of same interest reported less hospital stay and rapid return to physical activity . chronic postoperative pain is strongly related to two main patient - related factors ; age and body mass index or three surgery related factors such as surgery for recurrence with anterior approach , operations performed in specialist hernia centers , and finally the experience of the surgeon . the total laparoscopic preperitoneal ( tapp ) and then the open preperitoneal approach in the present study significantly reduced the final chronic pain score per patient in comparison with the anterior transinguinal approach and our data came in concordance with studies of same interest . , there are many studies traced patient age in relation to occurrence chronic pain and found that the risk of chronic pain decreased with increasing age . the bmi is another studied factor for chronic pain occurrence where many investigators found good correlation between less bmi values and chronic pain and our data in the present study supported these finding . the postoperative complications of hernia repair were estimated regarding the rate and traced regarding the type in similar previous studies as early and delayed forms . early complication , defined as that occurring within 1 month of surgery , are wound seroma , sepsis , scrotal edema and hematoma formation while the long - term complications , assessed at 3 months are testicular atrophy and recurrence . in published literatures , the overall postoperative complication rates were 18 - 38% and may reach as high as 49.7% in some series and in our study , the overall complication rate was 19.7% in both groups a and c and 34.36% in group b due to more tissue dissection and manipulation . wound hematoma and superficial wound infections are the most common problems in previous series and serious complications , such as major hemorrhage , pubic osteitis and testicular atrophy , occur in less than one percent of patients undergoing herniorrhaphy . minor complications such as seroma formation , wound sepsis , and scrotal hematoma were seen in the three groups in the present study but with more incidence in group b. this observation met with data reported by other investigators . long term complications such as testicular atrophy and recurrence were traced by many researchers who reported 0% incidence for testicular atrophy and 0% or very low incidence ( 1.5% ) for recurrence in their studies . while others found 4.38% hernia recurrence after posterior preperitoneal repair 10% in the open anterior approach while other data ranged between 2% and 14% . in a multicenter study , there was no significant difference in the recurrence rate between laparoscopic and open methods of hernia repair was revealed . however , while the laparoscopic technique has a significantly lower re - recurrence rate than the open technique , there is no statistical difference in the re - recurrence rate between the two techniques during short - term follow - up evaluation . accordingly , we found that recurrence rate was statistically insignificant in the three groups and this finding met with other data in published literatures . an indirect comparison between tapp and open ( ohr ) techniques by considering randomized , controlled trials in a network meta - analysis , the following outcomes were considered : operative time , postoperative complications , hospital stay , postoperative pain , time to return to work and recurrences . testicular dysfunction , atrophy and necrosis as a result of ischemic orchitis is a well - known complication after anterior inguinal hernia repair with 1% occurrence following primary herniorrhaphy and 5% in recurrent cases but in open preperitoneal repair , the procedure is safe as it effectively eliminates testicular complications . a significant decrease in testicular volume and less improvement in blood flow is seen after open repair where there is significant reduction in serum testosterone , follicle stimulating ( fsh ) , and leutinizing ( lh ) hormones level . testicular ischemia and necrosis are thought to be due to acute thrombosis of the pampiniform venous plexus rather than arterial injury , as there is collateral arterial flow to the testis from the inferior epigastric , vesical , prostatic , and scrotal arteries . testicular atrophy is thought to be more common after open procedures particularly recurrent inguinal hernias due to greater manipulation of the spermatic cord beyond the pubic tubercle and during dissection of the distal hernia sac . according to these finding , we found that no testicular atrophy was seen in patients of both groups a and c but seen in two patients of group b ( 4.25% ) due to operating within the fibrotic field with tissue reaction around the mesh . in recurrent inguinal hernia , the laparoscopic and open posterior approaches are equally effective in term of operative outcome . it is inexpensive , has a low recurrence rate , and allows the surgeon to cover all potential defects with one piece of mesh . however , while laparoscopic hernia repair requires a lengthy learning curve and is difficult to learn and perform , it has advantages of less post - operative pain , early recovery with minimal hospital stay , low post - operative complications , and recurrence .

introduction : the repair of the recurrent hernia is a daunting task because of already weakened tissues and distorted anatomy . open posterior preperitoneal approach gives results far superior to those of the anterior approach . laparoscopic transabdominal preperitoneal ( tapp ) inguinal hernia repair is an evolving technique associated with advantages of a minimally invasive approach . the present work aimed at comparing these three approaches for repair of recurrent inguinal hernia regarding complications and early recurrence.materials and methods : a total of 180 patients were divided randomly into three equal groups : a , b , and c. group a patients were subjected to open posterior preperitoneal approach , those of group b were subjected to transinguinal anterior tension - free repair and group c patients were subjected to tapp . the primary end point was recurrence and the secondary end points were time off from work , postoperative pain , scrotal swelling , and wound infections.results:the mean hospital stay , the mean time to return to work and the mean time off from work were less in group c then a and b. chronic postoperative pain was observed in eight patients in group a ( 13.33% ) , in 18 patients in group b ( 30% ) and six patients in group c ( 10% ) . the overall complication rate was 19.7% in both groups a and c and 34.36% in group b.conclusion:in recurrent inguinal hernia , the laparoscopic and open posterior approaches are equally effective in term of operative outcome . the open preperitoneal hernia repair is inexpensive , has a low recurrence rate . postoperative recovery is short and postoperative pain is minimal . this approach gives results far superior to those of the commonly used anterior approach . however , while laparoscopic hernia repair requires a lengthy learning curve and is difficult to learn and perform , it has advantages of less post - operative pain , early recovery with minimal hospital stay , low post - operative complications and recurrence.trial registrationactrn12613001050741

Trial Registration INTRODUCTION MATERIALS AND METHODS Materials Sample Size and Randomization Surgical Teams and Study Sites Operative Techniques End Points Ethical Consideration Statistical Analysis RESULTS DISCUSSION CONCLUSION

PMC4201802

adolescents constitute only a small percentage of treatment seekers in drug dependence treatment settings ( 15 years : 0.4% and 16 - 20 years : 4.6% ) . community surveys suggest that tobacco , alcohol and cannabis are prevalent substances of abuse among indian adolescents , but opioid use is quite uncommon in this age group ( about 1 in 1000 ) . heroin use was seen in 3.3% of drug - using adolescents in contact with nehru yuva kendras ( grass - root level voluntary organization for youth ) across the country . potentially serious complications , e.g. sexual risks , violence and other high risk behaviors are associated with adolescent substance use , which may pose a significant public health issue . the long - term pharmacotherapies for opioid dependence can be either in the form of an opioid agonist ( e.g. buprenorphine ) or antagonist ( e.g. naltrexone ) therapy . the use of pharmacotherapies , in conjunction with psychosocial support , is the mainstay of treatment of adult opioid users , but there is only a limited evidence base for their efficacy in adolescents with opioid dependence . very few reports or studies are available till date on long - term buprenorphine maintenance for adolescent opioid users and only one study has described the use of naltrexone ( antagonist ) in adolescent opioid dependence . buprenorphine is a partial agonist at mu - opioid receptor , which is used in low to moderate doses to enable the patient to abstain from the use of illicit opioids and prevent craving or withdrawals . it is available as sublingual ( 2 mg/4 mg ) tablets administered under supervision of staff usually on a daily basis in view of their abuse liability . a take - home combination of sublingual buprenorphine - naloxone ( 4:1 ) is available which is ineffective by intravenous route , thereby minimizing the risk of abuse . naltrexone is an opioid receptor antagonist which blocks the effects of opioids and prevents the positive reinforcement associated with the use of opioids . no prior study or case report from india has described the use of agonist maintenance or antagonist therapy for adolescent opioid users . we discuss two adolescent patients with opioid ( heroin ) dependence visiting national drug dependence treatment centre ( nddtc ) , all india institute of medical sciences ( aiims ) , new delhi for treatment , who were stablilized on buprenorphine and naltrexone respectively for a substantial period of time . master a , 15 years old , studied up to 6 std and belonged to a middle socio - economic status joint family living in a small town in the state of uttar pradesh ( 300 km from centre ) . he presented to adolescent clinic , nddtc , aiims with parents for the first time in january 2009 with chief complaints of the regular use of tobacco ( both smokeless and smoking ) for 8 years , occasional cannabis ( ganja ) use for 3 years and regular heroin ( smack ) use for 3 years . heroin , the primary drug of use , was initiated at the age of 12 years out of curiosity with some of his friends and later , continued for its pleasurable effects . there was evidence of tolerance , withdrawals and diminished control over the time and amount of heroin use . the usual daily dose was 2 - 3 pudias ( worth rs 100 - 150 ) . as a result of drug use , he started having frequent discord with parents and siblings . he engaged in stealing and working as an accomplice of pick - pocketers to sustain drug use . he spent many nights out of his house without informing his whereabouts and experienced a significant decline in physical health and social functioning . the heroin use continued uninterrupted for 3 years till he presented for treatment on parent 's initiative . the personal history suggested the presence of conduct symptoms ( truancy , lying , stealing from home ) since childhood prior to onset of drug use . the complete hemogram , blood sugar , liver and kidney function tests were within normal limits . patient was hospitalized for management of withdrawals with gradually tapering the dose of low strength buprenorphine ( 0.4 mg s / l tab ; 2 mg / day ) and subsequently , initiated on naltrexone ( 25 mg / day ; dispensed from centre 's pharmacy free of cost ) . psychosocial interventions aimed at rapport - building , motivation enhancement and psycho education were offered . however , patient was lost to follow - up after discharge restarting the heroin use and spending time with drug - using peers . there were two more admissions in ensuing months ( june 2009 and august 2009 ; about 2 weeks each ) which were considered for the purpose of detoxification and reinstatement of naltrexone in view of long distance of travel . the psychosocial intervention sessions were provided for patient ( to identify high risk situations , relapse prevention , coping skills enhancement , vocational skill building ) . efforts were also made to engage the family members more actively in treatment process ( and address mother 's overprotective behavior for patient and conflict between parents ) . however , after each of these admissions , there was no follow - up visit in opd and patient resumed the drug use . however , after the 4th admission ( 2 weeks ; october 2009 ) and subsequent discharge on naltrexone ( 25 mg / day ) , he followed up in out - patient clinic with parents as advised during the ward sessions . patient continued with the fortnightly follow - up , compliant to medication and maintaining abstinence ( as confirmed by parents and repeated urinalysis ) for a period of 10 months ( oct 2009-aug 2010 ) . throughout this duration , parents were actively involved in treatment process , supervising the compliance at home and participating in sessions . patient would intermittently report disinclination to take naltrexone , due to occasional body aches , discomfort and not feeling fit(protracted withdrawals ) . eventually , he refused to take naltrexone on his own , relapsed within a few days and was lost to follow up for nearly an year . he again presented to the out - patient clinic ( in july 2011 ) with the current use of 3 - 4 pudias ( small packets ) smack on a daily basis . he was not doing any meaningful activity , mostly staying away of home even during nights and engaged in anti - social activities with drug - using peers . plan for long term management was discussed with patient as well as both parents , during which patient reported disinclination to take naltrexone due to physical discomfort . after a re - assessment , patient was initiated and stabilized on agonist replacement regimen ( buprenorphine - naloxone combination 4 mg / day ) , which was dispensed biweekly to either of parents . patient felt comfortable on above medication and completely abstained from street heroin ( confirmed by negative results on urinalysis using thin layer chromatography or rapid cassette tests ) , though occasional cannabis use continued . he would not go out with drug - using peers and started remaining at home , leading to improvement in his relationships with family . his father involved him in their small - scale manufacturing unit ( packeted eatables / snacks ) and patient fulfilled various responsibilities , including financial handlings with no complaints . patient works for nearly 7 - 8 h in a day and spends time with family in evenings . currently , patient is on the same dose of buprenorphine - naloxone ( 4 mg / day ) for nearly 2 years ( dispensed fortnightly to him / his parents ) , during which he visits the center every month for follow - up continuing till date . master b , 17 years old , school drop - out after primary class , belonged to nuclear family of lower middle socioeconomic status residing in delhi . patient had a history of regular tobacco and occasional cannabis ( ganja ) use since 10 years of age , regular inhalant use ( correction fluid- 1bottle / day ) since 11 years of age and heroin use since 16 years of age . at the time of presentation , heroin was the primary drug of use for nearly a year , with a usual dose of 1 - 2 pudias ( small packets ; worth rs 50 - 100 ) per day . there was significant dysfunction in all the major domains of functioning and patient never tried to abstain ( except for 2 - 3 forced admissions in unauthorized privately run centers lasting few weeks ) . in the psychiatric history , at the age of 17 years , patient had a moderate depressive episode lasting 2 weeks and in the ensuing 6 months , there were further four depressive episodes ( each lasting 10 - 20 days ) and two hypo - manic episodes ( 3 - 5 days each ) , all of which had no apparent precipitating factor and remitted spontaneously . patient was using tobacco and heroin during this period , but no temporal relationship was observed with drug use . there was a childhood history suggestive of hyperactivity / impulsivity and inattentiveness since 5 - 6 years of age leading to poor academic performance and subsequent drop - out from school . no treatment was sought and symptoms improved after early adolescence . in addition , there is also a history of conduct symptoms prior to onset of drug use ( truancy , lying to parents , initiating quarrels , physical fights , throwing stones and kicking small animals , running away from home ) . patient fulfilled the icd-10 diagnostic criteria for tobacco dependence , opioid dependence and bipolar affective disorder - not otherwise specified , and a lifetime diagnosis of volatile solvent dependence and hyperkinetic conduct disorder . psychosocial treatment sessions focused on enhancing motivation and providing psycho education on various aspects of substance use and psychiatric disorders . after a detailed assessment , by the third out - patient visit , the patient was started on tab naltrexone ( 50 mg / day ) and tab valproate ( 1 gm / day ) . patient continued in regular follow up on the above medication for a period of 1 year and 4 months . the compliance was supervised by mother and he began assisting his father in tailoring in the initial few months . there were no further mood episodes during this period and patient was largely abstinent from illicit substances , confirmed by repeated cassette tests . the occasional use of cannabis , however , persisted along with a few behavioral problems . the psychosocial intervention sessions were delivered at each follow - up and some of the patient 's conflicts with parents were taken up during the sessions . master a , 15 years old , studied up to 6 std and belonged to a middle socio - economic status joint family living in a small town in the state of uttar pradesh ( 300 km from centre ) . he presented to adolescent clinic , nddtc , aiims with parents for the first time in january 2009 with chief complaints of the regular use of tobacco ( both smokeless and smoking ) for 8 years , occasional cannabis ( ganja ) use for 3 years and regular heroin ( smack ) use for 3 years . heroin , the primary drug of use , was initiated at the age of 12 years out of curiosity with some of his friends and later , continued for its pleasurable effects . there was evidence of tolerance , withdrawals and diminished control over the time and amount of heroin use . the usual daily dose was 2 - 3 pudias ( worth rs 100 - 150 ) . as a result of drug use , he started having frequent discord with parents and siblings . he engaged in stealing and working as an accomplice of pick - pocketers to sustain drug use . he spent many nights out of his house without informing his whereabouts and experienced a significant decline in physical health and social functioning . the heroin use continued uninterrupted for 3 years till he presented for treatment on parent 's initiative . the personal history suggested the presence of conduct symptoms ( truancy , lying , stealing from home ) since childhood prior to onset of drug use . the complete hemogram , blood sugar , liver and kidney function tests were within normal limits . patient was hospitalized for management of withdrawals with gradually tapering the dose of low strength buprenorphine ( 0.4 mg s / l tab ; 2 mg / day ) and subsequently , initiated on naltrexone ( 25 mg / day ; dispensed from centre 's pharmacy free of cost ) . psychosocial interventions aimed at rapport - building , motivation enhancement and psycho education were offered . however , patient was lost to follow - up after discharge restarting the heroin use and spending time with drug - using peers . there were two more admissions in ensuing months ( june 2009 and august 2009 ; about 2 weeks each ) which were considered for the purpose of detoxification and reinstatement of naltrexone in view of long distance of travel . the psychosocial intervention sessions were provided for patient ( to identify high risk situations , relapse prevention , coping skills enhancement , vocational skill building ) . efforts were also made to engage the family members more actively in treatment process ( and address mother 's overprotective behavior for patient and conflict between parents ) . however , after each of these admissions , there was no follow - up visit in opd and patient resumed the drug use . however , after the 4th admission ( 2 weeks ; october 2009 ) and subsequent discharge on naltrexone ( 25 mg / day ) , he followed up in out - patient clinic with parents as advised during the ward sessions . patient continued with the fortnightly follow - up , compliant to medication and maintaining abstinence ( as confirmed by parents and repeated urinalysis ) for a period of 10 months ( oct 2009-aug 2010 ) . throughout this duration , parents were actively involved in treatment process , supervising the compliance at home and participating in sessions . patient would intermittently report disinclination to take naltrexone , due to occasional body aches , discomfort and not feeling fit(protracted withdrawals ) . eventually , he refused to take naltrexone on his own , relapsed within a few days and was lost to follow up for nearly an year . he again presented to the out - patient clinic ( in july 2011 ) with the current use of 3 - 4 pudias ( small packets ) smack on a daily basis . he was not doing any meaningful activity , mostly staying away of home even during nights and engaged in anti - social activities with drug - using peers . plan for long term management was discussed with patient as well as both parents , during which patient reported disinclination to take naltrexone due to physical discomfort . after a re - assessment , patient was initiated and stabilized on agonist replacement regimen ( buprenorphine - naloxone combination 4 mg / day ) , which was dispensed biweekly to either of parents . patient felt comfortable on above medication and completely abstained from street heroin ( confirmed by negative results on urinalysis using thin layer chromatography or rapid cassette tests ) , though occasional cannabis use continued . he would not go out with drug - using peers and started remaining at home , leading to improvement in his relationships with family . his father involved him in their small - scale manufacturing unit ( packeted eatables / snacks ) and patient fulfilled various responsibilities , including financial handlings with no complaints . patient works for nearly 7 - 8 h in a day and spends time with family in evenings . currently , patient is on the same dose of buprenorphine - naloxone ( 4 mg / day ) for nearly 2 years ( dispensed fortnightly to him / his parents ) , during which he visits the center every month for follow - up continuing till date . master b , 17 years old , school drop - out after primary class , belonged to nuclear family of lower middle socioeconomic status residing in delhi . patient had a history of regular tobacco and occasional cannabis ( ganja ) use since 10 years of age , regular inhalant use ( correction fluid- 1bottle / day ) since 11 years of age and heroin use since 16 years of age . at the time of presentation , heroin was the primary drug of use for nearly a year , with a usual dose of 1 - 2 pudias ( small packets ; worth rs 50 - 100 ) per day . there was significant dysfunction in all the major domains of functioning and patient never tried to abstain ( except for 2 - 3 forced admissions in unauthorized privately run centers lasting few weeks ) . in the psychiatric history , at the age of 17 years , patient had a moderate depressive episode lasting 2 weeks and in the ensuing 6 months , there were further four depressive episodes ( each lasting 10 - 20 days ) and two hypo - manic episodes ( 3 - 5 days each ) , all of which had no apparent precipitating factor and remitted spontaneously . patient was using tobacco and heroin during this period , but no temporal relationship was observed with drug use . there was a childhood history suggestive of hyperactivity / impulsivity and inattentiveness since 5 - 6 years of age leading to poor academic performance and subsequent drop - out from school . no treatment was sought and symptoms improved after early adolescence . in addition , there is also a history of conduct symptoms prior to onset of drug use ( truancy , lying to parents , initiating quarrels , physical fights , throwing stones and kicking small animals , running away from home ) . patient fulfilled the icd-10 diagnostic criteria for tobacco dependence , opioid dependence and bipolar affective disorder - not otherwise specified , and a lifetime diagnosis of volatile solvent dependence and hyperkinetic conduct disorder . psychosocial treatment sessions focused on enhancing motivation and providing psycho education on various aspects of substance use and psychiatric disorders . after a detailed assessment , by the third out - patient visit , the patient was started on tab naltrexone ( 50 mg / day ) and tab valproate ( 1 gm / day ) . patient continued in regular follow up on the above medication for a period of 1 year and 4 months . the compliance was supervised by mother and he began assisting his father in tailoring in the initial few months . there were no further mood episodes during this period and patient was largely abstinent from illicit substances , confirmed by repeated cassette tests . the occasional use of cannabis , however , persisted along with a few behavioral problems . the psychosocial intervention sessions were delivered at each follow - up and some of the patient 's conflicts with parents were taken up during the sessions . these case descriptions add to the limited literature on long term pharmacological treatment of adolescent patients with opioid dependence . both the adolescent patients had some common clinical features , e.g. school drop - outs , presence of psychiatric risk factors , very early - onset of first substance use ( 7 and 10 years respectively ) , use of multiple substances , progression from licit to illicit substance , significant dysfunction in various domains and poor motivation at baseline . these are in accordance to the existing literature on the adolescent - onset substance use disorders . after initiation of pharmacological treatment in conjunction with psychosocial intervention sessions , both the patients could be retained in long term follow - up ( patient a : 2 years ; patient b : 1.3 years ) , were abstinent from heroin and engaged in meaningful work . the use of buprenorphine maintenance is quite uncommon among adolescent patients and very few studies have evaluated its efficacy in this age group , which is summarized here . a multi - site study of opioid - dependent youth ( of which 12 were adolescents ) examined buprenorphine - naloxone treatment vs. standard detoxification , and found significantly better outcomes in drug use and other measures at the end of 12 weeks . however , the adolescent sub - sample in this study was deemed to be inadequate for a separate analysis . a file review from an australian treatment setting compared first treatment episode of adolescents on buprenorphine ( n = 25 ) versus methadone ( n = 20 ) , and found that treatment retention was higher in methadone compared to buprenorphine ( mean : 354 days vs. 58 days ) in adolescents . another us study reported positive experiences of few adolescent patients on buprenorphine in a substance abuse treatment program that also focused on adolescent development . finally , a cochrane review of various databases ( 1966 - 2008 ) for randomized or controlled trials for effectiveness of any maintenance agent in adolescents , found only two trials ( only one of which involved buprenorphine ) . the cochrane review was inconclusive and meta - analysis could not be carried out with two heterogeneous trials . there is often a general policy of dispensing agonist medication only to adult patients due to concerns about starting youth on treatment that is often long - term and reluctance to bring young patients into daily contact with adult patients with extensive addiction histories and antisocial behaviors . however , a small percentage of adolescents with early - onset of opioid dependence may experience severe dysfunction , engage in high risk and anti - social behaviors and show a poor motivation to quit . it has been recommended elsewhere too that agonist maintenance may be considered for adolescents who have opioid dependence , age is at least 16 years or above , opioid use 1 year and two documented failed attempts . so far , only one study is available for naltrexone use in adolescent opioid dependence . in this preliminary non - controlled , extended - release naltrexone was found to be safe and efficacious in a sample of 16 adolescents and young adults ( mean age : 18.5 years ) . of total , 10 were retained in treatment for at least 4 months and 9 had a good outcome . in addition , few studies and case reports have evaluated the overall safety of naltrexone for adolescents with alcohol use disorders ( sample age : 16 - 18 years ; dose 50 mg / day ) . the patients discussed above , naltrexone was initiated after a discussion with patient , parents and a review of existing literature . naltrexone was tolerated well at the dose of 50 mg / day , with no significant adverse effects . compared to patient a , patient b had a shorter duration of heroin use and consequently , did not experience any protracted withdrawals , craving or need for an agonist . it is to be noted that adolescent opioid users frequently have a number of other problems , e.g. the use of drugs by peers , poor family support , living in a locality with easy availability of drugs and underlying psychiatric morbidity , some of which were present in one or both patients . failure to address those issues will reduce the chance of successful treatment and increase the risk of relapse . the comorbid bipolar disorder was managed with a mood stabilizer in patient b , with no recurrence during follow - up period . a good family support was present in both cases , which played a crucial role in ensuring compliance to medication and other treatment aspects . for example , the take - home doses of buprenorphine - naloxone were dispensed free of cost as a policy , but family members had to travel several hours by train to reach center on a fortnightly basis , thereby investing time and other resources over these years . in indian context , the active involvement of family in various aspects of treatment process contributes immensely toward improved functioning of the patient . some drug using adolescents may not have a family or family support , but wherever available , their participation should be actively sought at all treatment steps . to conclude , the case report adds to the limited literature on efficacy of pharmacotherapies for adolescent opioid use . however , there is a need to conduct systematically planned studies on the adolescent opioid use treatment .

adolescents constitute only a small percentage of treatment seekers in drug dependence treatment settings . little research evidence is available for pharmacological treatment of adolescent opioid dependence and no prior case report is available from india . we discuss two adolescent patients with opioid ( heroin ) dependence visiting a tertiary care center who have been stabilized on agonist ( sublingual buprenorphine - naloxone ) and antagonist ( oral naltrexone ) respectively for a substantial period of time . a comprehensive management approach , including intensive psychosocial interventions and family involvement , was followed in addition to pharmacotherapies . more research is needed on the efficacy of pharmacological treatment in adolescent opioid users .

INTRODUCTION CASE REPORTS Case 1 Case 2 DISCUSSION

PMC3263534

parkinson 's disease ( pd ) is a fatal and incurable neurodegenerative disorder characterized by resting tremors , postural instability , and bradykinesia . the death of midbrain substantia nigra neurons characterizes the pathology of both familial and sporadic forms of pd . two types of molecules accumulate within these dying cells : misfolded and aggregated -synuclein in cytoplasmic inclusions called lewy bodies and oxidative free radicals [ 1 , 3 ] . therefore , a major hypothesis is that -synuclein misfolding and aggregation is linked to neuronal toxicity in pd [ 46 ] . while the molecular basis of sporadic pd is still unclear , mutations in six genes cause familial pd , including three point mutations ( a53 t , a30p , and e46k ) within the -synuclein gene [ 810 ] . in addition to self - aggregation [ 1113 ] , several other -synuclein properties that may be relevant to pd include maintenance of neurotransmitter vesicular pools [ 14 , 15 ] , synaptic plasticity [ 16 , 17 ] , phospholipid binding and lipid metabolism [ 1821 ] , microtubule binding , chaperone - like ability [ 2325 ] , and er - golgi trafficking [ 26 , 27 ] . while the a30p and a53 t familial mutants are widely studied in how they alter the above properties to cause cytotoxicity and contribute to pd pathogenesis , less is known about the third and most recently discovered familial mutant , e46k . thus far , e46k is known to aggregate into fibrils more rapidly than wild - type -synuclein in vitro [ 29 , 30 ] and in cell culture . additionally , recent biochemical studies suggest that e46k enhances contact between the amino and carboxyl domains although whether this interaction increases the likelihood of aggregation is still being sorted . in contrast to the a30p and a53 t familial mutants , e46k exhibits a reduced accumulation of early oligomers [ 13 , 33 , 34 ] . like wild - type -synuclein , e46k can form ion channel - like pores in lipid bilayers although recent work suggests that these channels are less conductive than those formed by wild - type -synuclein . on the other hand , e46k binds phospholipids more readily than wild - type -synuclein [ 29 , 36 , 37 ] and is more potent in inducing toxicity in neuroblastoma cells that are cotreated with cytokines . in living cells , however , little is still known of e46k 's ability to bind either the plasma membrane or internal membrane systems . nor is it clear how e46k aggregation and/or membrane association might cause cellular toxicity . budding and fission yeast models have advanced our molecular understanding of several neurodegenerative diseases , including pd [ 21 , 3942 ] . yeasts revealed that -synuclein binds plasma membranes extensively in addition to its propensity to aggregate , but the relative importance of aggregation and membrane binding to toxicity is still unresolved [ 21 , 4346 ] . some models demonstrate -synuclein expression and dose - dependent aggregation itself causes toxicity [ 21 , 44 ] , while others report that -synuclein - dependent toxicity , especially when moderately expressed , requires additional factors , such as proteasomal inhibition and oxidative stress [ 43 , 45 ] , or tau coexpression . in 2003 , willingham et al . similarly , disruption of the secretory pathway and er - golgi trafficking [ 26 , 27 ] were first found in yeast to increase -synuclein toxicity . here , we exploited the utility of both budding yeast and fission yeasts to test the hypothesis that the e46k mutant aggregates and binds membranes more strongly than wt -synuclein in living organisms and that it accumulates to induce cellular toxicity . indeed , we found that e46k binds the plasma membrane in budding yeast and associates with endomembrane systems and form intracellular aggregations in fission yeast . while e46k is not toxic to budding yeast , it is toxic to fission yeast especially when associated with internal membrane systems . as described in our past studies [ 45 , 46 ] , we tagged -synuclein with gfp at its c - terminus and induced its expression with galactose in budding yeast and by removal of thiamine ( the repressor ) in fission yeast . our first goal was to assess whether e46k was toxic to budding and fission yeasts , and we report that e46k is toxic to yeasts , albeit in a species- and strain - specific manner . first , for budding yeast , we examined two haploid strains of opposite mating types ( by4741 mat a and by4742 mat ) and a diploid strain ( by4743 mat a/ ) . e46k was not toxic to any strain , as assessed by two assays : serial dilution spotting on solid growth plates ( figure 1(a ) ; by4742 not shown ) and growth curves from liquid culture ( data not shown ) . by4741 cells that expressed e46k grew equally well as cells that did not express -synuclein ( vector alone and gfp ) and those that expressed wt -synuclein or two other familial mutants ( a30p and a53 t ; figure 1(a ) , top ) . therefore , as we had previously reported for wt , a30p , and a53 t , e46k -synuclein did not adversely affect budding yeast growth . additionally , neither the mating type ( data not shown ) nor ploidy doubling ( figure 1(a ) , bottom ) altered the lack of e46k -synuclein - dependent toxicity in budding yeast . next , we examined how -synuclein affected the growth of two haploid fission yeast strains of opposite mating types : h ( tcp1 ) and h+ ( sp3 ) . in contrast to our budding yeast findings , we found that several -synuclein variants were toxic to fission yeast ( figure 1(b ) ) . firstly , when compared to vector alone and gfp expressing cells , wt , e46k and a53 t -synuclein expressing cells were each toxic in both strains , but not a30p ( figure 1(b ) ) . in tcp1 , e46k and wt were toxic to similar extents but less than a53 t ( figure 1(b ) , top ) . in sp3 , e46k was more toxic than wt yet still less than a53 t ( figure 1(b ) , bottom ) . to gain insight into e46k -synuclein 's differential toxicity in yeasts e46k -synuclein consistently localized to the plasma membrane of by4741 yeast during 48 hours of expression , similar to wt and a53 t -synuclein localization patterns ( figure 2(a ) ) that we previously had reported for by4741 . quantification of localization in fluorescing cells indicates that more than 90% expressed e46k -synuclein at the plasma membrane ( figure 2(a ) ) . unlike a53 t , however , e46k -synuclein was rarely seen aggregated in the cytoplasm ( figure 2(a ) ) . nor did we observe any e46k localization to internal endomembrane systems . in contrast , a30p -synuclein was mostly cytoplasmically diffuse , similar to gfp alone ( figure 2(a ) ) . e46k plasma membrane association and the patterns of localization of the other -synuclein variants were maintained in by4742 ( figure 2(b ) ) and by4743 ( figure 2(c ) ) strains . we next wondered if e46k bound the plasma membrane more extensively than wt -synuclein but found no difference in the extent of plasma membrane association between them over the first 24 hours of expression : both appeared at the plasma membrane as early as 6 hours to similar extents ( figure 2(d ) ) . thus , the lack of e46k -synuclein toxicity in budding yeasts correlated with plasma membrane association . in stark contrast to budding yeast , e46k -synuclein was prominently absent at the plasma membrane when examined in the two haploid fission yeast strains ( figure 3 ) . in tcp1 , e46k aggregated in the cytoplasm , again strikingly similar to wt and a53 t -synuclein ( figure 3(a ) ) that we previously had reported in tcp1 . -synuclein and gfp alone were cytoplasmically diffuse ( figure 3(a ) ) , also as previously reported . most cells had 13 aggregates , but some had up as many as 510 per cell ( figure 3(c ) ) . these aggregates were outside the nucleus , as determined by dapi staining ( figure 3(d ) , top ) . notably , dapi staining typically altered the pattern of -synuclein localization , reducing the size and location of -synuclein puncta , which suggests that some of the larger accumulations were either a collection of dispersible smaller aggregates or compact membranous vesicles ( figure 3(d ) , top ) . initially , up to 24 hours of expression , both e46k and wt -synuclein distributed to the membrane linings of many intracellular compartments , in addition to aggregating in the cytoplasm ( figure 3(b ) ) . over the next 24 hours , 76% wt -synuclein expressing cells contained aggregates similar to tcp1 , while e46k -synuclein was even more prominently localized to diverse intracellular locations ( 98% cells ) , with no cells containing just aggregates ( figure 3(b ) ) . over this same course of time , a30p -synuclein remained cytoplasmically diffuse while , a53 t was mostly aggregated by 48 hours ( 84% ) , with endomembrane localization less extensive than e46k ( 16% ; figure 3(b ) ) . this e46k intracellular localization pattern excluded the nucleus but not the nuclear envelope ( figure 3(d ) , lower ) , and prominently lit up perinuclear and vacuolar / prevacuolar compartments ( figure 3(b ) ) , and other components of the endomembrane system that need further identification . often , vesicular accumulations were observed within cells : mostly with e46k , less with wt , and rarely with a53 t ( figure 3(e ) ) . thus , the added toxicity of e46k -synuclein in sp3 fission yeast appears correlated best with its localization to diverse endomembrane organelles . we previously reported that wt and a53 t -synuclein aggregate in a time- and concentration - dependent manner in fission yeast , providing live cell support for the nucleation polymerization hypothesis . whether e46k does the same has not been reported . indeed , we found that e46k -synuclein localization in tcp1 was time - dependent ( figure 4(a ) ) . over a time course of 48 hours , e46k was initially diffuse in the cytoplasm for the first 12 hours , but thereafter aggregated into one or more punctate aggregates within individual cells ( figure 4(a ) ) . by 18 hours , less than 5% cells had cytoplasmically diffuse e46k while , wt -synuclein was cytoplasmically diffuse in 10% cells even at 30 hours ( figure 4(a ) ) . importantly , the e46k pattern of aggregation closely resembled a53 t -synuclein aggregation ( figure 4(a ) ) . remarkably , at no point over the time course was e46k -synuclein ( nor wt or a53 t ) localized at the plasma membrane ( figure 3(a ) ) . a30p -synuclein remained cytoplasmically diffuse and never aggregated over this same time course ( data not shown ) . similarly , e46k -synuclein also aggregated in tcp1 in a concentration dependent manner , which was demonstrated by growing e46k cells in the presence of decreasing amounts of the repressor , thiamine ( 10 , 1.0 , 0.1 , and 0 m ; figure 4(b ) , left ) . at the two highest thiamine concentrations , e46k -synuclein was diffusely localized in the cytoplasm ( figure 4(b ) , left ) . however , at the lower thiamine concentrations as more protein is expressed , e46k started aggregating in most cells , similar to wt and a53 t -synuclein ( figure 4(b ) , left ) . the low level of -synuclein observed by gfp fluorescence even at the highest thiamine concentration was confirmed by western blotting ( figure 4(c ) , right ) . thus , e46k -synuclein aggregated in a time- and concentration - dependent manner , supporting the nucleation polymerization model . alternatively , the data can also be explained by concentration - dependent membrane localization as well , if the e46k puncta are , in fact , compact membrane structures , rather than aggregates . we wondered if the strain - specific enhancement of e46k -synuclein toxicity in fission yeast that was lacking in budding yeast was partly due to its selective accumulation in fission yeast . in budding yeast , e46k and wt -synuclein were indeed either similar ( by4741 ) or even lower ( by4743 ) at 24 and 48 hours of expression , indicating a lack of accumulation ( figure 5(a ) ) . in fission yeast , however , we did observe a slight increase in e46k -synuclein expression relative to wt in both tcp1 ( 48 hrs ) and sp3 strains ( both 24 and 48 hrs ; figure 5(b ) ) . given that serine phosphorylation is linked to -synuclein related pd pathology [ 4850 ] , we next asked if e46k toxicity is linked to phosphorylation . firstly , we found that both e46k and wt -synuclein were phosphorylated at serine-129 in both budding and fission yeasts and to the same extent ( figure 5(c ) ) . importantly , the enhanced e46k toxicity in sp3 was not correlated with altered level of -synuclein phosphorylation ( figure 5(c ) ) . finally , we asked if the selective e46k -synuclein toxicity in fission yeast was linked to a reduction in organismal growth and survival . as shown in the schematic ( figure 6(a ) ) , 1000 yeast cells each expressing wt or e46k -synuclein or containing parent vector alone for either 8 ( early log phase of growth ) or 18 hours ( late log phase ) were plated onto repressing or inducing media . these survival plates were then first grown for two days , and the induced plates were grown even further for three more days . firstly , in the tcp1 strain both e46k and wt -synuclein expressing cells were significantly reduced in colony size compared to parent vector cells when they continued to express -synuclein ( induction = figure 6(b ) ) , but not when they stopped expressing -synuclein ( repression figure 6(b ) ) . this was true irrespective of whether -synuclein expressing cells were plated from either early or late log phase of growth . three days later , both wt and e46k -synuclein cells did gain the original colony sizes initially seen with parent vector cells ( figure 6(b ) ) . we did not observe a reliable difference in colony number between cells that contained parent vector and cells expressing wt or e46k -synuclein ( data not shown ) . in the sp3 strain , where e46k was shown to be more toxic than wt -synuclein , e46k -synuclein expressing cells formed even smaller colonies than wt -synuclein expressing cells when -synuclein was still being expressed ( induction figure 6(c ) ) . unlike in tcp1 , these e46k colonies did not reach the colony size of parent vector cells even after three days of addition growth even though wt colonies did ( figure 6(c ) ) . once again , we did not observe a reliable difference in colony number between cells that contained parent vector and expressing wt or e46k -synuclein ( data not shown ) . in by4741 budding yeast , similar survival assays did not demonstrate any difference in colony size or number between parent vector , wt , and e46k expressing cells under induced conditions at either two or five days of growth ( figure 6(d ) and data not shown ) . the initial difference in colony size of all yeast cells between repressed ( glucose ) and induced ( galactose ) plates was simply due to the slight general lag in yeast growth in galactose compared to glucose . thus , the wt and e46k -synuclein toxicity in tcp1 and the additional e46k selective toxicity in sp3 both correlated well with reduced yeast growth but not colony number . e46k is the most recently discovered of the three -synuclein point mutants that cause familial pd . since its discovery in 2004 , only a handful of mostly in vitro studies we demonstrate significant organismal evidence that may help explain its toxic potential in pd , and we discuss three notable findings below . ( 1 ) e46k extensively associates with membrane systems in both yeasts but does so in distinct ways : binding plasma membrane in budding yeast and endomembranes in fission yeast . ( 2 ) e46k aggregates in fission yeast , but not in budding yeast , in a time- and concentration - dependent manner . ( 3 ) e46k is toxic to fission yeast in a strain - specific manner that correlates with reduced cellular growth , survival , and modest accumulation . in support of our hypothesis , live cell gfp microscopy illustrates that e46k associates with the plasma membrane of budding yeast . thus , this study is the first to demonstrate e46k association with diverse membrane compartments in living cells , supporting its well - documented affinity for membrane phospholipids in vitro . since e46k binds liposomes more readily than wt , a30p , or a53 t , and solution nmr work also suggests that e46k adopts a structure that favors lipid binding more rapidly than these other -synuclein variants [ 29 , 37 ] , these studies provide a molecular explanation for why e46k ( more than wt or a53 t ) strongly associates with endomembrane systems in yeasts . e46k is clearly different from the a30p mutant , which does not associate with membranes within either yeast this study ; [ 45 , 46 ] nor bind lipids in vitro [ 14 , 5153 ] . since e46k enhances n- to c - terminal contacts within -synuclein domains and is within the n - terminal ktkegv amino acid repeats essential for membrane association [ 10 , 30 , 54 ] , our data strengthens the notion that the e46k impact on the n to c - terminal contact likely facilitates a helical conformation , the structural form required for -synuclein to interact with lipids [ 18 , 55 ] . in addition to cytoplasmic localization in neuronal tissue , -synuclein also associates with the secretory pathway [ 56 , 57 ] , localizes to the plasma membrane [ 16 , 5862 ] , and outside cells [ 6165 ] . thus , e46k 's association ( and to a lesser degree , wt and a53 t ) with internal membrane systems in fission yeast cells is noteworthy . thus , e46k -synuclein 's prominent presence in perinuclear compartments suggests that it may associate with er and disrupt er to golgi transport to generate toxicity , just as wt and a53 t -synuclein have been reported to do in budding yeast cells [ 26 , 27 ] in budding yeast , overexpression of the secretory pathway specific g protein rab1 also rescues yeasts from -synuclein - dependent [ 26 , 27 ] . while we do not know why e46k ( and to a lesser extent , wt and a53 t ) binds such distinct membrane compartments between the two yeasts , differences in -synuclein concentration in the two yeasts are unlikely to be a major reason , since e46k never associates significantly with the fission yeast plasma membrane even when expressed at low concentrations . since the two yeast species are separated by almost 400 million years of evolution , inevitable differences in plasma membrane and endomembrane composition are a more likely explanation . we speculate that differences in the plasma and endomembrane composition exist between budding yeast and fission yeast and that e46k -synuclein might have increased affinity for plasma membrane phospholipids in budding yeast and endomembranes in fission yeast . budding and fission yeasts do have modest differences : in phospholipid composition , in fission yeast being an inositol auxotroph , and fission yeast 's lipid composition varies with temperature and growth . specific differences between the yeasts may also exist in the composition of membrane proteins with which -synuclein variants interact . e46k aggregates more rapidly than wt -synuclein in fission yeast , again supporting our hypothesis . that this , e46k aggregation more closely matches a53 t -synuclein is supported by in vitro evidence that e46k and a53 t aggregate at similar rates [ 29 , 30 ] in cell culture , e46k readily forms intracellular aggregates . our work supports the notion that e46k enhances -synuclein aggregation due to its location within the n - terminal ktkegv repeats , which influence aggregation rates . synthetic e(xx)k mutations at these points decreased fibrillization lag time in vitro . rospigliosi et al . also demonstrated that the e46k mutant resulted in n to nac region contact , which , compounded by an overall decrease in negative charge , is hypothesized to increase aggregation rates , providing an explanation for e46k 's impact on aggregation . the absence of e46k aggregation in budding yeast may be due to two factors : the moderate expression of -synuclein in our lab and the membrane composition differences between the two yeasts . in budding and fission yeasts , other labs that express -synuclein at moderate levels also report plasma membrane association [ 42 , 44 ] . e46k 's enhanced toxicity in fission yeast supports our hypothesis that it is more toxic than wt -synuclein even though neither of them is toxic in budding yeast . the lack of -synuclein toxicity in budding yeast strain by4741 could be due to dosage - dependent and strain - specific toxicity , as -synuclein is toxic in the w303 strain with two copies expressed but not with one copy . our fission yeast data indicates that endomembrane association is a key contributor to toxicity although we do not exclude toxicity contributions from interactions with the plasma membrane or cytoplasmic aggregation . one of the most important questions in pd is the identification of the toxic -synuclein species , and protofibrils possess several features in vitro that make them excellent candidates for being this toxic species , including the ability to permeabilize synthetic vesicles . both a30p and a53 t form more protofibrils than wt -synuclein in vitro [ 4 , 11 , 73 ] , but little still is known whether e46k does the same , in vitro or in organisms . we speculate that the differential toxicity ( even within fission yeast strains ) may be due to how much e46k -synuclein protofibrils form within each strain and that endomembrane association may be a significant contributor . the degree and range of e46k association with endomembrane systems may determine how much it contributes to cytotoxicity . even though e46k localizes more to internal organelles in tcp1 than wt -synuclein does , their level of toxicity is similar . we speculate that the added toxicity in sp3 may partly be due to more e46k localization to vacuoles / prevacuoles . thus , disruption of vesicle integrity may mediate toxicity as much as added association with the er . strain differences in lipid composition may also underlie why sp3 differs from tcp1 in the extent of -synuclein association with intracellular organelles . since most tcp1 cells express e46k in aggregates , the few cells that do exhibit endomembrane localization likely produce too little toxicity to be detected by the growth and survival assays . lastly , the difference in toxicity could be due to the h ( tcp1 ) and h+ ( sp3 ) mating - type differences , including altered interactions of -synuclein with secretion of specific mating factors . our data confirms an initial report in budding yeast and extends it to fission yeast that yeasts possess protein kinases that can phosphorylate human -synuclein . we are also the first to report that wt and e46k -synuclein are phosphorylated in a fission yeast model ( for fuller description , see ) . although the extent of serine-129 phosphorylation does not correlate with enhanced e46k -synuclein toxicity in fission yeast , it provided us the opportunity to investigate the contribution of phosphorylation to -synuclein aggregation and membrane association in both models that we report elsewhere . future studies in fission yeast will determine whether -synuclein will disrupt vesicular pool distribution , accumulates in vesicles , and colocalizes with secretory pathway proteins , as it does in budding yeast [ 21 , 27 , 76 ] . it is possible that such -synuclein endomembrane association also impairs -synuclein degradation and toxicity via lysosmal pathways , as has been observed in budding yeast ( [ 47 , 77 ] , perez et al . both yeast models recapitulate and extend our in vitro and cell culture knowledge of e46k -synuclein familial mutant properties in two organismal model systems . together , they illustrate that e46k can both aggregate within cells and associate with its diverse membrane systems . importantly , the organismal context regulates both these features by varying the extent of aggregation and the extent of association with plasma membrane and endomembrane systems . our data indicates that -synuclein binding to endomembranes may be key to generating toxicity that negatively impacts cellular growth and survival . finally , our work further strengthens the relevance of fission yeast , alongside the more widely accepted budding yeast , to serve as a model organism for studying misfolded proteins linked to human neurodegeneration , opening doors for genetic screens and chemical treatments to dissect the role of genetic factors that regulate -synuclein misfolding and toxicity and contribute to deeper understanding of pd pathogenesis . cdnas of wild - type ( wt ) and a53 t -synuclein were provided by christopher ross at johns hopkins university . these -synuclein cdnas were first subcloned into the mammalian expression vector pcdna3.1/c - terminal gfp ( invitrogen ) , fusing -synuclein with gfp at the c - terminus . the -synuclein - gfp cdnas and gfp cdna were then pcr - amplified and subcloned into the pyes2.1/v5-his - topo budding yeast expression vector ( invitrogen ) or pnmt1/v5-his - topo fission yeast vector ( invitrogen ) . the -synuclein - gfp fusion yeast vectors were transformed into chemically competent e. coli cells for replication and storage . the a30p mutant was created using site - directed mutagenesis ( invitrogen ) on wt -synuclein - gfp pyes2.1/v5-his - topo budding yeast expression vector ( invitrogen ) or pnmt1/v5-his - topo as described in sharma et al . and brandis et al . , respectively [ 45 , 46 ] . the e46k mutant was made similarly created with the following primers : e46k fp : 5atgtaggctccaaaaacaagaagggagtggtgc 3 e46k rp : 5cttggttttggagcctacatagagaacacc 3 mutations were sequenced at university of chicago to confirm successful substitution . the parent pyes2.1 vector for budding yeast ( invitrogen ) and parent pnmt1 prep vector ( gift from judy potashkin , rosalind franklin university , ill , usa ) served as controls . parent budding yeast strains by4741 ( mata his31 leu20 met150 ura30 ) , by4742 ( mathis31 leu20 lys20 met150 ura30 ) , and by4743 ( mata/ his31 leu20 lys20 ura30 ) . fission yeast strains tcp1 ( h leu1 - 32 ) and sp3 ( h+ leu1 - 32 ) were provided by invitrogen and judy potashkin ( rosalind franklin university of medicine and science , north chicago , ill , usa ) , respectively . yeast cells were grown on synthetic - complete media lacking uracil ( sc - ura ) . in the pyes2 vector , -synuclein expression was controlled through a tightly regulated galactose - inducible promoter ( gal1 ) . yeast were grown to mid - log phase in sc - ura glucose ( 2% ) or sc - ura raffinose ( 2% ) media at 30c . cells were washed with water and diluted to log - phase ( 5 10 cells / ml ) in sc - ura galactose ( 2% ) media to induce -synuclein expression . s. pombe strains were transformed with pnmt1 vectors using the lithium - acetate transformation method . transformed cells were selected by growth on pombe dropout medium - leucine ( pdm - leu ) containing 10 m thiamine . budding or fission yeast cells at 2.5 10 cells / ml concentration were washed twice with 100 mm nan3 and solubilized in electrophoresis sample buffer ( esb ) . the esb contained 2% sodium dodecyl sulfate ( sds ) , 80 mm tris ( ph 6.8 ) , 10% glycerol , 1.5% dithiothreitol , 1 mg / ml bromophenol blue , and a cocktail of protease inhibitors and solubilizing agents ( 1% triton - x 100 , 1 mm phenylmethylsulfonyl fluoride , 1 mm benzamidine , 1 mm sodium orthovanadate , 0.7 mg / ml pepstatin a , 0.5 mg / ml leupeptin , 10 mg / ml e64 , 2 mg / ml aprotinin , and 2 mg / ml chymostatin ) . lysates were electrophoresed at 130 volts on a 10%20% tris - glycine gel ( invitrogen ) with 1x sds running buffer . gels were transferred to pvdf membranes using a semidry transfer method and probed using the desired antibodies . to detect -synuclein , a mouse monoclonal anti - v5-ap antibody ( invitrogen ) was used at 1 : 2000 . mouse antiphosphoglycerokinase ( pgk ; molecular probes ) was used at 1 : 1000 as a loading control for budding yeast and anti--actin ( abcam ) was used at 1 : 1000 as a loading control for fission yeast . for both serine-129 phosphorylation blots were probed with a rabbit -synuclein ( phospho s129 ) antibody ( ab59264 ; abcam ) at 1 : 500 , followed by a goat antirabbit secondary antibody ( santa cruz biotechnology ) . yeast cells were grown in either 10 ml sc - ura+glucose ( budding yeast ) or emm+t ( fission yeast ) overnight at 30c and 200 rpm . to collect cells they were washed twice with 5 ml h2o , re - suspended in 10 ml h2o , and counted using a hemocytometer to determine cell density . flasks containing 25 ml sc - ura+galactose ( budding yeast ) or emmt ( fission yeast ) were inoculated to a density of 2.0 10 cells / ml . duplicate spectrophotometer 600 nm absorbance measurements of 1 ml of cells in a plastic cuvette were taken at 0 , 3 , 6 , 12 , 18 , 24 , 36 , and 48 hours after - induction . a growth curve was generated by plotting the average absorbance readings of three separate experiments versus time in microsoft excel . yeast cells were grown in either 10 ml sc - ura+glucose ( budding yeast ) or emm+t ( fission yeast ) overnight at 30c and 200 rpm . to collect cells they were washed twice with 5 ml h2o , resuspended in 10 ml h2o , and counted using a hemocytometer to determine cell density . cells were diluted 5 fold in a 96 well microtitier plate and spotted onto sc - ura+glucose and sc - ura+galactose ( budding yeast ) or emm+t and emmt ( fission yeast ) growth plates . cells were grown for 24 hours , and pictures were taken using an hp canoscan scanner . yeast cells were grown in either 10 ml sc - ura+glucose ( budding yeast ) or emm+t ( fission yeast ) overnight at 30c and 200 rpm . to collect cells they were washed twice with 5 ml h2o , resuspended in 10 ml h2o , and counted using a hemocytometer to determine cell density . flasks containing 25 ml sc - ura+galactose ( budding yeast ) or emmt ( fission yeast ) were inoculated to a density of 2.0 10 cells / ml . 1 ml of cell culture was pelleted at 5000 rpm for 1 minute . the remaining 100 ul of cell culture was vortexed , and 510 ul of sample was pipetted onto a glass slide . cells were visualized using a nikon te2000-u fluorescent microscope , and images were collected and quantified using metamorph 4.0 software . all microscopy experiments were conducted twice . for each yeast sample , at each time point ~750 cells of each type were scored for all or some of these localization patterns ( depending on the yeast ) : cytoplasmically diffuse , plasma membrane , aggregate , membrane / diffuse , membrane / aggregate , endomembrane , endomembrane / aggregate , and endomembrane / diffuse ( bottom ) . yeast cells were grown in either 10 ml sc - ura+glucose ( budding yeast ) or emm+t ( fission yeast ) overnight at 30c and 200 rpm . to collect cells , yeast were pelleted at 1500 g for 5 minutes at 4c . they were washed three times with 5 ml h2o , resuspended in 10 ml h2o , and counted using a hemocytometer to determine cell density . flasks containing 25 ml sc - ura+galactose ( budding yeast ) or emmt ( fission yeast ) were inoculated to a density of 2.0 10 cells . at 8 and 18 hours after induction , cell density was determined . 1.8 10 cells were removed and washed once with 1 ml of h2o . the cells were resuspended in 1 ml of water , and diluted 1 : 1000 . 300 l of 1 : 1000 diluted cells ( 550 cells ) were spread on 150 mm plates containing sc - ura+galactose and sc - ura+glucose ( budding yeast ) or emmt and emm+t ( fission yeast ) , for either two days or three additional days at 30c , and images were taken of plates and analyzed for colony number and size .

in parkinson 's disease ( pd ) , midbrain dopaminergic neuronal death is linked to the accumulation of aggregated -synuclein . the familial pd mutant form of -synuclein , e46k , has not been thoroughly evaluated yet in an organismal model system . here , we report that e46k resembled wild - type ( wt ) -synuclein in saccharomyces cerevisiae in that it predominantly localized to the plasma membrane , and it did not induce significant toxicity or accumulation . in contrast , in schizosaccharomyces pombe , e46k did not associate with the plasma membrane . instead , in one strain , it extensively aggregated in the cytoplasm and was as toxic as wt . remarkably , in another strain , e46k extensively associated with the endomembrane system and was more toxic than wt . our studies recapitulate and extend aggregation and phospholipid membrane association properties of e46k previously observed in vitro and cell culture . furthermore , it supports the notion that e46k generates toxicity partly due to increased association with endomembrane systems within cells .

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

PMC3066704

in both the uk and the us , medical education , including assessment , has been transformed over the last 30 years , driven by changes to the medical environment in which graduates will work . there has been a shift away from the traditional apprenticeship model of learning in medicine : the student is no longer considered an empty vessel to be filled by those who have obtained the desired competencies instead it is now widely recognised that medical education is preparation for the multitude of demands of professional practice . traditionally , clinical competence has been assessed by measuring students ' ability to recall and apply knowledge . before they embark on careers as medical professionals , students must demonstrate their competence in terms of professional attitudes and skills as well as knowledge . this means medical schools need to have the tools and understanding to systematically , appropriately and reliably assess the competence of medical students . assessment is a term that refers to a spectrum of activities that take place in the learning environment to measure the learning that has taken place . in its broadest sense it can be considered as about getting to know our students and the quality of their learning . it can be formal , informal , formative or summative , objective or subjective , learning can be measured against peer or against a pre defined criteria and conducted by the learner , his peer or the teacher . every assessment that is provided gives a clear message to the learner about what they should learn and how they should go about that learning . learners can , to some extent , escape from the effects of poor teaching , by creating their own learning strategies and opportunities ; however , if they wish to graduate and practice , they can not escape the effects of inadequate assessment . it is a powerful driver of student learning and so it is important to get it right ( see supplementary material for the qualities of a good assessment ) . libyan doctors and medical students are calling for changes to teaching and assessment methods at both undergraduate and postgraduate levels . in december 2006 staff from university college london 's academic centre for medical education ( acme ) were invited by the libyan board of medical specialties ( lbms ) in tripoli to facilitate the first of a series of one - day workshops on assessment and to initiate the discussions about developing a contemporary assessment strategy . this article outlines the course and the outcomes of preliminary discussions between academics from the uk , participants in the assessment courses and representatives from the libyan board of medical specialties regarding the future of assessment in libyan medical education . the one day course aimed to increase participants ' knowledge of up - to - date assessment theory and methods and to promote discussion about the future for assessment in libyan medical education . the objectives of the course were that by the end of the training day participants would : have explored the definition and purpose of assessmentbe familiar with key concepts in assessmenthave a framework for selecting and using appropriate assessments in their own settinghave considered their role as an assessorbe familiar with the objective structured clinical examination ( osce ) assessment methodhave practiced three contemporary assessment techniques have explored the definition and purpose of assessment be familiar with key concepts in assessment have a framework for selecting and using appropriate assessments in their own setting have considered their role as an assessor be familiar with the objective structured clinical examination ( osce ) assessment method have practiced three contemporary assessment techniques the day consisted of a morning of interactive sessions facilitated by acme academics and practical workshops to develop skills in the afternoon , comprising the following elements : an overview of assessmentselecting appropriate assessmentsappropriate assessor behaviouran introduction to the oscea multiple choice questions ( mcq ) writing workshop . participants practiced writing useful , reliable and valid written examination questionsa mock osce workshop : participants played roles of students and examiners on a 4 station osce . an overview of assessment selecting appropriate assessments appropriate assessor behaviour an introduction to the osce a multiple choice questions ( mcq ) writing workshop . participants practiced writing useful , reliable and valid written examination questions a mock osce workshop : participants played roles of students and examiners on a 4 station osce . the day was rounded off with discussions between uk and libyan academics and medical educations on how assessment should be taken forward in libyan medical education . the group discussed : how assessment should change to fit in with contemporary medical education in libyawhat assessment toolkit might workhow assessors and candidates should be prepared for assessments how assessment should change to fit in with contemporary medical education in libya what assessment toolkit might work how assessors and candidates should be prepared for assessments in both the uk and the us , medical education , including assessment , has been transformed over the last 30 years , driven by changes to the medical environment in which graduates will work . there has been a shift away from the traditional apprenticeship model of learning in medicine : the student is no longer considered an empty vessel to be filled by those who have obtained the desired competencies instead it is now widely recognised that medical education is preparation for the multitude of demands of professional practice . traditionally , clinical competence has been assessed by measuring students ' ability to recall and apply knowledge . before they embark on careers as medical professionals , students must demonstrate their competence in terms of professional attitudes and skills as well as knowledge . this means medical schools need to have the tools and understanding to systematically , appropriately and reliably assess the competence of medical students . assessment is a term that refers to a spectrum of activities that take place in the learning environment to measure the learning that has taken place . in its broadest sense it can be considered as about getting to know our students and the quality of their learning . it can be formal , informal , formative or summative , objective or subjective , learning can be measured against peer or against a pre defined criteria and conducted by the learner , his peer or the teacher . every assessment that is provided gives a clear message to the learner about what they should learn and how they should go about that learning . learners can , to some extent , escape from the effects of poor teaching , by creating their own learning strategies and opportunities ; however , if they wish to graduate and practice , they can not escape the effects of inadequate assessment . it is a powerful driver of student learning and so it is important to get it right ( see supplementary material for the qualities of a good assessment ) . libyan doctors and medical students are calling for changes to teaching and assessment methods at both undergraduate and postgraduate levels . in december 2006 staff from university college london 's academic centre for medical education ( acme ) were invited by the libyan board of medical specialties ( lbms ) in tripoli to facilitate the first of a series of one - day workshops on assessment and to initiate the discussions about developing a contemporary assessment strategy . this article outlines the course and the outcomes of preliminary discussions between academics from the uk , participants in the assessment courses and representatives from the libyan board of medical specialties regarding the future of assessment in libyan medical education . the one day course aimed to increase participants ' knowledge of up - to - date assessment theory and methods and to promote discussion about the future for assessment in libyan medical education . the objectives of the course were that by the end of the training day participants would : have explored the definition and purpose of assessmentbe familiar with key concepts in assessmenthave a framework for selecting and using appropriate assessments in their own settinghave considered their role as an assessorbe familiar with the objective structured clinical examination ( osce ) assessment methodhave practiced three contemporary assessment techniques have explored the definition and purpose of assessment be familiar with key concepts in assessment have a framework for selecting and using appropriate assessments in their own setting have considered their role as an assessor be familiar with the objective structured clinical examination ( osce ) assessment method have practiced three contemporary assessment techniques the day consisted of a morning of interactive sessions facilitated by acme academics and practical workshops to develop skills in the afternoon , comprising the following elements : an overview of assessmentselecting appropriate assessmentsappropriate assessor behaviouran introduction to the oscea multiple choice questions ( mcq ) writing workshop . participants practiced writing useful , reliable and valid written examination questionsa mock osce workshop : participants played roles of students and examiners on a 4 station osce . an overview of assessment selecting appropriate assessments appropriate assessor behaviour an introduction to the osce a multiple choice questions ( mcq ) writing workshop . participants practiced writing useful , reliable and valid written examination questions a mock osce workshop : participants played roles of students and examiners on a 4 station osce . the day was rounded off with discussions between uk and libyan academics and medical educations on how assessment should be taken forward in libyan medical education . the group discussed : how assessment should change to fit in with contemporary medical education in libyawhat assessment toolkit might workhow assessors and candidates should be prepared for assessments how assessment should change to fit in with contemporary medical education in libya what assessment the deans of the libyan medical schools have agreed that there is a need for change in assessment practices . the lmbs endorsed this need for change and a lively discussion produced agreement on the following principles : libya needs to update assessment methodsplanning is key to successful developmentsthose in positions of responsibility for assessment must commit to the process of changeosces should be used throughout medical education ( undergraduate , postgraduate and education of professions allied to medicine)training and support to write and validate mcqs are requireda single comprehensive question bank should be created and used by all medical schools . for each mcq , a standard should be set when it is entered into the bank and stored in the question bank as well , in order to identify hard and easy questionsassessors should be trained in assessment behaviors , particularly to help them behave in an objective and standardized manner . crucially they need to understand the curriculum being tested and need to be able to give effective feedback.candidates should be given opportunities to learn about and practice assessments so that they understand assessments that they are expected to take , and have access to learning tools and resources to prepare themassessments must be fairresources are necessary to implement change on a wide scaleall key assessors must attend the acme assessment coursedespite the challenges to be faced , changes in assessment must be made as soon as possible libya needs to update assessment methods planning is key to successful developments those in positions of responsibility for assessment must commit to the process of change osces should be used throughout medical education ( undergraduate , postgraduate and education of professions allied to medicine ) training and support to write and validate mcqs are required a single comprehensive question bank should be created and used by all medical schools . for each mcq , a standard should be set when it is entered into the bank and stored in the question bank as well , in order to identify hard and easy questions assessors should be trained in assessment behaviors , particularly to help them behave in an objective and standardized manner . crucially they need to understand the curriculum being tested and need to be able to give effective feedback . candidates should be given opportunities to learn about and practice assessments so that they understand assessments that they are expected to take , and have access to learning tools and resources to prepare them assessments must be fair resources are necessary to implement change on a wide scale all key assessors must attend the acme assessment course despite the challenges to be faced , changes in assessment must be made as soon as possible it was agreed that concrete steps need to be taken and that the faculty of libyan medical schools need to identify : the time frame for change and key personnel who will take a lead in the change processresources and requirements to implement these changesa time frame for piloting the osce at one medical school the time frame for change and key personnel who will take a lead in the change process resources and requirements to implement these changes a time frame for piloting the osce at one medical school medical education is changing the world over and it is essential for libyan medical educators to keep abreast of local and international demands that are made of today 's healthcare providers . medical educators have a responsibility to train professionals who are able to provide excellent clinical care . today this means they need to understand and integrate clinical knowledge , skills and attitudes in order to be able to diagnose effectively and perform safely in the context of developing good relationships with patients and colleagues . participants on this pilot course overwhelmingly agreed that there was much to be done and that change needed to happen . participants debated how to take things forward and agreed that libya is ready to make changes to assessment . this will require tools , equipment , time to change , training for all involved and , above all , it needs commitment to change .

assessment is a powerful driver of student learning : it gives a message to learners about what they should be learning , what the learning organisation believes to be important , and how they should go about learning . assessment tools allow measurement of student achievement and thereby give teachers insight into their students ' learning , and enable teachers to make systematic judgements about progress and achievement . it is vital then that assessment tools drive students to learn the right things as well as measure student learning appropriately . any attempts to reform curricula and teaching methods must consider the role of assessment in the learning process.libyan doctors and medical students have been calling for changes to teaching and assessment methods at undergraduate and postgraduate levels . a team from the academic centre for medical education at university college , london have been running workshops in conjunction with the libyan board of medical specialties since 2006 to discuss strategic aims of assessment in medical education in libya for the 21st century and to deliver an assessment skills course to libyan educators . this article outlines the course and the outcomes of preliminary discussions between academics from the uk , participants in the assessment courses and representatives from the libyan board of medical specialties . as a result of these discussions it was agreed by all that libyan medical school assessment methods need updating and , despite significant challenges , changes in assessment must be made as soon as possible . there is a real need for support in both addressing these changes and for practical training for assessors in contemporary assessment methods .

Introduction Current context Assessment course aims and objectives Course content and format of the day Result of the discussion regarding the future of undergraduate assessment in Libya Conclusion

PMC2212485

madin darby canine kidney ( mdck ) cells , stably transfected with the cdna encoding pigr derived from rabbit ( obtained from keith mostov , university of california , san francisco ) , were maintained in mem containing 10% fetal bovine serum ( hyclone , logan , ut ) , 100 m nonessential amino acids , 2 mm glutamine , 20 g / ml gentamicin , and 15 mm hepes ( all from gibco brl , gaithersburg , md ) , and were grown in 5% co2 at 37c . cells were grown to confluence on nitrocellulose filter inserts ( millicell ; millipore , bedford , ma ) ; polarization of the cells was confirmed by the electric potential determined by a millicell ers resistance meter ( millipore ) . sendai virus strain 52 was grown in 10-d - old chicken embryos and the virus was harvested from allantoic fluid after extensive centrifugation ( 18 ) . viral - specific iga and igg antibodies were produced and purified as previously described ( 14 , 16 , 19 , 20 ) . in brief , mice were immunized , twice intraduodenally and once orally by feeding tube with 100 g of whole virus plus 5 g of cholera toxin ( list laboratories , campbell , ca ) . mice were boosted by intravenous injection of 30 g of antigen , and splenocytes of immunized animals were fused with sp2/0 cells as per published protocols ( 14 , 16 , 19 , 20 ) . clones secreting antibody to the specific antigen were selected , subcloned , expanded , and frozen . selected hybridoma cells were injected into balb / c mice primed with 2,6,10,14-tetramethylpentadecane ( pristane ; sigma chemical co. , st . gold particles ( bb international , cardiff , uk ) were dialyzed in pbs before use . antibody to be labeled was dialyzed at a concentration of 1 mg / ml against a 2 mm borax ( sigma chemical co. ) buffer ( ph 7.2 ) and centrifuged at 100,000 g for 1 h. after addition of antibody to the gold , bsa was added to a final concentration of 1% and the gold - antibody slurry was centrifuged at 60,000 g for 1 h. the sediment was washed twice by resuspending in tris - buffered saline ( tbs ) with 1% bsa , ( ph 7.2 ) , followed by recentrifugation . the final pellet was resuspended in tbs/1% bsa/0.1% sodium azide and stored at 4c until used . when confluent , polarized cell monolayers were apically infected with 1030 pfu / cell of sendai virus . 4 h later , ascites containing equivalent elisa titers of iga specific for the viral hemagglutinin neuraminidase ( hn ) protein , igg specific for the viral hn protein , or an irrelevant iga ( mineral oil plasmacytoma line 315 ) were added to the basolateral surface . 24 h after inoculation , cells were fixed in 2% paraformaldehyde for 30 min at 4c and permeabilized by subsequent incubation in 0.25% saponin for 1 h at room temperature . to detect viral hn , monolayers experimentally treated with iga were stained with a primary biotin - conjugated murine mab for 1 h at room temperature and then 20 h at 4c , followed by 5 nm gold - labeled sheep anti - biotin for 1 h at room temperature before embedding ( amersham pharmacia biotech , arlington , il ) ; the primary antibody ( murine igg ) was directed against a different epitope on the viral hn protein than the iga antibody added to the basolateral surface during the experiment . experimentally added iga was detected by staining , initially with the igg fraction of rabbit anti mouse iga for 1 h at room temperature and 20 h at 4c , followed by 15 nm gold - labeled goat anti similarly , detection of viral protein in cells experimentally treated with igg used staining with a murine monoclonal iga directed against a different epitope on the hn protein than the original igg applied basolaterally to the cells . this was followed by treatment with the igg fraction of rabbit anti mouse iga for 1 h at room temperature and 20 h at 4c and 15 nm gold - labeled goat anti the monolayers were then embedded in epon 812 , ultra - thin sectioned , and counterstained with uranyl acetate and lead citrate . sections were then examined with an electron microscope ( zeiss cem 902 ; carl zeiss inc . polarized mdck monolayers were apically infected with 30 pfu / cell and 4 h later incubated with 15 nm gold - labeled iga anti - hn or igg anti - hn or irrelevant iga applied basolaterally . additional control polarized mdck cultures were also infected with 30 pfu / cell apically , but 4 h later were treated basolaterally with unlabeled iga anti - hn or apically with 15 nm gold - labeled iga anti - hn . a total of 100 coded electron micrographs from four separate experiments were subjected to morphometric analysis with computer assistance ( image ii program ; national institutes of health , bethesda , md ; running on a centris 650 ; apple computer , cupertino , ca ) . for each micrograph , the area of the field of view , excluding extracellular area and areas occupied by cell nuclei or grid bars , was determined by digital planimetry ; the cytoplasmic area depicted on each print was then calculated from the calibrated magnification of the particular micrograph ( ranging from 7,000 to 30,000 ) and the print magnification ( calibrated at 2.6 ) . next , the total number of small ( 5-nm ) and large ( 15-nm ) gold particles were enumerated separately in each micrograph ; these values were then divided by the cytoplasmic area in that micrograph to determine total particle density ( expressed as particles / square micrometer ) . finally , the number of small particles within a 225-nm radius of at least one large particle , and the number of large particles within a 225-nm radius of at least one small particle , were enumerated ; again , the density of small particles near to large particles and of large particles near to small particles was calculated by dividing the number of each category of colocalized particle on each print by the cytoplasmic area within that print . the morphometric density data were subjected to two - way analysis of variance , classified according to the incubation conditions of the cells and the particular experiment ; since interexperimental variation was not significant , the data of the four individual experiments were pooled , and a one - way classification ( according to incubation conditions ) is reported . in addition to expressing the morphometric data normalized to cytoplasmic area ( particle densities ) , colocalization was also assessed by the fraction of all antibody label that lies in proximity ( within 225 nm ) to virus label , and vice versa , expressed as percentages of the total particles of the appropriate size in that micrograph . two - way analysis of variance ( classified by experiment and by incubation ) of the percentages indicated no significant difference among experiments ; all four experiments were pooled , and the results of a one - way classification are reported . madin darby canine kidney ( mdck ) cells , stably transfected with the cdna encoding pigr derived from rabbit ( obtained from keith mostov , university of california , san francisco ) , were maintained in mem containing 10% fetal bovine serum ( hyclone , logan , ut ) , 100 m nonessential amino acids , 2 mm glutamine , 20 g / ml gentamicin , and 15 mm hepes ( all from gibco brl , gaithersburg , md ) , and were grown in 5% co2 at 37c . cells were grown to confluence on nitrocellulose filter inserts ( millicell ; millipore , bedford , ma ) ; polarization of the cells was confirmed by the electric potential determined by a millicell ers resistance meter ( millipore ) . sendai virus strain 52 was grown in 10-d - old chicken embryos and the virus was harvested from allantoic fluid after extensive centrifugation ( 18 ) . viral - specific iga and igg antibodies were produced and purified as previously described ( 14 , 16 , 19 , 20 ) . in brief , mice were immunized , twice intraduodenally and once orally by feeding tube with 100 g of whole virus plus 5 g of cholera toxin ( list laboratories , campbell , ca ) . mice were boosted by intravenous injection of 30 g of antigen , and splenocytes of immunized animals were fused with sp2/0 cells as per published protocols ( 14 , 16 , 19 , 20 ) . clones secreting antibody to the specific antigen were selected , subcloned , expanded , and frozen . selected hybridoma cells were injected into balb / c mice primed with 2,6,10,14-tetramethylpentadecane ( pristane ; sigma chemical co. , st . gold particles ( bb international , cardiff , uk ) were dialyzed in pbs before use . antibody to be labeled was dialyzed at a concentration of 1 mg / ml against a 2 mm borax ( sigma chemical co. ) buffer ( ph 7.2 ) and centrifuged at 100,000 g for 1 h. after addition of antibody to the gold , bsa was added to a final concentration of 1% and the gold - antibody slurry was centrifuged at 60,000 g for 1 h. the sediment was washed twice by resuspending in tris - buffered saline ( tbs ) with 1% bsa , ( ph 7.2 ) , followed by recentrifugation . the final pellet was resuspended in tbs/1% bsa/0.1% sodium azide and stored at 4c until used . when confluent , polarized cell monolayers were apically infected with 1030 pfu / cell of sendai virus . 4 h later , ascites containing equivalent elisa titers of iga specific for the viral hemagglutinin neuraminidase ( hn ) protein , igg specific for the viral hn protein , or an irrelevant iga ( mineral oil plasmacytoma line 315 ) were added to the basolateral surface . 24 h after inoculation , cells were fixed in 2% paraformaldehyde for 30 min at 4c and permeabilized by subsequent incubation in 0.25% saponin for 1 h at room temperature . to detect viral hn , monolayers experimentally treated with iga were stained with a primary biotin - conjugated murine mab for 1 h at room temperature and then 20 h at 4c , followed by 5 nm gold - labeled sheep anti - biotin for 1 h at room temperature before embedding ( amersham pharmacia biotech , arlington , il ) ; the primary antibody ( murine igg ) was directed against a different epitope on the viral hn protein than the iga antibody added to the basolateral surface during the experiment . experimentally added iga was detected by staining , initially with the igg fraction of rabbit anti mouse iga for 1 h at room temperature and 20 h at 4c , followed by 15 nm gold - labeled goat anti rabbit igg for 1 h at room temperature ( amersham pharmacia biotech ) . similarly , detection of viral protein in cells experimentally treated with igg used staining with a murine monoclonal iga directed against a different epitope on the hn protein than the original igg applied basolaterally to the cells . mouse iga for 1 h at room temperature and 20 h at 4c and 15 nm gold - labeled goat anti the monolayers were then embedded in epon 812 , ultra - thin sectioned , and counterstained with uranyl acetate and lead citrate . sections were then examined with an electron microscope ( zeiss cem 902 ; carl zeiss inc . polarized mdck monolayers were apically infected with 30 pfu / cell and 4 h later incubated with 15 nm gold - labeled iga anti - hn or igg anti - hn or irrelevant iga applied basolaterally . additional control polarized mdck cultures were also infected with 30 pfu / cell apically , but 4 h later were treated basolaterally with unlabeled iga anti - hn or apically with 15 nm gold - labeled iga anti - hn . a total of 100 coded electron micrographs from four separate experiments were subjected to morphometric analysis with computer assistance ( image ii program ; national institutes of health , bethesda , md ; running on a centris 650 ; apple computer , cupertino , ca ) . for each micrograph , the area of the field of view , excluding extracellular area and areas occupied by cell nuclei or grid bars , was determined by digital planimetry ; the cytoplasmic area depicted on each print was then calculated from the calibrated magnification of the particular micrograph ( ranging from 7,000 to 30,000 ) and the print magnification ( calibrated at 2.6 ) . next , the total number of small ( 5-nm ) and large ( 15-nm ) gold particles were enumerated separately in each micrograph ; these values were then divided by the cytoplasmic area in that micrograph to determine total particle density ( expressed as particles / square micrometer ) . finally , the number of small particles within a 225-nm radius of at least one large particle , and the number of large particles within a 225-nm radius of at least one small particle , were enumerated ; again , the density of small particles near to large particles and of large particles near to small particles was calculated by dividing the number of each category of colocalized particle on each print by the cytoplasmic area within that print . the morphometric density data were subjected to two - way analysis of variance , classified according to the incubation conditions of the cells and the particular experiment ; since interexperimental variation was not significant , the data of the four individual experiments were pooled , and a one - way classification ( according to incubation conditions ) is reported . in addition to expressing the morphometric data normalized to cytoplasmic area ( particle densities ) , colocalization was also assessed by the fraction of all antibody label that lies in proximity ( within 225 nm ) to virus label , and vice versa , expressed as percentages of the total particles of the appropriate size in that micrograph . two - way analysis of variance ( classified by experiment and by incubation ) of the percentages indicated no significant difference among experiments ; all four experiments were pooled , and the results of a one - way classification are reported . although our previous data support an intersection of iga antibody with a viral envelope glycoprotein ( 1417 ) , we here document definitively an intracellular interaction between iga and viral proteins by immunoelectron microscopy ( figs . 13 ) . the hn glycoprotein , which is responsible for adherence of virions to host epithelial cells , is synthesized in infected cells on the rough endoplasmic reticulum , glycosylated , processed through the golgi apparatus , and finally transported to the apical surface , where it is inserted into the host cell membrane in anticipation of virion assembly and budding ( 2123 ) . as shown in fig . 1 , basolateral addition of iga anti - hn ( a ) , but not igg anti - hn ( b ) or irrelevant iga ( c ) , reduces the expression of immunodetectable hn protein on the apical host cell membrane , and virion budding after 24 h. collaterally , there is a reduction in the density of total immunostainable viral hn protein ( table 1 ) and in viral titer in the apical supernatant ( data not shown ) only if iga anti - hn is added to the cells . as seen in fig . 2 , specific iga ( a ) , but not specific igg ( b ) or irrelevant iga ( c ) , colocalizes with viral hn protein within multilamellar membrane - bound inclusions in the cell cytoplasm . indeed , quantitative morphometry reveals a 10-fold higher density of specific iga colocalized with viral protein relative to colocalized specific igg ( table 1 ) . moreover , when expressed as a percentage of total ig label , colocalized specific iga was four times more abundant than colocalized specific igg ( table 1 ) . as these data imply , the total density of detectable intracellular specific iga ( 430 23 particles/100 m ) was more than three times that of specific igg ( 127 83 particles/100 m ) . in contrast , irrelevant iga did not accumulate within infected cells ( total density = 27.7 9.4 particles/100 m ) , and did not colocalize to viral label ( table 1 ) . in parallel experiments using antibodies labeled directly with gold particles before application to the basolateral surface of the cell ( fig . 3 ) , infected cells treated with iga anti - hn demonstrate massive accumulation of gold particles within innumerable membrane - delimited inclusions ( fig . smaller but similar organelles are rarely visible in infected cells treated with irrelevant iga ( fig . 3 b ) , specific igg ( data not shown ) , or no antibody ( data not shown ) . the relative abundance of these structures in cells treated with specific iga indicates that iga anti - hn promotes their formation . these inclusions are not the result of intracellular gold , since they are visible to a similar degree in infected cells treated with unlabeled iga anti - hn , albeit without the gold particles ( fig . 3 c ) . to determine if accumulation of iga results from endocytosis of free antibody or immune complexes formed on the apical cell surface by transported antibody , gold - labeled iga anti - hn was added to the apical supernatant of infected cells . as shown ( fig . 3 d ) , only a few gold particles accumulate within the cells . the large quantities of gold and palisaded bodies seen in fig . 3 a are not visualized , indicating that specific iga is retarded in the infected cell during transcytosis , rather than being subject to significant re - uptake after release into the apical medium . scanning laser confocal microscopy with fluorescent antibodies discloses colocalization of antibody and viral hn protein in essentially all infected cells treated with polymeric iga anti - hn applied basolaterally . the colocalization of specific iga antibody and hn protein is seen only in the apical third of the polarized monolayer ( data not shown ) , suggesting that the multilamellar inclusions arise from apical recycling endosomes ( 2428 ) . colocalization is never observed in uninfected cells , nor in infected cells treated with irrelevant polymeric iga or igg anti - hn . finally , additional studies compared iga mabs directed against the hn viral envelope protein to those against the viral nucleoprotein ( np ) . in contrast to the hn protein , which is synthesized in the endoplasmic reticulum , the synthesis of np occurs on free cytoplasmic ribosomes ( 29 , 30 ) . upon addition to the basolateral surface , iga anti - hn but not iga anti - np colocalizes with the respective viral protein by immunoelectron microscopy ( data not shown ) despite the fact that both iga antibodies undergo effective transcytosis . furthermore , the addition of iga anti - hn reduces viral titers in the apical supernatants from infected monolayers , whereas iga anti - np does not ( data not shown ) . these differences between iga anti - hn and anti - np antibodies are consistent with the different sites of synthesis and processing of the two viral proteins relative to the transcytotic pathway of the iga antibody . the current observations , in conjunction with our prior findings ( 1417 ) , strongly support the hypothesis that during transcytosis , specific iga can complex with some viral proteins within polarized epithelial cells and thereby prevent virion assembly and release . the current model of epithelial transcytosis does not postulate a unique receptor ligand endosomal pathway for the transport of iga from the basolateral to the apical cell surface . rather , the pigr iga complex travels through common endosomal compartments with other recycling proteins that undergo endocytosis ( 2428 ) . iga complex is delivered to early basolateral endosomes , but is later routed to apical recycling endosomes , which are thought to be a key site of protein sorting . thus , apical recycling endosomes are a potential location for iga to intercept viral proteins , a view compatible with our confocal microscopic observations . as demonstrated by these studies , the ability of specific iga to interrupt viral replication depends on the mode of replication of the particular virus in question , and the viral protein that is recognized by the antibody . viral glycoproteins that are synthesized on the rough endoplasmic reticulum and subsequently transported to the apical cell surface are probably most vulnerable to intracellular neutralization . prevention of virion assembly and budding by iga acting intracellularly may potentially forestall cytopathic effects and spare the cell , at least during some viral infections . preservation of the integrity of the mucous membrane in this manner could maintain the epithelial barrier and retard systemic dissemination of viral antigens . thus , the ability of iga antibody to act within epithelial cells would synergistically reinforce its traditional extracellular function in affording humoral antiviral defense . these issues impact upon the strategy to develop mucosal vaccines and argue for continued investigation into humoral immune defense mechanisms in the mucosa . treatment with specific iga , but not specific igg or irrelevant iga , reduces the appearance of viral protein on the cell surface . polarized monolayers of mdck cells in culture well inserts , stably transfected with the pigr derived from rabbit , were infected with sendai virus at 10 pfu / cell . 4 h later , ascites containing equivalent elisa titers of iga specific for the viral hn protein ( clone 37 hn ; a ) , igg specific for the viral hn protein ( clone 20 hn ; b ) , or an irrelevant iga ( mineral oil plasmacytoma line 315 ; c ) were added to the basolateral surface as previously described ( 6 ) . productive viral infection is apparent in cells treated with specific igg ( b ) or irrelevant iga ( c ) , with dense accretions of viral protein in patches on the apical portion of the cytoplasmic membrane ( arrows ) , sometimes forming domed buds containing fibrillar chromatin - like material ( arrowheads ) . note that in a and c , small ( 5 nm ) gold particles label the viral proteins , whereas in b , viral protein is detected by large ( 15 nm ) gold particles . infected cells treated with specific iga ( a ) show little viral protein at the cell surface and no accretions of viral protein or bud formations . colocalization of specific iga and viral protein within the cytoplasm of sendai virus infected cells . in polarized mdck cells infected with sendai virus and treated by basolateral application of iga anti - hn 4 h later ( a , a replicate culture of fig . 1 a ) , there is colocalization of numerous large ( 15 nm ) gold particles labeling iga ( large arrows ) and numerous small ( 5 nm ) gold particles labeling viral protein ( small arrows ) to form multilamellar membrane structures , located deep within the cytosol . in infected cells treated with specific igg ( b ) or irrelevant iga ( c ) , budding virions arising from the cell surface ( indicated by asterisks ) are identified by anti - viral hn protein staining ( 15-nm gold particles in b , large arrow ; 5-nm gold particles in c , small arrow ) . the intracellular inclusions which develop in infected cells treated with specific iga are only rarely identified in cells treated with specific igg , and ig does not frequently colocalize with viral protein . neither infected cells treated with irrelevant iga nor uninfected cells ever contain these structures . specific iga promotes the formation of membrane - delimited inclusions of multiple palisaded layers . polarized mdck cells were infected with 30 pfu / cell and 4 h later were incubated with 15 nm gold - labeled iga anti - hn ( a ) , igg ( data not shown ) , or irrelevant iga ( b ) , applied basolaterally . other ( control ) polarized mdck cells were also infected with 30 pfu / cell but 4 h later were treated basolaterally with unlabeled iga anti - hn ( c ) or apically with 15 nm gold - labeled iga anti - hn ( d ) . cells treated basolaterally with gold - labeled iga anti - hn contain aggregates of numerous gold particles within numerous vesicular structures ( a ) that contain multi - lamellar structures ( a , inset ) . in contrast , although a few gold particles are seen within vesicles in cells treated with igg anti - hn ( data not shown ) or irrelevant iga ( b ) , the massive aggregates of gold associated with multilamellar structures , seen in a , are not visible . infected cells treated with unlabeled iga anti - hn ( c ) demonstrate vesicles similar in appearance to those in cells receiving gold - labeled specific iga ( a ) but without gold particles , indicating that the formation of these structures is not due to the presence of gold . although infected cells apically treated with gold - labeled iga anti - hn exhibit a few gold particles in vesicles ( d ) , large aggregates of gold and inclusions containing lamellae are not visualized , indicating that the initial reaction between iga antibody and viral protein occurs within the cell during transcytosis and not near the cell surface after release into the apical supernatant upon subsequent re - uptake . bars : a , 1 m ; inset to a , 0.25 m ; b d , 0.5 m . morphometric assessment of colocalization of ig and viral hn protein significantly ( f > 4.8 , p < 0.002 ) higher than all other groups . significantly ( f = 6.4 , p < 0.001 ) higher than infected cells treated with specific iga and higher than both groups of uninfected cells . significantly ( f > 4.8 , p < 0.002 ) lower than infected cells treated with specific iga and higher than all other groups .

immunoglobulin ( ig)a provides the initial immune barrier to viruses at mucosal surfaces . specific iga interrupts viral replication in polarized epithelium during receptor - mediated transport , probably by binding to newly synthesized viral proteins . here , we demonstrate by immunoelectron microscopy that specific iga monoclonal antibodies ( mabs ) accumulate within sendai virus infected polarized cell monolayers and colocalize with the hemagglutinin neuraminidase ( hn ) viral protein in a novel intracellular structure . neither igg specific for hn nor irrelevant iga mabs colocalize with viral protein . treatment of cultures with viral - specific iga but not with viral - specific igg or irrelevant iga decreases viral titers . these observations provide definitive ultrastructural evidence of a subcellular compartment in which specific iga and viral envelope proteins interact , further strengthening our hypothesis of intracellular neutralization of virus by specific iga antibodies . our results have important implications for intracellular protein trafficking , viral replication , and viral vaccine development .

Materials and Methods Cell and Virus Culture. mAbs. Gold Labeling of Antibodies. Incubations and Immunoelectron Microscopy. Morphometric Analysis of Cytoplasmic Viral Protein and Antibody, and Assessment of Colocalization. Results and Discussion Figures and Tables

PMC3517956

the performance of definitive cements and the longevity of a restoration are affected by preparation coarseness , and the type of provisional cement , and cleaning technique used to remove provisional cement remnants.1 due to the negative effect that residual provisional cement , and debris on prepared abutment teeth may have on the performance of definitive cement,2 all remnants must be removed prior to definitive cementation.1 mechanical methods for removing provisional cement remnants include the use of a dental explorer , pumice or probes : however , none of these methods are fully effective ; terata3 showed that the removal of provisional cement from bovine enamel and dentin with an explorer was incomplete . cement remnants have been observed microscopically on surfaces that appeared macroscopically clean , thus provoking a search for alternative methods for eliminating provisional cement remnants.4 for removal of debris and remnants from the dentin surface dentin surface , different cleaning agents containing ethanol , ethyl acetate , acetone , or chlorhexidine digluconate have been marketed . kanakuri et al.5 reported that the use of a rotational brush with running water was the best method . button et al.6 reported that higher retentive strengths for glass ionomer and zinc polycarboxylate cements were obtained with tooth preparations cleansed with plain flour pumice than those cleansed with an explorer only . bachmann et al.7 investigated the bond strength of dentin bonding agents after teeth were cleaned with a scaler , a cotton pellet with pumice , and different soaps . the authors reported that the use of soap to remove remnants of provisional cements prior to adhesive cementation was not recommended for clinical use . kanakuri et al.5 reported that the use of a rotational brush with running water was the best method . for removal of debris and remnants from the dentin surface , different cleaning agents containing ethanol , ethyl acetate , acetone , or chlorhexidine digluconate have been marketed . laser devices have been used in dentistry for soft - tissue surgery , root - end sealing and sterilization , and for altering enamel and dentin surfaces , in order to increase resistance to decay or to facilitate the bonding of composites.8 because laser etching is painless and involves neither heat nor vibration , this method is attractive to patients as well as clinicians . studies have been performed using different kinds of lasers , including er : yag , nd : yag , and co2 lasers with attention focused particularly on er : yag lasers , due to their effectiveness in removing dental hard tissue.9 however , the literature contains no information about structural changes in dentin following the use of an er : yag laser to clean temporary cement remnants from dentin surfaces . therefore , the purpose of this in vitro aimed to measure and compare the sbs values of ceramic discs to dentin surfaces cleaned with a dental explorer , pumice , cleaning bur and er : yag laser . the hypothesis tested was that the sbs would be higher after er : yag laser dentin cleaning with a dental explorer , pumice , or cleaning bur . a total of , 36 caries - free unrestored human third - molar teeth that had been stored in a 0.5% cloramin t solution at 4 for up to 1 month following extraction were selected as tooth specimens . soft tissue was removed with a scaler ( h6/h7 , hu - friedy , chicago , usa ) . after cleaning the teeth with pumice and then each tooth was embedded in autopolymerizing acrylic resin ( palapress , heraeus kulzer , wehrheim , germany ) using a cylindrical plastic mold ( 20 20 mm ) . the occlusal third of each crown was sectioned with a slow - speed diamond section ( minitom , struers gmbh nederland ) under water cooling and the exposed dentin surfaces were ground with 320-grit silicon - carbide abrasive paper under running water to ensure a smooth surface . provisional restorations were simulated by pouring an acrylic medium ( dentalon plus , wehrheim , germany ) in a teflon mold to produce thirty - six 10 2 mm discs . the acrylic was prepared and polymerized according to the manufacturer 's instructions . following polymerization , discs were removed from the mold , examined for size and air bubbles , and then cemented to the dentin surfaces of tooth specimens using eugenol - free provisional cement ( cavex , haarlem , holland ) and a special loading device ( algol instrument co , hsin - chuang , taipei , taiwan ) under a load of 9.2 n. all specimens were stored in distilled water at 37 2 for 2 days before use . specimens were randomly assigned to 1 of 4 groups different dentin cleaning protocols ( n = 9 ) , as follows : group 1 ( control ) : provisional cements were mechanically removed with a dental explorer using moderate pressure until the dentin surface was macroscopically clean . group 2 : provisional cement was removed from dentin surfaces by cleaning with a brush and pumice for 1 min using a rotary instrument at 5,000 rotations / min under water cooling . group 3 : provisional cement was removed from dentin surfaces by cleaning with a bur ( opticlean , kerr , california , usa ) for 1 min using a rotary instrument at 5,000 rotations / min under water cooling . group 4 : provisional cement was removed from dentin surfaces by cleaning with an er : yag laser ( fidelis plus 3 , fotona , ljubljana , slovenia ) under an air water spray at 200 mj , 20 hz a pulse duration of 100 s , tip diameter of 800 nm and a working distance of 0.5 mm between tip and dentin surface until the dentin surface was macroscopically clean . thirty - six 7 3 mm pressed lithium disilicate glass - ceramic discs ( ivoclar - vivadent ag , schaan , liechtenstein ) were fabricated by the lost wax technique and ingots were injected into the furnace ( ep 600 , ivoclar - vivadent , schaan , liechtenstein ) . the surfaces of the discs were ground with 220 , 360 , and 600 grit silicon - carbide sandpaper to standardize the bonding surface . ground ceramic discs were air - abraded with 50 m al2o3 particles ( korox , bego , bremen , germany ) for 14 seconds from a distance of approximately 10 mm at 400 kpa with a sand - blasting device ( ar - ge dental , denizli , turkey ) . the discs were then cleaned in distilled water for 10 min in an ultrasonic bath ( healthsonics , livermore , ca , usa ) to ensure a contaminant - free surface . self - adhesive luting cement ( sa cement , kuraray , tokyo , japan ) was used to bond the ceramic discs to the dentin surfaces . cement was mixed and then applied to ceramic and dentin surfaces with no primer or adhesive . a special loading device was designed to apply a constant load of 9.2 n to the ceramic discs ( fig . 1 ) . this load was used to create a uniform resin luting layer of approximately 100 m to simulate the range of film thickness for all ceramic crowns . initial light curing ( light intensity of - 1000 mw / cm , elipar freelight led 2 , 3 m espe , mn , usa ) was performed for 10 seconds . the luting agent was polymerized from each direction ( mesial , distal , buccal , lingual , and occlusal ) with the same curing device for 40 seconds . after cementation , the specimens were removed from the alignment device and stored in deionized water and thermocycled for 5,000 cycles between 5 2 and 55 2 , with a dwell time of 20 seconds and a transfer time of 5 seconds . finally , luted specimens were placed in a jig for sbs testing , as described under iso / tr 11405 . they were loaded to failure with a crosshead speed of 0.05 mm / min until decementation occurred , using a universal testing machine ( instron , canton , ma , usa ) . sbs values were calculated and recorded using the following formula10 : ( p = maximum force ( n ) and a = interfacial area ( mm ) ) . after sbs testing , the dentin surfaces of the de - bonded specimens were examined with a stereomicroscope ( wild m3b ; heerbrugg , switzerland ) at 25 magnification to identify the mode of bond failure . the failure mode was classified according to one of three types : adhesive failure at interface ( ad ) , cohesive fracture of resin cement ( co ) , or mixed fracture at the interface of resin cement ( mi ) . two specimens were randomly selected from each group and prepared for sem analysis after the sbs test . the de - bonded specimens from each group were gold sputter - coated ( bal - tec scd 050 sputter coater ; bal - tec ag , liechtenstein ) and observed by sem ( leo 440 , leica - zeiss , cambridge , uk ) . all data sets were subjected to normality tests using the kolmogorov smirnov method ; data are presented as medians and 25 and 75 percentiles ( for skewed data ) . one - way anova and tukey hsd tests were used to perform multiple comparisons with a level of p>.05 significance level . all analyses were performed with the statistical package for scientists ( sigmastat , aspire software international , wa , usa ) windows version 3.10 . a total of , 36 caries - free unrestored human third - molar teeth that had been stored in a 0.5% cloramin t solution at 4 for up to 1 month following extraction were selected as tooth specimens . soft tissue was removed with a scaler ( h6/h7 , hu - friedy , chicago , usa ) . after cleaning the teeth with pumice and then each tooth was embedded in autopolymerizing acrylic resin ( palapress , heraeus kulzer , wehrheim , germany ) using a cylindrical plastic mold ( 20 20 mm ) . the occlusal third of each crown was sectioned with a slow - speed diamond section ( minitom , struers gmbh nederland ) under water cooling and the exposed dentin surfaces were ground with 320-grit silicon - carbide abrasive paper under running water to ensure a smooth surface . provisional restorations were simulated by pouring an acrylic medium ( dentalon plus , wehrheim , germany ) in a teflon mold to produce thirty - six 10 2 mm discs . following polymerization , discs were removed from the mold , examined for size and air bubbles , and then cemented to the dentin surfaces of tooth specimens using eugenol - free provisional cement ( cavex , haarlem , holland ) and a special loading device ( algol instrument co , hsin - chuang , taipei , taiwan ) under a load of 9.2 n. all specimens were stored in distilled water at 37 2 for 2 days before use . specimens were randomly assigned to 1 of 4 groups different dentin cleaning protocols ( n = 9 ) , as follows : group 1 ( control ) : provisional cements were mechanically removed with a dental explorer using moderate pressure until the dentin surface was macroscopically clean . group 2 : provisional cement was removed from dentin surfaces by cleaning with a brush and pumice for 1 min using a rotary instrument at 5,000 rotations / min under water cooling . group 3 : provisional cement was removed from dentin surfaces by cleaning with a bur ( opticlean , kerr , california , usa ) for 1 min using a rotary instrument at 5,000 rotations / min under water cooling . group 4 : provisional cement was removed from dentin surfaces by cleaning with an er : yag laser ( fidelis plus 3 , fotona , ljubljana , slovenia ) under an air water spray at 200 mj , 20 hz a pulse duration of 100 s , tip diameter of 800 nm and a working distance of 0.5 mm between tip and dentin surface until the dentin surface was macroscopically clean . thirty - six 7 3 mm pressed lithium disilicate glass - ceramic discs ( ivoclar - vivadent ag , schaan , liechtenstein ) were fabricated by the lost wax technique and ingots were injected into the furnace ( ep 600 , ivoclar - vivadent , schaan , liechtenstein ) . the surfaces of the discs were ground with 220 , 360 , and 600 grit silicon - carbide sandpaper to standardize the bonding surface . ground ceramic discs were air - abraded with 50 m al2o3 particles ( korox , bego , bremen , germany ) for 14 seconds from a distance of approximately 10 mm at 400 kpa with a sand - blasting device ( ar - ge dental , denizli , turkey ) . the discs were then cleaned in distilled water for 10 min in an ultrasonic bath ( healthsonics , livermore , ca , usa ) to ensure a contaminant - free surface . self - adhesive luting cement ( sa cement , kuraray , tokyo , japan ) was used to bond the ceramic discs to the dentin surfaces . cement was mixed and then applied to ceramic and dentin surfaces with no primer or adhesive . a special loading device was designed to apply a constant load of 9.2 n to the ceramic discs ( fig . 1 ) . this load was used to create a uniform resin luting layer of approximately 100 m to simulate the range of film thickness for all ceramic crowns . initial light curing ( light intensity of - 1000 mw / cm , elipar freelight led 2 , 3 m espe , mn , usa ) was performed for 10 seconds . the luting agent was polymerized from each direction ( mesial , distal , buccal , lingual , and occlusal ) with the same curing device for 40 seconds . after cementation , the specimens were removed from the alignment device and stored in deionized water and thermocycled for 5,000 cycles between 5 2 and 55 2 , with a dwell time of 20 seconds and a transfer time of 5 seconds . finally , luted specimens were placed in a jig for sbs testing , as described under iso / tr 11405 . they were loaded to failure with a crosshead speed of 0.05 mm / min until decementation occurred , using a universal testing machine ( instron , canton , ma , usa ) . sbs values were calculated and recorded using the following formula10 : ( p = maximum force ( n ) and a = interfacial area ( mm ) ) . after sbs testing , the dentin surfaces of the de - bonded specimens were examined with a stereomicroscope ( wild m3b ; heerbrugg , switzerland ) at 25 magnification to identify the mode of bond failure . the failure mode was classified according to one of three types : adhesive failure at interface ( ad ) , cohesive fracture of resin cement ( co ) , or mixed fracture at the interface of resin cement ( mi ) . two specimens were randomly selected from each group and prepared for sem analysis after the sbs test . the de - bonded specimens from each group were gold sputter - coated ( bal - tec scd 050 sputter coater ; bal - tec ag , liechtenstein ) and observed by sem ( leo 440 , leica - zeiss , cambridge , uk ) . all data sets were subjected to normality tests using the kolmogorov smirnov method ; data are presented as medians and 25 and 75 percentiles ( for skewed data ) . one - way anova and tukey hsd tests were used to perform multiple comparisons with a level of p>.05 significance level . all analyses were performed with the statistical package for scientists ( sigmastat , aspire software international , wa , usa ) windows version 3.10 . the mean sbs values and the results of one - way anova are shown in fig . 2 . the dentin cleaning methods did not significantly affect the sbs of resin cement to dentin ( p>.05 ) : dental explorer ( 2.04 0.78 mpa ) , pumice ( 3.30 1.49 mpa ) , cleaning bur ( 2.60 1.50 mpa ) , and laser ( 2.59 0.92 mpa ) . however , the specimens in the groups cleaned with the dental explorer or laser showed mixed failures . on the dentin surfaces of the specimens cleaned with the dental explorer or cleaning bur , the dentin surfaces of the specimens cleaned with a brush and pumice showed tubule obliteration . laser irradiation at 200 mj with a pulse repetition rate of 20 hz provided the cleanest dentin surface , with open dentin tubules and the absence of cracked areas ( fig . , mix fractures observed resin cement debris was observed on all dentin surfaces ( fig . 3 ) except the group cleaned with the dental explorer , which had smooth and clean surfaces ( fig . however , the specimens in the groups cleaned with the dental explorer or laser showed mixed failures . on the dentin surfaces of the specimens cleaned with the dental explorer or cleaning bur , significantly more temporary cement remnants were found versus the other groups . the dentin surfaces of the specimens cleaned with a brush and pumice showed tubule obliteration . laser irradiation at 200 mj with a pulse repetition rate of 20 hz provided the cleanest dentin surface , with open dentin tubules and the absence of cracked areas ( fig . , mix fractures observed resin cement debris was observed on all dentin surfaces ( fig . 3 ) except the group cleaned with the dental explorer , which had smooth and clean surfaces ( fig . a durable and predictable bond strength between dental materials and teeth is important for clinical success.11 one problem associated with ill - fitting restorations is related to remnants of temporary cement on abutment surfaces.5 this is the first study to report on the use of the er : yag laser as a cleaning method . the results did not support the research hypothesis that the sbs between resin cement and dentin would be higher after cleaning with an er : yag laser when compared to cleaning with a dental explorer , pumice , or cleaning bur . carvalho et al.12 showed that application of eugenol - containing provisional cement decreased the bond strength between resin cement and dentin . some authors have indicated that eugenol - containing materials have adverse effects , including altered wettability and reactivity of dentin.1 for this reason , in the current study eugenol - free temporary cement was used as the temporary cement . yap et al.13 observed elevated bond strength and resin infiltration to dentin after mechanical removal of remnants using an ultrasonic scaler followed by cleaning of dentin surfaces with a pumice - water slurry . however , in the present study , sem observation and sbs tests results gave contradictory findings . sem showed that a dental explorer alone was unable to remove all temporary cement from the dentin surfaces and that further cleaning was necessary . some cleaning protocols have been found to alter dentin surface texture.14 however , reducing roughness is not the only factor in improving bond strength . factors such as the chemical composition of the dentin surface may have a stronger influence on adhesion , and taken together with surface roughness and physical parameters such as capillary action will be decisive for the dentin surface free energy and consequently adhesive diffusion into the deminerilized dentin surface.3 sem observation following sbs testing in the present study showed that while laser cleaning produced effective dentin surface microstructures ( fig . 3d ) , resin cement particles were not present to the extent expected ( fig . 4d ) . while laser application may have certain advantages , the use of an er : yag laser instead of conventional techniques for cleaning provisional cement was not found to significantly improve sbs . furthermore , er : yag lasers are still too expensive to be cost - effective.8 according to a previous study , the use of cleaning bur also removes the dentin that is in direct contact with temporary cement , making this a reliable method for removing temporary cement , especially in inaccessible areas.3 the current study has a number of limitationsthat only one type of temporary cement and adhesive resin cement were used and that the tests were performed under in vitro conditions . differences in cleaning protocols did not affect the sbs between dentin and ceramic surfaces . the use of a cleaning bur to remove temporary cement may be the most effective cleaning method .

purposethe purpose of this study was to evaluate the effect of provisional cement removal by different dentin cleaning protocols ( dental explorer , pumice , cleaning bur , er : yag laser ) on the shear bond strength between ceramic and dentin.materials and methodsin total , 36 caries - free unrestored human third molars were selected as tooth specimens . provisional restorations were fabricated and cemented with eugenol - free provisional cement . then , disc - shaped ceramic specimens were fabricated and randomly assigned to four groups of dentin cleaning protocols ( n = 9 ) . group 1 ( control ) : provisional cements were mechanically removed with a dental explorer . group 2 : the dentin surfaces were treated with a cleaning brush with pumice group 3 : the dentin surfaces were treated with a cleaning bur . group 4 : the provisional cements were removed by an er : yag laser . self - adhesive luting cement was used to bond ceramic discs to dentin surfaces . shear bond strength ( sbs ) was measured using a universal testing machine at a 0.05 mm / min crosshead speed . the data were analyzed using a kolmogorov smirnov , one - way anova and tukey hsd tests to perform multiple comparisons ( =0.05).resultsthe dentin cleaning methods did not significantly affect the sbs of ceramic discs to dentin as follows : dental explorer , pumice , cleaning bur , and er : yag laser.conclusionthe use of different cleaning protocols did not affect the sbs between dentin and ceramic surfaces .

INTRODUCTION MATERIALS AND METHODS Tooth preparation Cementation of provisional restorations Experimental design Ceramic disc preparation Luting procedures Shear bond strength (SBS) test Stereomicroscope and scanning electron microscope (SEM) evaluation Statistical analyses RESULTS Fracture patterns SEM observation DISCUSSION CONCLUSION

PMC3658219

a 57-year - old caucasian male was admitted with suspected acute coronary syndrome , primarily because of pronounced st segment elevations in the precordial ekg leads ( figure 1 ) . the day before admission he experienced two episodes of chest pain while walking . the pain was dull , at a maximum intensity 5/10 on the visual analogue scale , located to the left side of the sternum , and did not radiate elsewhere , but was accompanied by dyspnea and lightheadedness . on both occasions the patient sought medical assistance the next day upon his wife s insistence , because he had reportedly suffered an anterior wall mi in 1987 and again in 1999 , but had never been hospitalized in the acute phase . both times , he completed an outpatient program of cardiac rehabilitation , and had good exercising capacity ( 10 met ) that allowed him to work up until a few years ago . he had a severe kyphoscoliotic deformation of the spine and chest due to an injury in childhood . three years ago he had been diagnosed with syderopenic anemia because of persistent , low - grade blood loss due to gastroesophageal reflux disease , documented by endoscopy , and bleeding hemorrhoids . his medication consisted of metoprolol 50 mg twice a day , and oral iron supplements . his blood pressure was 125/70 mmhg , with a respiratory rate of 24/min , and a regular heart rate of 58/min . the thorax was severely kyphoscoliotic with a right - sided thoracic spine convexity ( figure 2 ) , but lung sounds were normal . the apical impulse of the heart was not palpable because of the chest wall deformity . heart sounds were of low intensity and a faint precordial systolic murmur radiating towards the axilla was heard . the cardiac troponin i was normal both in the emergency room ( 0.006 g / l ) and 6 hours later ( 0.008 g / l ) . also , the n - terminal - pro - b - type natriuretic peptide ( 412 ng / l ) was normal . g / l with a mean corpuscular volume of 77 10/l and a mean corpuscular hemoglobin of 27 10/g . ekg results are shown in figure 1 . in the patient s past medical records , deep precordial q waves and st segment elevation were noted in 1987 as having occurred at which point he was told he had suffered a mi . an old ekg record was obtained from 1995 showing very similar findings as upon the current admission , thus ruling out dynamic ekg changes . there were no changes in the ekg during the hospitalization and no rhythm disturbances were recorded . there were no signs of impaired contraction of the anterior wall , but a severely thickened interventricular septum was found . the systolic function of the left ventricle was normal with an ejection fraction of 0.60 . a moderate mitral valve insufficiency was noted due to degenerative changes of the mitral valve leaflets , and only a mild transaortic gradient of up to 25 mmhg was noted . magnetic resonance imaging of the heart ( gradient echo , cine technique ) confirmed that the patient had hcm ( figure 3 ) . the thickness of the interventricular septum was 3.9 cm in systole and 3.5 cm in diastole . no significant dynamic outflow obstruction was found in the supine position , since the outflow tract remained at least 2 cm wide during systole . myocardial perfusion scintigraphy with dipyridamole - induced stress did not show reversible myocardial ischemia or irreversible ischemia , ie , myocardial scarring ( figure 4 ) . no provocation tests with acetylcholine or hyperventilation were carried out to specifically rule out vasospastic angina , but the absence of ekg dynamics did not suggest that it was the cause of the patient s chest pain . immediately upon admission , while still under observation for suspected acute coronary syndrome , the patient received a transfusion of two units of packed red cells , raising his hemoglobin level to 116 g / l . oral iron and a beta - blocker were continued , a proton - pump inhibitor ( omeprazole ) was started due to gastroesophageal reflux , and an angiotensin - converting enzyme inhibitor ( perindopril ) was prescribed because of mitral insufficiency . although hypertrophic cardiomyopathy may cause chest pain and dyspnea , we had no proof of regional septal ischemia during dipyridamole - induced stress . therefore we presumed the chest pain to be noncardiac in origin , while the accompanying dyspnea and lightheadedness was thought to be due to hyperventilation caused by anxiety . the patient was discharged on the 5th day of hospitalization and scheduled for follow up by a cardiologist . however , in addition to the possibility of acute anterior wall mi , the list of differential diagnoses is quite extensive,7 and should include hcm ( table 1 ) . the hallmark of hcm is myocardial hypertrophy that is often asymmetric , and occurs in the absence of an obvious stimulus.8 although any region of the left ventricle can be affected , hypertrophy frequently involves the interventricular septum , which may result in obstruction of the left ventricular outflow tract.8 hcm is a genetically heterogeneous disease that may be inherited or may arise from a spontaneous mutation . over 300 dominant mutations in genes on several different chromosomes , encoding various components of the myocardial contractile system have been reported to cause hcm.9 patients with hcm may be asymptomatic , or may present with symptoms such as dyspnea , presyncope / syncope , chest pain , palpitations , orthopnea , paroxysmal nocturnal dyspnea , congestive heart failure , arrhythmias , or sudden cardiac death . abnormal q waves are often associated with hcm1011 especially in younger patients , where they have a 50% sensitivity and > 90% specificity for the diagnosis of hcm.1011 although st and t wave abnormalities are often present , most often in the context of left ventricular hypertrophy , persistent st segment elevation is a rare finding , described only in individual patients with hcm . as late as 2000 , welsh and tymchak thought they were the first to describe persistent st elevation in a man with hcm.4 in fact , the phenomenon of reciprocal changes in the precordial ekg leads , reflecting hypertrophy of the interventricular septum , was described by braudo et al as early as 196412 and was reviewed by goldberger in 1979 in the context of explaining st segment elevation,1 but no explanation has been given for why the finding is so rare . in our patient , the kyphoscoliotic deformation of the chest positioned the severely hypertrophied interventricular septum nearly parallel to the left ventricular free wall , which was of normal thickness , and the deformed left side of the chest wall ( figure 2 ) , which allowed for maximal expression of the reciprocal ekg changes . our patient s ekg reading from lead v4 showed a classical ventricular hypertrophy pattern when inverted by 180 ( figure 5 ) . the patient with hcm and anterior lead st elevation , described by welsh and tymchak , was morbidly obese,4 which likely brought the heart into a horizontal position and also allowed for notable reciprocal ekg expression of the septal hypertrophy . in conclusion , we hypothesize that st segment elevation is most strongly manifested in those patients with hcm where the hypertrophied septum and left ventricular free wall , of normal thickness , are positioned in parallel with the left side of the chest wall , allowing for best reciprocal ekg expression of the septal hypertrophy in anterior leads . thus , we believe ekg findings should be interpreted keeping in mind the chest wall shape of the patient .

a 57-year - old male was admitted with suspected acute coronary syndrome . he reported experiencing moderate chest pain when walking during the day prior to admission , but had very prominent st segment elevations in the precordial electrocardiography ( ekg ) leads . a physical examination revealed remarkable severe kyphoscoliosis with chest deformity . the patient s cardiac troponin levels remained normal , while cardiac ultrasound and magnetic resonance imaging of the chest confirmed hypertrophic cardiomyopathy ( hcm ) with severe thickening of the interventricular septum . ischemic heart disease was ruled out by myocardial perfusion imaging with 99mtc - mibi during rest and dipyridamole - induced stress without showing irreversible or reversible myocardial ischemia . our diagnosis was that the chest pain was noncardiac in origin and that the pronounced st segment elevations in the precordial ekg leads reflected the severely hypertrophic interventricular septum through the normally thick left ventricular free wall . the patient s chest wall deformity brought his septum and the ventricular free wall nearly parallel to the left side of the chest wall , allowing for complete expression of the reciprocal ekg pattern of septal hypertrophy . we suggest that ekg findings should always be interpreted with the chest wall shape being kept in mind .

Case report Discussion

PMC3302021

traditional laboratory - based cancer research involves expensive trial and error experimental strategies applied to humans , animals , and their harvested tissues . in silico experimentation , the coupling of current computing technologies with mathematical or theoretical characterizations of cancer cell biology , provides a novel approach to guiding the early stages of hypothesis development and experimental design that has the potential to create subsequent efficiencies and cost savings in the laboratory . this computational approach is advantageous because it allows vast numbers of experiments to be carried out that are easily observed at any desired level of detail and can be repeated and controlled at will . such studies involving in vitro and in vivo animal experiments involve hypothesis generation and testing to determine whether further trials are warranted and are extremely costly both in terms of researchers ' time and the associated financial investment . costs , such as laboratory setup , equipment and space , time spent by academics training others , and the time , equipment , and materials costs involved in repetitive , hands - on experimental work , all contribute to the expense of laboratory - based experimental research . our contention in this paper , a view shared by many researchers in the closely related fields of computational , theoretical and mathematical biology , is that in silico experiments can be used as precursors to , or in combination with , preclinical experimental studies to provide guidance for the development of more refined hypotheses and experimental studies . in silico and mathematical modeling lends itself to the determination of preliminary information such as toxicity , pharmacokinetics , and efficacy , which can then be used to guide preclinical and clinical studies . in silico experimentation involves the combination of biological data and expert opinion with mathematical and computer - based representations to construct models of biology . computer - based experiments can then be carried out using these models rather than , or in combination with , laboratory research . using parameter distributions based on current expert opinion ( fuzzy inputs ) or actual biological data ( random variables ) as inputs into the in silico models , it is possible to create what are effectively computational patients upon which to experiment . it is of course also possible to consider smaller - scale experiments and even multiscale experiments , conducted on molecular , cellular , and tissue / organ levels . appropriate use of in silico models involves making predictions based on experimental data and expert information and allows the models to be effectively used to inform clinical trials with a view to reducing costs and increasing efficiency . to provide an example , consider the study of cell transfer therapy for metastatic melanoma patients of rosenberg et al . . the authors commented on the difficulty of deriving meaningful results from human experiments because of the variations in cell types , tumor types , immune states , and more fundamentally the human subjects themselves . while rosenberg et al . suggest a solution to such a problem is to treat the same patient in differing ways over a period of time , another more ethical and flexible , and less hazardous method is through the use of in silico models and experimentation . there is a rich history of theoretical studies involving mathematical and computational approaches to studying cancer . burton and greenspan pioneered the mathematical modeling of tumor growth with models of growth dynamics explained as a problem of diffusion [ 25 ] . since that time , theoretical studies of most aspects of tumor growth and related processes have been investigated at least to some extent , using various different methodologies including differential equations , stochastic models , and cellular automata . araujo and mcelwain provide an excellent review of the mathematical modeling work carried out up to middle of the last decade . more recently , alarcn et al . , mallet and coworkers [ 8 , 9 ] , and ferreira et al . [ 10 , 11 ] have used a new paradigm that of spatiotemporal , stochastic models using hybrid cellular automata techniques to represent computational patients or in silico experiments in a new direction for cancer research . this experimental paradigm extends the traditional mathematical modeling of cancer to incorporate computational simulations that are parameterized in such a way to represent different patients or different experiments . it is also becoming more common to find mathematical studies appearing in the cancer literature . utley et al . , for example , discuss improvement in survival rates resulting from postoperative chemotherapy for lung cancer patients . they note that the marginal ( 5% ) survival rate improvement due to chemotherapy may be outweighed for some patients by the morbidity due to the treatment and that further trials do not actually improve information provided to patients , but rather improve the certainty of that prediction . propose the use of a mathematical model , utilizing patient - specific pathological cancer stage data combined with existing techniques , to arrive at better evidence for informing patients regarding their postoperative treatment choices . in a study more at the preclinical stage of research , de pillis et al . describe a differential equation - based model for the interactions between a growing tumor , natural killer cells , and cd8 t cells of the host immune system . with a view to understanding how the immune system assists in rejecting growing tumors , de pillis et al . present mathematical descriptions of key mechanisms in the immune response before fitting the model to data from published mouse and human studies . a parameter sensitivity analysis reveals the key role of a patient - specific variable and that the model may in fact provide a means to predict positive response of particular patients to treatment . mallet and de pillis and later de pillis et al . explored a particular type of in silico model known as a hybrid cellular automata - partial differential equation ( ca - pde ) model to describe the interactions between a growing tumor and the host immune response . a hybrid ca - pde model combines the traditional continuum methods of applied mathematics , such as macroscale reaction - diffusion equations describing chemical concentrations , with more modern , individual , or grid - based automaton methods , which are used for describing individual cell - level phenomena . the hybrid ca - pde modeling approach has been successfully used in the past to model tumor growth , chemotherapeutic treatment , and the effects of vascularization on a growing tumor [ 7 , 10 , 11 , 14 ] . in section 3 we discuss this model in some detail , explaining how the model is constructed as well as typical outputs of an in silico model of this type . while in vitro and in vivo models use actual biological materials and/or actual animals to investigate hypotheses and , for example , predict effectiveness of treatment strategies , in silico models use specifically designed computer programs to mimic these real experimental environments and to conduct computational experiments . there exist a number of different types of in silico model including differential equation models that track changes in quantities over time and/or space , network models that trace lines of probabilistic causation and/or correlation , discrete cellular automata- or individual - based models , and hybrids of all of these models . rather than providing models of real biological phenomena and structures that have a basis in some sort of extracted tissue or a somehow related animal species , these in silico models are comprised of mathematical and computational representations such as formulae , equations , and/or computer programs . parameterized so that quantities or rates not known in the real world or which are specific to different experiments can be investigated via computational experiments , or as we dub them in silico trials . the concept of the in silico trial can be thought of as akin to clinical trials . just as each patient in a clinical trial has their own set of characteristics such as height , age , and status with regard to smoking and alcohol consumption so too we can run the program of an in silico model multiple times with varied parameters to produce computational patients in an in silico trial . the development of in silico model is often a process of cross - disciplinary collaboration between cancer biologists and mathematicians or modelers . generally , the initial stages involve the model builder obtaining an understanding of the tumor biology required for developing the in silico model . this will be a period of intense collaborative work involving discussions between all investigators and a review of the theoretical and experimental literature . the next stage involves abstraction of biological information into a mathematical or computational form , that is , building the update rules . this requires the creation of mathematical representations of relevant micro level biological phenomena and mechanisms ( such as rates and results of cell division , methods for representing distributions of chemical molecules , and interactions between antigen and antigen presenting cells ) and the compilation of these into a macrolevel description of the real experimental situation . following the development of the update rules , the algorithm for the entire process is computerized usually employing generic programming languages such as c++ or with mathematical software such as matlab . this algorithm allows for the solution of the in silico model and facilitates easy simulation of large numbers of experiments , that is , repeated simulation of the model using many different parameter sets in order to mimic running slightly different experiments in the laboratory . this could reflect , for example , an investigation of the effect of different quantities of gold nanoparticles on effectiveness of radiotherapy or the effect of different concentrations of chemotherapeutic treatments . while largely automated via the computer program , the simulation of the in silico model requires careful and continuous monitoring to ensure that computations converge ( i.e. , solutions are obtained rather than computational errors ) and to make adjustments to investigations when results of interest are observed . following simulation of the in silico model , the results of the computational experiments are analyzed and interpreted . the investigators use the outputs of the model to determine what results are already useful for informing any associated experimental studies as well as what parts of the in silico model are deficient and require refinement along with a follow - up round of in silico experiments . the whole process can be repeated , with refinement , as often as new information is required , and in general the costs of follow - up in silico experimentation decrease as the fundamental computational framework has already been developed . in the remainder of this section , we present an oversimplified and generic model along with the computational algorithm to further illuminate this concept . a cellular automaton ( ca ) is a type of mathematical model , discrete in both space and time . here we consider a two - dimensional ca , such as that which could be used to model the surface of the skin or possibly a petri dish , but note that three - dimensional models are simple , if computationally expensive , extensions of the same concepts . a two - dimensional ca consists of a lattice or grid of ca elements covering a region of space ( see figure 1 ) . applied in the biological context , each element is allowed to house one or more biological cells and , depending on the experimental situation being modeled , may also hold other matter such as molecules , debris , fluid , or bacteria . the cells in the ca elements are allowed to interact with one another via update rules . the set of update rules defines how the state of each element changes in response to its current state and the current state of its neighbors the definition of these rules is the fundamental modelling stage in the development of the in silico model ( see figure 2 ) . the accuracy of the model is heavily dependent on designing rules that adequately reflect the real interactions between cells . the system is first initialized so that the computational representation presented in the cellular automata grid matches some initial condition for the ensuing computational experiment . next , a sequence of time steps is carried out such that the model time is incremented by a small amount at each step . within each time step , every spatial location or element in the ca grid is investigated to identify its contents . depending on the contents , an appropriate update rule is applied which may involve the states of the neighboring elements . updates are made throughout the grid , time is incremented , and the process continues . to extend this model to allow in silico trials , the computer program for the algorithm described in the above paragraph is wrapped in a further program . this involves providing a collection of two or more ( depending on the number of experiments or trials required ) parameter value sets to the algorithm and running the algorithm once with each set . the output data , for example , cell counts over time , for each trial is exported to memory at the completion of each trial . as mentioned earlier , with regard to developing an accurate description of the biological process of interest , the specification of the update rules for a cellular automata - based in silico model is the most important part of the modelling process . to demonstrate this , consider the seemingly simple case of the movement of one cell to a neighboring location and the following increasingly complex but increasingly accurate rules . rule 1if there is one or more empty ca elements surrounding a cell , move to a randomly chosen empty element , otherwise , do not move . if there is one or more empty ca elements surrounding a cell , move to a randomly chosen empty element , otherwise , do not move . rule 2if there is one or more empty ca elements surrounding a cell and moving to one would increase the cell 's satisfaction in some way , move to a randomly chosen element of this type , otherwise , do not move . if there is one or more empty ca elements surrounding a cell and moving to one would increase the cell 's satisfaction in some way , move to a randomly chosen element of this type , otherwise , do not move . rule 3if there is one or more empty ca elements surrounding a cell , consider moving to one of these locations with a probability that depends on factors such as cell adhesion levels , nutrient supply , and chemoattractants , otherwise , do not move . if there is one or more empty ca elements surrounding a cell , consider moving to one of these locations with a probability that depends on factors such as cell adhesion levels , nutrient supply , and chemoattractants , otherwise , do not move . each of these rules could be implemented in an in silico model as the determining factor regarding whether or not a cell moves . clearly moving from rule 1 to rule 3 , the amount of realism increases , but , simultaneously , the amount of information required to design the rule also increases . rule 1 does not require any information about the cells of interest the cell simply moves if it can , and the location it moves to is randomly chosen . on the other hand , rule 3 requires that the modeler has some preexisting or obtainable understanding regarding how cells respond to chemoattractants , how cell adhesion affects motility , and what impact nutrient levels have on the decision of a cell to move from location to location . thus we note that with more information about the biological process , the modeler can construct more realistic update rules , but at the same time , a lack of information by no means rules out in silico modeling . in fact , in silico models can yield rich information when they are used from the very early stages as part of hypothesis generation and testing when there is a dearth of biological information . mallet and de pillis presented a so - called hybrid cellular automata model of the interactions between the cells of a growing tumor and those of the host immune system . mallet and de pillis successfully designed a computational method for investigating the interactions between an idealized host immune system and a growing tumor . the simulated tumor growth experiments were found to be in qualitative agreement with both the experimental and theoretical literature . it was found that even with quite simple mathematical descriptions of the biological processes and with an overly simplified description of the host immune system , the computational model had the potential to produce the behavior observed in laboratory experiments including spherical and papillary tumor growth geometries , stable and oscillatory tumor growth dynamics , and the infiltration of the tumor by immune cells . it was also possible to show the dependence of these different morphologies on key model parameters related to the immune response . numerical solutions produced using the mallet and de pillis model agreed qualitatively with the experimental results demonstrated by zhang et al . , schmollinger et al . , and soiffer et al . while a laboratory model is usually designed to focus on a particular stage of a process or a specific event , in silico models can be designed to focus on arbitrarily small or large - scale phenomena . mallet and de pillis chose to focus on the early stages of tumor growth during which the tumor is adjacent to , but not yet infiltrated by , nutrient supplying vasculature in order to allow for an investigation of the initial interactions between the immune system and the emerging tumor . the simple model incorporated a simplified immune system comprised of two cell types , namely , the natural killer ( nk ) cells of the innate immune system and the cytotoxic t lymphocytes ( ctls ) of the specific immune system . a hybrid cellular automata and partial differential equation model was constructed with an aim to demonstrate the combined effects of the innate and specific immune systems on the growth of a two - dimensional representation of a growing tumor . this was accomplished by constructing a model with computerized cell behaviors built from descriptions in the experimental literature and findings of dynamic models of tumor immune system interactions developed in the theoretical literature such as the work of kuznetsov and knott and de pillis and radunskaya [ 19 , 20 ] . mallet and de pillis ' hybrid cellular automata model employed a coupled deterministic - stochastic approach that had the benefit of being conceptually accessible as well as computationally straightforward to implement . the authors used reaction - diffusion equations , to describe chemical species such as growth nutrients , and a cellular automata strategy to track the tumor cells and two distinct immune cell species . together , these elements simulated the growth of the tumor and the interactions of the immune cells with the tumor growth . the model tracked cells both through time and through space a clear advantage over dynamic models that assume a spatially well - mixed population of cells , which is not often the case in reality . unlike continuum - based spatiotemporal models , which are generally comprised entirely of partial differential equations , the hybrid cellular automata approach allows for the consideration of individual cell behavior and associated randomness , rather than applying a general rule to a collection of cells , as is the case with continuum models . while the mallet and de pillis model considered only four cell species with an overly simplistic view of the immune system , it is easily modified to cater for the inclusion of more cell types or new chemical species . the evolution of the cell species considered in the mallet and de pillis model proceeds according to a combination of probabilistic and deterministic rules , developed in an attempt to describe the phenomena considered important in the theoretical model . in particular , mallet and de pillis imposed a simplifying assumption to the host cells such that , other than their consumption of nutrients , they allow tumor cells to freely divide and migrate and were more or less passive bystanders to tumor growth . tumor cells on the other hand were able to move , divide , die due to nutrient levels and die because of the immune response , each with a probability that depended on some combination of nutrient levels , local immune response , and crowding due to the presence of other tumor cells . level by recruitment from outside the domain of interest whenever the local density dropped too far below its equilibrium level . both natural killer cells and cytotoxic t cells were able to lyse tumor cells , although ctls could do so more than once and were able to attract other ctls to the local area . ctls were also subject to removal from the local region with a probability depending on the local tumor cell density . the rules used to represent these phenomena are developed as approximations of reality and involve considering individual events , such as an interaction between a cell on the periphery of a tumor and a natural killer cell , and attempting to quantify what happens as a result of this interaction . this act of quantifying is guided by accepted results in the experimental and theoretical literature , expert elicitation , and simple physical arguments . as mentioned in the previous section , the development of these rules is the most important step in model development . while the design and statement of all the ca rules are presented in the original paper , here we expand on the design of one of the rules to elucidate how such objects are constructed . just as an experimentalist in the laboratory does not consider each of these explicitly , we do not attempt to represent each of them in the computational model either . instead , we distil what information is available in the literature and from collaborators to arrive at a model representation of the chance that the event occurs given certain conditions . this distilled model representation is the cellular automata rule . for the case of cell division , mallet and de pillis consider that given a tumor cell , the probability of division increases with the ratio of nutrient concentration to the number of tumor cells already present in the local region . note that there is no mention of subcellular signal processing and neither is there any consideration of macrolevel pressure fields . instead , the chance of the occurrence of a cell division is condensed into a consideration of whether or not there are sufficient nutrients nearby and whether or not the region is already crowded with tumor cells . this rule is interesting because it also incorporates a second subrule that of the placement of the daughter cell . the model dictates that the grid location upon which the daughter cell is placed depends upon the cells occupying the neighborhood of the mother cell . for example , a dividing cell with at least one host cell or necrotic space surrounding it will place its daughter cell randomly in one of those noncancerous locations and either destroy the host cell or simply replace the necrotic material . on the other hand , if all elements around the dividing cell are filled with tumor cells , the daughter cell will be placed in the neighboring element containing the fewest tumor cells . in silico models such as that of mallet and de pillis can produce an array of different outputs . in this particular work , the authors focused on presenting growth curves and two - dimensional spatial snapshots in time of growing tumors that were compared with experimental results . figures 3 and 4 , for example , show a growth curve and two - dimensional snapshot of a tumor growing in the absence of the immune system . this result was used as a baseline to compare with experimental and previous mathematical results prior to investigating the effects of the immune system with this new model . note , in figure 3 , the initially exponential growth phase ( cycle 0200 ) , before a phase of linear growth ( cycle 200800 ) . these growth characteristics mimic the growth rates described in the experimental work of folkman and hochberg and mathematically by greenspan . figure 4 is a snapshot in time ( 800 cell cycles ) of the same simulation where we see a roughly circular tumor with a radius of about 200 cells growing steadily outward toward the sources of the nutrient . higher tumor cell densities are seen at the periphery of the tumor while in the center , a necrotic core is beginning to form with some necrotic material already appearing . mallet and de pillis also presented a particularly interesting application of their model that produced qualitatively similar simulated tumors to the results of some recent experimental studies of immune response to tumor growth . and kuznetsov and knott discussed the relationship between increased survival rates of cancer patients , tumor necrosis , and fibrosis , and the presence of intratumoral t cells or infiltrated t lymphocytes . in figures 5(a ) and 5(b ) , immune cells are shown to have infiltrated a growing tumor . in particular , the darker regions in figure 5(a ) are evidence of tumor necrosis while lighter regions of figure 5(b ) are indicative of high immune cell populations . , soiffer et al . , and kuznetsov and knott where strings of immune cells are moving into the tumor , surrounding individual cells , and causing tumor cell necrosis . the simulation results showed employee parameters for a compact tumor ( in the absence of the immune system ) , low - level ctl recruitment , and low ctl death probability . we emphasize again that the same computer program is used to implement these simulations as those considered in the previous figures ; varying system parameters is all that is required to consider quite a different experiment when using the in silico modeling technology . the example of an in silico model presented in this section employed a moderately complex , hybrid cellular automata - partial differential equation methodology to describe interactions between the host immune system and a growing tumor . in the absence of a simulated immune system , the model was capable of reproducing both compact - circular and wild papillary tumor morphologies . morphology change was directly related to the relative rates of consumption of the survival and mitosis nutrients by both tumor and host tissue cells , and the results presented correspond qualitatively with the experimental literature ( such as folkman and hochberg ) . when the model allowed for a simulated immune system , with different choices of t - lymphocyte recruitment and/or death parameters , oscillatory growth curves were observed for nearly all parameter sets . depending on the strength of the immune system recruitment and death parameters , the tumor growth either increased without bound or resulted in destruction of the invasive growth . the model was also able to reproduce experimentally observed immune cell infiltration of growing tumors . the different sets of parameter values used in the simulation of the mallet and de pillis model are the primary method for computationally mimicking different strengths of immune systems of , for example , healthy individuals , capable of early tumor detection and destruction , and individuals in poor immune health , for whom tumors grow easily . in summary , even though the update rules proposed in the mallet and de pillis model were relatively simple and the number of cell types considered was far from exhaustive , the authors created an in silico model that was able to produce results in qualitative agreement with both the experimental and theoretical literature and which could be improved upon to provide useful preclinical results of relevance for further model development for guiding experimental work related to various treatment and vaccination strategies . in silico experimental modeling of cancer involves combining findings from biological literature with computer - based models of biological systems in order to conduct investigations of hypotheses entirely in the computer laboratory . in this paper we have presented a discussion of the concept of in silico modeling and how in silico models are constructed in practice . we have presented an example of in silico modeling that is relevant to the study of cancer and discussed its application and use as a hypothesis - generating tool as a precursor to or in combination with traditional clinical and laboratory research . this type of computational tool , when used in transdisciplinary research teams , has the potential to allow researchers to refine their experimental programs with an aim to reducing costs and increasing research efficiency , and we advocate increased use of such strategies by research groups .

in silico experimental modeling of cancer involves combining findings from biological literature with computer - based models of biological systems in order to conduct investigations of hypotheses entirely in the computer laboratory . in this paper , we discuss the use of in silico modeling as a precursor to traditional clinical and laboratory research , allowing researchers to refine their experimental programs with an aim to reducing costs and increasing research efficiency . we explain the methodology of in silico experimental trials before providing an example of in silico modeling from the biomathematical literature with a view to promoting more widespread use and understanding of this research strategy .

1. Introduction 2. Methods 3. An Example in Cancer Biology 4. Conclusions

PMC4004211

soluble amyloid oligomers are now thought to be the main toxic species that cause neurodegeneration in alzheimer s and other amyloid diseases . small assemblies made up of dimers , trimers , and tetramers of the -amyloid peptide ( a ) , as well as larger assemblies such as dodecamers , have been shown to disrupt synaptic activity and cause neuronal cell death . atomic - level details of the structures of amyloid oligomers are desperately needed in order to understand how the oligomers form and the molecular basis by which they cause neurodegeneration . the oligomers are polymorphic and dynamic , forming as different species and equilibrating slowly with the monomer and with -amyloid fibrils , which are generally more stable . while the structures of amyloid oligomers are still largely unknown , a number of approaches have been taken to gain insights into their structures . -sheet structure and interactions a common feature of amyloid fibril formation are generally thought to be important in the structures and interactions of amyloid oligomers . incorporation of amyloidogenic peptides into larger proteins can control amyloid supramolecular assembly and allow observation of oligomeric assemblies at atomic resolution . peptide fragments can also serve as chemical models of oligomers ; x - ray crystallographic studies of these peptide fragments have provided insights into the structures of amyloid oligomers . chemical cross - links within amyloidogenic monomers that stabilize folded -sheet conformations can promote oligomer formation and help prevent fibril formation . these cross - linked systems are more amenable to study and can provide simpler and more stable chemical models of the unstable oligomers formed by amyloidogenic peptides and proteins . computational models of oligomers have been constructed from atomic - level structures of amyloid fibrils , which are understood far better at atomic resolution than the oligomers . our laboratory is gaining insights into the structures and interactions of amyloid oligomers by combining fragments of amyloidogenic peptides and proteins with molecular templates to create macrocycles that promote -sheet structure and interactions while blocking amyloid fibril formation . we recently reported the x - ray crystallographic structures of oligomers of a peptide from -amyloid . we incorporated the nonapeptide sequence qklvffaed ( a1523 ) into macrocyclic -sheet peptide 1 , with -linked ornithine turn units and a template strand that features an unnatural amino acid , hao . in the solid state , the -sheet forms a hydrogen - bonded dimer , which assembles face - to - face to make a cruciform tetramer , which is a key subunit of the lattice . the cruciform tetramers assemble into triangular dodecamers , and the triangular dodecamers further assemble into the lattice . the hydrogen - bonded dimers are antiparallel , and the -strands are fully aligned , with residues 1723 of one of the macrocycles aligned with residues 2317 of the other . the resulting four - stranded -sheet forms a plane , with the side chains projecting from the upper and lower faces of the plane . residues k16 , v18 , f20 , and e22 of each macrocycle project from one face of the plane ( the vf face ) , and residues q15 , l17 , f19 , a21 , d23 of each macrocycle project from the other face of the plane ( the lfa face ) . the vf face has the hydrophobic residues v18 and f20 flanked by the polar residues k16 and e22 . the lfa face has the hydrophobic residues l17 , f19 , and a21 flanked by the polar residues q15 and d23 . the hydrogen - bonded dimers assemble in a crisscross fashion through hydrophobic interactions between the vf faces to give the cruciform tetramers . figure 1 illustrates the faces of the macrocycle and the structure of the cruciform tetramer . cartoon illustrating the lfa and vf faces of macrocyclic -sheet 1 and the cruciform tetramer formed in the solid state . the vf faces form the inner hydrophobic core of the cruciform tetramer , and the lfa faces form the outer surface . in the current study , we set out to determine how macrocyclic -sheet peptides containing the a1523 nonapeptide assemble in solution . we began by using h nmr spectroscopy to study how macrocyclic -sheet peptide 2a folds and oligomerizes in aqueous solution . the two molecules differ only in that 1 contains a p - bromophenylalanine ( f ) in the template strand , for single anomalous dispersion ( sad ) phasing in x - ray crystallographic structure determination , while 2a contains a tyrosine . as our studies of macrocyclic -sheet 2a unfolded , we prepared additional homologues ( 2b , 2c , 3 , and 4 ) to interrogate the assembly process . the following describes these studies and elucidates how the tetramer that forms in solution differs from that which forms in the solid state . we investigated the folding and assembly of the macrocyclic -sheets in d2o and in h2o the h nmr spectrum of macrocyclic -sheet 2a is disperse , with methyl resonances from l17 and a21 unusually upfield ( 0.35 and 0.49 ppm ) , aromatic resonances from f19 unusually upfield ( 6.28 and 6.52 ppm ) , and many of the amino acid -protons unusually downfield ( 5.0 ppm ) . one of the resonances from one of the hao amino acids ( the h4 resonance of hao1 ) appears unusually downfield at 9.17 ppm . the upfield shifting of the aromatic and aliphatic resonances is characteristic of the formation of an oligomer with a well - packed hydrophobic core comprising aromatic residues ( hao , phe , etc . ) and aliphatic residues ( leu , ala , etc . ) . minor additional resonances , associated with a monomer lacking a hydrophobic core are also present , most notably at 0.690.79 ppm ( l17 and v18 ) . figure 2 illustrates the h nmr spectrum of macrocyclic -sheet 2a at 2.0 mm in d2o solution . h nmr spectra of macrocyclic -sheet peptides 1 , 2a , and 2b at 2.0 mm in d2o at 500 mhz and 298 k. noteworthy resonances that reflect important shared features of the folding and assembly of these peptides are labeled and highlighted with dashed lines . the h nmr spectrum of macrocyclic -sheet 1 is virtually identical to that of macrocyclic -sheet 2a , indicating that both peptides fold and oligomerize in a similar fashion in solution . the h nmr spectrum of macrocyclic -sheet 1 also exhibits additional minor resonances from l17 and v18 associated with a monomer lacking a hydrophobic core . these resonances are similar in intensity to those of macrocyclic -sheet 2a , indicating that the oligomers formed by both macrocycles are similar in association constant ( kassoc ) as well as in structure . h nmr noesy studies establish the formation of hydrogen - bonded dimers that are antiparallel , with the -strands of residues 1723 shifted out of alignment by two residues toward the c - termini ( figure 3 ) . notably , the noesy spectrum in d2o exhibits strong noes between the -protons of l17 and d23 and between the -protons of f19 and a21 ( figure 4 ) . additional strong noes associated with -sheet folding of the macrocycles occur between the -protons of k16 and y and between the -protons of f20 and k ( figure 4 ) . other noes characteristic of folding are described in detail in the si , as are additional noes associated with folding and dimerization that are seen in the noesy spectrum in h2o macrocyclic -sheet 1 exhibits similar patterns of noes , indicating that it folds and dimerizes in a fashion similar to that of macrocycle 2a ( figure s2 in the si ) . the shifted structure of the dimers formed by the macrocycles in solution stands in sharp contrast to the aligned structure of macrocycle 1 in the solid state ( figure 3 ) . cartoons and chemical structures illustrating the hydrogen - bonded dimers formed by macrocyclic -sheet peptide 1 in the solid state ( left ) and by both macrocyclic -sheet peptides 2a and 1 in solution ( right ) . both hydrogen - bonded dimers are antiparallel : in the solid - state dimer , residues 1723 of one of the macrocycles align with residues 2317 of the other ; in the solution - state dimers , these -strands are shifted out of alignment by two residues toward the c - termini . key noes associated with solution - state dimerization and folding of 2a are shown with red and blue arrows . selected expansions of the noesy spectrum of macrocyclic -sheet peptide 2a at 8.0 mm in d2o at 500 mhz and 300.5 k. key intermolecular interstrand noes associated with dimerization are highlighted in red ; key intramolecular interstrand noes associated with folding are highlighted in blue . at low concentrations ( e.g. , 0.1 mm ) , the monomer predominates in the h nmr spectrum of macrocyclic -sheet 2a . the methyl resonances from l17 and v18 of the monomer are prominent at 0.690.79 ppm , and the methyl resonances from l17 and a21 of the oligomer at 0.35 and 0.49 ppm are small . as the concentration of 2a is increased , the relative intensities of the resonances from the oligomer increase and the relative intensities of the resonances from the monomer decrease ( figure 5 and figure s3 in the si ) . at 0.2 mm , the resonances of the monomer and oligomer are roughly equal in intensity . at high concentrations ( e.g. , 8.0 mm ) , the resonances of the monomer are barely visible . the strong concentration dependence of the monomer dimer equilibrium , but rather reflects cooperative association in which the dimers are a subunit of a higher - order oligomer in this case a tetramer consisting of a dimer of dimers . expansions of the h nmr spectra of macrocyclic -sheet peptide 2a at various concentrations in d2o at 500 mhz and 298 k. noteworthy characteristic resonances of the monomer and the oligomer are labeled and highlighted with dashed lines . the noesy spectrum of macrocyclic -sheet 2a shows additional crosspeaks that are consistent with a tetramer in which two hydrogen - bonded dimers form a sandwich - like assembly . notably , the noesy spectrum in d2o exhibits noes between hao2 and threonine and between hao2 and hao1 that only make sense as interlayer noes between the hydrogen - bonded dimers . specifically , the methoxy group of hao2 gives noes with the methyl group of threonine , and the h3 and h4 protons of hao2 give noes with the h3 and h4 protons of hao1 . figure 6 illustrates these interlayer noe crosspeaks in the noesy spectrum ; figure 7 illustrates the sandwich - like assembly consistent with these noes . figure s4 and table s1 ( si ) provide additional data . selected expansions of the noesy spectrum of macrocyclic -sheet peptide 2a at 8.0 mm in d2o at 500 mhz and 300.5 k. key interlayer noes associated with tetramerization illustration of the tetramer formed as a sandwich - like assembly of two hydrogen - bonded dimers of macrocyclic -sheet peptide 2a in aqueous solution . macrocyclic -sheet peptide 1 forms a similar sandwich - like tetramer in solution . the four threonines of the tetramer point toward the interior of the sandwich - like assembly , as do all of the residues on the lfa faces of the -sheets ( q15 , l17 , f19 , a21 , and d23 ) . the magnetic anisotropy from the packed aromatic groups of the resulting hydrophobic core shift the methyl resonances of l17 and a21 upfield . thus , the structure of this solution - state tetramer , in which the lfa faces make up the hydrophobic core , differs markedly from the structure of the solid - state tetramer , in which the vf faces make up the hydrophobic core . in the solid - state structure , the lfa faces are on the exterior of the tetramer and the vf faces are on the interior ; in the solution - state structure , the vf faces are on the exterior and the lfa faces are on the interior . to probe the assembly of the tetramer , we studied macrocyclic -sheet peptide 3 . macrocyclic -sheet 3 is a homologue of 2a with a lysine in place of the threonine in the template strand . at 1.0 mm essentially no tetramer is observed in the h nmr spectrum of 3 ( figure 8) . as the concentration is increased to 2.0 and 4.0 mm , resonances for the tetramer appear ; at 8.0 mm the tetramer predominates . the tetramerization is far weaker than that of macrocyclic -sheet 2a , in which the tetramer is observed at 0.1 mm and predominates at 0.3 mm . expansions of the h nmr spectra of macrocyclic -sheet peptide 3 at various concentrations in d2o at 500 mhz and 298 k. noteworthy characteristic resonances of the monomer and the oligomer are labeled and highlighted with dashed lines . addition of salt ( nacl ) augments tetramer formation , suggesting that intermolecular ionic repulsion is partially responsible for the diminished tetramerization of macrocyclic -sheet 3 . without nacl , macrocylic -sheet 3 is 46% tetramerized at 4.0 mm ; with 25 mm nacl , it is 70% tetramerized ; with 150 mm nacl , it is 80% tetramerized ( figure s6 and table s3 in the si ) . the loss of hydrophobic interactions between the methyl group of threonine and the methoxy group of hao2 may also contribute to the diminished stability of the tetramer of macrocyclic -sheet 3 . diffusion - ordered spectroscopy ( dosy ) nmr studies support the formation of a tetrameric species . measurement of the dosy spectrum of macrocyclic -sheet peptide 2a in d2o at 2.0 mm and 8.0 mm and 298 k gave a diffusion coefficient of 10.0 10 cm / s and 10.1 10 cm / s , respectively , for the oligomer . the diffusion coefficient does not vary from 2.0 mm to 8.0 mm , suggesting the presence of a single oligomerization state . measurement of the dosy spectrum of macrocyclic -sheet peptide 3 in d2o at 2.0 mm and 298 k gave a diffusion coefficient of 16.4 10 cm / s for the corresponding monomer . consistent with tetramer formation , the diffusion coefficient of the oligomer of macrocyclic -sheet peptide 2a is 0.61 times that of the monomer of macrocyclic -sheet peptide 3 . to further study the assembly of the tetramer , we mutated residues on the lfa and vf faces to examine how the hydrophobic residues on each face control tetramer formation . we created two double mutants of 2a , in which either the hydrophobic residues v18 and f20 or the hydrophobic residues f19 and a21 were rendered more hydrophilic by hydroxylation . in double mutant 2b , v18 was replaced with threonine and f20 was replaced with tyrosine ( v18t , f20y ) . in double mutant 2c , f19 was replaced with tyrosine and a21 was replaced with serine ( f19y , a21s ) . the h nmr spectrum of the v18t , f20y double mutant 2b is strikingly similar to that of macrocyclic -sheet 2a ( figure 2 ) , indicating that 2a and 2b fold and oligomerize in a similar fashion in aqueous solution . the methyl resonances from l17 and a21 appear unusually upfield , the aromatic resonances from f19 also appear unusually upfield , and many of the amino acid -protons appear unusually downfield . the h nmr spectra of both compounds reflect similar monomer oligomer equilibria . at 0.1 mm , the monomer predominates and only small resonances from the tetramer are present ; at 1.0 mm , the resonances from the tetramer predominate and only small resonances from the monomer are present . thus , v18t , f20y double mutation does not substantially alter the equilibrium constant for tetramer formation . the h nmr spectrum of the f19y , a21s double mutant 2c differs markedly from those of 2a and 2b ( figure s7 in the si ) . the methyl resonances from l17 do not appear unusually upfield and the amino acid -protons do not appear unusually downfield . these observations indicate that f19y , a21s double mutation disrupts the formation of the tetramer . the h nmr spectrum of macrocyclic -sheet 2c shows some minor broadened resonances at 2.0 mm , which diminish at lower concentrations , suggesting that some weaker nonspecific self - association may persist when tetramer formation is disrupted . the dramatic differences between macrocyclic -sheets 2b and 2c further demonstrate the importance of hydrophobic interactions of the lfa face of the macrocycle in tetramer formation . when the lfa face is hydroxylated , tetramer formation is disrupted , but when the vf face is hydroxylated , tetramer formation is not affected . to probe the role of hydrogen bonding in tetramer formation , we blocked the hydrogen - bonding edge of the macrocycle by n - methylation . macrocyclic -sheet 4 is a homologue of macrocyclic -sheet 2a with n - methylphenylalanine in place of phenylalanine at position 20 . the f20f mutation is designed to block formation of the hydrogen - bonded dimer and thus the assembly of a tetramer comprising a dimer of hydrogen - bonded dimers . the h nmr spectrum of macrocyclic -sheet 4 also differs markedly from those of 2a and 2b ( figure s7 in the si ) . the methyl resonances from l17 and a21 do not appear unusually upfield and the amino acid -protons do not appear unusually downfield . the disruption of tetramer formation by n - methylation demonstrates that hydrogen bonding is also essential for tetramer formation . dosy nmr studies of macrocyclic -sheets 14 suggest that 1 , 2a , and 2b are tetrameric at millimolar concentrations , while 2c , 3 , and 4 are monomeric . as mentioned above , the oligomeric 2a exhibits a diffusion coefficient of 10.0 10 cm / s in d2o at 298 k , while monomeric 3 exhibits a diffusion coefficient of 16.4 10 cm / s . the ratio of these diffusion coefficients about 0.6 is consistent with tetramer formation . macrocyclic -sheets 1 and 2b exhibit diffusion coefficients similar to that of 2a , while macrocyclic -sheets 2c and 4 exhibit diffusion coefficients similar to that of 3 ( table 1 ) . diffusion coefficient measured at 8.0 mm . to corroborate the dosy studies , we performed analytical ultracentrifugation ( auc ) sedimentation velocity studies on macrocyclic -sheet 2b . the auc studies are best performed in nonzero ionic strength to avoid nonideality resulting from charge interactions between the large cationic molecules . thus , we performed auc sedimentation velocity studies in the presence of salt , using 0.10 , 0.30 , and 0.66 mm solutions of macrocycle 2b in h2o containing 25 mm nacl at 293 k. the sedimentation velocity data fit well to a reversible monomer tetramer equilibrium with slow exchange on the time scale of the experiment ( hours ) . the tetramer predominated at all three concentrations , with the greatest fraction of monomer present at 0.10 mm . analysis of the data from the 0.10 mm experiment gave a good fit to a monomer tetramer equilibrium with a 2.14 kda monomer and a 8.55 kda tetramer and a kassoc of 1.93 10 m. ( for details see the si . ) the magnetic anisotropy of the diastereotopic -protons of the -linked ornithine turn units in the h nmr spectra reflect that the tetramers of 1a , 2a , and 2b form well - folded -sheets , while the monomers of 2c , 3 , and 4 are only partially folded . in a well - folded macrocyclic -sheet , the difference in the chemical shifts ( ) of the diastereotopic pro - s and pro - r -protons of the -linked ornithine turn units ( orn ) is about 0.6 ppm in aqueous solution . values substantially lower than 0.6 ppm reflect the formation of partially folded macrocyclic -sheet structures . at 2.0 mm and 298 k in d2o , the tetramers of 1 , 2a , and 2b exhibit large magnetic anisotropies , while the monomers of 2c , 3 , and 4 exhibit smaller magnetic anisotropies ( table 2 ) . to further investigate the folding and oligomerization of macrocylic -sheet 2a , we compared the h nmr chemical shifts of the -protons of the 2a tetramer to those of acyclic control peptide 5 . peptide 5 contains the a1523 nonapeptide and two -linked ornithine turn units but lacks the lower template strand . the -proton resonances of a1523 in the 2a tetramer appear 0.041.04 ppm downfield of those of acyclic control , with an average downfield shifting of 0.66 ppm ( figures 9 and s8 in the si ) . the large downfield shifting of the -protons suggests the formation of a well - folded -sheet structure . downfield shifting of the h nmr -proton resonances of the 2a tetramer and the 3 monomer , relative to acyclic control 5 . the h nmr spectrum of 2a was recorded at 8.0 mm in d2o at 500 mhz and 300.5 k. the h nmr spectra of 3 and 5 were recorded at 2.0 and 1.2 mm , respectively , in d2o at 500 mhz and 298 k. in contrast , the -proton resonances of the monomer of 2a are not nearly as far downfield shifted . although it is not feasible to identify all of the -proton resonances of the monomer of 2a because the tetramer predominates even at submillimolar concentrations , it is possible to do so in the close homologue 3 , which is largely monomeric at low millimolar concentrations . the -proton resonances of a1523 in the 3 monomer show far less downfield shifting , with an average of only 0.13 ppm ( figures 9 and s8 in the si ) . the smaller downfield shifting of the -protons of the monomers of 2a and 3 reflects the formation of -sheet structures that are only partially folded . although the individual peptide monomer units are only partially folded , the tetramers that form exhibit secondary , tertiary , and quaternary structure reminiscent of proteins . the unusually well - defined structures of the tetramers are reflected in the strong noes observed and in the large magnetic anisotropies of the l17 , f19 , and a21 side chains and many of the -protons in the h nmr spectra . to gain further insight into the structure of the tetramers formed by the macrocyclic -sheets in aqueous solution , we used the x - ray crystallographic structure of the tetramer of macrocyclic -sheet 1 to create a model of the solution - state tetramer of macrocyclic -sheet 2a . we generated the initial coordinates for the model in pymol by ( 1 ) changing the p - bromophenylalanine of 1 to tyrosine , ( 2 ) shifting the crystallographic dimers out of alignment by two residues toward the c - termini , ( 3 ) moving the dimers to pack through the lfa faces instead of the vf faces , ( 4 ) selecting appropriate rotamers of f20 , and ( 5 ) orienting the dimers to approximately match the observed interlayer noes between the methoxy group of hao2 and the methyl group of threonine . we then generated a minimum - energy structure ( local minimum ) of the tetramer in macromodel with the maestro user interface using the mmffs force field with gb / sa water solvation , minimizing first with distance constraints to match the observed noes between -protons ( figure 3 ) and between the layers of the -sheets ( figures 6 and 7 ) and then without constraints . the tetramer consists of a dimer of hydrogen - bonded dimers and is essentially symmetrical , consisting of four roughly symmetrical monomers arranged in roughly d2 symmetry . f19 , and a21 of the dimers pack tightly to form a hydrophobic core within the tetramer ( figure 10b and c ) . the methyl group of a21 sits over the phenyl group of f19 in the opposing layer of the sandwich - like structure , consistent with the observed upfield shifting of the methyl resonance of a21 in the h nmr spectrum . the pro - s methyl group of l17 sits over the aromatic ring of hao2 in the opposing layer , consistent with the pronounced upfield shifting of one of the methyl resonances of l17 in the h nmr spectrum . the methyl group of the threonine is close to the methoxy group of hao2 , and hao1 is close to hao2 , consistent with the observed noes between these groups ( figure 10d and figure 6 ) . model of macrocyclic -sheet peptide 2a as a tetramer , based on the noe cross peaks of 2a and the x - ray crystallographic structure of 1 . ( a ) the hydrogen - bonded dimers are antiparallel and shifted out of alignment by two residues toward the c - termini . residues l17 , f19 , and a21 of the hydrophobic core are shown ( the lfa face ) . ( d ) detail of the contacts between threonine , hao1 , and hao2 , which give rise to the interlayer noe crosspeaks that are shown in figure 6 . the solution - state tetramers formed by macrocyclic -sheets 1 , 2a , and 2b differ from the solid - state tetramer observed for macrocyclic -sheet 1 in three notable ways : although both tetramers comprise antiparallel -sheet dimers , the solution - state dimers are out of register , shifted out of alignment by two residues toward the c - termini , while the solid - state dimers are in register , with all residues aligned ( figure 11 ) . the solution - state dimers are sandwiched through the lfa faces , while the solid - state dimers are sandwiched through the vf faces ( figure 12 ) . the two solution - state dimers that form the tetramer are nearly parallel to each other , while the two solid - state dimers are nearly orthogonal ; the former are oriented at roughly 15 , while the latter are oriented at roughly 83 ( figure 12 ) . model of macrocyclic -sheet peptide 2a and the x - ray crystallographic structure of 1 as dimers . ( a ) x - ray crystallographic structure of hydrogen - bonded dimers of 1 that are antiparallel and fully aligned . ( b ) solution - state structure of hydrogen - bonded dimers of 2a that are antiparallel and shifted out of alignment by two residues toward the c - termini . model of macrocyclic -sheet peptide 2a and the x - ray crystallographic structure of 1 as tetramers . ( a ) top view of the x - ray crystallographic structure of 1 as a tetramer . residues v18 and f20 of the hydrophobic core are shown ( the vf face ) . ( b ) top view of solution - state structure of 2a as a tetramer . residues l17 , f19 , and a21 of the hydrophobic core are shown ( the lfa face ) . the differences between the solution - state tetramer and the solid - state tetramer may reflect the need to maximize hydrophobic contacts in aqueous solution . the lfa face of the dimer presents six hydrophobic residues from a1523 , while the vf face presents only four ( figure 12 ) . hydrophobic contact is maximized in the aqueous tetramer through contact between these six residues . the bulky hydrophobic side chains of l17 and f19 pack well with the small hydrophobic side chain of a21 in the opposing dimer of the tetramer . in the solid state , the tetramer is part of a lattice in which there are additional intermolecular contacts . the tetramers are in contact with other tetramers , as well as with water and organic cocrystallants , and these contacts likely help stabilize the tetramer . differences in ph and protonation state may also be important in the differences between the solution - state and solid - state tetramers . the differing morphology of the solution - state and solid - state tetramers is significant , because it may provide a glimpse into some of the structural bases for polymorphism among a oligomers in alzheimer s disease . polymorphism has previously been observed at atomic resolution in a fibrils , but not in oligomers . because little is known about the structures of amyloid oligomers , little is known about the structural bases of oligomer polymorphism . much of what is currently known about amyloid oligomer polymorphism focuses on differences in reactivity toward oligomer - specific antibodies or differences in size and shape that can be observed by electron microscopy , atomic - force microscopy , gel electrophoresis , or mass spectrometry . the contrasting structures of the solution - state and solid - state tetramers described here demonstrate subtle differences among oligomers that can be observed at atomic resolution . differing alignment of the -strands within the -sheets also gives rise to unique structures . while not seen in the two types of tetramers here , both parallel and antiparallel -sheet structures may also be possible . macrocyclic -sheet peptides containing the a1523 nonapeptide exhibit rich supramolecular chemistry , forming tetramers with well - defined structures in aqueous solution and in the solid state . the solution - state and solid - state tetramers exhibit noteworthy polymorphism , differing in the alignment of the monomers within the hydrogen - bonded dimers , the faces of the hydrogen - bonded dimers involved in tetramer formation , and the rotational orientation of the hydrogen - bonded dimers within the tetramers ( figure 13 ) . both hydrogen bonding and hydrophobic interactions are important in tetramer formation . residues l17 , f19 , and a21 are critical in the formation of the hydrophobic core of the tetramers in solution , and the size complementarity of the small a21 residue and large l17 and f19 residues may play a special role in their stability . cartoon illustrating the structure of the solid - state tetramer of macrocyclic -sheet 1 ( top ) , and the solution - state tetramer of macrocyclic -sheets 1 and 2a ( bottom ) . the vf faces form the inner hydrophobic core of the solid - state tetramer of 1 , and the lfa faces form the outer surface . the lfa faces form the inner hydrophobic core of the solution - state tetramer of 1 and 2a , and the vf faces form the outer surface . the supramolecular assembly of amyloidogenic peptides to form soluble oligomers is almost impossible to study at atomic resolution with natural full - length amyloidogenic peptides , because the oligomers that form are heterogeneous in size and morphology and because the oligomers are dynamic and can ultimately form insoluble amyloid . chemical model systems that limit uncontrolled supramolecular assembly and contain important segments of the amyloidogenic peptides can help identify modes in which the peptides interact . we anticipate that chemical model systems based on macrocyclic peptides will prove widely useful in elucidating the supramolecular assembly and oligomer formation of other amyloidogenic peptides .

this contribution reports solution - phase structural studies of oligomers of a family of peptides derived from the -amyloid peptide ( a ) . we had previously reported the x - ray crystallographic structures of the oligomers and oligomer assemblies formed in the solid state by a macrocyclic -sheet peptide containing the a1523 nonapeptide . in the current study , we set out to determine its assembly in aqueous solution . in the solid state , macrocyclic -sheet peptide 1 assembles to form hydrogen - bonded dimers that further assemble in a sandwich - like fashion to form tetramers through hydrophobic interactions between the faces bearing v18 and f20 . in aqueous solution , macrocyclic -sheet peptide 1 and homologue 2a form hydrogen - bonded dimers that assemble to form tetramers through hydrophobic interactions between the faces bearing l17 , f19 , and a21 . in the solid state , the hydrogen - bonded dimers are antiparallel , and the -strands are fully aligned , with residues 1723 of one of the macrocycles aligned with residues 2317 of the other . in solution , residues 1723 of the hydrogen - bonded dimers are shifted out of alignment by two residues toward the c - termini . the two hydrogen - bonded dimers are nearly orthogonal in the solid state , while in solution the dimers are only slightly rotated . the differing morphology of the solution - state and solid - state tetramers is significant , because it may provide a glimpse into some of the structural bases for polymorphism among a oligomers in alzheimer s disease .

Introduction Results Discussion Conclusion

PMC3179882

as the primary fuel for abiding sports , carbohydrate intake before , during , and after exercise , has a positive effect on endurance performance [ 16 ] . apart from being an energy substrate , the sight , smell , and taste of food may act as positive reinforcements . by generating promises for food intake , these senses play a role in reward prediction . as a result , the body starts to function as if it is going to receive food [ 710 ] . recent studies suggest that simple mouth rinses with a carbohydrate solution can improve endurance performance even for performances that last about an hour , that is , in conditions where glycogen stores should not be limiting [ 1115 ] . both complex and simple sugars can elicit a mouth rinse effect [ 1114 ] . intravenous infusion of glucose with a similar effect on blood sugar regulation parameters , as compared to a swallowed glucose solution , did not affect performance in a ~1-hour - time trial . it seems that specific oropharyngeal receptors , linked to brain centers that are involved in motivation and reward , play a role in the ergogenic effect of carbohydrate mouth rinsing . the first experiments were done on cycle ergometers and involved young well - trained subjects who were asked to finish a specific amount of work in a minimum of time ( time trial ) . when rinsing their mouths with a nonsweet carbohydrate solution subjects were faster as compared to rinsing with water [ 1214 ] . not all subsequent mouth - rinse protocols successfully increased endurance performance , as some studies showed a lack of enhancement of time - trial performance in the fed state on a cycle ergometer while another study reported no improvement when using a treadmill . all those studies involved well - trained subjects and used similar time - trial experimental paradigms . given the rather surprising nature of the potential underlying mechanisms for these findings and the need to further probe the hypothesis of exercise enhancement through oropharyngeal receptor stimulation , we therefore decided to extend the existing evidence base by investigating the effect of an oral carbohydrate stimulus on exercise capacity using a time - to - exhaustion test , comparing fed and fasted states . and since no other studies tested this effect in a nonathletic population , we hypothesized that a carbohydrate mouth rinse would affect endurance capacity in a nonathletic population and would have more effect in the fasted state as compared to the fed state . the study was approved by the ethical committee of the university of balamand in beirut , lebanon . subjects signed an informed consent form and could withdraw at any moment from the study . exclusion criteria were a sedentary lifestyle , active training for endurance sports , and any preexisting illness . a fan was set 2 m away from the participants to provide cooling during exercise . subjects were asked to refrain from any strenuous activity , alcohol , caffeine , or any other form of stimulant during the last 24 hours preceding tests . each participant did 5 tests on a cycle ergometer ( monark , ergomedic 839e , used in power setting , rpm independent ) . on their first visit , the participants did an incremental test to exhaustion , based on kuiper 's protocol [ 14 , 19 ] . after a 5 min warm - up at 100 watts , the power was increased 50 watts every 2.5 min until the heart rate reached 160 bpm . maximal workload ( wmax ) was then calculated as wout + ( t/150 ) 25 , where wout refers to the last completed stage and t to the time of the unfinished stage . on separate days , time between tests was not less than 72 hrs and not more than 96 hrs . after 5 min warm - up at 50 watts , power was set at 60%wmax and subjects would cycle until exhaustion . based on preliminary experiments in subjects of similar training status , we chose 60%wmax since higher percentages yielded exercise times that were too short . subjects were required to keep the pedaling rate between 70 and 100 rpm ; when they failed to pedal at least at 70 rpm , the test ended . heart rate was monitored continuously with telemetry ( nike triax c5 , ny , usa ) and subjects were asked to rate on a 10 point borg scale [ 20 , 21 ] every 5 minutes , just after each mouth rinsing , their rate of perceived exertion ( rpe ) . on 2 occasions , subjects would rinse their mouth with a 25 ml maltodextrin solution ( 6.4% , cho ) and on the 2 other with water ( pla ) for 510 seconds every 5 min . subjects were not allowed to swallow the solution and had to spit it out ; they were however allowed to drink water from a separate bottle ad libitum . for both solutions , subjects would once start the test after an overnight fast ( fcho and fpla ) and once after a standardized carbohydrate - rich breakfast ( cho and pla ) . breakfast included sandwiches / cereals / oatmeal along with fat - free milk / fruit juice ( no coffee or tea ) , fresh fruits ( bananas / figs ) , and dried fruits ( raisins / apricots / dates ) . tests were done between 09 h 30 and 11 h 30 am and subjects did the paired ( fasted versus fed ) trials at the same time of the day . after each test , the subjects were asked to indicate what solution they thought they had received . they could listen to music of their choice during the test and were able to see the time elapsed . heart rate was not given to them . during the tests , the researcher and the participant were the only two people inside the laboratory . the order of tests was done in a randomized balanced order and the subjects were blinded to the mouth - rinse solution . data were analyzed using pasw statistics ( version 18.0.0 , chicago , ill , usa ) . effects of mouth rinsing with maltodextrin and prandial state on time - to - exhaustion , heart rate , and rpe were analysed using a repeated measures two - by - two way anova . average age ( sd ) was 21 3 yrs , body mass 83 8 kg , stature 180 6 cm . maximum exercise capacity was 200 18 watt corresponding to an estimated maximum aerobic capacity of 31 7 ml o2/kg / min or 8.9 2.0 mets . resting heart rate was 72 6 bpm , maximum heart rate was 167 10 bpm , 75 4% of expected ( karvonen ) . subjects did the time - to - exhaustion trials at 120 11 watt ( 60% wmax ) . average values for time - to - exhaustion , mean exercise heart rate , maximum exercise heart rate , mean exercise rpe , and maximum rpe are shown in table 1 . the repeated measures two - by - two anova revealed a main effect of maltodextrin mouth rinsing on time - to - exhaustion ( p = 0.020 ) , no effect of prandial state ( p = 0.220 ) and a tendency for interaction ( p = 0.075 ) ( see also figure 1 ) . there was no main effect of mouth rinsing or prandial state on mean exercise heart rate but a significant interaction ( p < 0.0001 ) . there was a significant main effect of mouth rinsing on average exercise rpe ( p = 0.032 ) but not of prandial state . similar results were found for maximum rpe with a main effect of mouth rinsing ( p = 0.035 ) and no effect of prandial state or interaction . for the fcho trial , 8 subjects out of 13 identified the correct solution , for the fpla trial 9 subjects out of 13 . for both the pla and cho trials , 6 out of 13 identified the correct solution . chi - square was 0.692 ( p = 0.405 ) , preventing the rejecting of the null - hypothesis of 50 - 50% chance to guess correctly . the main finding was that a mouth rinse with a maltodextrin solution improved time - to - exhaustion in both pre- and postprandial states by an average 7 3% and 3 2% , respectively . this improvement was accompanied by an average 6 1% drop in the level of perceived exertion when compared to placebo . the ergogenic effect of carbohydrate presence in the mouth tended to be more evident on an empty stomach . our results complement those reported in earlier studies [ 1215 ] , by adding time - to - exhaustion on a cycle ergometer , as another exercise performance testing paradigm in which oral carbohydrate rinsing , has an effect and by reporting that this effect is present in young and apparently healthy but nonathletic subjects . we chose to use the original solutions used by carter et al . in their first study , a watery nonsweet tasting maltodextrin solution and just plain water as placebo . saliva contains amylase , which breaks down starch into low - molecular - weight maltodextrins , but very few glucose units . there may have been some release of single glucose units while rinsing with the maltodextrin solution , which is probably what was needed in order to activate the purported cho receptors in the oropharynx . since the subjects were not allowed to swallow ( and did not swallow ) the rinse solution , very little carbohydrate made its way to the stomach and the intestinal system . we are not aware of any glucose transporters in the oropharynx , so there will be very little uptake of glucose in the condition of mouth rinsing with the maltodextrin solution . it can not be totally excluded that some subjects had an idea of what they were taking since they indicated slight differences in texture for the two solutions , and in the fasted state , 65% of the subjects correctly distinguished between them . also for practical reasons , but since in the fed state only 46% guessed correctly , with an overall ( fed and fasted together ) 56% correct guess not significantly different from pure chance ( 50% ) , placebo effects would rather seem unlikely . the interpretation of this finding is not straightforward and can be a chance finding and should probably only be interpreted as an absence of a main effect . were the first to report an effect of carbohydrate mouth rinsing solution on time - trial performance lasting ~1 hour . they came to their original experimental design from the observation that an oral glucose solution improved a ~1 hr cycling - time trial , while intravenous glucose administration did not . since then , several other studies from the same and from other laboratories were published , but not always reporting increased performance ( see table 2 ) . beelen et al . looked specifically at the effects of carbohydrate mouth rinsing in the fed state and did not find any difference with placebo . they suggested that oral perception of carbohydrates perhaps only plays a role when muscle and liver glycogen stores are reduced . our findings would suggest that also in postprandial state the effect might occur , at least in our experimental settings . to explain their original findings , carter et al . mentioned a parasympathetic reflex triggered by the taste , smell , and sight of food and an associated cephalic phase of insulin release ( cpir ) , a sudden rise in insulin concentrations within minutes following gustatory stimulation resulting from a rapid mobilization of previously stored insulin in beta cells . this causes a small increase ( ~5 u / ml ) in arterial plasma insulin which would improve glucose uptake and maintain carbohydrate oxidation rates . this cephalic phase of insulin release is well described for the presence of sweetness in the oral cavity but has not been described for nonsweet - tasting carbohydrate such as used in the original experiment of carter et al . or our setup and remains to be investigated . combined a time - trial setup with fmri experiments looking for the brain areas associated with on one hand oral presence of carbohydrate and on the other hand the presence of sweetness . apart from confirming the performance enhancing effect of mouth rinsing with a carbohydrate solution , the authors found in a separate experimental setup that sweetness and carbohydrate sensing did not activate the same brain areas on fmri and that carbohydrate sensing passed through the primary taste cortex and the putative secondary taste cortex and then activated brain areas involved in reward and motor control including the anterior cingulate cortex ( acc ) , parts of the basal ganglia ( right caudate ) , and the ventral striatum . it would thus seem that the performance enhancing effect of the presence of cho in the oropharynx is related not to sweetness but to some other quality . this contention is corroborated by the recent findings of gant et al . who used an artificial sweetener in both the placebo and the maltodextrin solution and showed only an ergogenic effect for the latter . they reported that oral presence of carbohydrate facilitates corticomotor output to activated muscle not only in the fatigued state but also in the nonfatigued state and concluded that this type of sensorimotor integration probably also occurs without severe exercise - induced metabolic perturbations . the oropharynx contains many receptors , including taste receptors distributed on the surface of the tongue , epiglottis , palate , and oesophagus , recognizing sweet , salt , bitter , sour , and umami tastes , and other kinds of oral somatosensory receptors , identifying viscosity , fat texture and temperature . these receptors are now complemented with hitherto unknown oropharyngeal receptors that apparently sense the presence of complex and simple sugars , independently from sweetness . since food intake is indispensable for survival , it elicits a strong affective response , and in case of good palatability , a hedonic experience . because it is so difficult to investigate the relationship between physical phenomena , such as brain processes , and subjective experience [ 8 , 10 ] . also competitions , trophies , sport media , fame , and fortune represent positive reinforcement creating appetitive motivation . mouth rinse carbohydrate solutions can apparently activate this appetitive motivation in order to increase endurance performance [ 1214 ] , force production , or endurance capacity ( this study ) . the observation that this effect is perhaps more pronounced in the fasted state suggests that these receptors signal the cns that exercise - related energy expenditure is possible without jeopardizing the organism 's integrity . there is also evidence from animal studies of the presence of carbohydrate receptors independent of sweetness sensation . these are presented as heterodimers and serve as natural - sugar and artificial - sweeteners detectors . this suggests that the presence of t1r3-independent detection pathways expressed in homodimers like t1r2 or other non - t1r receptors [ 15 , 2729 ] . despite these advances in animal and human research the brain circuitry involved in these regulatory mechanisms the human oropharynx is equipped with receptors that can signal the presence of simple and complex carbohydrate . in ~1 hr duration maximum endurance exercise ( i.e. , before depletion of endogenous carbohydrate stores ) , carbohydrate mouth rinsing significantly improves performance , especially in the fasted state . our results show that this effect is also present in time - to - exhaustion type effort in young but nonathletic subjects .

background . oropharyngeal receptors signal presence of carbohydrate to the brain . mouth rinses with a carbohydrate solution facilitate corticomotor output and improve time - trial performance in well - trained subjects in a fasted state . we tested for this effect in nonathletic subjects in fasted and nonfasted state . methods . 13 healthy non - athletic males performed 5 tests on a cycle ergometer . after measuring maximum power output ( wmax ) , the subjects cycled four times at 60% wmax until exhaustion while rinsing their mouth every 5 minutes with either a 6.4% maltodextrin solution or water , one time after an overnight fast and another after a carbohydrate rich breakfast . results . mouth rinsing with maltodextrin improved time - to - exhaustion in pre- and postprandial states . this was accompanied by reductions in the average and maximal rates of perceived exertion but no change in average or maximal heart rate was observed . conclusions . carbohydrate mouth rinsing improves endurance capacity in both fed and fasted states in non - athletic subjects .

1. Background 2. Methods 3. Results 4. Discussion 5. Conclusions

PMC5276766

thanks to dynamic stability , a subject displaces and controls the center of gravity ( cog ) within the base of support in dynamic functional conditions , performing complex combined movements such as striding forward ( forward - lunge , fl ) , going up and down a step ( step - up - over , suo ) , and standing up from a seated position ( sit - to - stand , sts)1 . many researchers have studied the ability of the computerized dynamic posturography to characterize several features of balance in different populations2 , 3 . this test provides accurate values regarding parameters such as impact , rising , distance , time , impulse and cog sway , during the aforementioned movements in dynamic functional conditions3 . using computerized dynamic posturography , these tests show the balance impairments of patients who could benefit from rehabilitation treatment during activities of living involving dynamic conditions4 . however , only one study has reported the reliability of the fl and suo dynamic functional tests and none have reported the reliability of the sts test5 , which makes the clinical applicability of these dynamic balance tests controversial . in this study , the intrasession and intersession reliability of measurements obtained in the fl , suo and sts tests were assessed . this study also calculated the standard error of measurement ( sem ) , which shows the ability of the tests to exclude the noise from measurements6 , 7 . the absolute and relative minimal detectable changes ( mdc ) were also calculated to estimate the real change between two measurements of the same subject across time , as this parameter is clinically more relevant8 . the sample size was determined considering a two - tailed hypothesis , a 0.05 type i error , a 0.93 intraclass correlation coefficient ( icc ) estimated for the impulse variable in the fl test and a 0.075 accuracy level9 . forty - five healthy subjects were randomly selected from the general university population ( 24 males and 21 females ; 23.89 5.49 years ; 1.69 0.08 cm ; 68.43 10.97 kg ; 32 right - handed , 12 left - handed , 2 ambidextrous ) . subjects with balance affectation10 , musculoskeletal diseases or related surgery , or previous balance training were excluded . before beginning the tests , each participant signed an informed consent form . the ethics committee of seville university approved this study ( code : m2012/024/20130226 ) , which was carried out at the physiotherapy unit of a sports medicine center . measurements were carried out using the platform balance master system 8.2 ( usa ) under controlled laboratory conditions by an experienced examiner , and following a standardized protocol so all the participants were given directions in the same way . data were collected in two assessment sessions , 68 days apart . during the first session , initially , the participants practiced each dynamic test to acquaint themselves with the movement exertion when receiving a visual prompt from a screen10 . , the three tests were performed only once , with three trials performed for each . forward - lunge ( fl ) test : the participants stayed as upright and as still as possible on the platform . on seeing the prompt go on the screen , they moved their foot forward as far and fast as they could , bending the knee . the trunk was kept in a vertical position while the cog transferred from one foot to the other quickly . the subjects were instructed not to use their arms for help during the movement . the outcome variables were the distance reached ( distance ) defined as the stride length , measured from the cog location during the standing position to the furthest point reached by the cog while stepping forward , expressed as a percentage of the subject s height ( % ) . the impact index was defined as the maximal reaction force generated by the swinging forward leg when it contacted the ground , expressed as a percentage of the body weight ( % ) . the contact time ( contact time ) was defined as the lapse of time ( s ) between the moment when the swinging forward leg left the ground to when it made contact with the ground again ; and the impulse force was defined as the total amount of work done by the swinging forward leg during the stance phase , and the push back force of the movement , expressed as a percentage of the body weight multiplied by the time that the force was exerted ( % s ) . the mean of three trials per test was used following the manufacturer s protocol and previous recommendations regarding the highest relative and absolute reliabilities of biomechanical variables in the forward - lunge test11 . step - up - over ( suo ) test : the participants stayed as upright and as still as possible on the platform . on seeing the prompt go on the screen , they put one foot on a 21-cm high step and then lifted the other foot , passed it over the step without touching it and placed it back on the ground . the outcome variables were : the lift - up index , defined as the maximal strength exerted by the lower limb on the step during the lift - up phase , expressed as a percentage of the body weight ( % ) ; the movement time , defined as the lapse of time when it contacted the ground again ; and the impact index , defined as the maximal reaction force generated by the swinging forward leg when it contacted the ground again , expressed as a percentage of the body weight ( % ) . although the platform manufacturer states that this test should be carried out quickly , in our prior experience we observed that this instruction resulted in performance of the movement while running . this caused the other variables the lift - up index and impact index to exhibit characteristics not related to the subjects behaviors in activities of daily living . as a consequence , the participants were instructed to mount the step in a spontaneous way , as they would in normal daily life . sit - to - stand ( sts ) test : the participants sat quickly on a 41-cm high box placed on the platform . on seeing the prompt go on the screen , they stood up as quickly as possible without using their arms and stood still . the outcome variables were the rising index , defined as the maximal strength exerted by the lower limbs during the rising phase , expressed as a percentage of the body weight ( % ) , and the cog sway velocity ( cog sway velocity ) , defined as the velocity of the cog during the rising phase and the next five seconds ( /s ) . each test was carried out three times and the mean was used the statistical analysis . the mean as well as the differences between each pair of measurements were calculated ( the first was compared to the second one for intrasession reliability , and the first to the third , for intersession reliability ) giving the sixteen variables , and the skewness coefficient and the kurtosis were also calculated also . the relative reliability was analyzed considering the icc , which was calculated following two procedures : a two factor mixed - effect model ( icc a ) with absolute agreement ; and a one factor random - effect model ( icc b ) . the absence of a systematic error in the measurements was verified due to the equality of icc a and the icc b12 . the sem was calculated following the stratford and goldsmith procedure7 . the absolute reliability or the repeatability , also known as minimal detectable change ( mdc95 ) because it refers to the minimal change that falls outside the measurement error of an instrument used to measure a symptom14 , was calculated using the equations : sem 1.96 2 , 95% confidence level . according to one - way analysis of variance ( one - way anova ) , the square root of the quadratic mean of the residual from the intrasubject effect test is the intrasubject standard deviation , which allows calculation of the systematic error12 , 13 . the sem and the mdc95 were also calculated as percentages of the measurement mean6 , 8 . in this way , therefore , mdc95 indicates a real change with 95% confidence for individual participants and demonstrates the repeatability of the test . the statistical tests were performed using a 95% confidence interval ( ci ) ( p<0.05 ) . fifty subjects were enrolled , but five of them dropped out from the second session due to issues not related to the research . for that reason , the estimated icc value for a 0.075 accuracy level was 0.902 . table 1table 1.mean values , standard deviations and ranges of unilateral and bilateral variables in the forward - lunge , step - up - over and sit - to - stand testssession 1 : trial 1session 1 : trial 1session 2forward - lungemean ( sd)rangemean ( sd)rangemean ( sd)rangedistance ( % ) right56.5 ( 6.3)(4069)56.6 ( 6.2)(3969)56.8 ( 6.2)(4369)left56.3 ( 5.8)(4167)55.8 ( 6.0)(4269)56.3 ( 6.0)(4470)impact index ( % ) right27.4 ( 7.6)(1250)26.3 ( 7.1)(1546)27.3 ( 6.8)(1549)left26.7 ( 6.7)(1241)25.8 ( 6.3)(1541)27.9 ( 6.8)(1745)contact time ( s)right1.0 ( 0.2)(0.51.3)1.0 ( 0.2)(0.61.4)1.0 ( 0.2)(0.61.4)left1.0 ( 0.2)(0.61.4)1.0 ( 0.2)(0.61.3)1.0 ( 0.2)(0.61.4)impulse force ( % xs)right106.8 ( 16.3)(69139)106.6 ( 17.2)(64144)107.4 ( 16.1)(73148)left104.3 ( 16.4)(71142)103.8 ( 15.3)(72134)104.6 ( 15.4)(75145)step - up - overlift - up index ( % ) right43.0 ( 9.3)(1860)48.9 ( 11.2)(2377)48.3 ( 11.8)(2280)left40.2 ( 9.7)(1861)43.8 ( 10.0)(1968)45.4 ( 12.0)(1979)movement time ( s)right1.3 ( 0.2)(1.01.7)1.2 ( 0.2)(0.91.5)1.2 ( 0.2)(1.01.6)left1.3 ( 0.2)(1.01.7)1.2 ( 0.1)(0.91.5)1.2 ( 0.2)(0.91.6)impact index ( % ) right42.1 ( 13.1)(1174)44.4 ( 13.7)(1579)43.5 ( 13.6)(1176)left44.1 ( 12.7)(2070)48.0 ( 13.6)(1580)47.6 ( 14.4)(1785)sit - to - standrising index ( % ) 29.6 ( 8.5)(1250)31.9 ( 8.4)(1749)32.3 ( 8.9)(1854)cog swayvelocity ( /s)3.1 ( 1.1)(1.05.4)3.4 ( 1.0)(1.25.3)3.4 ( 1.0)(1.25.4)cog : center of gravity ; sd : standard deviation ; distance ( % subject s height : cm ) , impact index ( % body weight : kg ) , contact time ( s ) , force impulse ( % body weight : kg time : s ) lift - up index ( % body weight : kg ) , movement time ( s ) , rising index ( % body weight : kg ) , cog sway velocity ( /s ) gives a descriptive analysis of the collected data . the normality of all the variables registering a skewness coefficient and kurtosis 1.5 was calculated . in all cases , icc a was not different from icc b , implying that there were no systematic errors . accordingly , only icc a is shown in tables 2table 2.forward-lunge testintrasessionintersessionicc ( 95% ci)sem ( % ) mdc95 ( % ) icc ( 95% ci ) sem ( % ) mdc95 ( % ) distance ( % ) right0.9 ( 0.90.9)1.8 ( 3.1%)4.9 ( 8.6%)0.9 ( 0.80.9)2.2 ( 3.8%)6.0 ( 10.6%)left0.9 ( 0.91.0)1.7 ( 3.0%)4.7 ( 8.3%)0.9 ( 0.70.9)2.3 ( 4.2%)6.5 ( 11.5%)impact index ( % ) right0.8 ( 0.60.9)3.4 ( 12.6%)9.4 ( 34.8%)0.6 ( 0.40.7)4.3 ( 15.9%)11.9 ( 44.1%)left0.8 ( 0.60.9)3.1 ( 11.4%)8.5 ( 31.7%)0.7 ( 0.50.8)3.8 ( 14.4%)10.7 ( 39.8%)contact time ( s)right0.9 ( 0.80.9)0.1 ( 6.1%)0.2 ( 16.2%)0.7 ( 0.50.8)0.1 ( 10.1%)0.3 ( 27.3%)left0.9 ( 0.80.9)0.1 ( 5.2%)0.1 ( 13.5%)0.8 ( 0.60.9)0.1 ( 8.3%)0.2 ( 22.9%)impulse forceright0.9 ( 0.80.9)5.8 ( 5.4%)16.1 ( 15.1%)0.7 ( 0.50.8)9.4 ( 8.8%)26.0 ( 24.3%)(%xs)left0.9 ( 0.80.9)5.1 ( 4.9%)14.2 ( 13.6%)0.8 ( 0.70.9)7.2 ( 6.9%)20.0 ( 19.2%)ci : confidence interval ; icc : intraclass correlation coefficient ; mdc : minimal detectable change ; sem : standard error mean ; distance ( % subject s height : cm ) , impact index ( % body weight : kg ) , contact time ( s ) , impulse force ( % body weight : kg time : s ) p<0.001 , 3table 3.step-up-over testintrasessionintersessionicc ( 95% ci ) sem ( % ) mdc95 ( % ) icc ( 95% ci ) sem ( % ) mdc95 ( % ) lift - up index ( % ) right0.6 ( 0.20.8)5.1 ( 11.0%)14.2 ( 30.5%)0.7 ( 0.30.9)5.0 ( 10.7%)13.8 ( 29.5%)left0.7 ( 0.40.8)5.3 ( 12.4%)14.7 ( 34.2%)0.6 ( 0.30.8)6.0 ( 14.0%)16.7 ( 38.8%)movement time ( s)right0.7 ( 0.30.9)0.1 ( 5.7%)0.2 ( 15.3%)0.6 ( 0.40.8)0.1 ( 8.1%)0.3 ( 21.8%)left0.5 ( 0.10.8)0.1 ( 7.1%)0.3 ( 19.8%)0.5 ( 0.10.8)0.1 ( 7.9%)0.3 ( 21.4%)impact index ( % ) right0.8 ( 0.70.9)5.0 ( 11.5%)13.8 ( 31.8%)0.9 ( 0.80.9)5.0 ( 11.4%)13.7 ( 31.7%)left0.7 ( 0.50.8)6.7 ( 14.3%)18.5 ( 39.7%)0.7 ( 0.50.8)7.0 ( 15.0%)19.3 ( 46.6%)ci : confidence interval ; icc : intraclass correlation coefficient ; mdc : minimal detectable change ; sem : standard error of mean ; lift - up index ( % body weight : kg ) , movement time ( s ) , impact index ( % body weight : kg ) p<0.001 and 4table 4.sit-to-stand testintrasessionintersessionicc ( 95% ci)sem ( % ) mdc95 ( % ) icc ( 95% ci)sem ( % ) mdc95 ( % ) rising index ( % ) 0.8 ( 0.60.9)3.4 ( 10.8%)9.4 ( 30.0%)0.7 ( 0.50.8)4.5 ( 14.5%)12.5 ( 40.0%)cog swayvelocity ( /s)0.7 ( 0.50.8)0.6 ( 17.7%)1.6 ( 48.8%)0.6 ( 0.30.7)0.7 ( 21.7%)2.0 ( 59.8%)ci : confidence interval ; cog : center of gravity ; icc : intraclass correlation coefficient ; mdc : minimal detectable change ; sem : standard error of mean ; rising index ( % body weight : kg ) , cog sway velocity ( /s ) p<0.001 . the weir classification for icc was used15 : scores from 0.50 to 0.69 , good ; from 0.70 to 0.89 , very good ; and from 0.90 , excellent ; sem% lower than 35% , good ; mdc95% lower than 30% , reasonable ; and mdc95% lower than 10% , excellent16 . cog : center of gravity ; sd : standard deviation ; distance ( % subject s height : cm ) , impact index ( % body weight : kg ) , contact time ( s ) , force impulse ( % body weight : kg time : s ) lift - up index ( % body weight : kg ) , movement time ( s ) , rising index ( % body weight : kg ) , cog sway velocity ( /s ) ci : confidence interval ; icc : intraclass correlation coefficient ; mdc : minimal detectable change ; sem : standard error mean ; distance ( % subject s height : cm ) , impact index ( % body weight : kg ) , contact time ( s ) , impulse force ( % body weight : kg time : s ) p<0.001 ci : confidence interval ; icc : intraclass correlation coefficient ; mdc : minimal detectable change ; sem : standard error of mean ; lift - up index ( % body weight : kg ) , movement time ( s ) , impact index ( % body weight : kg ) p<0.001 ci : confidence interval ; cog : center of gravity ; icc : intraclass correlation coefficient ; mdc : minimal detectable change ; sem : standard error of mean ; rising index ( % body weight : kg ) , cog sway velocity ( /s ) p<0.001 table 2 shows icc and 95% confidence intervals of the fl test . they were excellent to very good for the intrasession reliabilities ( range 0.90.8 ) , and very good to good for the intersession reliabilities ( range 0.90.7 ) . the sem% scores for all the parameters were much lower than 35% and the mdc95% of most of the parameters were lower than 30% ( except for the impact index ) , with excellent scores ( around 10% ) for the distance variable , which was the most reliable , followed by the impulse and the contact time . they were very good to good for the intra- and inter - session reliabilities ( range 0.90.5 ) . the sem% scores of all the parameters were much lower than 35% and the mdc95% scores of most of all the parameters were around 30% , though the lift - up and impact index of the suo showed poorer ability to detect real change than the fl variables . table 4 shows the iccs ( 95% ci ) of the sts test , which were very good for the intrasession reliability ( range 0.80.7 ) and very good to good for the intersession reliability ( range 0.70.6 ) . the sem% scores of both parameters were much lower than 35% and the most reliable variable was the rising index , which had an intersession mdc95% of 30% , whereas the cog sway exceeded this limit in intra- and inter - session reliabilities . the main finding of this study was the good absolute reliability of most fl variables , which means that this test is a potential diagnosis tool for musculoskeletal diseases . however , comparisons with other research are difficult due to the icc context - dependence17 , 18 the icc being affected by factors such as the number of subjects , the number of repetitions , the lapse of time between assessment sessions , the protocol followed , the number of examiners and systematic errors7 . however , the 0.9 icc hypothesis for the impulse variable of the fl test was not been proven , as the intersession icc for the impulse values of the fl in the present study were 0.7 and 0.8 for the right and left legs , respectively . the relative variability of the fl measurements made with a 1-week lapse of time ( intersession ) is bigger than that observed between the measurements made during the same session ( intrasession ) . this shows that time is a factor influencing on the fl parameters intersubject variability . furthermore , fl contact time may differ with dynamic movement exertion due to anterior cruciate ligament ( acl ) pathology11 . fl contact time data collected in this present study were longer than those of a study following the same protocol and methods5 , even though subjects were specifically asked in both studies to perform fl as quickly as possible . the age range and nature of the sedentary subjects enrolled in the present study ( 1840 years compared to 1826 ) might explain the longer contact time duration . thus , these slight differences may show the impact of age or physical condition on this variable . the fl contact time was longer in another previous study , possibly because a greater number of test repetitions and the absence of a specific instruction to perform fl as quickly as possible11 . also , a previous study reported a 270 ms difference in the fl contact time between control and experimental subjects who had not fully recovered from acl surgery19 . in our study , the absolute reliability of the fl contact time was calculated using the sem values of 50 to 100 ms . the mdc95 was lower ( 130270 ms ) indicating the ability of the fl test ability to discriminate between subjects with or without acl pathologies . the fl test was also able to highlight the variation in the fl contact time between two measurements made on the same subject suffering from this kind of pathology . furthermore , our results for the fl and suo impact when down a step ( similar to a short stride ) are higher than the impact values of a long stride . since a longer stride elicits less stress than a shorter one , previous studies have reported that the distances achieved ( the stride length ) in the fl test affects the impact force and the stress borne by the patellofemoral joint20 . therefore , the shorter the step , the bigger the force that the lower limb absorbs . also , the fl test mainly involves the hip extensors ; therefore , it would be worthwhile to investigate whether this test is sensitive and specific to pathologies involving these muscles21 . similarly , our results for the movement times of the suo test were longer than those of similar studies5 . in the present study , the subjects were not asked to perform this test quickly as the manufacturer recommends ; rather , they were asked to perform it at that in their daily life . the difference in instructions may explain the longer times observed in the present study as well as the differences seen in the impact and rising indices , which were lower in the present study . parameters based on the vertical reaction forces during descending stairs tests are able to monitor the rehabilitation progress after acl surgery22 . patients suffering from patellofemoral pain minimize the reaction forces both in ascent and descent phase of stairs to maintain normal levels of stress in the joint23 . accordingly , the suo lift - up and impact index ( derived from the reaction forces ) may show these adaptations . it is our opinion that this test should be accompanied by other functional tests to help to determine their limitations . although the relative variabilities of these parameters in our present research were high , their absolute reliabilities persuaded us that this test is a useful tool . the sts cog velocity exhibited relative and absolute reliability values lower than the sts rising index . in agreement with our present findings , goldie et al.25 determined that measurements based on ground reaction forces in a unipedal stance were more reliable than those based on centers of pressure ( cop)25 . the sts and suo rising and lift - up indices theoretically represent similar aspects of the ground reaction forces in two movements of daily living ( going upstairs and standing up ) . thus , they may complement each other and enhance the possibility of discriminating between subjects with and without a musculoskeletal disease . a limitation of this study was that the intrasession and intratest reliabilities were calculated from measurements made within 1 hour , which features the internal consistency of the measurements ( i.e. , the ability of the tool to reproduce measurements and the accuracy of the measurements in stable subjects ) . moreover , although the test - retest and intraexaminer reliabilities were calculated , the interexaminer reliability was not . however , this factor affects the variability of measurements made in tests that depend on the explanation that an examiner gives to subjects ; i.e. results that are based on subjects understanding17 . consequently , these aspects should be considered when discussing the reliability of the results . in conclusion , the contact time , distance and force impulse variables of the fl test showed excellent absolute and relative reliabilities . the variables based on cop changes in the sts tests did not show enough reliability in a healthy population . the collected data establishes a range of normal values for the variables of the tests , as well as relative and absolute reliabilities and mdc95% ( minimal detectable change ) of dynamic postural stability measurements of a healthy population .

[ purpose ] to examine the intrasession and intersession reliability and the absolute reliability of three functional dynamic tests forward - lunge , step - up - over and sit - to - stand tests using computerized dynamic posturography . [ subjects and methods ] an intra - test and test - retest , repeated measure study was designed . forty - five healthy subjects twice carried out the forward - lunge test , step - up - over test , and sit - to - stand test on two days , one week apart . the intrasession and intersession reliabilities as judged by the intraclass correlation coefficient ( icc ) and the minimal detectable change of the three functional tests were calculated . [ results ] excellent to very good intrasession reliability of the forward - lunge test ( icc range of 0.90.8 ) was found . very good to good intrasession reliability of the step - up - over test ( icc range of 0.90.5 ) was found and very good intrasession reliability of the sit - to - stand test ( icc range of 0.80.7 ) was found . the minimal detectable change at the 95% confidence level of most of the measures was lower than 30% . [ conclusion ] the forward - lunge , step - up - over and sit - to - stand tests are reliable measurement tools .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2121196

science is a notorious example of what economists call increasing returns to scalegroups of productive people feed off each other to create something that is more than the sum of its parts . the reverse is also true : isolated individuals will have a hard time generating momentum and are less productive . so how can you get a new hotbed of research started in a country with a limited history of biomedical research ? in the 40 years since gaining independence , singapore has become one of the wealthiest nations in the asia pacific region . per capita income , for example , has increased an average of 6.4% per year from 1965 to 2000 . yet that change came in the late 1990s with the decision to emphasize knowledge industries , including biomedical science . the decision was spurred in part by the worldwide electronics slump , which drove jobs in the electronics industry a mainstay of the country 's economy to cheaper locations in asia . and with no natural resources to bolster the country 's economy in times of hardship , the singapore economy needed to diversify , to become like a table with many legs , says philip yeo , chairman of singapore 's agency for science , technology , and research ( a*star ) . the additional leg of biomedical science would stand us in good stead in riding the next big wave . philip yeo , chairman of a*star and driving force for singapore 's biomedical science initiative . this need to diversify spawned the biomedical sciences initiative ( bmsi ) , a plan masterminded by yeo and hashed out in an all - night session with three of singapore 's top doctors , tan chorh chuan , then dean of medicine at the national university of singapore ( nus ) , and oncologists john wong and kong hwai loong a group yeo refers to as the biomedical sciences gang of 4. although the idea met with considerable skepticism , says yeo , the government eventually ( and reluctantly ) gave him the green light . the plan was announced in 2000one day before the unveiling of the human genome project and devoted nearly us$2 billion over five years to the development of public and private sector biomedical research . for the government , failure of the bmsi is not an option . when we say that singapore is serious about science , we are dead serious , said second minister for trade and industry vivian balakrishnau in his opening remarks at an october 2005 keystone symposium on stem cells , senescence , and cancer the first keystone meeting to be held outside north america . for singapore , success is a matter of life and death.when we say that singapore is serious about science , we are dead serious.-vivian balakrishnau , second minister for trade and industry when we say that singapore is serious about science , we are dead serious . -vivian balakrishnau , second minister for trade and industry yeo immediately set out to lure some of the world 's best scientists to singapore . the idea was that top - tier scientists will help make singapore a more enticing option for the junior faculty , postdocs , and students required to get the cooperative machinery of science up and running . it 's a circle , says kong peng lam , acting executive director of a*star 's biomedical research council ( bmrc ) , if you get the best to come here , they will attract the best . thanks to yeo 's aggressive recruitment efforts , singapore is now home to a bevy of acclaimed scientists . among them are sir david lane , discoverer of the p53 tumor suppressor gene , who is serving a two - year stint as the executive director of the institute for molecular and cell biology ( imcb ) , and edison liu , former head of the division of clinical sciences at the national cancer institute ( nci ) in the us , who became the executive director of the genome institute of singapore ( gis ) in 2001 . japanese cancer researcher yoshiaki ito , who identified runx3 as a tumor suppressor gene associated with stomach cancer , relocated to the imcb in 2002with his entire laboratory staff in tow after reaching japan 's mandatory university retirement age of 63 . also on board is molecular biologist alan colman , formerly with ppl therapeutics the scotland - based pharmaceutical company that cloned dolly the sheep . colman , who received a s$6 million grant to relocate to singapore , is now chief scientific officer of es cell international , a government - owned biotech company that is developing human embryonic stem cells for disease therapy . nobel laureate sydney brenner , a long - time advisor to the singaporean government ( he served as chairman of singapore 's scientific advisory board from 1987 to 1997 ) , is now chairman of the bmrc . brenner splits his time between singapore and the salk institute for biological studies in san diego . if you ask what attracted these scientific heavy hitters to singapore a country with a steamy climate , authoritarian government and a notoriously draconian penal code the common denominator seems to be the vision of philip yeoa phrase heard with an almost eerie regularity in singapore . in fact , the universally glowing reports from singapore 's expat scientists ( along with a*star 's apparently strict requirement for scientists to obtain permission before speaking to the press ) leave one wondering if the picture is a bit too rosy.if you get the best to come here , they will attract the best . -kong peng lam , acting executive director of the bmrc but the few complaints one can coax out of singapore 's imported scientists tend to be cultural rather than scientific . the government 's fear of criticism , for example , has dampened artistic expression , meaning that singapore 's cultural scene does n't measure up to that of most cosmopolitan cities . others are put off by the lack of political opposition to the ruling people 's action party , who have been in power since singapore gained independence . but for those already in singapore , the scientific bounty clearly outweighed the cultural drawbacks . and with researchers in the us and europe complaining that funding is becoming increasingly difficult to secure , the stellar reports from singapore 's expats along with the no - strings - attached money given to new principal investigators ( pis)might prove too tantalizing to pass up . this was true for two of the most recent defectors to singapore : cancer geneticists neal copeland and nancy jenkins , who recently relocated to singapore 's imcb from the us nci . in addition to the funding crunch in the us , the duo cite restrictions on stem cell research and frustration with national institutes of health 's ( nih ) new ethics policy which forbids nih employees from consulting for or holding financial interests in private companies as part of the impetus to end their two - decade - long stint at the nci . we did n't even negotiate a budget [ at imcb ] , we just told them how many people and how many mice ( thousands of cages) then sent them a list of equipment we needed . the husband - and - wife team who developed a technique to accelerate the identification of cancer - causing genes in mice turned down offers from stanford university and memorial sloan kettering cancer center in new york , opting instead for what they saw as an opportunity to conduct science in a less restrictive environment than in the us . indeed , whereas the us nih frowns upon scientists ' involvement in the private sector , singapore 's a*star does just the opposite . knowing that successful biotech companies generate both revenue and jobs , the government encourages researchers to apply for patents and , based on those patents , to launch biotech start - up companies offering three - year leaves of absence and s$150,000 in seed money as incentive . the free - flowing money is also part of singapore 's allure for younger scientists , along with the potential for upward mobility . because we are at ground zero , says lam , there are many opportunities [ for young scientists ] and many positions available . a good example of this upward mobility is thomas dick , head of the tuberculosis group at the novartis institute for tropical diseases , who moved to singapore in 1990 when biomedical research was still in its infancy . fresh out of graduate school at the university of heidelberg , dick heard that the newly founded imcb was looking for postdoctoral fellows and thought why not asia , why not the tropics for a couple of years ? six years later , when the time came for dick to venture out on his own , he questioned whether to stay in singapore or return to europe . as it turned out , he says , this step was quite easy here in singapore , and the lab head position was attractive : it came with funding ! since then , dick has risen through the ranks from laboratory head to assistant professor to associate professor in regular three - year intervals.immunology picks up speedsingapore may have lagged behind other countries in the immunology arena in recent years , but they 're now trying to make up for it . the immunology program at the nus still in its infancy at a mere two years old is headed by michael kemeny , an allergy expert who was recruited in 2004 from king 's college hospital in london . although king 's college was then being made into a national center for allergy and immunology ( along with imperial college london ) , kemeny was enticed by the idea of launching a new program in singapore.figure 3michael kemeny ( top ) and paul macary.the fledgling program , which currently has 16 full and 14 associate members drawn from various departments and institutes at nus and biopolis , will be expanding its ranks in the next few years , with faculty positions available at the full , associate , and assistant levels.on offer for those considering a move east are the program 's lavish new digs in the refurbished imcb building ( where it will relocate later this year ) , a collaboration - friendly research environment and an outstanding start - up package . within 1 - 2 years of coming to singapore , says kemeny , new pis typically hold up to s$1 million of competitive funding.kemeny also stresses the program 's proximity to a medical school and tertiary referral hospital , and its access to investigators across a multitude of disciplines among them computational biology , epidemiology , neuroscience , microbiology , pharmacology , imaging , bioengineering , structural biology , and stem cell biology which breeds a unique collaborative environment . no other group in singapore is able to work so closely with so many different specialities and experts , says kemeny.this solid financial support helped convince paul macary , an assistant professor in the immunology program , to join kemeny in singapore . the start - up package i was offered was significantly better than those from uk and us institutions , says macary , whose offer included not only research funds and a competitive salary , but also subsidized living accommodations and an education allowance for his children.but macary was initially skeptical . as a postdoc in the uk , he says , there is a general consensus that the us and europe are the only places where you can establish yourself as a good biomedical researcher . on top of that was concern about feeling isolated as one of only a small number of immunologists in singapore . macary , who moved to singapore with his wife and young children , also praises singapore as a safe and child - friendly place with a world - class education system.with enthusiastic salesmen like kemeny and macary and plenty of cash to import and train new talent , the immunology program seems poised for success . singapore may have lagged behind other countries in the immunology arena in recent years , but they 're now trying to make up for it . the immunology program at the nus still in its infancy at a mere two years old is headed by michael kemeny , an allergy expert who was recruited in 2004 from king 's college hospital in london . although king 's college was then being made into a national center for allergy and immunology ( along with imperial college london ) , kemeny was enticed by the idea of launching a new program in singapore . the fledgling program , which currently has 16 full and 14 associate members drawn from various departments and institutes at nus and biopolis , will be expanding its ranks in the next few years , with faculty positions available at the full , associate , and assistant levels . on offer for those considering a move east are the program 's lavish new digs in the refurbished imcb building ( where it will relocate later this year ) , a collaboration - friendly research environment and an outstanding start - up package . within 1 - 2 years of coming to singapore , says kemeny , new pis typically hold up to s$1 million of competitive funding . kemeny also stresses the program 's proximity to a medical school and tertiary referral hospital , and its access to investigators across a multitude of disciplines among them computational biology , epidemiology , neuroscience , microbiology , pharmacology , imaging , bioengineering , structural biology , and stem cell biology which breeds a unique collaborative environment . no other group in singapore is able to work so closely with so many different specialities and experts , says kemeny . this solid financial support helped convince paul macary , an assistant professor in the immunology program , to join kemeny in singapore . the start - up package i was offered was significantly better than those from uk and us institutions , says macary , whose offer included not only research funds and a competitive salary , but also subsidized living accommodations and an education allowance for his children . but macary was initially skeptical . as a postdoc in the uk , he says , there is a general consensus that the us and europe are the only places where you can establish yourself as a good biomedical researcher . on top of that was concern about feeling isolated as one of only a small number of immunologists in singapore . macary , who moved to singapore with his wife and young children , also praises singapore as a safe and child - friendly place with a world - class education system . with enthusiastic salesmen like kemeny and macary and plenty of cash to import and train new talent , singapore may have lagged behind other countries in the immunology arena in recent years , but they 're now trying to make up for it . the immunology program at the nus still in its infancy at a mere two years old is headed by michael kemeny , an allergy expert who was recruited in 2004 from king 's college hospital in london . although king 's college was then being made into a national center for allergy and immunology ( along with imperial college london ) , kemeny was enticed by the idea of launching a new program in singapore . . the fledgling program , which currently has 16 full and 14 associate members drawn from various departments and institutes at nus and biopolis , will be expanding its ranks in the next few years , with faculty positions available at the full , associate , and assistant levels . on offer for those considering a move east are the program 's lavish new digs in the refurbished imcb building ( where it will relocate later this year ) , a collaboration - friendly research environment and an outstanding start - up package . within 1 - 2 years of coming to singapore , says kemeny , new pis typically hold up to s$1 million of competitive funding . kemeny also stresses the program 's proximity to a medical school and tertiary referral hospital , and its access to investigators across a multitude of disciplines among them computational biology , epidemiology , neuroscience , microbiology , pharmacology , imaging , bioengineering , structural biology , and stem cell biology which breeds a unique collaborative environment . no other group in singapore is able to work so closely with so many different specialities and experts , says kemeny . this solid financial support helped convince paul macary , an assistant professor in the immunology program , to join kemeny in singapore . the start - up package i was offered was significantly better than those from uk and us institutions , says macary , whose offer included not only research funds and a competitive salary , but also subsidized living accommodations and an education allowance for his children . but macary was initially skeptical . as a postdoc in the uk , he says , there is a general consensus that the us and europe are the only places where you can establish yourself as a good biomedical researcher . on top of that was concern about feeling isolated as one of only a small number of immunologists in singapore . macary , who moved to singapore with his wife and young children , also praises singapore as a safe and child - friendly place with a world - class education system . with enthusiastic salesmen like kemeny and macary and plenty of cash to import and train new talent , although biomedicine in singapore seems to be off to a flying start , the country will need more than just manpower to ensure that their costly gamble pays off . to succeed , singapore needs to turn basic research into revenue a feat that will require other ingredients , including state - of - the - art facilities , support for manufacturing and research and development ( r&d ) , and corporate partnerships . for top - notch research facilities , one need look no further than biopolis , a futuristic two - million - square - foot biomedical research complex that houses the sparkling new laboratories of a*star 's five research institutes plus dedicated space for biotech start - ups and pharmaceutical companies not to mention restaurants , a day care center , fitness center , pub , and even a 7-eleven convenience store . biopolis which went from concept to reality in an astonishing 20 months was designed to foster cross - disciplinary collaboration and bridge the gap between academic and industrial research . construction of biopolis phase 2 , which will add another 120,000 square feet of research space , is currently underway and is targeted for completion at the end of this year . also under construction is phase 1 of a 390,000-square - foot center for physical sciences called fusionopolis , slated for completion in mid-2007 . it recently launched a preclinical research facility for neurodegenerative diseases , the first such facility in the asia pacific region , and its next investment in singapore will be a us$70 million r&d pilot plant dedicated to converting experimental molecules into commercially viable drugs . schering plough , merck , pfizer , and eli lilly also have active subsidiaries in singapore . in 2004 , swiss pharmaceutical company novartis teamed up with the singaporean government to create the novartis institute for tropical diseases , which is focused on studying neglected tropical diseases such as dengue fever and drug - resistant tuberculosis . singapore 's biomedical initiative is also bolstered by partnerships with overseas universities including stanford and johns hopkins universities in the us , sweden 's karolinska institute , and japan 's riken institutes . one example is the recent development of a diagnostic kit for avian influenza ( h5n1 ) . the kit , developed by local start - up company veredus laboratories pte ltd using nucleic acid primers designed at a*star 's gis , allows for rapid , accurate diagnosis of human cases of avian flu and is currently being used in indonesia . but the real question is whether the biomedical science push is translating into jobs and revenue . 10,200 people were employed in the country 's biomedical science industry , a figure the economic development board hopes will increase to 15,000 by 2015 . the biggest revenue source so far is the bulk drug manufacturing rather than the more recently established biotech sector . biomedical manufacturing output is also on the rise , surpassing its 2005 target of s$12 billion a year ahead of schedule . another part of keeping the biomedical ball rolling is keeping the money flowing a nonissue for the singaporean government , whose financial commitment to biomedical research has been unwavering . on the heels of the initial bms initiative comes the science and technology 2010 plan , announced in february of this year , which will commit another us$5 billion over five years toward bolstering public and private sector r&d . with its chunk of the change , the bmrc will focus on translational research with the hope of turning basic research discoveries into clinically useful and commercially viable products . but money alone will not ensure success . according to yeo , the most important elements for maintaining momentum in the biomedical sciences are two - legged ones , people ! indeed , singapore needs to maintain a critical mass of scientists , preferably while lessening its reliance on imported talent currently about one third of all scientists in singapore are foreigners . another brainchild of yeo , this endeavor involves a complete revamping of the country 's education system from overhauling the primary school curriculum to offering scholarship programs that fund undergraduate and ph.d . this training comes with strings attached : a*star scholars are committed to six years of research at an a*star research institute after graduating . a*star is also fishing out younger students with its new young researchers attachment program , in which pre - university students from around the globe study at singapore 's secondary schools and junior colleges , spending vacations on stints at a*star 's research institutes . those that do well are then eligible for a*star scholarships.whereas the us nih frowns upon scientists ' involvement in the private sector , singapore 's a*star does just the opposite . whereas the us nih frowns upon scientists ' involvement in the private sector , singapore 's a*star does just the opposite . biopolis boasts a complete work live play atmosphere . as with his recruitment efforts those who are funded are allowed only three years to complete their undergraduate degree and five for their ph.d . when you first start out at 18 years old , says pearline teo , an a*star scholar who earned her undergraduate degree in biology at johns hopkins university , teo , who is now working toward a ph.d . in immunology at stanford university , says the gpa requirement is a source of grumbling among her fellow a*star scholars , particularly at stanford , where only about 10% of the class receives an a grade . perhaps the biggest hurdle that singapore faces in revamping its education system is cultural . in a country where education is notoriously rote and rigid , it remains to be seen whether a*star students will measure up in terms of scientific creativity . as the first crop of ph.d . former singapore prime minister lee kuan yew famously stated that air conditioning was the most important invention for lifting tropical developing countries out of their heat - induced lethargy . now , singapore is taking the next step attempting to make the inventing process itself an integral part of the economy . we have the funds , single - minded focus , long - term planning , and industrial investment experience and the daring to go for it .

figure 1biopolis , singapore 's futuristic research hub.how does a country one - fourth the size of rhode island with little history in biomedical science become one of the world 's biomedical research giants ? the answer : with a pile of money and a large dose of chutzpah . since 2000 , singapore has dumped more than us$2 billion into developing a biomedical research industry from scratch . is the gamble paying off ?

A matter of life and death The personnel payoff IMMUNOLOGY PICKS UP SPEED Recipe for success Keeping the ball rolling

PMC4713815

scoliosis is a 3-dimensional vertebral transformation , accompanied by rotation and lateral deviation of each vertebra1 . scoliosis most often develops in childhood ( at around 10 years of age ) becoming apparent in adolescence , and is reported to deteriorate rapidly2 . an especially fast progression of scoliosis is shown during the adolescent period , when growth is the most active before it is completed , which has worsened in recent years due to a lack of physical activity and due to the prolonged use of smartphones and pcs in awkward postures3 . in cases of scoliosis that escape detection , patients complain of decreased social activity and backache , primarily due to changes in body shape , which are often accompanied by breathing difficulties . in extreme cases , the possibility of death due to a decline in cardiac and pulmonary function and respiratory failure increases , and surgery is inevitably necessary4 , 5 . thus , early detection is paramount , because the prognosis of undetected scoliosis is poor6 . the main methods used to diagnose scoliosis at clinical sites are cobb s angle , risser sign , vertebral stability and rotation of vertebrae ( perdriolle or nash - moe method)7 . most of these methods use expensive magnetic resonance imaging ( mri ) equipment . in view of the characteristics of scoliosis , patients do not complain of limitations to physical movement or discomfort at early stages , and only receive a diagnosis at clinical sites after vertebral arrangement progression becomes serious . many studies have found that examination at an early age ( 11 or 12 years ) is appropriate because early screenings for scoliosis enable treatment to be given at a critical point in time8 . although there are many diagnostic methods that use high - priced cutting - edge equipment , active treatment following quick early detection at schools or other youth facilities is the most efficient evaluation method and means of enabling treatment9 . in addition to examination methods that use harmful radiation , location comparisons of the shoulders , chest , waist , and pelvis by ocular inspection ; pulmonary function tests ; and measurements using a scoliometer along with forward bending , are also reliable measurement methods10 . this study carried out a correlation analysis based on quick predictive methods with the naked eye , such as the calcaneal valgus angle , the calcaneal angle and adam s forward bending test , in front - line facilities such as schools , then checked those methods preciseness and reliability in order to present the most effective evaluation method . the purpose of this study was to detect adolescent scoliosis early , to prevent unnecessary surgeries and aid proper treatment . this study targeted adolescents who received diagnoses of idiopathic scoliosis with 10 or more of cobb s angle on whole spine ap x - ray examination from rehabilitation medicine specialists . after potential subjects were informed of the objectives and procedures of the experiment , those who understood the purpose of the study , and agreed to actively participate in the work were enrolled in the study . this study received approval from the institutional review board ( irb ) of clinical test at gwangju oriental hospital of dongshin university on september 9 , 2013 ( irb no : dsgoh-017 ) . this study selected those subjects who had no experience with conservative treatment for scoliosis in the past , those who had no other problems in terms of orthopedics , and those with no history of cardiac problems and/or pulmonary disease . the participants physical properties are shown in table 1table 1.physical characteristics of subjectsitem classageheightweightbmifemale ( n=96)15.22.4160.75.547.96.018.51.9male ( n=22)16.52.4174.97.364.310.220.92.4total ( n=118)15.52.4163.48.050.99.518.92.2bmi : body mass index . for the adam s forward - bending test , participants placed their feet together , allowing their arms to hang naturally , and bent their upper bodies forward 90 from the standing position . examiners used visual evaluations , with their eyes at levels equal to those of the participants backs , and measured asymmetrical thoracic rotation angles ( atra ) and asymmetrical lumbar rotation angles ( alra ) at the rib hump and lumbar hump using a scoliometer ( usa ) ( table 2table 2.the mean and standard deviation of scoliosis related variablesitem classatraalralllcvarcvafemale ( n=96)7.13.46.94.041.66.67.93.87.43.9male ( n=22)6.52.95.23.841.67.56.73.76.94.5total ( n=118)7.03.36.64.041.66.77.73.87.34.0atra : asymmetry thoracic rotation angle ( ) ; alra : asymmetry lumbar rotation angle ( ) ; ll : lumbar lordosis ( ) ; lcva : left calcaneal valgus angle ( ) ; rcva : right calcaneal valgus angle ( ))11 . atra : asymmetry thoracic rotation angle ( ) ; alra : asymmetry lumbar rotation angle ( ) ; ll : lumbar lordosis ( ) ; lcva : left calcaneal valgus angle ( ) ; rcva : right calcaneal valgus angle ( ) participants were instructed to stand straight in order to view lumbar lordosis angles from the sagittal plane , bending the upper limbs forward by 90 , and keeping the eyes forward . lumbar lordosis angles were used to calculate crossed angles by drawing each line on the upper end - plate of the first lumbar vertebra and the upper end - plate of the first sacral vertebra , giving the calculated angle of the size of the curvature as shown in fig . total lumbar lordosis ( ll ) measurement for the analysis of the calcaneal valgus angles , this study performed a plumb line test13 , as shown in fig . 2.calcaneal valgus angle measurement ( a : left calcaneal valgus angle [ ] , b : right calcaneal valgus angle [ ] ) , using the body balance index system , steps , korea . this study measured the angle between the mid - calcaneal axis and the mid - talar axis . calcaneal valgus angle measurement ( a : left calcaneal valgus angle [ ] , b : right calcaneal valgus angle [ ] ) this study computed the mean and standard deviation of the factors related with scoliosis , and extracted variables with correlation at significance level of 0.05 . this set the degree of scoliosis as a dependent variable to predict thoracic and lumbar scoliosis using ankle angles and physique factors , and set height , weight , and left and right calcaneal ankle angles as independent variables , and then performed a multiple regression analysis to find meaningful explanatory variables . variables were extracted at a significance level ( ) of 0.05 by applying a stepwise method , and then the regression equation was calculated . tables 3table 3.summary of stepwise regression procedure for dependent variable atrasteppartial r2model r2c(p)bmi0.130.134.48lcva0.040.171.32bmi : body mass index ; lcva : left calcaneal valgus angle ( ) ; * * * p<0.0001 ( f=16.11 ) , * p < 0.05 ( f=5.24 ) and 4table 4.multiple regression analysis for predicting atravariableparameter estimatestandard errortype ii ssintercept14.192.72245.88bmi0.450.13106.79lcva0.180.0847.51atra = 14.190.45228 bmi + 0.17 lcvabmi : body mass index ; atra : asymmetry thoracic rotation angle ( ) ; lcva : left calcaneal valgus angle ( ) ; * * * p < 0.0001 ( f=27.13 ) , * * p < 0.001 ( f=11.78 ) , * p < 0.05 ( f=5.24 ) show the results of variable extraction and regression equations using a stepwise method of multiple regression analysis in order to predict thoracic scoliosis through height , weight , bmi , and left and right calcaneal valgus angles . bmi : body mass index ; lcva : left calcaneal valgus angle ( ) ; * * * p<0.0001 ( f=16.11 ) , * p < 0.05 ( f=5.24 ) bmi : body mass index ; atra : asymmetry thoracic rotation angle ( ) ; lcva : left calcaneal valgus angle ( ) ; * * * p < 0.0001 ( f=27.13 ) , * * p < 0.001 ( f=11.78 ) , * p < 0.05 ( f=5.24 ) as shown in table 3 , the variables that could predict only thoracic scoliosis were selected by applying the stepwise method ; thereby , bmi and left calcaneal valgus angles were selected . bmi and left calcaneal valgus angles explained 17% of atra , and were statistically significant variables ( p<0.05 ) . to estimate atra as shown in table 4 , this study set bmi and left calcaneal valgus angles as independent variables , and thoracic scoliosis angles as dependent variables , and constructed a regression equation . the root mean square error ( rmse ) was 1.48 ; therefore , bmi and left calcaneal valgus angles were effective variables to predict thoracic scoliosis ( p<0.05 ) . tables 5table 5.summary of stepwise regression procedure for dependent variable alrasteppartial r2model r2c(p)ll**0.070.071.46bmi0.020.091.35bmi : body mass index ; ll : lumbar lordosis ( ) , * * p < 0.001 ( f=7.47 ) and 6table 6.multiple regression analysis for predicting alravariableparameter estimatestandard errortype ii ssintercept17.233.72324.68bmi0.270.1832.49ll0.130.0676.61alra = 17.230.2696 bmi0.13 llbmi : body mass index ; alra : asymmetry lumbar rotation angle ( ) ; ll : lumbar lordosis ( ) , * * * p < 0.0001 ( f=21.48 ) , * p < 0.05 ( f=5.07 ) show the results of extraction of variables and the regression equation using stepwise multiple regression analysis methods in order to predict the degree of lumbar scoliosis through height , weight , bmi and left and right ankle valgus angles . bmi : body mass index ; ll : lumbar lordosis ( ) , * * p < 0.001 ( f=7.47 ) bmi : body mass index ; alra : asymmetry lumbar rotation angle ( ) ; ll : lumbar lordosis ( ) , * * * p < 0.0001 ( f=21.48 ) , * p < 0.05 ( f=5.07 ) as shown in table 5 , as the result of extracting the variables that can predict lumbar scoliosis by applying stepwise multiple regression analysis , lumbar lordosis and bmi were selected . the two variables explained 9% of alra , and were statistically significant variables ( p<0.05 ) . bmi and lumbar lordosis were as independent variables , and alra was set as a dependent variable to estimate alra , formulating the regression equation shown in table 6 . the rmse of estimation was 1.68 , therefore , the y - intercept was not zero ( p<0.0001 ) , and lumbar lordosis was an effective variable for predicting lumbar scoliosis ( p<0.05 ) . this study sought to identify degrees of scoliosis through the left and right calcaneal valgus angles , which can be observed with the naked eye without radiation techniques , and by using a scoliometer , with which measurements can easily be conducted . to enhance the reliability of this study , correlations were analyzed with each variable by additionally associating lumbar lordosis in the sagittal plane . ultimately , a negative correlation between lumbar lordosis ( ll ) and alra was revealed ( r=0.266 ) . compensation for this can be revealed through observing the degree of asymmetry in the tuberosity of the calcaneus . this is consistent with other studies14 which found that imbalances are revealed in the sagittal plane . compensation can be divided into two , namely , intraspinal compensation and extraspinal compensation . muscle function changes occur , resulting in hip , lumbar and cervical pain , while proper standing positions can not be maintained15 , 16 . the greatest degree of ll is found at the third lumbar vertebra , fourth lumber vertebra , or at the disk between them . lumbar segment lordosis starts between the first and second lumbar vertebrae , and gradually increases in the lower section . it was found that the greatest degree of segment lordosis is found between the fourth and fifth lumbar vertebrae , and the angle between the fifth and first lumbar vertebrae accounts for 60% of total lordosis12 . the pelvic tilt is the angle made by the line connecting the center of the sacral vertebral superior endplate and hip axis with the central plumb line , and is a location indicator reflecting pelvic compensation mechanisms when there are sagittal plane imbalances17 . from recent studies , it was revealed that the shape of the pelvis is important in in determining the curve of the sagittal plane17,18,19 . vialle et al.19 revealed that some correlation exists between pelvic incidence angles and ll in normal individuals . this assertion nearly matched the findings of this study , in that significant correlation exists between lumbar lordosis angles and alra . this study revealed that the left calcaneal valgus angle has a correlation with atra ( r=0.282 ) . when the left ankle arch collapses causing flat feet , and both shoulders become imbalanced , scoliosis appears to develop . namely , imbalance from the feet affects the legs , pelvis and vertebrae ; therefore , scoliosis is believed to be resultant . according to a preceding study , when load is added to a specific lower limb , energy consumption increases this energy consumption increase was reported to increase more when it was added to the end of the lower limb , rather than close to the trunk , despite the same weight20 . scoliosis is a disease in which 3-dimensional transformation of the vertebrae bend together to one side with vertebral rotation of one foot s load , arising from an imbalance of the feet ; therefore , it was reported that a change of the angle supporting the body weight , including shoes , can affect the prognosis of the disease21 . the pelvic obliquity attributable to rotational malalignment results in compensatory curves of the spine or the accentuation of any pre - existing curves . carole et al.23 , tested the reliability of a quantitative clinical posture assessment tool among persons with idiopathic scoliosis . the dependability coefficients ( ) for posture indices are presented in the tibiocalcaneal angles ( l and r ; =0.51 and 0.53 , respectively ) . according to 3-dimensional analysis results , it was reported that functional shoes are helpful to vertebrae , especially for lumbar muscular strength consolidation and vertebral health , through the improvement of feet stability25 . based on the results of this study and the many preceding studies , the progress of scoliosis can be predicted to some degree , depending on visual checks of the sole arch shape , tuberosity of the calcaneus , adam s forward bending test , and the use of a scoliometer . idiopathic scoliosis develops during adolescence and progresses rapidly26 , and therefore correction becomes difficult if the treatment window passes . therefore , it is important to diagnose scoliosis early27 . if scoliosis is diagnosed in its early stages through prompt examination and evaluation at schools or other facilities , curvature progression and unnecessary surgeries can be prevented through conservative treatment with walking aids and/or exercise , when curvatures are slight28 . first , calcaneal valgus angles and asymmetrical thoracic - lumbar rotation angles were only assessed in patients with adolescent scoliosis ; there was no control group . moreover , direct analysis of the correlations between cobb angles and calcaneal valgus angles and asymmetrical thoracic - lumbar rotation angles was not performed . future studies will be required to classify major curvatures in detail , and to analyze the relationship between cobb angles , asymmetrical thoracic - lumbar rotation angles and calcaneal valgus angles , the results of which may contribute to streamlined predictions and evaluations of idiopathic scoliosis .

[ purpose ] this study aimed to provide a predictable evaluation method for the progression of scoliosis in adolescents based on quick and reliable measurements using the naked eye , such as the calcaneal valgus angle of the foot , which can be performed at public facilities such as schools . [ subjects and methods ] idiopathic scoliosis patients with a cobb s angle of 10 or more ( 96 females , 22 males ) were included in this study . to identify relationships between factors , pearson s product - moment correlation coefficient was computed . the degree of scoliosis was set as a dependent variable to predict thoracic and lumbar scoliosis using ankle angle and physique factors . height , weight , and left and right calcaneal valgus angles were set as independent variables ; thereafter , multiple regression analysis was performed . this study extracted variables at a significance level ( ) of 0.05 by applying a stepwise method , and calculated a regression equation . [ results ] negative correlation ( r=0.266 ) was shown between lumbar lordosis and asymmetrical lumbar rotation angles . a correlation ( r=0.281 ) was also demonstrated between left calcaneal valgus angles and asymmetrical thoracic rotation angles . [ conclusion ] prediction of scoliosis progress was revealed to be possible through ocular inspection of the calcaneus and adams forward bending test and the use of a scoliometer .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2427366

mutagenesis and preparation of rna in vitro transcripts a plasmid encoding adenovirus type 2 ( ad2 ) va rnai was created with a 5-t7 rna polymerase ( t7 rnap ) promoter and 3-hepatitis delta virus ribozyme sequence followed by a drai restriction site for run - off transcription , as described previously ( 27 ) . va rnai mutants were generated in this plasmid by quikchange site - directed mutagenesis ( stratagene ) and confirmed by automated dna sequencing . the va rnai terminal stem ( ts ) was first deleted in three successive segments of 5 bp each to create the mutants ts5 , ts10 , and ts15 . for these rnas , the 3 terminal bp of the wild - type sequence were maintained to provide a strong promoter for t7 rna polymerase ( t7 rnap ) . a final deletion of the entire terminal stem , including the asymmetric bulge of nucleotides 2130 , created the construct ts21 . in ts21 the t7 rnap promoter was generated by reversal of the new terminal base pair , g c . a final mutation in the apical stem of full - length va rnai , 78cu , generated base pair rna . drai - linearized plasmid dna templates ( 100 g / ml ) were used in run - off transcription reactions ( 0.5 or 1.0 ml volume ) under optimal conditions for va rnai ( 28 ) using t7 rnap expressed from plasmid pt7 - 911 and purified by ni affinity chromatography ( 29 ) . rna transcripts were purified by preparative denaturing page with gels containing 50% urea and 8% acrylamide . va rnai bands were identified by uv shadowing , excised from the gel , and eluted using a biotrap device ( schleicher & schuell ) before ethanol precipitation and resuspension in te buffer . samples contained 2025 g of rna in a solution containing 10 mm mops buffer , ph 7.0 , and 50 mm kcl . for experiments with va rnai ( ts21 ) at different ph values , the following buffers were used in otherwise identical conditions : mes ( ph 5.5 , 6.0 , and 6.5 ) , mops ( 6.5 , 7.0 , and 7.5 ) , and hepes ( 7.5 and 8.0 ) . experiments performed with different buffers at the same ph gave identical results . uv melting curves were collected on a varian cary 400 uv - visible spectrophotometer with a 6-cell multichanger , running in dual beam mode . up to five melting curves were collected in each experiment with the sixth cell containing only buffer and fitted with an in - sample temperature probe . 3a ) , referred to as melting profiles , were calculated using a savitsky - golay algorithm as implemented in the program od deriv . pkr protein expression and purification pkr was expressed in escherichia coli in a nonphosphorylated form using plasmids encoding both the kinase and -protein phosphatase ( 30 , 31 ) . purification was accomplished using established procedures as follows : chitin affinity chromatography for pkr expressed from plasmid ptyb2-pkr(-pp ) ( 30 ) or sequential purification by heparin affinity , poly(i)poly(c ) rna - sepharose affinity , and gel filtration chromatographies on an ktapurifier 100 system for untagged pkr expressed from ppet - pkr / ppase ( 31 ) . poly(i)poly(c ) rna - sepharose was prepared using single - stranded poly(c ) ( 4 mg / ml ; ge healthcare ) and poly(i ) ( 4 mg / ml ; sigma ) rnas coupled to cnbr - activated sepharose 4b ff ( ge healthcare ) as described by wagner et al . pkr produced from ppet - pkr / protein phosphatase was used for all other experiments . pkr autophosphorylation inhibition assays purified pkr was dialyzed into 2 reaction buffer ( 100 mm tris , ph 7.8 , 100 mm kcl , 10% glycerol , 5 mm dithiothreitol ) . wild - type va rnai and each terminal stem deletion mutant rna were diluted into the same buffer to generate a 5 stock for each point in the inhibition assay ( see fig . pkr ( 2 l ; 0.1 g ) was preincubated with the va rnai ( 3 l ) at room temperature for 5 min . an equal volume of 2 pkr / activation reaction mixture was added , and each sample was incubated at room temperature for a further 10 min . the 2 pkr / activation reaction mixture contained 0.6 g / ml poly(i)poly(c ) , 4 m mgcl2 , 40 m atp , and 0.2 mci / ml [ p]atp ( 6000 ci / mmol , 10 mci / ml ; perkinelmer life sciences ) . samples were heated at 90 c for 25 min and fractionated by 10% acrylamide sds - page at 150200 v for 1 h. gels were fixed , dried , and exposed to an imaging plate ( ge healthcare ) and viewed using typhoon 8600 phosphorimager . control experiments without poly(i)poly(c)-activating rna showed that none of the va rnai samples activated pkr . isothermal titration calorimetry ( itc)the thermodynamics of va rnai - pkr interactions were measured using a vp - itc microcalorimeter following established procedures for va rnai ( 33 , 34 ) . rna and protein were dialyzed exhaustively into 10 mm sodium phosphate , ph 6.5 , 100 mm nacl , and 5 mm -mercaptoethanol . the sample cell contained ts21 rna at a concentration of 3 m , and the injection syringe contained pkr at 60 m . titration experiments were performed at 30 c and involved a single 2-l injection followed by 28 10-l injections of 24 s duration with 360 s spacing . titration curves were fit by a nonlinear least squares method in microcal origin software using a model for one or two binding sites . a model with two binding sites was found to give the optimal fit and was used to extract thermodynamic parameters kd , h , s , and n table 1thermodynamic parameters of pkr - rna interactionrnakdnhsgnmkcal / molcal / molkkcal / mol ts21 83 20.1 1.16 6.7 10.4 9.8 va rna1 79 9 nr 11.3 4.8 9.8 adata are from ref . 38bnr indicates not reported thermodynamic parameters of pkr - rna interaction data are from ref . gel filtration experiments were performed using a superdex 10/300 gl column ( ge healthcare ) attached to an ktapurifier 100 system and equilibrated with 10 mm sodium phosphate buffer , ph 6.5 , and 100 mm nacl . the pkr concentration was 3 m for all experiments that contained protein , whereas the rna concentration was 0.75 m ( 4:1 protein to rna ratio ) , 1.5 m ( 2:1 ) , 3 m ( rna alone and 1:1 ) , or 6 m ( 1:2 ) . to examine its role in creating or maintaining a functional va rnai structure that efficiently inhibits pkr , the ad2 va rnai terminal stem ( ts ) was progressively shortened . three successive deletions of 5 bp each and a final deletion of the entire terminal stem , including the asymmetric bulge of nucleotides 2130 , were made to create ts5 , ts10 , ts15 , and ts21 rnas , respectively ( fig . 1 ) . pkr kinase inhibition assays pkr autophosphorylation correlates well with subsequent activity against its primary cellular target eif2 ( 35 ) and was used as a convenient assay of efficacy of our mutant va rnai inhibitors . we anticipated that the activity of va rnai would decrease with each successive deletion to the terminal stem . the ability of each mutant rna to inhibit pkr autophosphorylation upon addition of dsrna activator rna was therefore measured and compared with wild - type va rnai ( fig . wild - type va rnai inhibited pkr completely at a concentration of < 5 g / ml , in agreement with previous studies ( 23 , 36 ) . as expected , the mutant rnas showed a decrease in activity for each successive deletion of 5 bp from the terminal stem , with 5 , 10 , and 50 g / ml rna required for full pkr inhibition by the ts5 , ts10 , and ts15 rnas , respectively ( fig . this most severely deleted rna displayed wild - type activity , completely inhibiting pkr autophosphorylation at a concentration of < 5 g / ml . to confirm this , the experiment was repeated at equimolar concentrations of wild - type and ts21 rnas ( because for an equal mass of rna in each reaction , the shorter va rnai ( ts21 ) would contain 50% more molecules ) . quantitation of these assays confirmed that va rnai ( ts21 ) inhibits pkr autophosphorylation at least as efficiently as wild - type rna despite lacking the entire terminal stem ( fig . impact of terminal stem deletions on va rnai structure rna uv melting analysis can be used to assess the impact of mutations upon the folding and stability of an rna molecule . the unfolding of large rnas can , however , be a complex process that occurs in a number of overlapping transitions ( 37 ) . we therefore describe unfolding transitions in the melting profile , the first derivative of the uv melting curve , as apparent transitions , each with an associated apparent melting temperature ( tm ) . we have shown that wild - type va rnai unfolds in two independent apparent transitions ( fig . 3a ) and assigned the lower ( 60 c ) and higher ( 85 c ) temperature apparent transitions to the unfolding of the terminal stem / central domain and apical stem , respectively ( 25 ) . this assignment is further confirmed by the present data as deletions in the terminal stem affect only the lower temperature apparent transition ( fig . the second unfolding transition , corresponding to the apical stem unfolding , is identical to that of wild - type va rnai in each case . proposed secondary structure for ad2 va rnai ( 16 ) , including a highly conserved pair of complementary tetranucleotide sequences ( outline typeface ) . shaded boxed regions indicate helical segments progressively deleted from the terminal stem to create rnas : ts5 ( nts 48 and 148152 ) , ts10 ( nts 413 and 143152 ) , ts15 ( nts 418 and 138152 ) , and ts21 ( nts 132 and 131155 ) . the white boxed region ( nts 13 and 153155 ) was retained in ts5 , ts10 , and ts15 rnas to maintain a strong promoter for in vitro transcription by t7 rnap ; in ts21 , base pair c g was reversed for this purpose , as indicated . a final point mutation , 79cu , was made to create an apical stem mutant rna ( bp rna ) with a watson - crick a proposed secondary structure for ad2 va rnai ( 16 ) , including a highly conserved pair of complementary tetranucleotide sequences ( outline typeface ) . shaded boxed regions indicate helical segments progressively deleted from the terminal stem to create rnas : ts5 ( nts 48 and 148152 ) , ts10 ( nts 413 and 143152 ) , ts15 ( nts 418 and 138152 ) , and ts21 ( nts 132 and 131155 ) . the white boxed region ( nts 13 and 153155 ) was retained in ts5 , ts10 , and ts15 rnas to maintain a strong promoter for in vitro transcription by t7 rnap ; in ts21 , base pair c g was reversed for this purpose , as indicated . a final point mutation , 79cu , was made to create an apical stem mutant rna ( bp rna ) with a watson - crick a a , assays of pkr autophosphorylation inhibition by wild - type va rnai and terminal stem deletion mutants ts5 , ts10 , ts15 , and ts21 . pkr ( 0.1 g ) was preincubated with 0 , 0.5 , 1 , 5 , 10 , and 50 g / ml of va rna prior to activation by poly(i)poly(c ) rna ( 0.3 g / ml ) . a progressive reduction in inhibitory activity is observed as the terminal stem is shortened , but this trend is sharply reversed for the largest deletion ts21 . b , assays of pkr autophosphorylation inhibition by wild - type ( wt ) va rnai and ts21 rna performed as in a but using equimolar rna concentrations ( 0 , 0.01 , 0.02 , 0.04 , 0.1 , 1.0 , and 10 m ) . c , quantification of the data shown in b. each experiment in a and b was repeated in at least three independent experiments . va rnai ts21 is an active inhibitor of pkr autophosphorylation . a , assays of pkr autophosphorylation inhibition by wild - type va rnai and terminal stem deletion mutants ts5 , ts10 , ts15 , and ts21 . pkr ( 0.1 g ) was preincubated with 0 , 0.5 , 1 , 5 , 10 , and 50 g / ml of va rna prior to activation by poly(i)poly(c ) rna ( 0.3 g / ml ) . a progressive reduction in inhibitory activity is observed as the terminal stem is shortened , but this trend is sharply reversed for the largest deletion ts21 . b , assays of pkr autophosphorylation inhibition by wild - type ( wt ) va rnai and ts21 rna performed as in a but using equimolar rna concentrations ( 0 , 0.01 , 0.02 , 0.04 , 0.1 , 1.0 , and 10 m ) . c , quantification of the data shown in b. each experiment in a and b was repeated in at least three independent experiments . the unfolding of the wild - type va rnai terminal stem and central domain are coupled and highly cooperative and result in the sharp apparent transition in the melting profile at 61 c ( fig . our previous studies demonstrated that even very subtle alterations in sequence , such as compensatory base pair exchanges , can be sufficient to uncouple these unfolding events into two or more apparent transitions ( 25 ) . such uncoupling of the first apparent transitions is observed for all the terminal stem deletion rna mutants ( fig . the ts5 rna terminal stem and central domain unfold as two apparent transitions , one at the original apparent tm ( 60 c ) and one at lower temperature ( tm 50 c ) . both ts10 and ts15 rna have a similar profile shape but , surprisingly , in addition to uncoupling the unfolding process , the apparent tm of each new apparent transition is also increased with the highest stability of the remaining terminal stem / central domain observed for ts15 rna . the melting profile for ts21 mutant is the most remarkable and distinct from wild - type va rnai . deletion of the terminal stem creates an rna with a very loose central domain structure that unfolds in two very broad apparent transitions centered around 45 and 65 c ( fig . 3b ) . a range of uv melting experiments conducted in the presence of various concentrations and types of monovalent cation or with mg did not indicate any specific stabilization of any domain of the rna ( data not shown ) . however , significant stabilization of the first broad apparent transition ( tm 45 c ) is observed at low ph ; over the range 7.5 to 5.5 a stabilization of 11 c is observed ( fig . 3c ) . in sharp contrast , the second broad transition ( tm 65 c ) corresponding to the remaining central domain structure is unaffected by changes in ph . 3d ) show that the central domain stability is also dependent upon ph in this context , although the tm is smaller , presumably as the structure is already stabilized close to the maximum extent possible by the intact terminal stem . specific protonation - dependent stabilization within the central domain most likely arises from a component of tertiary structure within the rna ( see discussion ) . the apical stem is also moderately stabilized at lower ph with a tm 4 c over the same range for both ts21 and wild - type va rnai ( fig . we hypothesized that the ph dependence of apical stem unfolding might arise from the presence of an ( ac ) mismatch pair predicted from the current secondary structure model ( fig . this was confirmed using a 79cu mutation to create a watson - crick a - u base pair at this position ( bp rna ) , removing the potential protonation site . this resulted in an apical stem structure that was most stable at neutral ph ( fig . analysis of wild - type and ts21 va rnai interaction with pkr we next examined the interaction of ts21 rna with pkr to explore potential differences in molecular recognition between this new mini - va rnai and the wild - type sequence . analytical gel filtration chromatography of pkr , va rnai , and pkr - va rnai complexes at various molar ratios was used to investigate the nature and stoichiometry of binding ( fig . wild - type va rnai eluted considerably earlier from the column than ts21 rna as expected based on the molecular size ( fig . pkr and both rnas in isolation showed a single major peak in the chromatogram characterized by strong 230 nm / no 260 nm and strong 260 nm / weak 230 nm absorbances , respectively . the protein and rna content of each peak for various complex ratios could therefore be determined using the relative absorbances . at an input molar ratio of 1:2 pkr to ts21 rna , a single peak corresponding to a complex of 1:1 stoichiometry is observed well resolved from a second later eluting peak corresponding to free rna . wild - type va rnai exhibits the same behavior , but the peaks are not well resolved because of the larger size of the rna . at an input ratio of 1:1 , for both rnas a single major peak is observed that elutes at the volume corresponding to the 1:1 complex . in contrast , at higher protein to rna ratios a difference in binding behavior is observed . for ts21 , although some higher molecular weight species are visible as a leading shoulder , a significant portion of the protein - rna complex still elutes at the volume corresponding to the complex of 1:1 stoichiometry ( fig . 4 , marked by dotted vertical line between 1:1 and 2:1 panels in the right column ) . in contrast , with a 2:1 input ratio , the pkr - wild - type va rnai complex elutes significantly earlier , corresponding to higher molecular weight complex , with no 1:1 stoichiometry complex remaining . for both rnas , a further increase in protein to rna input ratio decreases the elution volume for both complexes ( or mixtures of complexes ) , but at no concentration a , uv melting curves ( dashed lines ) and melting profiles ( first derivatives of the melting curves ; solid lines ) for wild - type va rnai collected at 260 ( blue ) and 280 nm ( black ) . the assignment shown of rna domain unfolding to each region of the melting profile was determined using compensatory base pair changes in each helical region of the rna ( 25 ) . b , melting profiles of wild - type va rnai ( black ) , ts5(blue ) , ts10 ( green ) , ts15 ( orange ) , and ts21 ( red ) at 280 nm . all terminal stem mutations affect only the first apparent transition ( tm 60 c ) as expected . ts21 rna unfolds in three apparent transitions with the first two corresponding to the remaining central domain structure . the first central domain apparent unfolding transition is dramatically stabilized by low ph ( tm 11 c between ph 5.5 and 7.5 ) , whereas the second apparent transition is unaffected ( tm 0 ) . the final apparent transition ( tm 85 c ) corresponds to the apical stem and shows a small increase in stability at lower ph ( tm 4 c ) . d , comparison of uv melting profiles at 280 nm for full - length wild - type va rnai and bp rna . as for ts21 rna , both the central domain ( tm 60 c ) and apical stem ( tm 85 c ) unfolding exhibit dependence upon ph in the context of the full - length wild - type rna , with greater stability at lower ph . in contrast , the ph dependence is reversed for bp rna , which lacks the ( ac ) mismatch pair ( fig . a , uv melting curves ( dashed lines ) and melting profiles ( first derivatives of the melting curves ; solid lines ) for wild - type va rnai collected at 260 ( blue ) and 280 nm ( black ) . the assignment shown of rna domain unfolding to each region of the melting profile was determined using compensatory base pair changes in each helical region of the rna ( 25 ) . b , melting profiles of wild - type va rnai ( black ) , ts5(blue ) , ts10 ( green ) , ts15 ( orange ) , and ts21 ( red ) at 280 nm . all terminal stem mutations affect only the first apparent transition ( tm 60 c ) as expected . ts21 rna unfolds in three apparent transitions with the first two corresponding to the remaining central domain structure . the first central domain apparent unfolding transition is dramatically stabilized by low ph ( tm 11 c between ph 5.5 and 7.5 ) , whereas the second apparent transition is unaffected ( tm 0 ) . the final apparent transition ( tm 85 c ) corresponds to the apical stem and shows a small increase in stability at lower ph ( tm 4 c ) . d , comparison of uv melting profiles at 280 nm for full - length wild - type va rnai and bp rna . as for ts21 rna , both the central domain ( tm 60 c ) and apical stem ( tm 85 c ) unfolding exhibit dependence upon ph in the context of the full - length wild - type rna , with greater stability at lower ph . in contrast , the ph dependence is reversed for bp rna , which lacks the ( ac ) mismatch pair ( fig . 5 ) under conditions used previously to characterize the binding of various rnas to pkr ( 34 , 38 ) . in line with previous observations , the titration curve for ts21 rna was best fit using a model for two binding sites , and the values derived for parameters associated with the high affinity binding site are given in table 1 . most strikingly , although the enthalpic and entropic contributions to binding differ dramatically for ts21 and wild - type va rnai , the resulting binding affinity and overall free energy change are identical . va rnai has long been recognized as a potent inhibitor of the cellular anti - viral defenses mediated by pkr . a large body of evidence has identified the apical stem of va rnai as the primary binding site and its complex central domain as the major determinant of inhibitory activity ( 1324 ) . although far less extensively examined , prior mutagenesis suggested that an intact terminal stem structure , particularly adjacent to the central domain , might also contribute to inhibition of pkr ( 21 ) . our previous studies of va rnai unfolding supported this idea because the central domain stability was found to be coupled to and dependent upon that of the terminal stem ( 25 ) . thus , in addition to carrying promoter sequences , the terminal stem might play an indirect role in va rnai activity by acting as a structural clamp to stabilize the functional rna tertiary fold of the central domain or to protect against rna unwinding or nuclease attack . elution of wild - type ( wt ) va rnai ( left column ) and ts21 rna ( right column ) from a gel filtration column , alone and with pkr at various molar ratios ( indicated in the top right of each panel as input protein : rna ratio ) . uv absorbance was measured at both 260 and 230 nm to allow the relative protein and rna content of each peak to be determined . elution of wild - type ( wt ) va rnai ( left column ) and ts21 rna ( right column ) from a gel filtration column , alone and with pkr at various molar ratios ( indicated in the top right of each panel as input protein : rna ratio ) . uv absorbance was measured at both 260 and 230 nm to allow the relative protein and rna content of each peak to be determined . to directly assess its contribution to va rnai structure and pkr inhibition , we used site - directed mutagenesis to systematically shorten the terminal stem helix . in doing so , we also hoped to identify an rna with a significantly shortened terminal stem that might be beneficial for in vitro biochemical and biophysical analyses of pkr - rna interactions . for example , deletion of a significant length of the helix would remove the potential for the terminal stem to provide secondary , or creation of a minimal va rnai inhibitor of pkr the terminal stem of va rnai was deleted in three sequential steps of 5 bp and a final deletion of the entire helix ( fig . we anticipated that a progressive reduction in pkr inhibitory activity would be observed but that it might be possible to identify a point at which the terminal stem could be deleted without significant loss of function . for the first three rnas , ts5 , ts10 , and ts15 , this expectation was met , with each requiring more inhibitor rna to provide complete inhibition of pkr . remarkably , however , the trend was completely reversed for ts21 rna , which exhibited wild - type activity ( fig . 2 ) despite completely lacking a terminal stem domain . as the apical stem in isolation is a weak activator of pkr and the two conserved tetranucleotide sequences in the central domain immediately adjacent to the 21 deletion are critical for efficient inhibition , the ts21 rna created here is the shortest possible va rna inhibitor of pkr ( mini - va rnai ) . structural impact of terminal stem deletions uv melting experiments were used to assess the global impact on the rna structure of each terminal stem deletion . in line with previous observations ( 25 ) , deletions in the terminal stem affected only the lower temperature apparent transition that corresponds to the coupled unfolding of the terminal stem and central domain . even the smallest deletion , ts5 , uncouples the unfolding of these domains such that two apparent transitions are observed , one at lower apparent tm and the other at the original tm . similarly shaped profiles are observed for ts10 and ts15 rnas but with variation in both the hypochromicity and tm of both apparent transitions ( fig . the changes observed in apparent tm might reflect a reorganization of the base pairing within the terminal stem , e.g. to incorporate nucleotides in the 2430 loop as the lower stem is shortened ( and presumably destabilized ) . terminal stem structures corresponding to the wild - type and each deletion mutant sequence , but with a short stable stem - loop above base pair 39120 in place of the central domain and apical stem , were assessed using mfold ( 39 ) . two identical low energy structures were identified for each rna , with the secondary structure of fig . 1 corresponding to a marginally less stable structure ( data not shown ) . it is therefore plausible that the changes observed in the melting profiles correspond to a switch in the pairing scheme to the alternative structure identified as the stem is shortened . regardless of the precise pairing , however , such alterations clearly do not have a major impact on the remaining central domain structure ; for deletions up to ts15 , the melting profiles indicate a folded structure and the steady decrease in pkr inhibition activity correlates with the size of the deletion . itc analysis of rna - protein interaction for titration of pkr ( 60 m ) into ts21rna ( 3 m ) . the titration heats measured on binding ( top panel ) were integrated to produce the binding isotherm ( lower panel ) , and this fit to a model for two binding sites using the origin software package . the thermodynamic parameters determined from the fit for the first , high affinity , binding site are shown in table 1 . itc analysis of rna - protein interaction for titration of pkr ( 60 m ) into ts21rna ( 3 m ) . the titration heats measured on binding ( top panel ) were integrated to produce the binding isotherm ( lower panel ) , and this fit to a model for two binding sites using the origin software package . the thermodynamic parameters determined from the fit for the first , high affinity , binding site are shown in table 1 . the melting profile for the remaining central domain structure of ts21 rna is , however , dramatically different with two very broad peaks of low hypochromicity ( fig . we initially took this result to indicate that the central domain of va rnai was largely unfolded in the absence of an intact terminal stem , supporting the idea of its role as a structural clamp to secure the functional central domain structure ( 19 ) . although this may be true , from the high activity of the ts21 rna the terminal stem is clearly not essential for pkr inhibition . it is also interesting to note that in the itc analysis of the ts21 rna - pkr interaction , there is a large change in the enthalpic and entropic contributions to binding despite an overall identical g and binding affinity as for wild - type - va rnai . although we are cautious in attempting to interpret the physical meaning of these changes , both the uv melting profiles and other spectroscopic analyses we have conducted with ts21 rna5 point to a flexible , loosely folded central domain structure . earlier structure probing experiments on the full - length rna have shown that both pkr and mg can induce similar conformational changes upon binding ( 15 ) . these observations suggest that va rnai has a dynamic flexible structure , most pronounced in the absence of a terminal stem in ts21 rna , that is folded or stabilized into the correct functional tertiary structure upon interaction with pkr . the va rnai central domain contains a ph - dependent tertiary structure removal of the terminal stem allowed us to assess in more detail other factors that might influence the va rnai central domain tertiary folding . as for wild - type va rnai ( 25 ) , no significant specific stabilization of any apparent unfolding transition was observed in melting experiments containing various concentrations of mg or different monovalent ions ( data not shown ) . one component of the central domain was , however , specifically stabilized by low ph with a striking difference in apparent tm of 11 c over the ph range 5.57.5 . this result provides clear evidence for the involvement of a protonated base in creating the va rnai central domain structure , such as a protonated cytosine in forming base triple interactions as observed in dna ( 40 , 41 ) and rna triplexes ( 42 , 43 ) , and other rna tertiary structures ( 4446 ) . the most obvious candidates for the site(s ) of protonation within the central domain are cytosines 104105 , 107 , or 116 in an interaction with base pairs in the lower stem of the rna ( nts 120130 ) . interestingly , c is immediately adjacent to the conserved pair of tetranucleotides also proposed to be involved in the rna tertiary structure ( 20 ) . however , point mutations at each of these nucleotides ( to either g or a ) did not dramatically reduce the inhibitory function of the resulting rna ( 24 ) . therefore , confirmation of the site of protonation and whether this is critical for central domain tertiary structure folding and/or pkr inhibition will require further detailed investigation . implications for analysis of pkr - rna interaction the terminal stem is an imperfectly paired helix that could potentially provide a secondary binding site for the dsrna binding domain of pkr . our gel filtration data shows differences in complex formation with excess pkr between wild - type and ts21 rnas . for both rnas similar large ( nonspecific ) complexes are ultimately formed , but unlike the wild - type rna , ts21 rna initially maintains a predominantly 1:1 complex stoichiometry suggesting that it does lack some lower affinity binding site for pkr . itc data for va rnai and other inhibitor and activator rnas are best fit using a model for two sites , where the lower affinity site is ascribed to nonspecific binding and otherwise ignored ( 33 , 34 , 38 ) . our itc analysis of ts21 rna , which lacks the terminal stem , shows two distinct binding events . the kd and g values for the high affinity site are in excellent agreement with previous values for va rnai ( table 1 ) although , as noted above , with large compensating changes in h and s . more curiously , even though the second weak affinity binding site is well resolved in the titration and the data apparently well fit by a two - site model , the thermodynamic parameters obtained for this site are highly unrealistic . the shape of the titration suggests a binding site of moderate affinity , but a kd of 0.5 nm is obtained . we hypothesize that this binding site in fact arises from conformational heterogeneity in the va rnai apical stem ( 20 ) . rather than a single rna molecule with two binding sites , the data reflect a mixed population of rna structures with the fully extended apical stem predominant and producing the high affinity site . we predict that rnas with modified apical stems that remove this heterogeneity will produce a pkr binding titration curve fit optimally with a single high affinity site , and experiments to test this hypothesis are currently underway . va rnai is processed by dicer to produce short single - stranded rnas from the ts that are incorporated into risc , and an apical stem - central domain ( as - cd ) rna , equivalent to ts21 rna , that remains fully active against pkr . thus , each transcript from the va rnai gene is able to both saturate cellular rna interference mechanisms and block the pkr - mediated down - regulation of general translation of cellular and viral proteins via eif2 phosphorylation . abbreviations used for proteins are as follows : pkr , dsrna binding motifs ( 1 and 2 ) ; kd , kinase domain va rnai is processed by dicer to produce short single - stranded rnas from the ts that are incorporated into risc , and an apical stem - central domain ( as - cd ) rna , equivalent to ts21 rna , that remains fully active against pkr . thus , each transcript from the va rnai gene is able to both saturate cellular rna interference mechanisms and block the pkr - mediated down - regulation of general translation of cellular and viral proteins via eif2 phosphorylation . abbreviations used for proteins are as follows : pkr , dsrna binding motifs ( 1 and 2 ) ; kd , kinase domain . va rnai function in vivo the va rnai terminal stem has been suggested to play a role in stabilizing or protecting the critical central domain against degradation in vivo ( 21 ) . although our results do agree that the terminal stem provides a large degree of structural stabilization , it is clearly not necessary for pkr inhibition . the active mini - va rnai we have produced in vitro is strikingly similar to the product of dicer processing of the full - length rna in vivo ( 26 ) . thus , adenovirus may both inhibit pkr and saturate dicer using a single gene product where each rna transcript contributes to both mechanisms ( fig . va rnai may therefore be a truly bifunctional rna and exemplify a remarkably parsimonious use of short rna transcripts to counteract host cell defenses against viral infection .

adenoviruses use the short noncoding rna transcript virus - associated ( va ) rnai to counteract two critical elements of the host cell defense system , innate cellular immunity and rna interference , mediated by the double - stranded rna - activated protein kinase ( pkr ) and dicer / rna - induced silencing complex , respectively . we progressively shortened the va rnai terminal stem to examine its necessity for inhibition of pkr . each deletion , up to 15 bp into the terminal stem , resulted in a cumulative decrease in pkr inhibitory activity . remarkably , however , despite significant apparent destabilization of the rna structure , the final rna mutant that lacked the entire terminal stem ( ts21 rna ) efficiently bound pkr and exhibited wild - type inhibitory activity . ts21 rna stability was strongly influenced by solution ph , indicating the involvement of a protonated base within the va rnai central domain tertiary structure . gel filtration chromatography and isothermal titration calorimetry analysis indicated that wild - type va rnai and ts21 rna form similar 1:1 complexes with pkr but that the latter lacks secondary binding site(s ) that might be provided by the terminal stem . although ts21 rna bound pkr with wild - type kd , and overall change in free energy ( g ) , the thermodynamics of binding ( h and s ) were significantly altered . these results demonstrate that the va rnai terminal stem is entirely dispensable for inhibition of pkr . potentially , va rnai is therefore a truly bi - functional rna ; dicer processing of the va rnai terminal stem saturates the rna interference system while generating a mini - va rnai molecule that remains fully active against pkr .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION

PMC4550659

research data repositories based on platforms such as dspace were introduced about 10 years ago , and their use in domains such as chemistry and molecular sciences has gradually increased [ 2 , 3 ] . their importance has recently come to the fore with funding agencies in the usa , europe and asia all indicating that open deposition of research data will become a mandatory aspect of their funding , and many universities are now starting to consider the implications of implementing research data management , or rdm [ 46 ] . an early example of such rdm is illustrated with a project to produce a library of quantum - mechanically - optimised molecular coordinates derived from a computable subset of the national cancer institutes ( nci ) collection of small molecules . the information for each molecule was originally annotated by optimising the coordinates with respect to the energy obtained using the semi - empirical pm5 parameter set in mopac ( then the most current parameter set ) and creating a dspace collection . at the commencement of the present project , the original deposition of this information for 175,356 molecules into the institutional repository of the university of cambridge represented the only openly accessible copy . an issue frequently raised in the context of research data management relates to the prospects of being able to access and use such digitally held information in the future . relatively recently , such questions were largely directed towards the expected longevity of physical media such as punched cards and floppy disks ( both now effectively extinct ) , hard drives , cdroms , dvds , magnetic tape etc . few of these media have proven lifetimes exceeding 20 years and the real problem would be locating working devices capable of reading such physical media in the future . quite different problems are associated with virtual collections , where the physical medium is less important than the information associated with the data itself . in this context , it is becoming increasingly accepted that successful long - term preservation of digital data depends upon repeated incremental improvements or curations taking place in 510 year cycles . such operations can in principle be repeated indefinitely , thus creating a long - term mechanism with an anticipated lifetime of 100 + years if required . these curation cycles can track the evolution of data storage hardware , data formats and introduction of new software , so ensuring that the data remains accessible and in a usable form . the purpose of this project was to explore the viability of the long - term preservation of the 10 year old cambridge dataset through such an incremental curation by performing its migration to the spectra repository hosted at imperial college london . specific benefits of undertaking such a curation include re - filtering the original source data for errors not previously eliminated , to produce an enhanced metadata record for each entry , and to recompute the optimised molecular coordinates by using the newer pm7 method . the original pm5 method used to obtain the molecular geometries was never formally published and is now unavailable , whereas the succeeding pm7 method has been formally peer reviewed and published . we will also compare our approach with two other examples drawn from computational chemistry . the first is typical of how almost all datasets derived from molecular computations are currently curated ; in this case the stochastic generation of all possible stable molecular structures from an initial set of specified atoms . the trend in scientific publication in recent years has required authors reporting such studies to include more extensive data in the form of supporting information ( si ) to accompany the scientific narrative from which their models are constructed and their conclusions drawn . we will argue here that these si - based mechanisms for depositing , retrieving and re - using the data components of journal articles are no longer fit for this purpose ( if indeed they ever were ) and should be urgently replaced by repositories of data and closely - coupled metadata as a fundamentally different model for research data management . the second example describes such a deposition of a dataset containing the quantum mechanically computed structures and properties of 134,000 molecules into the figshare digital repository . we will ask here what the attributes of such a deposition must be in order to enable efficient formal re - curation 10 years after the original creation of the dataset , arguing that there are some essential structures and standards that must be fulfilled for such a process to be properly enabled . the migration of the original dataset was performed in three sequential phases , retrieval from the original repository , a technical validation and re - deposition into the spectra repository . although this software contains a component that can provide structured data representations of entries for harvesting ( oai - ore resource maps ) , this was not enabled on the cambridge repository when we started our migration in july 2014 . however , since the human - readable landing pages for each entry all conform to a structured html template , it nevertheless proved possible to extract all the data using ad hoc scripting and html processing ( a process often informally referred to as web - scraping ) . this process was markedly inefficient , requiring three separate http requests to the server per record , and took several days to complete . this approach is by no means unique ; most large existing collections of ( chemical ) data require similar processes whereby a human has to initially read the documentation ( if available ) for the templates used to access the items and then to write appropriate custom codes or scripts to retrieve them . such a method means that any unexpected change in the template resulting from , for example , the release of a new version of the dataset then inherits the risk of breaking these scripts . stated more formally , the inferred uniform resource locators ( urls ) for such collections of data are not persistent . the principal aim of our curation objectives therefore was to eliminate the need for such ad hoc scripting and replace it with a more efficient and standards - based workflow for achieving this persistence . the following were retrieved from the original deposition at cambridge : the source url for 175,356 records.175,356 documents in xml - cml syntax encoded using chemical mark - up language ( cml ) , containing a molecular structure from the nci database and some metadata describing the entry.158,879 xml - cml documents containing the pm5 optimized coordinates of the nci database structure and basic metadata , including the nci identifier for version 3 of the nci open database and the computed inchi and inchikey . of these these entries were previously identified as having additional complexities such as the presence of metal atoms or problems with correctly adding hydrogen atoms and charges , and so a pm5 calculation had not been attempted . here we have adopted the same strategy of not recovering these entries in the present curation . 175,356 documents in xml - cml syntax encoded using chemical mark - up language ( cml ) , containing a molecular structure from the nci database and some metadata describing the entry . 158,879 xml - cml documents containing the pm5 optimized coordinates of the nci database structure and basic metadata , including the nci identifier for version 3 of the nci open database and the computed inchi and inchikey . of these these entries were previously identified as having additional complexities such as the presence of metal atoms or problems with correctly adding hydrogen atoms and charges , and so a pm5 calculation had not been attempted . here we have adopted the same strategy of not recovering these entries in the present curation . no metadata were provided in the original depositions that gave an unambiguous description of the two xml - cml documents present in the form of a cml schema declaration , and no mopac version information or mopac input or output files were saved to act as alternative sources of this information . the cml syntax corresponding to the annotation derived from pm5 optimisation in the form of files named e.g. nsc138467_post - mopac.cml in the original collection was incomplete ; bond connection terms were missing and the cml documents failed validation according to the cml schema version 2.4 . the first task was therefore to develop a protocol to produce a reliable and valid input file suitable for re - calculating the properties using the newer pm7 method . many entries in the nci collection comprise two or more disconnected components , of which only the larger component was retained in the original editing . the resulting missing component in the starting structure was predominantly a counter ion and its removal requires a charge to be assigned to the remaining fragment . this information was originally captured in both original xml - cml documents , the first as part of an identifier element containing an early form of the inchi string : < identifier version=0.932beta tautomeric=0 > < basic > c13h21n2o,1h3 - 12h(2h3)15(13h(3h3)4h3)11(16)10 - 7h-6h-8h-14(5h3)9h-10</basic > < charge>+1</charge > < /identifier > the second is declared more formally in the cml molecule element associated with the pm5 calculation : < molecule id=nsc138467 formalcharge=1 name=mol1 > of the 158,122 unique documents in the latter category , the formalcharge declarations were distributed as follows ; 153,127 ( 0 ) , 28 ( 1 ) , 4,456 ( + 1 ) , 18 ( 2 ) , 483 ( + 2 ) , 2 ( 3 ) , 3 ( + 3 ) , 1 ( + 4 ) , 4 ( 5 ) . manual inspection of the species with very large formal charges ( > |3| ) indicates these are all errors arising from the original curation process because of incorrect interpretations of e.g. metal centres . our original attempt to transform this information into a mopac input involved the standard openbabel program , version 2.3.2 . it transpired openbabel did not correctly propagate the charge information in either of the original cml files by transformation into an appropriate mopac keyword declaration such as charge = 1 . instead the generic statement put keywords here was the only content of the mopac keyword line . this raises some interesting issues : absolute fidelity in any syntactic transformation of data from one format to another is very difficult to achieve . thus there are often multiple syntaxes for any given information field , such as the two shown above for expressing the charge on a molecule , and all such variations must be honoured with complete fidelity to achieve reliability . although some forms can be quickly deprecated ( such as the first example above ) , these forms can not be ignored and they must be processed.the mopac program does not mandate the presence of all keywords . a calculation may still succeed on the assumption that a missing keyword simply defaults to a pre - determined value . in this case , mopac will assume that the value of an undeclared charge keyword corresponds to zero , which is a clear error if the charge was intended to be non - zero . it can be very difficult , if not impossible to find complete definitions of what implicit assumptions are made in any system . often the only source of these is the actual computer code itself.a further implicit rule for mopac keywords is that the spin - multiplicity of the system is computed from the total electron count after the appropriate charge is applied . for a system where a charge of e.g. + 1 is left undeclared , that will result in a molecule with an odd number of electrons , and this is then treated implicitly as a molecule with a doublet spin state . we also note that these implicit rules are not universal ; other programs such as gaussian use different conventions.if the explicit keyword singlet ( spin state ) is declared , a safe assumption for virtually all real molecules that exist as physical samples , this can act as a checksum . the mopac program will then throw an error and the calculation will not proceed if this spin state conflicts with any declared or undeclared / implicit charge . absolute fidelity in any syntactic transformation of data from one format to another is very difficult to achieve . thus there are often multiple syntaxes for any given information field , such as the two shown above for expressing the charge on a molecule , and all such variations must be honoured with complete fidelity to achieve reliability . although some forms can be quickly deprecated ( such as the first example above ) , these forms can not be ignored and they must be processed . a calculation may still succeed on the assumption that a missing keyword simply defaults to a pre - determined value . in this case , mopac will assume that the value of an undeclared charge keyword corresponds to zero , which is a clear error if the charge was intended to be non - zero . it can be very difficult , if not impossible to find complete definitions of what implicit assumptions are made in any system . a further implicit rule for mopac keywords is that the spin - multiplicity of the system is computed from the total electron count after the appropriate charge is applied . for a system where a charge of e.g. + 1 is left undeclared , that will result in a molecule with an odd number of electrons , and this we also note that these implicit rules are not universal ; other programs such as gaussian use different conventions . if the explicit keyword singlet ( spin state ) is declared , a safe assumption for virtually all real molecules that exist as physical samples , this can act as a checksum . the mopac program will then throw an error and the calculation will not proceed if this spin state conflicts with any declared or undeclared / implicit charge . instead of using openbabel , we made a custom conversion of the original post - mopac pm5 calculation into cml files to ensure the correct keywords were written to the mopac input file . this is not a critical decision , since the final atom positions do not in general depend on the initial coordinate system selected . a pm7 geometry optimisation was then performed using the resources of the imperial college high performance computing service . the majority of calculations completed within tens of seconds and the total required approximately 20 cpu days of computer time . an inchi identifier is a canonicalization based on the atom connectivity of a molecule , which in turn is derived from cartesian coordinates for each atom using simple heuristic rules specifying a range of atom pair distances for any element combination . unfortunately , atom connection distances are not formally defined as accepted standards , and the precise values are ultimately the choice of the designers of any program implementing them . the limits however are usually sufficient tolerant to cover the vast majority of real molecules without any disagreement , and this would especially be true of the nci set which cover real systems rather than hypothetical or computed molecules . this does not entirely exclude there being a very small number of molecules where specific atom - pair distances might fall within e.g. a bond range using pm5-optimised coordinates but which are e.g. outside such a range using pm7 values . we note that whilst it is possible to replace these relatively arbitrary rules by using a quantum mechanically derived property of the electron density topology called the bcp ( bond critical point ) to define an atom - pair connectivity , this is not currently used for determining inchi identifiers . we proceeded to derive inchi identifiers using the following openbabel commands : babel -i cml nsc383508_original.cml -o xyz out.xyz --canonical ( for the original nci - based data)babel -i cml nsc383508_post - mopac.cml -o xyz out.xyz --canonical ( for the pm5-computed data ) babel -i mopout mopac-pm7.out -o xyz out.xyz --canonical ( for the newly generated pm7-computed data).babel -i xyz in.xyz -o inchi out.inchi babel -i cml nsc383508_original.cml -o xyz out.xyz --canonical ( for the original nci - based data ) babel -i cml nsc383508_post - mopac.cml -o xyz out.xyz --canonical ( for the pm5-computed data ) babel -i mopout mopac-pm7.out -o xyz out.xyz --canonical ( for the newly generated pm7-computed data ) . babel -i xyz in.xyz -o inchi out.inchi commands 13 convert all the data into cartesian coordinates to remove any possible atom connection data that might have been generated by mopac or other sources . this process ensures that the connectivities created using the last command and then used to create the inchi are normalised against a single connection algorithm ( being the one contained in openbabel , version 2.3.2 ) . these inchi strings are then compared with those derived in a similar manner using the original nci and the original pm5 computed coordinates ( table 1).table 1comparison of generated inchi identifiersnci = pm5 = pm7(pm5 = pm7 ) nci(pm7 = nci ) pm5(pm5 = nci ) pm7pm7 pm5 nci154,5522,041573470486 comparison of generated inchi identifiers of the 158,122 unique values ( table 1 ) , 97.7 % matched for all three instances , which provides a great measure of confidence that the atom - connection algorithm is robust . to identify the origin of the 2.3 % of inchi mis - matches , we have to dissect the inchi identifier itself into its component layers : the molecular formula layer ( 1131).the pairwise atom connectivity layer , determined as described above ( 127).the hydrogen layer , in which hydrogen atoms are added to all heavy atoms where a valence is perceived to be unsatisfied if the hydrogens are not already declared . because we have subjected all the systems to computational quantum modelling , all hydrogen atoms are already explicitly defined in our coordinates ( 1252).a charge layer , also defined for all the molecules in our collection ( 9).a stereochemical layer . because our coordinates are all specified in 3d space , the stereochemistry is always defined . this layer includes double - bond isomerism ( 292 ) and tetrahedral configurations ( 267).an isotope layer ( 22 ) . the molecular formula layer ( 1131 ) . the pairwise atom connectivity layer , determined as described above ( 127 ) . the hydrogen layer , in which hydrogen atoms are added to all heavy atoms where a valence is perceived to be unsatisfied if the hydrogens are not already declared because we have subjected all the systems to computational quantum modelling , all hydrogen atoms are already explicitly defined in our coordinates ( 1252 ) . a charge layer , also defined for all the molecules in our collection ( 9 ) . a stereochemical layer . because our coordinates are all specified in 3d space , the stereochemistry is always defined . this layer includes double - bond isomerism ( 292 ) and tetrahedral configurations ( 267 ) . an isotope layer ( 22 ) . the distribution of the 2,997 differences between the pm7 and the nci inchi identifiers ( 2,041 + 470 + 486 , table 1 ) are shown in parenthesis in the listing above , and each is very briefly discussed below : the discrepancies in the formula layer originate from mismatches in the hydrogen count . this is because , historically , molecules were not always defined with explicit coordinates for all hydrogen atoms . instead they were inferred from residual valences , these in turn inferred from bonding angles and other geometric and heuristic information . the process of replacing such implicit hydrogens with explicit ones is not always exact.the connection layer mismatch originates from bonds that are on the verge of connection and derives from ( possibly small ) geometric changes from the quantum mechanical re - optimisation . s bonds in sulfur species .this and the formula layer together account for the great majority of the mis-matches.the small number of mis - matches in the charge layer may result from the inchi code heuristic for deciding the appropriate charge for a molecule . as noted above , we detected some unreasonable high charges resulting from this process.because traditionally molecules were expressed in the molfile v2 format which allows just 2d coordinates to be defined , stereochemistry had to be added using an additional parameter associated with each bond connection and equivalent to the stereochemical wedge notations used in organic chemistry . this information is not free of ambiguity , since the stereochemistry is defined relative to other atoms and can lead to logical contradictions . when such two dimensional coordinates and this additional information is converted into 3d coordinates ( a process carried out during the original deposition ) , ambiguities can result.isotopes were not included in the mopac - pm7 calculation . this is because , historically , molecules were not always defined with explicit coordinates for all hydrogen atoms . instead they were inferred from residual valences , these in turn inferred from bonding angles and other geometric and heuristic information . the connection layer mismatch originates from bonds that are on the verge of connection and derives from ( possibly small ) geometric changes from the quantum mechanical re - optimisation . this and the formula layer together account for the great majority of the mis - matches . the small number of mis - matches in the charge layer may result from the inchi code heuristic for deciding the appropriate charge for a molecule . because traditionally molecules were expressed in the molfile v2 format which allows just 2d coordinates to be defined , stereochemistry had to be added using an additional parameter associated with each bond connection and equivalent to the stereochemical wedge notations used in organic chemistry . this information is not free of ambiguity , since the stereochemistry is defined relative to other atoms and can lead to logical contradictions . when such two dimensional coordinates and this additional information is converted into 3d coordinates ( a process carried out during the original deposition ) , ambiguities can result . isotopes were not included in the mopac - pm7 calculation . for each remaining entry , the pm7-derived inchi strings and keys were added to the smiles strings and the nci and cas accession identifiers obtained from the original data and propagated as metadata . we note that the nci identifiers themselves may not necessarily persist across different versions of the nci database , which was version 3 at the time of the original curation and has subsequently been updated to version 4 in 2012 . prior to import to spectra , each entry was packaged individually to produce an archive file , termed a sword [ 21 , 22 ] bundle . sword ( simple web - service offering repository deposit ) is an interoperability standard for data ingest into digital repositories , rendering these bundles suitable for import into any sword - compliant repository , not just dspace - based spectra . the bundles contains a mets manifest and data files and were created using a locally written tool . the mets manifest contained the following metadata : inchi and inchikey and smiles string.cas and nci accession ids , nci entry name.back-link back to the entry in the cambridge repository.doi link to the published description .orcid identifiers for the contributing authors.link to creative commons license terms . the datafiles included within the bundle were : two cml files containing unaltered copies of the nci coordinates and pm5 computed mopac output documents obtained from the original source repository.a third cml file conflating the three previous structures , containing the original nci structure , the original pm5 structure from the original repository and the newly computed pm7 structure.mopac input and output files for the new pm7 calculation . two cml files containing unaltered copies of the nci coordinates and pm5 computed mopac output documents obtained from the original source repository . a third cml file conflating the three previous structures , containing the original nci structure , the original pm5 structure from the original repository and the newly computed pm7 structure . mopac input and output files for the new pm7 calculation . import of this fileset to the destination spectra repository was performed using the sword web service interface . owing to a limitation of the dspace - spectra sword interface , no bulk - import function was available and all of the new packages had to be to uploaded individually , a process that took approximately 60 days . doubtless this exceptionally long time resulted from some undiagnosed server misconfiguration and should not be considered a representative characteristic . the outcome of the curation process resides in a new collection on the spectra repository comprising 158,122 entries . the new curation has two persistent identifiers for the collection itself and within that collection , individual molecular entries are themselves also assigned two persistent identifiers , as for example the entry shown in figs . 1 and 2 [ 26 , 27 ] . the first of these is the handle with a registered prefix 10042 associated with the spectra dspace server . the second is the datacite doi associated with the prefix 10.11469 as registered to imperial college , with individual entries prefixed with the common string ch/ to indicate the chemistry department at that institution . the individual items in the collection also have a full set of associated metadata descriptors ( fig . 1).fig . newly introduced metadata since the creation of the original collection include the following : the contributors are listed individually , with each name linked to their corresponding orcid entry page.the computational resource used for annotation is also linked with a non - persistent identifier ; currently to the web landing page for the organisation.several chemical identifiers are included such as smiles , inchi and the cas accession number . the significance of including such metadata is that it is registered automatically with datacite.org , and hence available for fielded searches .the orcid entries for all collaborators are explicitly listed , and again become available for searching .a back - link to the original item deposition allows comparison of the original and the newly curated entry . because this handle prefix ( 1810 ) is unregistered with cnri , the central authority for handle registration , it can not be treated formally for resolution as a persistent identifier . this is one of the aspects we wished to rectify in the current curation.there is also a persistent identifier link to the journal article describing the original work . in due time , the present article could itself be so - referenced in a future curation.a pair of new persistent identifiers for each molecule has been minted as part of the curation . the first is a handle assigned using the handle manager tool in dspace itself and which can be resolved using either of the services http://hdl.handle.net/ or http://doi.org/. this handle is internally annotated with 10320/loc records to enable automated retrieval of individually requested files from the deposition . the prefix 10042 is registered to the spectra server.the second persistent identifier is assigned using the datacite api , and serves as a mechanism to allow datacite to acquire the metadata for this entry . the prefix 10.14469 and the suffix ch are as described above.a ( non - persistent ) link to the original publisher is included.a ( non - persistent ) link to the open license for the data , in this instance creative commons attribution ( cc0 ) . it is perhaps surprising that this license is itself not identified by its own persistent identifier , but the uris for the cc licenses and the corresponding resources are however machine - processable . the contributors are listed individually , with each name linked to their corresponding orcid entry page . the computational resource used for annotation is also linked with a non - persistent identifier ; currently to the web landing page for the organisation . several chemical identifiers are included such as smiles , inchi and the cas accession number . the significance of including such metadata is that it is registered automatically with datacite.org , and hence available for fielded searches . the orcid entries for all collaborators are explicitly listed , and again become available for searching . a back - link to the original item deposition allows comparison of the original and the newly curated entry . because this handle prefix ( 1810 ) is unregistered with cnri , the central authority for handle registration , it can not be treated formally for resolution as a persistent identifier . there is also a persistent identifier link to the journal article describing the original work . in due time a pair of new persistent identifiers for each molecule has been minted as part of the curation . the first is a handle assigned using the handle manager tool in dspace itself and which can be resolved using either of the services http://hdl.handle.net/ or http://doi.org/. this handle is internally annotated with 10320/loc records to enable automated retrieval of individually requested files from the deposition . the second persistent identifier is assigned using the datacite api , and serves as a mechanism to allow datacite to acquire the metadata for this entry . a ( non - persistent ) link to the original publisher is included . a ( non - persistent ) link to the open license for the data , in this instance creative commons attribution ( cc0 ) . it is perhaps surprising that this license is itself not identified by its own persistent identifier , but the uris for the cc licenses and the corresponding resources are however machine - processable . this metadata describes the contents of the data files resident for each entry ( fig . 2 ) . this compressed archive contains the mets manifest for the deposition , expressed syntactically as an xml file containing a number of declared namespaces defining various metadata schemas . the mets manifest , along with another internal xml document , the oai - ore resource map defines the contents , locations and properties of the documents comprising the collection . the second item ( fig . 2 ) includes three xml files expressed syntactically as xml documents declaring the cml schema . also included in this fileset are three files relating to the mopac program : the input file , the corresponding pm7 output and pm7 archive file summarising the computed properties . in principle , all the information in these files could also be absorbed into the cml descriptors , although this has not been done in the present instance . these files in turn have associated mime types , information that allows automated retrieval of the files using one of the mechanisms briefly described below . in curating the original collection at cambridge by relocating it to a separate dspace server , we wished to ensure that new persistent identifiers for each entry could be minted using datacite . that in turn required the metadata follows the dublin core schema held on the dspace - spectra repository to be mapped onto the datacite schema using an xslt - based crosswalk transform . the following procedure was used to achieve this.a recent release of dspace ( dspace4 ) largely automates the minting of dois using datacite . our target dspace ( spectra ) is running version 1.8 ; the doi module for dspace 4 is confined to a few distinct packages that were implemented into version 1.8 without affecting the other components . the following java packages were extracted from dspace4 and used within dspace 1.8:org.dspace.identifier.doi , org.dspace.services , org.dspace.versioning , org.apache , httpcomponents , httpclient-4.2.jar , org.apache.httpcomponents.httpcore-4.3.1.jardoi-specific properties in the existing install were configured via dspace.cfg . an auxiliary configuration file spring-dspace-addon-identifier-services.xml is packaged within the org.dspace.identifier package and used for connection details.configuring the xml schema transformation that translates or crosswalks between the dspace dublin core metadata schema and the datacite metadata schema . dspace4 delivered the requisite crosswalk , dim2datacite.xsl , for version 2 of the datacite schema.a requirement was to provide metadata that described the locations , filenames and file types of the individual datafiles associated with each doi , in order to provide a machine discoverable and operable path from the doi directly to the files containing chemical data . to achieve this , the dspace 4 xml schema transformation ( crosswalk ) was extended to include the locations of the mets and oai - ore metadata files that are generated by dspace , as relatedidentifiers . these related identifiers used the hasmetadata relation type which was introduced in version 3.0 of the datacite schema : a recent release of dspace ( dspace4 ) largely automates the minting of dois using datacite . our target dspace ( spectra ) is running version 1.8 ; the doi module for dspace 4 is confined to a few distinct packages that were implemented into version 1.8 without affecting the other components . the following java packages were extracted from dspace4 and used within dspace 1.8 : org.dspace.identifier.doi , org.dspace.services , org.dspace.versioning , org.apache , httpcomponents , httpclient-4.2.jar , org.apache.httpcomponents.httpcore-4.3.1.jar doi - specific properties in the existing install were configured via dspace.cfg . an auxiliary configuration file spring-dspace-addon-identifier-services.xml is packaged within the org.dspace.identifier package and used for connection details . crosswalks between the dspace dublin core metadata schema and the datacite metadata schema . dspace4 delivered the requisite crosswalk , dim2datacite.xsl , for version 2 of the datacite schema . a requirement was to provide metadata that described the locations , filenames and file types of the individual datafiles associated with each doi , in order to provide a machine discoverable and operable path from the doi directly to the files containing chemical data . to achieve this , the dspace 4 xml schema transformation ( crosswalk ) was extended to include the locations of the mets and oai - ore metadata files that are generated by dspace , as relatedidentifiers . these related identifiers used the hasmetadata relation type which was introduced in version 3.0 of the datacite schema : < relatedidentifier relatedidentifiertype=url relationtype=hasmetadata relatedmetadatascheme=mets schemeuri=http://www.loc.gov / mets/ > https://spectradspace.lib.imperial.ac.uk:8443/metadata/handle/10042/159060/mets.xml both the mets and oai - ore files contain the desired metadata and can be processed as required . as an example , the filesec section of the mets is show in part below : < mets : file checksumtype=md5 groupid=group_file_1367638 id=file_1367638 mimetype=chemical / x - cml > < mets : flocat loctype=url xlink : title=pm7.xml xlink : type=locator xlink : href=/bitstream / handle/10042/159060/pm7.xml?sequence=3/ > the crosswalk was also extended to add metadata for orcid as name identifiers for the contributors and various chemical identifiers ( inchi , inchikey , cas , nci and smiles ) as a set of alternate identifiers.in addition , the pm7.xml file containing the newly computed structures and properties was registered against its chemical mime type , chemical / x - cml for each doi , using the datacite media api . the datacite content resolver then allows this cml file to be directly retrieved from the associated doi through content negotiation using the resource http://www.crosscite.org/cn/ or directly by url . the crosswalk was also extended to add metadata for orcid as name identifiers for the contributors and various chemical identifiers ( inchi , inchikey , cas , nci and smiles ) as a set of alternate identifiers . in addition , the pm7.xml file containing the newly computed structures and properties was registered against its chemical mime type , chemical / x - cml for each doi , using the datacite media api . the datacite content resolver then allows this cml file to be directly retrieved from the associated doi through content negotiation using the resource http://www.crosscite.org/cn/ or directly by url . it took around 8 h to mint dois for an initial run of 23,240 items , and further subsequent updates took only a few hours . each update required about 24 h to become visible in datacite . new items in the repository are now synchronised hourly using the dspace4 programme , registerdoi . at this stage , the datacite search api proved to be a useful tool for checking the quality and validity of the curation and its metadata . search queries were used to retrieve lists of all entries belonging to the new dspace - spectra collection in an easily parsed format and with the necessary metadata to identify discrepancies , such as duplicate dspace depositions , duplicate assigned datacite dois or corrupted or invalid metadata . some examples of such use are collected in table 2 and are also described below . an advantage is that this kind of analysis can be done without privileged access to the host repository and its underlying databases , which makes it easier for peers and users to scrutinize the quality of large open data collections and flag any potential errors.table 2examples of data discovery and datametrics using metadataentryurlpurpose of search1 http://search.datacite.org/api?&q=prefix:10.14469&alternateidentifier:nci\:*&fl=doi,title,relatedidentifier&wt=xml&rows=3 returns the first three entries for which any nci descriptor is specified , restricted to the imperial college prefix , containing values for the title , doi and relatedidentifier and expressed in xml syntax2 http://search.datacite.org / ui?&q = alternateidentifier : smiles\:*.*+alternateidentifier : nci\ : * this returns all entries for which both a smiles and nci molecular descriptor is specified and which contains a period in the smiles string3 http://search.datacite.org/ui?q=orcid:0000-0002-8635-8390+publicationyear:[2014+to+2014 ] this returns the metadata about the orcid associated with the creator of a data set , along with a specified period for its creation4 http://search.datacite.org/ui?q=orcid:*+prefix:10.14469 a variation of the preceding example , illustrating all entries at imperial college that have an associated orcid for their creator5 http://search.datacite.org/ui?q=orcid:*+doi:10.14469\/ch\/ * a second variation of the preceding example , illustrating all entries at imperial college that have an associated orcid for their creator and a doi assigned to the chemistry department6 http://search.datacite.org/ui?q=has_media:true&fq=prefix:10.14469 searches for any entry associated with a declared media type . the prefix is that registered by imperial college london ; the media type found for this prefix is chemical / x - cml7 http://search.datacite.org/ui?q=inchikey=lqposwkbqvcbks-pgmhmlkasa-n a search using a specified value for the inchi chemical identifier associated with the dataset . our repository was constructed along the lines that each deposition describes a single molecular collection , where a single inchi descriptor relates to the important molecular entity in that collection8 http://search.datacite.org / ui?q = alternateidentifier : inchikey\ : * a variation on the preceding specific search , where all entries containing an inchikey are returned9 http://stats.datacite.org/?fq=datacentre_facet%3a%22bl.imperial+-+imperial+college+london%22&fq=allocator_facet%3a%22bl+-+the+british+library%22&q=#tab-resolution-report this provides a url resolution report for all dois associated with the imperial college london prefix10 http://stats.datacite.org/?fq=datacentre_facet%3a%22bl.imperial+-+imperial+college+london%22&q=#tab-prefixes this returns the number of datasets associated with imperial college as a whole examples of data discovery and datametrics using metadata although this software contains a component that can provide structured data representations of entries for harvesting ( oai - ore resource maps ) , this was not enabled on the cambridge repository when we started our migration in july 2014 . however , since the human - readable landing pages for each entry all conform to a structured html template , it nevertheless proved possible to extract all the data using ad hoc scripting and html processing ( a process often informally referred to as web - scraping ) . this process was markedly inefficient , requiring three separate http requests to the server per record , and took several days to complete . this approach is by no means unique ; most large existing collections of ( chemical ) data require similar processes whereby a human has to initially read the documentation ( if available ) for the templates used to access the items and then to write appropriate custom codes or scripts to retrieve them . such a method means that any unexpected change in the template resulting from , for example , the release of a new version of the dataset then inherits the risk of breaking these scripts . stated more formally , the inferred uniform resource locators ( urls ) for such collections of data are not persistent . the principal aim of our curation objectives therefore was to eliminate the need for such ad hoc scripting and replace it with a more efficient and standards - based workflow for achieving this persistence . the following were retrieved from the original deposition at cambridge : the source url for 175,356 records.175,356 documents in xml - cml syntax encoded using chemical mark - up language ( cml ) , containing a molecular structure from the nci database and some metadata describing the entry.158,879 xml - cml documents containing the pm5 optimized coordinates of the nci database structure and basic metadata , including the nci identifier for version 3 of the nci open database and the computed inchi and inchikey . of these these entries were previously identified as having additional complexities such as the presence of metal atoms or problems with correctly adding hydrogen atoms and charges , and so a pm5 calculation had not been attempted . here we have adopted the same strategy of not recovering these entries in the present curation . the source url for 175,356 records . 175,356 documents in xml - cml syntax encoded using chemical mark - up language ( cml ) , containing a molecular structure from the nci database and some metadata describing the entry . 158,879 xml - cml documents containing the pm5 optimized coordinates of the nci database structure and basic metadata , including the nci identifier for version 3 of the nci open database and the computed inchi and inchikey . of these these entries were previously identified as having additional complexities such as the presence of metal atoms or problems with correctly adding hydrogen atoms and charges , and so a pm5 calculation had not been attempted . here we have adopted the same strategy of not recovering these entries in the present curation . no metadata were provided in the original depositions that gave an unambiguous description of the two xml - cml documents present in the form of a cml schema declaration , and no mopac version information or mopac input or output files were saved to act as alternative sources of this information . the cml syntax corresponding to the annotation derived from pm5 optimisation in the form of files named e.g. nsc138467_post - mopac.cml in the original collection was incomplete ; bond connection terms were missing and the cml documents failed validation according to the cml schema version 2.4 . the first task was therefore to develop a protocol to produce a reliable and valid input file suitable for re - calculating the properties using the newer pm7 method . many entries in the nci collection comprise two or more disconnected components , of which only the larger component was retained in the original editing . the resulting missing component in the starting structure was predominantly a counter ion and its removal requires a charge to be assigned to the remaining fragment . this information was originally captured in both original xml - cml documents , the first as part of an identifier element containing an early form of the inchi string : < identifier version=0.932beta /identifier > the second is declared more formally in the cml molecule element associated with the pm5 calculation : < molecule id=nsc138467 formalcharge=1 name=mol1 > of the 158,122 unique documents in the latter category , the formalcharge declarations were distributed as follows ; 153,127 ( 0 ) , 28 ( 1 ) , 4,456 ( + 1 ) , 18 ( 2 ) , 483 ( + 2 ) , 2 ( 3 ) , 3 ( + 3 ) , 1 ( + 4 ) , 4 ( 5 ) . manual inspection of the species with very large formal charges ( > |3| ) indicates these are all errors arising from the original curation process because of incorrect interpretations of e.g. metal centres . our original attempt to transform this information into a mopac input involved the standard openbabel program , version 2.3.2 . it transpired openbabel did not correctly propagate the charge information in either of the original cml files by transformation into an appropriate mopac keyword declaration such as charge = 1 . instead the generic statement put keywords here was the only content of the mopac keyword line . this raises some interesting issues : absolute fidelity in any syntactic transformation of data from one format to another is very difficult to achieve . thus there are often multiple syntaxes for any given information field , such as the two shown above for expressing the charge on a molecule , and all such variations must be honoured with complete fidelity to achieve reliability . although some forms can be quickly deprecated ( such as the first example above ) , these forms can not be ignored and they must be processed.the mopac program does not mandate the presence of all keywords . a calculation may still succeed on the assumption that a missing keyword simply defaults to a pre - determined value . in this case , mopac will assume that the value of an undeclared charge keyword corresponds to zero , which is a clear error if the charge was intended to be non - zero . it can be very difficult , if not impossible to find complete definitions of what implicit assumptions are made in any system . often the only source of these is the actual computer code itself.a further implicit rule for mopac keywords is that the spin - multiplicity of the system is computed from the total electron count after the appropriate charge is applied . for a system where a charge of e.g. + 1 is left undeclared , that will result in a molecule with an odd number of electrons , and we also note that these implicit rules are not universal ; other programs such as gaussian use different conventions.if the explicit keyword singlet ( spin state ) is declared , a safe assumption for virtually all real molecules that exist as physical samples , this can act as a checksum . the mopac program will then throw an error and the calculation will not proceed if this spin state conflicts with any declared or undeclared / implicit charge . absolute fidelity in any syntactic transformation of data from one format to another is very difficult to achieve . thus there are often multiple syntaxes for any given information field , such as the two shown above for expressing the charge on a molecule , and all such variations must be honoured with complete fidelity to achieve reliability . although some forms can be quickly deprecated ( such as the first example above ) , these forms can not be ignored and they must be processed . a calculation may still succeed on the assumption that a missing keyword simply defaults to a pre - determined value . in this case , mopac will assume that the value of an undeclared charge keyword corresponds to zero , which is a clear error if the charge was intended to be non - zero . it can be very difficult , if not impossible to find complete definitions of what implicit assumptions are made in any system . a further implicit rule for mopac keywords is that the spin - multiplicity of the system is computed from the total electron count after the appropriate charge is applied . for a system where a charge of e.g. + 1 is left undeclared , that will result in a molecule with an odd number of electrons , and this we also note that these implicit rules are not universal ; other programs such as gaussian use different conventions . if the explicit keyword singlet ( spin state ) is declared , a safe assumption for virtually all real molecules that exist as physical samples , this can act as a checksum . the mopac program will then throw an error and the calculation will not proceed if this spin state conflicts with any declared or undeclared / implicit charge . instead of using openbabel , we made a custom conversion of the original post - mopac pm5 calculation into cml files to ensure the correct keywords were written to the mopac input file . this is not a critical decision , since the final atom positions do not in general depend on the initial coordinate system selected . a pm7 geometry optimisation was then performed using the resources of the imperial college high performance computing service . the majority of calculations completed within tens of seconds and the total required approximately 20 cpu days of computer time . an inchi identifier is a canonicalization based on the atom connectivity of a molecule , which in turn is derived from cartesian coordinates for each atom using simple heuristic rules specifying a range of atom pair distances for any element combination . unfortunately , atom connection distances are not formally defined as accepted standards , and the precise values are ultimately the choice of the designers of any program implementing them . the limits however are usually sufficient tolerant to cover the vast majority of real molecules without any disagreement , and this would especially be true of the nci set which cover real systems rather than hypothetical or computed molecules . this does not entirely exclude there being a very small number of molecules where specific atom - pair distances might fall within e.g. a bond range using pm5-optimised coordinates but which are e.g. outside such a range using pm7 values . we note that whilst it is possible to replace these relatively arbitrary rules by using a quantum mechanically derived property of the electron density topology called the bcp ( bond critical point ) to define an atom - pair connectivity , this is not currently used for determining inchi identifiers . we proceeded to derive inchi identifiers using the following openbabel commands : babel -i cml nsc383508_original.cml -o xyz out.xyz --canonical ( for the original nci - based data)babel -i cml nsc383508_post - mopac.cml -o xyz out.xyz --canonical ( for the pm5-computed data ) babel -i mopout mopac-pm7.out -o xyz out.xyz --canonical ( for the newly generated pm7-computed data).babel -i xyz in.xyz -o inchi out.inchi babel -i cml nsc383508_original.cml -o xyz out.xyz --canonical ( for the original nci - based data ) babel -i cml nsc383508_post - mopac.cml -o xyz out.xyz --canonical ( for the pm5-computed data ) babel -i mopout mopac-pm7.out -o xyz out.xyz --canonical ( for the newly generated pm7-computed data ) . babel -i xyz in.xyz -o inchi out.inchi commands 13 convert all the data into cartesian coordinates to remove any possible atom connection data that might have been generated by mopac or other sources . this process ensures that the connectivities created using the last command and then used to create the inchi are normalised against a single connection algorithm ( being the one contained in openbabel , version 2.3.2 ) . these inchi strings are then compared with those derived in a similar manner using the original nci and the original pm5 computed coordinates ( table 1).table 1comparison of generated inchi identifiersnci = pm5 = pm7(pm5 = pm7 ) nci(pm7 = nci ) pm5(pm5 = nci ) pm7pm7 pm5 nci154,5522,041573470486 comparison of generated inchi identifiers of the 158,122 unique values ( table 1 ) , 97.7 % matched for all three instances , which provides a great measure of confidence that the atom - connection algorithm is robust . to identify the origin of the 2.3 % of inchi mis - matches , we have to dissect the inchi identifier itself into its component layers : the molecular formula layer ( 1131).the pairwise atom connectivity layer , determined as described above ( 127).the hydrogen layer , in which hydrogen atoms are added to all heavy atoms where a valence is perceived to be unsatisfied if the hydrogens are not already declared . because we have subjected all the systems to computational quantum modelling , all hydrogen atoms are already explicitly defined in our coordinates ( 1252).a charge layer , also defined for all the molecules in our collection ( 9).a stereochemical layer . because our coordinates are all specified in 3d space , the stereochemistry is always defined . this layer includes double - bond isomerism ( 292 ) and tetrahedral configurations ( 267).an isotope layer ( 22 ) . the molecular formula layer ( 1131 ) . the pairwise atom connectivity layer , determined as described above ( 127 ) . the hydrogen layer , in which hydrogen atoms are added to all heavy atoms where a valence is perceived to be unsatisfied if the hydrogens are not already declared . because we have subjected all the systems to computational quantum modelling , all hydrogen atoms are already explicitly defined in our coordinates ( 1252 ) a charge layer , also defined for all the molecules in our collection ( 9 ) . a stereochemical layer . because our coordinates are all specified in 3d space , the stereochemistry is always defined . this layer includes double - bond isomerism ( 292 ) and tetrahedral configurations ( 267 ) . an isotope layer ( 22 ) . the distribution of the 2,997 differences between the pm7 and the nci inchi identifiers ( 2,041 + 470 + 486 , table 1 ) are shown in parenthesis in the listing above , and each is very briefly discussed below : the discrepancies in the formula layer originate from mismatches in the hydrogen count . this is because , historically , molecules were not always defined with explicit coordinates for all hydrogen atoms . instead they were inferred from residual valences , these in turn inferred from bonding angles and other geometric and heuristic information . the process of replacing such implicit hydrogens with explicit ones is not always exact.the connection layer mismatch originates from bonds that are on the verge of connection and derives from ( possibly small ) geometric changes from the quantum mechanical re - optimisation . s bonds in sulfur species .this and the formula layer together account for the great majority of the mis-matches.the small number of mis - matches in the charge layer may result from the inchi code heuristic for deciding the appropriate charge for a molecule . as noted above , we detected some unreasonable high charges resulting from this process.because traditionally molecules were expressed in the molfile v2 format which allows just 2d coordinates to be defined , stereochemistry had to be added using an additional parameter associated with each bond connection and equivalent to the stereochemical wedge notations used in organic chemistry . this information is not free of ambiguity , since the stereochemistry is defined relative to other atoms and can lead to logical contradictions . when such two dimensional coordinates and this additional information is converted into 3d coordinates ( a process carried out during the original deposition ) , ambiguities can result.isotopes were not included in the mopac - pm7 calculation . this is because , historically , molecules were not always defined with explicit coordinates for all hydrogen atoms . instead they were inferred from residual valences , these in turn inferred from bonding angles and other geometric and heuristic information . the connection layer mismatch originates from bonds that are on the verge of connection and derives from ( possibly small ) geometric changes from the quantum mechanical re - optimisation . this and the formula layer together account for the great majority of the mis - matches . the small number of mis - matches in the charge layer may result from the inchi code heuristic for deciding the appropriate charge for a molecule . because traditionally molecules were expressed in the molfile v2 format which allows just 2d coordinates to be defined , stereochemistry had to be added using an additional parameter associated with each bond connection and equivalent to the stereochemical wedge notations used in organic chemistry . this information is not free of ambiguity , since the stereochemistry is defined relative to other atoms and can lead to logical contradictions . when such two dimensional coordinates and this additional information is converted into 3d coordinates ( a process carried out during the original deposition ) , ambiguities can result . an inchi identifier is a canonicalization based on the atom connectivity of a molecule , which in turn is derived from cartesian coordinates for each atom using simple heuristic rules specifying a range of atom pair distances for any element combination . unfortunately , atom connection distances are not formally defined as accepted standards , and the precise values are ultimately the choice of the designers of any program implementing them . the limits however are usually sufficient tolerant to cover the vast majority of real molecules without any disagreement , and this would especially be true of the nci set which cover real systems rather than hypothetical or computed molecules . this does not entirely exclude there being a very small number of molecules where specific atom - pair distances might fall within e.g. a bond range using pm5-optimised coordinates but which are e.g. outside such a range using pm7 values . we note that whilst it is possible to replace these relatively arbitrary rules by using a quantum mechanically derived property of the electron density topology called the bcp ( bond critical point ) to define an atom - pair connectivity , this is not currently used for determining inchi identifiers . we proceeded to derive inchi identifiers using the following openbabel commands : babel -i cml nsc383508_original.cml -o xyz out.xyz --canonical ( for the original nci - based data)babel -i cml nsc383508_post - mopac.cml -o xyz out.xyz --canonical ( for the pm5-computed data ) babel -i mopout mopac-pm7.out -o xyz out.xyz --canonical ( for the newly generated pm7-computed data).babel -i xyz in.xyz -o inchi out.inchi babel -i cml nsc383508_original.cml -o xyz out.xyz --canonical ( for the original nci - based data ) babel -i cml nsc383508_post - mopac.cml -o xyz out.xyz --canonical ( for the pm5-computed data ) babel -i mopout mopac-pm7.out -o xyz out.xyz --canonical ( for the newly generated pm7-computed data ) . babel -i xyz in.xyz -o inchi out.inchi commands 13 convert all the data into cartesian coordinates to remove any possible atom connection data that might have been generated by mopac or other sources . this process ensures that the connectivities created using the last command and then used to create the inchi are normalised against a single connection algorithm ( being the one contained in openbabel , version 2.3.2 ) . these inchi strings are then compared with those derived in a similar manner using the original nci and the original pm5 computed coordinates ( table 1).table 1comparison of generated inchi identifiersnci = pm5 = pm7(pm5 = pm7 ) nci(pm7 = nci ) pm5(pm5 = nci ) pm7pm7 pm5 nci154,5522,041573470486 comparison of generated inchi identifiers of the 158,122 unique values ( table 1 ) , 97.7 % matched for all three instances , which provides a great measure of confidence that the atom - connection algorithm is robust . to identify the origin of the 2.3 % of inchi mis - matches , we have to dissect the inchi identifier itself into its component layers : the molecular formula layer ( 1131).the pairwise atom connectivity layer , determined as described above ( 127).the hydrogen layer , in which hydrogen atoms are added to all heavy atoms where a valence is perceived to be unsatisfied if the hydrogens are not already declared . because we have subjected all the systems to computational quantum modelling , all hydrogen atoms are already explicitly defined in our coordinates ( 1252).a charge layer , also defined for all the molecules in our collection ( 9).a stereochemical layer . because our coordinates are all specified in 3d space , the stereochemistry is always defined . this layer includes double - bond isomerism ( 292 ) and tetrahedral configurations ( 267).an isotope layer ( 22 ) . the molecular formula layer ( 1131 ) . the pairwise atom connectivity layer , determined as described above ( 127 ) . the hydrogen layer , in which hydrogen atoms are added to all heavy atoms where a valence is perceived to be unsatisfied if the hydrogens are not already declared . because we have subjected all the systems to computational quantum modelling , all hydrogen atoms are already explicitly defined in our coordinates ( 1252 ) . a charge layer , also defined for all the molecules in our collection ( 9 ) . a stereochemical layer . because our coordinates are all specified in 3d space , the stereochemistry is always defined . this layer includes double - bond isomerism ( 292 ) and tetrahedral configurations ( 267 ) . the distribution of the 2,997 differences between the pm7 and the nci inchi identifiers ( 2,041 + 470 + 486 , table 1 ) are shown in parenthesis in the listing above , and each is very briefly discussed below : the discrepancies in the formula layer originate from mismatches in the hydrogen count . this is because , historically , molecules were not always defined with explicit coordinates for all hydrogen atoms . instead they were inferred from residual valences , these in turn inferred from bonding angles and other geometric and heuristic information . the process of replacing such implicit hydrogens with explicit ones is not always exact.the connection layer mismatch originates from bonds that are on the verge of connection and derives from ( possibly small ) geometric changes from the quantum mechanical re - optimisation . s bonds in sulfur species .this and the formula layer together account for the great majority of the mis-matches.the small number of mis - matches in the charge layer may result from the inchi code heuristic for deciding the appropriate charge for a molecule . as noted above , we detected some unreasonable high charges resulting from this process.because traditionally molecules were expressed in the molfile v2 format which allows just 2d coordinates to be defined , stereochemistry had to be added using an additional parameter associated with each bond connection and equivalent to the stereochemical wedge notations used in organic chemistry . this information is not free of ambiguity , since the stereochemistry is defined relative to other atoms and can lead to logical contradictions . when such two dimensional coordinates and this additional information is converted into 3d coordinates ( a process carried out during the original deposition ) , ambiguities can result.isotopes were not included in the mopac - pm7 calculation . this is because , historically , molecules were not always defined with explicit coordinates for all hydrogen atoms . instead they were inferred from residual valences , these in turn inferred from bonding angles and other geometric and heuristic information . the connection layer mismatch originates from bonds that are on the verge of connection and derives from ( possibly small ) geometric changes from the quantum mechanical re - optimisation . this and the formula layer together account for the great majority of the mis - matches . the small number of mis - matches in the charge layer may result from the inchi code heuristic for deciding the appropriate charge for a molecule . because traditionally molecules were expressed in the molfile v2 format which allows just 2d coordinates to be defined , stereochemistry had to be added using an additional parameter associated with each bond connection and equivalent to the stereochemical wedge notations used in organic chemistry . this information is not free of ambiguity , since the stereochemistry is defined relative to other atoms and can lead to logical contradictions . when such two dimensional coordinates and this additional information is converted into 3d coordinates ( a process carried out during the original deposition ) , ambiguities can result . for each remaining entry , the pm7-derived inchi strings and keys were added to the smiles strings and the nci and cas accession identifiers obtained from the original data and propagated as metadata . we note that the nci identifiers themselves may not necessarily persist across different versions of the nci database , which was version 3 at the time of the original curation and has subsequently been updated to version 4 in 2012 . prior to import to spectra , each entry was packaged individually to produce an archive file , termed a sword [ 21 , 22 ] bundle . sword ( simple web - service offering repository deposit ) is an interoperability standard for data ingest into digital repositories , rendering these bundles suitable for import into any sword - compliant repository , not just dspace - based spectra . the bundles contains a mets manifest and data files and were created using a locally written tool . the mets manifest contained the following metadata : inchi and inchikey and smiles string.cas and nci accession ids , nci entry name.back-link back to the entry in the cambridge repository.doi link to the published description .orcid identifiers for the contributing authors.link to creative commons license terms . the datafiles included within the bundle were : two cml files containing unaltered copies of the nci coordinates and pm5 computed mopac output documents obtained from the original source repository.a third cml file conflating the three previous structures , containing the original nci structure , the original pm5 structure from the original repository and the newly computed pm7 structure.mopac input and output files for the new pm7 calculation . two cml files containing unaltered copies of the nci coordinates and pm5 computed mopac output documents obtained from the original source repository . a third cml file conflating the three previous structures , containing the original nci structure , the original pm5 structure from the original repository and the newly computed pm7 structure . mopac input and output files for the new pm7 calculation . import of this fileset to the destination spectra repository was performed using the sword web service interface . owing to a limitation of the dspace - spectra sword interface , no bulk - import function was available and all of the new packages had to be to uploaded individually , a process that took approximately 60 days . doubtless this exceptionally long time resulted from some undiagnosed server misconfiguration and should not be considered a representative characteristic . the outcome of the curation process resides in a new collection on the spectra repository comprising 158,122 entries . the new curation has two persistent identifiers for the collection itself and within that collection , individual molecular entries are themselves also assigned two persistent identifiers , as for example the entry shown in figs . 1 and 2 [ 26 , 27 ] . the first of these is the handle with a registered prefix 10042 associated with the spectra dspace server . the second is the datacite doi associated with the prefix 10.11469 as registered to imperial college , with individual entries prefixed with the common string ch/ to indicate the chemistry department at that institution . the individual items in the collection also have a full set of associated metadata descriptors ( fig . 1).fig . newly introduced metadata since the creation of the original collection include the following : the contributors are listed individually , with each name linked to their corresponding orcid entry page.the computational resource used for annotation is also linked with a non - persistent identifier ; currently to the web landing page for the organisation.several chemical identifiers are included such as smiles , inchi and the cas accession number . the significance of including such metadata is that it is registered automatically with datacite.org , and hence available for fielded searches .the orcid entries for all collaborators are explicitly listed , and again become available for searching .a back - link to the original item deposition allows comparison of the original and the newly curated entry . because this handle prefix ( 1810 ) is unregistered with cnri , the central authority for handle registration , it can not be treated formally for resolution as a persistent identifier . this is one of the aspects we wished to rectify in the current curation.there is also a persistent identifier link to the journal article describing the original work . in due time , the present article could itself be so - referenced in a future curation.a pair of new persistent identifiers for each molecule has been minted as part of the curation . the first is a handle assigned using the handle manager tool in dspace itself and which can be resolved using either of the services http://hdl.handle.net/ or http://doi.org/. this handle is internally annotated with 10320/loc records to enable automated retrieval of individually requested files from the deposition . the prefix 10042 is registered to the spectra server.the second persistent identifier is assigned using the datacite api , and serves as a mechanism to allow datacite to acquire the metadata for this entry . the prefix 10.14469 and the suffix ch are as described above.a ( non - persistent ) link to the original publisher is included.a ( non - persistent ) link to the open license for the data , in this instance creative commons attribution ( cc0 ) . it is perhaps surprising that this license is itself not identified by its own persistent identifier , but the uris for the cc licenses and the corresponding resources are however machine - processable . the contributors are listed individually , with each name linked to their corresponding orcid entry page . the computational resource used for annotation is also linked with a non - persistent identifier ; currently to the web landing page for the organisation . several chemical identifiers are included such as smiles , inchi and the cas accession number . the significance of including such metadata is that it is registered automatically with datacite.org , and hence available for fielded searches . the orcid entries for all collaborators are explicitly listed , and again become available for searching . a back - link to the original item deposition allows comparison of the original and the newly curated entry . because this handle prefix ( 1810 ) is unregistered with cnri , the central authority for handle registration , it can not be treated formally for resolution as a persistent identifier . there is also a persistent identifier link to the journal article describing the original work . in due time a pair of new persistent identifiers for each molecule has been minted as part of the curation . the first is a handle assigned using the handle manager tool in dspace itself and which can be resolved using either of the services http://hdl.handle.net/ or http://doi.org/. this handle is internally annotated with 10320/loc records to enable automated retrieval of individually requested files from the deposition . the second persistent identifier is assigned using the datacite api , and serves as a mechanism to allow datacite to acquire the metadata for this entry . a ( non - persistent ) link to the original publisher is included . a ( non - persistent ) link to the open license for the data , in this instance creative commons attribution ( cc0 ) . it is perhaps surprising that this license is itself not identified by its own persistent identifier , but the uris for the cc licenses and the corresponding resources are however machine - processable . this metadata describes the contents of the data files resident for each entry ( fig . 2 ) . this compressed archive contains the mets manifest for the deposition , expressed syntactically as an xml file containing a number of declared namespaces defining various metadata schemas . the mets manifest , along with another internal xml document , the oai - ore resource map defines the contents , locations and properties of the documents comprising the collection . the second item ( fig . 2 ) includes three xml files expressed syntactically as xml documents declaring the cml schema . also included in this fileset are three files relating to the mopac program : the input file , the corresponding pm7 output and pm7 archive file summarising the computed properties . in principle , all the information in these files could also be absorbed into the cml descriptors , although this has not been done in the present instance . these files in turn have associated mime types , information that allows automated retrieval of the files using one of the mechanisms briefly described below . in curating the original collection at cambridge by relocating it to a separate dspace server , we wished to ensure that new persistent identifiers for each entry could be minted using datacite . that in turn required the metadata follows the dublin core schema held on the dspace - spectra repository to be mapped onto the datacite schema using an xslt - based crosswalk transform . the following procedure was used to achieve this.a recent release of dspace ( dspace4 ) largely automates the minting of dois using datacite . our target dspace ( spectra ) is running version 1.8 ; the doi module for dspace 4 is confined to a few distinct packages that were implemented into version 1.8 without affecting the other components . the following java packages were extracted from dspace4 and used within dspace 1.8:org.dspace.identifier.doi , org.dspace.services , org.dspace.versioning , org.apache , httpcomponents , httpclient-4.2.jar , org.apache.httpcomponents.httpcore-4.3.1.jardoi-specific properties in the existing install were configured via dspace.cfg . an auxiliary configuration file spring-dspace-addon-identifier-services.xml is packaged within the org.dspace.identifier package and used for connection details.configuring the xml schema transformation that translates or dspace4 delivered the requisite crosswalk , dim2datacite.xsl , for version 2 of the datacite schema.a requirement was to provide metadata that described the locations , filenames and file types of the individual datafiles associated with each doi , in order to provide a machine discoverable and operable path from the doi directly to the files containing chemical data . to achieve this , the dspace 4 xml schema transformation ( crosswalk ) was extended to include the locations of the mets and oai - ore metadata files that are generated by dspace , as relatedidentifiers . these related identifiers used the hasmetadata relation type which was introduced in version 3.0 of the datacite schema : a recent release of dspace ( dspace4 ) largely automates the minting of dois using datacite . our target dspace ( spectra ) is running version 1.8 ; the doi module for dspace 4 is confined to a few distinct packages that were implemented into version 1.8 without affecting the other components . the following java packages were extracted from dspace4 and used within dspace 1.8 : org.dspace.identifier.doi , org.dspace.services , org.dspace.versioning , org.apache , httpcomponents , httpclient-4.2.jar , org.apache.httpcomponents.httpcore-4.3.1.jar doi - specific properties in the existing install were configured via dspace.cfg . an auxiliary configuration file spring-dspace-addon-identifier-services.xml is packaged within the org.dspace.identifier package and used for connection details . crosswalks between the dspace dublin core metadata schema and the datacite metadata schema . dspace4 delivered the requisite crosswalk , dim2datacite.xsl , for version 2 of the datacite schema . a requirement was to provide metadata that described the locations , filenames and file types of the individual datafiles associated with each doi , in order to provide a machine discoverable and operable path from the doi directly to the files containing chemical data . to achieve this , the dspace 4 xml schema transformation ( crosswalk ) was extended to include the locations of the mets and oai - ore metadata files that are generated by dspace , as relatedidentifiers . these related identifiers used the hasmetadata relation type which was introduced in version 3.0 of the datacite schema : < relatedidentifier relatedidentifiertype=url relationtype=hasmetadata relatedmetadatascheme=mets schemeuri=http://www.loc.gov / mets/ > https://spectradspace.lib.imperial.ac.uk:8443/metadata/handle/10042/159060/mets.xml both the mets and oai - ore files contain the desired metadata and can be processed as required . as an example , the filesec section of the mets is show in part below : < mets : file checksumtype=md5 groupid=group_file_1367638 id=file_1367638 mimetype=chemical / x - cml > < mets : flocat loctype=url xlink : title=pm7.xml xlink : type=locator xlink : href=/bitstream / handle/10042/159060/pm7.xml?sequence=3/ > the crosswalk was also extended to add metadata for orcid as name identifiers for the contributors and various chemical identifiers ( inchi , inchikey , cas , nci and smiles ) as a set of alternate identifiers.in addition , the pm7.xml file containing the newly computed structures and properties was registered against its chemical mime type , chemical / x - cml for each doi , using the datacite media api . the datacite content resolver then allows this cml file to be directly retrieved from the associated doi through content negotiation using the resource http://www.crosscite.org/cn/ or directly by url . the crosswalk was also extended to add metadata for orcid as name identifiers for the contributors and various chemical identifiers ( inchi , inchikey , cas , nci and smiles ) as a set of alternate identifiers . in addition , the pm7.xml file containing the newly computed structures and properties was registered against its chemical mime type , chemical / x - cml for each doi , using the datacite media api . the datacite content resolver then allows this cml file to be directly retrieved from the associated doi through content negotiation using the resource http://www.crosscite.org/cn/ or directly by url . it took around 8 h to mint dois for an initial run of 23,240 items , and further subsequent updates took only a few hours . each update required about 24 h to become visible in datacite . new items in the repository are now synchronised hourly using the dspace4 programme , registerdoi . at this stage , the datacite search api proved to be a useful tool for checking the quality and validity of the curation and its metadata . search queries were used to retrieve lists of all entries belonging to the new dspace - spectra collection in an easily parsed format and with the necessary metadata to identify discrepancies , such as duplicate dspace depositions , duplicate assigned datacite dois or corrupted or invalid metadata . some examples of such use are collected in table 2 and are also described below . an advantage is that this kind of analysis can be done without privileged access to the host repository and its underlying databases , which makes it easier for peers and users to scrutinize the quality of large open data collections and flag any potential errors.table 2examples of data discovery and datametrics using metadataentryurlpurpose of search1 http://search.datacite.org/api?&q=prefix:10.14469&alternateidentifier:nci\:*&fl=doi,title,relatedidentifier&wt=xml&rows=3 returns the first three entries for which any nci descriptor is specified , restricted to the imperial college prefix , containing values for the title , doi and relatedidentifier and expressed in xml syntax2 http://search.datacite.org / ui?&q = alternateidentifier : smiles\:*.*+alternateidentifier : nci\ : * this returns all entries for which both a smiles and nci molecular descriptor is specified and which contains a period in the smiles string3 http://search.datacite.org/ui?q=orcid:0000-0002-8635-8390+publicationyear:[2014+to+2014 ] this returns the metadata about the orcid associated with the creator of a data set , along with a specified period for its creation4 http://search.datacite.org/ui?q=orcid:*+prefix:10.14469 a variation of the preceding example , illustrating all entries at imperial college that have an associated orcid for their creator5 http://search.datacite.org/ui?q=orcid:*+doi:10.14469\/ch\/ * a second variation of the preceding example , illustrating all entries at imperial college that have an associated orcid for their creator and a doi assigned to the chemistry department6 http://search.datacite.org/ui?q=has_media:true&fq=prefix:10.14469 searches for any entry associated with a declared media type . the prefix is that registered by imperial college london ; the media type found for this prefix is chemical / x - cml7 http://search.datacite.org/ui?q=inchikey=lqposwkbqvcbks-pgmhmlkasa-n a search using a specified value for the inchi chemical identifier associated with the dataset . our repository was constructed along the lines that each deposition describes a single molecular collection , where a single inchi descriptor relates to the important molecular entity in that collection8 http://search.datacite.org / ui?q = alternateidentifier : inchikey\ : * a variation on the preceding specific search , where all entries containing an inchikey are returned9 http://stats.datacite.org/?fq=datacentre_facet%3a%22bl.imperial+-+imperial+college+london%22&fq=allocator_facet%3a%22bl+-+the+british+library%22&q=#tab-resolution-report this provides a url resolution report for all dois associated with the imperial college london prefix10 http://stats.datacite.org/?fq=datacentre_facet%3a%22bl.imperial+-+imperial+college+london%22&q=#tab-prefixes this returns the number of datasets associated with imperial college as a whole examples of data discovery and datametrics using metadata the configured metadata infrastructures now associated with each item in the collection enable individual datafiles to be accessed based only on knowledge of the persistent identifiers and media type , which can be allowed to default to specific type . we have implemented three procedures for doing this ; these are fully described elsewhere with discussion of the pros and cons of each approach [ 33 , 34 ] . here we provide only a summary of these methods.the first access method to be developed is based on extensions to cnri handle record types known as 10320/loc . these allow the handle record to be retrieved using the handle rest api , which allows programmatic access to handle resolution using http . a typical invocation would be using a url of the type http://doi.org/10042/31117?locatt=mimetype:chemical/x-cml where the string 10042/31117 is the assigned handle identifier and chemical / x - cml the requested media type.the datacite media api also allows a doi to be resolved based on the media type of the required document , typically a url of form http://data.datacite.org/chemical/x-cml/10.14469/ch/153690 , where the string 10.14469/ch/153690 is the assigned datacite identifier , and chemical / x - cml the requested media type . this url can be passed to any requesting program and the file associated with this information will then be retrieved from the repository.oai-ore resource maps exposed through datacite metadata . we have made the oai - ore resource map and the mets manifest ( both generated internally by dspace ) discoverable by including their locations as relatedidentifiers within the datacite metadata for the dataset , as described above . this allows a script to query for example the resource map to retrieve the url associated with the data file . again , the only information required by the script is datacite_jmol(10.14469/ch/153690?chemical / x - cml ) , where datacite_jmol is the javascript function written to process the responses . the first access method to be developed these allow the handle record to be retrieved using the handle rest api , which allows programmatic access to handle resolution using http . a typical invocation would be using a url of the type http://doi.org/10042/31117?locatt=mimetype:chemical/x-cml where the string 10042/31117 is the assigned handle identifier and chemical / x - cml the requested media type . the datacite media api also allows a doi to be resolved based on the media type of the required document , typically a url of form http://data.datacite.org/chemical/x-cml/10.14469/ch/153690 , where the string 10.14469/ch/153690 is the assigned datacite identifier , and chemical / x - cml the requested media type . this url can be passed to any requesting program and the file associated with this information will then be retrieved from the repository . we have made the oai - ore resource map and the mets manifest ( both generated internally by dspace ) discoverable by including their locations as relatedidentifiers within the datacite metadata for the dataset , as described above . this allows a script to query for example the resource map to retrieve the url associated with the data file . again , the only information required by the script is datacite_jmol(10.14469/ch/153690?chemical / x - cml ) , where datacite_jmol is the javascript function written to process the responses . any of the above methods can be used in conjunction with e.g. a visualisation program which can convert the data contained in the retrieved file into a graphical representation , or as part of a script which could retrieve a greater number of files for the purpose of e.g. data mining . enhancement of the original cambridge dataset with the features described above greatly improves the discoverability of the data . enriching metadata and then exposing it in a manner that allows the datacite organisation to harvest it enables exploitation using the datacite interface and allows statistics to be collected . the current datacite search resource is still styled beta , and it is probable that the features offered in the future will become greatly enhanced . perhaps the most significant technical improvement realised as a result of this activity is the facilitation of future curation efforts , as part of a strategy to address the issue of what has graphically been described as link rot , whereby a worryingly large proportion of non - persistent identifiers used to cite data and associated information are found not to link correctly after just a few years or in some cases months . digital repositories are intrinsically designed to enable replication of content to other locations whilst preserving essential information such as persistent identifiers . here we focus on the dspace repository , which provides an oai - ore endpoint implementing the open archives initiative s object reuse and exchange standards to achieve such replication . the ore manifest for the deposition illustrated in figs . 1 or 2 for example is declared in metadata as : https://spectradspace.lib.imperial.ac.uk:8443/metadata/handle/10042/159060/ore.xml or https://spectradspace.lib.imperial.ac.uk:8443/metadata/handle/10042/159060/mets.xml for the mets manifest ( see above ) . these locators derive from the assigned handle for this entry as 10042/159060 . for each entry , a structured xml representation of the data ( for example pm7.xml ) , including a declared standard xml schema ( cml 2.4 ) is included . this allows the data to be directly parsed using a generic xml import / export tool , so enhancing any future wholesale export of the dataset . the use of xml is to be preferred to older legacy chemical formats , for which no explicit schemas are , or indeed can be , declared . the following illustrates a programmatic method for a curation procedure that could be employed if starting from a sword [ 21 , 22 ] /oai - ore and/or mets - enabled endpoints.obtain a list of all the individual entries for the collection . this is accomplished by using datacite to search for any unique identifier associated with the collection , which is defined in this example by the string alternateidentifier : nci : this can be accomplished using the command : curl http://search.datacite.org/api?&q=prefix:10.14469&alternateidentifier:nci\:*&fl=doi,title,relatedidentifier&wt=csv&rows=170000-onci.csv.the value 170,000 in this string is the expected upper bound . the prefix 10.14469 restricts the search to collections at imperial college only ( to disambiguate from any other collections with the same name elsewhere).this returns the following information for each entry ( in this example in csv format , with other options being xml , oai - pmh or json ) : doi , title , relatedidentifier 10.14469/ch/123315,nsc5396 , hasmetadata : url : https://spectradspace.lib.imperial.ac.uk:8443/metadata / handle/10042/130536/mets.xml , hasmetadata : url : https://spectradspace.lib.imperial.ac.uk:8443/metadata / handle/10042/130536/ore.xml , ispartof : handle:10042/31117 obtain a list of all the individual entries for the collection . this is accomplished by using datacite to search for any unique identifier associated with the collection , which is defined in this example by the string alternateidentifier : nci : this can be accomplished using the command : curl http://search.datacite.org/api?&q=prefix:10.14469&alternateidentifier:nci\:*&fl=doi,title,relatedidentifier&wt=csv&rows=170000-onci.csv . the prefix 10.14469 restricts the search to collections at imperial college only ( to disambiguate from any other collections with the same name elsewhere ) . this returns the following information for each entry ( in this example in csv format , with other options being xml , oai - pmh or json ) : doi , title , relatedidentifier 10.14469/ch/123315,nsc5396 , hasmetadata : url : https://spectradspace.lib.imperial.ac.uk:8443/metadata / handle/10042/130536/mets.xml , hasmetadata : url : https://spectradspace.lib.imperial.ac.uk:8443/metadata / handle/10042/130536/ore.xml , ispartof : handle:10042/31117 this reveals that ore and mets manifests are associated with the related identifier metadata element , and the direct path to each is obtained from the value of the hasmetadata child.these provide programmatic access ( using xslt transforms or other methods ) to the mets bitstream itself , which contains all the files in the deposition as a compressed archive . the mets bitstream has the url : https://spectradspace.lib.imperial.ac.uk:8443/dspace/bitstream/handle/10042/31117/mets.zip?sequence=8this is retrievable using : curlhttps://spectradspace.lib.imperial.ac.uk:8443/dspace / bitstream / handle/10042/31117/mets.zip?sequence=8.each mets manifest can then be injected into the destination repository , with the string 10042/31118 defining the handle for the entire new collection ( not that for the individual entries ) : these provide programmatic access ( using xslt transforms or other methods ) to the mets bitstream itself , which contains all the files in the deposition as a compressed archive . the mets bitstream has the url : https://spectradspace.lib.imperial.ac.uk:8443/dspace/bitstream/handle/10042/31117/mets.zip?sequence=8 this is retrievable using : curlhttps://spectradspace.lib.imperial.ac.uk:8443/dspace / bitstream / handle/10042/31117/mets.zip?sequence=8 . each mets manifest can then be injected into the destination repository , with the string 10042/31118 defining the handle for the entire new collection ( not that for the individual entries ) : curl -si data - binary @mets.zip \ -h content - disposition : filename = mets.zip \ -h content - type : application / zip \ -h x - packaging : http://purl.org / net / sword - types / metsdspacesip \ -h x - no - op : false -h x - verbose : true finally in this section we note premis , another international standard for metadata supporting the preservation of digital objects to help ensure their long - term usability . currently , the premis schema is only used in dspace instances to represent technical metadata about dspace bitstreams ( i.e. files ) , being generated by a premis crosswalk . here we compare our approach for data deposition with that of two alternative existing data repositories , one of which is also based on dspace ( dryad ) and a second figshare that is not . the first is run as a not - for - profit organisation that offers data deposition services , with persistent identifiers provided by both the dspace handle manager and also via datacite . dryad deploys a subset of the metadata configured for our spectra server , but significantly this does include an oai - pmh based programmatic method for access to the data object via the mets manifest , allowing a procedure similar to the oai - ore resource map outlined above to be used to access the datafile . dryad differs in one significant regard from our approach in terms of the granularity of the deposition . since our data is based on the computed properties of discrete molecules , we have adopted the strategy of one data record per molecule , and hence the dataset for each molecule is also assigned its own persistent identifiers . in contrast , the primary model used by dryad offers coarser granularity of one data record per associated publication whereby the complete dryad data set is linked with a peer reviewed journal publication . the net result is a pair of persistent identifiers , one for the article and one for the data , with the data component embargoed until the article itself is released after peer - review into the public . we do not regard this approach as an optimal one when dealing with molecular data , since it can not permit any discovery process for individual molecules contained in such a collection . dois can also be minted using the current ( 2015 ) version of figshare using the datacite api . this commercial repository is not currently oai - pmh / oai - ore compliant and so no standard ore or mets resource maps are declared to datacite using e.g. the related identifier element of the datacite metadata schema . this lack of oai - pmh / oai - ore compliance would render a lossless curation of our spectra collection to e.g. figshare more difficult to achieve programmatically , but such an operation is not excluded in the future when the functionality becomes available . we first return to discussing the article reporting the results of a stochastic exploration of the structures predicted using quantum mechanical procedures . the molecular coordinates for unexpected , unusual or interesting outcomes from this procedure were then deposited into the supporting information ( si ) associated with the published article . this contains just 10 species , although clearly far more molecules were computed at various levels of theory and these now appear lost to science . the si itself takes the form of a paginated pdf file downloadable from the article landing page , and which contains no exposed associated metadata for any individual entry . discussion is included here because it is very typical of the data associated with studies of this type . curation of such data is really only worthwhile if it is first aggregated into a larger collection , a process that is never attempted because of this formal lack of metadata . the resulting fragmentation and hence loss of valuable data is , we argue , one of the broken aspects of current publishing models that require urgent attention . as with the previous example , the next article describes quantum mechanics based procedures to obtain the molecular structures of a much larger collection of 134 kilo - molecules and the subsequent methods involved in creating a digital repository based collection of these . depositing all the calculations recovered from this process goes one important stage beyond the previous example , and is therefore to be welcomed . however , an important unanswered question is how easy would it be to curate this collection in a decade from now . in fact , several fundamental design features have made such an operation unnecessarily difficult.the entire dataset is associated with a single persistent identifier expressed as a compressed archive that a user can download and expand into a folder containing 133,886 individual files . the collected metadata however does not refer to these files , but to the folder containing them , which in turn means that the contents of this folder are in effect not discoverable using the mechanisms described above.in general , it is quite difficult on most computer systems to navigate a single folder containing 133,886 items . one would have to resort to using specialised software to do this , and this would probably restrict inspection to individual files and not to a sub - collection with specified properties.the individual entries adopt the original xmol xyz syntax . that syntax has then been annotated with a number of other properties , both to the individual atoms and to the molecule as a whole , the latter including both smiles and inchi strings . unfortunately , this annotation is in effect ad hoc in a manner that was not envisaged for the original xyz format . a human has to read the associated documentation to establish the precise meaning of the annotations , and then write suitable code to extract the annotations to render them useable for e.g. metadata . it is in general uncertain whether software that has been written to process standard xyz files lacking annotations could successfully cope with this additional content . at best , one might expect the annotations to be simply discarded , since their semantics are not accessible to such a program , only to a human . at worst , it could render the document entirely unreadable by standard software.the individual files themselves contain no information about the procedure used to compute the coordinates . in this regard , it would be quite difficult to use these files to reproduce the original calculation ; thus the original program inputs are not available , nor indeed are the original program outputs from the quantum mechanical calculation . curating such a collection therefore would require bespoke interpretation by a human , which always tends to be an expensive and error - prone solution . the entire dataset is associated with a single persistent identifier expressed as a compressed archive that a user can download and expand into a folder containing 133,886 individual files . the collected metadata however does not refer to these files , but to the folder containing them , which in turn means that the contents of this folder are in effect not discoverable using the mechanisms described above . in general , it is quite difficult on most computer systems to navigate a single folder containing 133,886 items . one would have to resort to using specialised software to do this , and this would probably restrict inspection to individual files and not to a sub - collection with specified properties . the individual entries adopt the original xmol xyz syntax . that syntax has then been annotated with a number of other properties , both to the individual atoms and to the molecule as a whole , the latter including both smiles and inchi strings . unfortunately , this annotation is in effect ad hoc in a manner that was not envisaged for the original xyz format . a human has to read the associated documentation to establish the precise meaning of the annotations , and then write suitable code to extract the annotations to render them useable for e.g. metadata . it is in general uncertain whether software that has been written to process standard xyz files lacking annotations could successfully cope with this additional content . at best , one might expect the annotations to be simply discarded , since their semantics are not accessible to such a program , only to a human . the individual files themselves contain no information about the procedure used to compute the coordinates . in this regard , it would be quite difficult to use these files to reproduce the original calculation ; thus the original program inputs are not available , nor indeed are the original program outputs from the quantum mechanical calculation . curating such a collection therefore would require bespoke interpretation by a human , which always tends to be an expensive and error - prone solution . the harvard clean energy project is another recent deposition based on quantum chemical calculations , with a claimed 2.3 million molecules associated with an even more impressive 150,000,000 dft calculations . access to any individual calculation on any specific molecule however is available only via a search front - end to the database based on specified search parameters . no metadata is exposed on any molecule or its calculation parameters in any standard form and it is difficult to envisage any type of curation that could be successfully applied to such a collection . we think it unlikely that enabling open curation was a design feature of the system , although we also believe that this should be included in future designs of such collections . the recently announced cern opendata portal is also included here , since the data described is very different from the chemical information described above , both in terms of the cost of its acquisition , and of its size and granularity . the organisational prefix for the collection is 10.7483 and this reveals ( in december 2014 ) 53 entries . a typical entry itself contains 3211 datafiles totalling 3.4 tb in size . analysing this data the software is distributed as a virtual image and is designed to be used in the form of a virtual machine containing all the tools required to acquire and analyse the data . the equivalent in our own implementation is the virtual jsmol container for the chemical data , that is made available indirectly in the web browser document object model ( dom ) as an html5 canvas , rather than as a virtual instance on a computer . working outside the virtual containers provided by the cern data portal is unlikely to be useful , whereas for chemistry the jsmol container could be replaced by other containers such as e.g. avogadro 2 . this brief survey of two recently published molecular data collections indicates that each subject domain will benefit from specifically optimising the features of repository collections for its own needs . we believe that in the chemistry domain , it is useful to adopt a molecular granularity and to develop metadata , search and acquisition mechanisms appropriate for this granularity , even at a scale of 2.3 million molecules . we think it less useful to aggregate the molecules into single containers for which metadata about individual molecules is not exposed . it is also essential that the procedures adopted are programmatic , in that all the required information to re - curate the dataset is available for machine processing . if this is so , then there is no reason why the process could not scale well beyond 2.3 million molecules if required . the mopac software , including the latest pm7 parameter set can be obtained and licensed from http://openmopac.net . the javascript routines implementing the functionality described in the results section are available via the repository entries cited in ref 36 . one of us ( jjps ) thanks the national institute of general medical sciences of the national institutes of health ( award number r44gm108085 ) for funding .

backgroundthe desirable curation of 158,122 molecular geometries derived from the nci set of reference molecules together with associated properties computed using the mopac semi - empirical quantum mechanical method and originally deposited in 2005 into the cambridge dspace repository as a data collection is reported.resultsthe procedures involved in the curation included annotation of the original data using new mopac methods , updating the syntax of the cml documents used to express the data to ensure schema conformance and adding new metadata describing the entries together with a xml schema transformation to map the metadata schema to that used by the datacite organisation . we have adopted a granularity model in which a datacite persistent identifier ( doi ) is created for each individual molecule to enable data discovery and data metrics at this level using datacite tools.conclusionswe recommend that the future research data management ( rdm ) of the scientific and chemical data components associated with journal articles ( the supporting information ) should be conducted in a manner that facilitates automatic periodic curation . graphical abstract : standards and metadata - based curation of a decade - old digital repository dataset of molecular information .

Background Methods Retrieval Technical validation InChI identifiers Re-deposition Exposing the metadata structures on DSpace-SPECTRa Metadata interfaces to DataCite Results and discussion Data discovery and datametrics The benefits of achieving SWORD/OAI-ORE and METS-enabled endpoints Comparison with other repositories Comparison with two other collections of molecular quantum mechanical calculation data Conclusions Code availability Acknowledgements Competing interests

PMC2855079

erythropoietin ( epo ) is know as a key - regulator of erythropoiesis stimulating proliferation , differentiation , and survival growth of erythroid precursor resulting in the increased production of red blood cells . in both men and mice exposed to altitude , plasma epo concentration reached a peak after a 24-hour period of exposure and subsequently declining in a progressive manner within days or weeks [ 3 , 4 ] . it has been demonstrated the relative importance of altitude epo - induced polycythemia in the process of acclimatisation to altitude hypoxia performed between days 4 and 9 of exposure . through specific binding to its receptor ( epor ) , epo triggers a chain of intracellular signaling events that depends on activation of jak2 tyrosine kinase . studies dealing with the expression of epor revealed a role for epo , in addition to erythropoiesis , on various tissues such as those of the central nervous system , pancreas , liver , lung , smooth , cardiac and skeletal muscles ( for review see ) . as a result , the effects of epo as growth factor , antiapoptotic factor or angiogenic factor on these tissues are considered of a great interest . indeed , the epor has been identified in mouse c2c12 primary myoblast cultures as well as in human skeletal muscle . on both c2c12 cells and primary cultures of mouse myoblasts more recently , the injection of recombinant human epo ( rhuepo ) has been shown to improve the regeneration process of rat soleus muscle . taken together , these results suggest a role for epo on the development and regeneration of skeletal muscle . epo was shown to have the same angiogenic power as vascular endothelial growth factor ( vegf ) . kertesz et al . demonstrated the role of epo and vegf in the formation of new blood vessels during the early development of tissues . using epo knock - out mutant , these authors showed that angiogenesis was defective while the vasculogenesis was relatively normal . on the other hand , suzuki et al . observed that the nonhematopoietic role of epo is dispensable to organism development and angiogenesis under normal conditions . from those data , . then we may hypothesise that the default of oxygen transport to tissues leads to local hypoxia which induces the accumulation of hypoxia - inducible factor-1 ( hif-1 ) and then the expression of the angiogenic factor vegf as shown in anemic brain . the vegf - a ( also referred as vegf ) protein comprises various isoforms of 121 to 206 amino acids in humans ( 120 to 205 in rodents ) , produced by alternative splicing during transcription [ 14 , 15 ] . the 120 , 164 and 188 isoforms are the most frequently expressed ones although each one has specific biological activities [ 16 , 17 ] . vegf121 is a freely diffusible protein whereas vegf189 is almost completely sequestered in the extracellular matrix ( ecm ) . vegf165 has intermediate properties , as it is moderately diffusible but a significant fraction remains bound to the cell surface and ecm . actually , the longer forms of vegf are stably incorporated to the ecm but can be released in a diffusible form by several agents . skeletal muscles of these patients present structural and functional abnormalities that contribute to explain , at least partly , their impaired exercise capacity , muscle deconditioning and decreased resistance to fatigue . there is a reduction in muscle mass associated with myofiber atrophy , mainly in type ii fibers [ 19 , 20 ] . moreover , reductions in muscle capillarity , as well as abnormalities of mitochondria are contributing factors for muscle fatigability [ 1921 ] . although muscle deconditioning in those patients could also be due to physical inactivity , those results suggest that epo deficiency could have consequences on muscle structure and capillarisation . in order to understand whether epo is an important factor for muscle and capillary development and maintenance in hypoxia , we propose to use a model of erythropoietin - sv40 t antigen ( epo - tag ) transgenic mice with a targeted disruption in the 5 untranslated region of the epo gene that dramatically reduces its expression [ 22 , 23 ] and then induces severe anaemia . we aim to assess whether epo deficiency alters skeletal muscle structure in either normoxia or hypoxia . for this purpose , epo - tag and wild - type ( wt ) mice were exposed to acute and chronic hypobaric hypoxia ( equivalent to 4500 m ) . we measured plasma and muscle epo concentrations , active hif-1 and vegf protein by enzyme - linked immunosorbent assay ( elisa ) . to analyse the expression profile of each vegf isoform , vegfr1 , vegfr2 and epor mrna , we used the real - time reverse transcriptase - polymerase chain reaction ( rt - pcr ) . the expression of epo mrna was evaluated first by classical rt - pcr and second by real - time rt - pcr . moreover , the microvessel network was analysed in gastrocnemius muscles as described by charifi et al . using the specific platelet endothelial cell adhesion molecule ( pecam ) antibody directed against cd31 . the capillary - to - fiber ratio ( c / f ) , the number of capillaries around a single fiber and the index of capillary tortuosity ( lc / pf ) were measured to evaluate the development of elongated vessels running parallel to the muscle fibers and the new sprouts from loops and cross connections . for this study , male wild - type f1 bl6/cba ( charles river , l'arbresle , france ) and male epo deficient sv-40 t antigen ( epo - tag ) mice were divided randomly into six groups : ( a ) normoxic wild - type ( nx wt ) ; ( b ) normoxic epo - tag ( nx epo - tag ) ; ( c ) acute hypoxic wild - type ( ahx wt ) ; ( d ) acute hypoxic epo - tag ( ahx epo - tag ) ; ( e ) chronic hypoxic wild - type ( chx wt ) ; and ( f ) chronic hypoxic epo - tag ( chx epo - tag ) . at the moment of dissection the experimental procedures were performed in agreement with the guide for the care and use of laboratory animals published by the national institutes of health ( nih publication no . 8523 , revised 1996 ) and were approved by the french ministre de l'agriculture guidelines ( authorisation number a-93 - 008 - 01 ) . mice undergoing ahx and chx were housed for 24 hours and 14 days , respectively , in a hypoxic chamber connected to a gas pump where air circulated at a pressure of 420 mm hg ( ~4500 m ) . hypobaric hypoxia was maintained by a vacuum source at flow rates sufficient to prevent co2 build - up . chamber pressure was interrupted 2030 minutes three times a week for cleaning , and food and water replacement . pressure changes were achieved slowly and the renewal of air in the chamber was sufficient to ensure the composition of atmospheric air . normoxic groups were kept in a normobaric normoxic environment ( 760 mm hg ) outside the hypobaric chamber . all animals were housed in standard conditions ( temperature 2023c with a 12 hours light : 12 hours dark cycle ) and had free access to tap water and food . at the end of time of each experiment two intra cardiac blood samples were taken for hematocrit determination with a microcentrifuge ( microspin ames , germany ) and hemoglobin measurement ( osm 3 , radiometer , copenhagen , denmark ) . under sterile conditions , gastrocnemius muscles were removed , rinsed in sterile phosphate buffered saline solution ( pbs ) . the muscles were snap frozen in liquid nitrogen or mounted in tissue freezing embedding medium ( cryoblock , labonord , france ) for immunohistochemistry techniques and then immediately placed in liquid nitrogen cooled isopentane . the quantitative determination of mouse epo concentration in plasma and muscle extracts was assayed by enzyme - linked immunosorbent assay ( elisa ) by using the quantikine mouse epo immunoassay ( r&d systems europe , abingdon , uk ) . for determination of epo plasma concentration , nx wt , nx epo - tag , ahx wt and ahx epo - tag were used ( n = 4 in each group ) . the plasma was collected using heparin as an anticoagulant , centrifuged at 13,000 g , 15 minutes at 4c and stored at 20c . because nothing is known about epo concentration in skeletal muscle , the gastrocnemius , soleus , plantaris , extensor digitorum longus and tibialis anterior muscles ( hindlimb muscles ) were mixed to ensure an adequate extraction volume and a sufficient protein quantity to ensure the detection by the kit . for this experiment these five muscles were homogenized together in a very small volume of pbs ( 1 : 4 weight / volume ) to concentrate total proteins , centrifuged for 10 minutes , 5,000 g at 4c and directly stored at 20c . after 2 freeze - thaw cycles to break - up the cell membranes , muscle homogenates were assayed without prior dilution whereas plasma samples required a 2-fold dilution in a calibrator diluent provided in the kit . a standard curve was done in duplicate using a stock solution of epo standard to produce a 2-fold dilution series ranging from 3000 to 0 pg / ml . in parallel , 50 l of sample were added to 50 l of assay diluent specific for mouse samples into each well of a microplate coated with a monoclonal antibody against mouse epo and incubated for 2 hours on a microplate shaker at room temperature . a positive control consisting of a recombinant epo solution , provided in the kit , was also loaded . after washing , 100 l of a monoclonal antibody against mouse epo conjugated to horse radish peroxidase ( hrp ) were added to each well and incubated for 2 hours on a microplate shaker at room temperature . finally , 100 l of substrate solution were added after 4 washes and incubated for 30 minutes at room temperature on the benchtop in the dark . after 30 minutes , the reaction was stopped using 100 l of a stop solution in each well . the concentration of epo ( pg / ml ) detected in each sample was compared to the epo standard curve . finally , data were expressed as the ratio of the quantity of epo ( in pg ) to that of total protein ( in mg ) for muscles . nuclear extracts of gastrocnemius of nx wt , nx epo - tg , ahx wt , ahx epo - tag , chx wt and chx epo - tag mice ( n = 5 per group ) were obtained by using lysis buffer a : 10 mm hepes ( ph 7.9 ) , 1.5 mm mgcl2 , 10 mm kcl , 0.5 mm dtt , 0.1% np-40 , 2 mm na3vo4 , 5 mm naf , an antiproteinase coktail and lysis buffer b : 20 mm hepes ( ph 7.9 ) , 1.5 mm mgcl2 , 420 mm nacl , 0.5 mm dtt , 25% glycerol , 2 mm na3vo4 , 5 mm naf and antiprotease coktail as described in the protocol of duoset ic mouse active hif-1 ( r&d system , europe , abingdon , uk ) . the determination of active hif1- has been carried out as described by the manufacturer ( r&d system , europe , abingdon , uk ) . total rna was extracted from the gastrocnemius muscle of mice from different groups ( nx , ahx and chx from wt and epo - tag mice ; n = 5 in each group ) by using the rneasy mini kit and it was digested with rnase free dnase set ( qiagen , courtaboeuf , france ) following the method provided by the manufacturer . rna preparations were quantified by using an ultraviolet visible recording spectrophotometer ( lkb pharmacia ultrospec ) using absorbencies at 260 and 280 nm . to evaluate the purity of the extracted rna , absorbance ratios at 260 nm/280 nm ( rna / protein ) were determined . we controlled the 260/280 ratio which was between 1.8 and 1.9 , indicative of pure rna . reverse transcription ( rt ) was carried out to synthesize the first strand cdna from 1.9 g of mrna with the superscript iii first - strand synthesys system ( invitrogen , carlsbad , ca , usa ) . polymerase chain reaction ( pcr ) techniques were used to identify the expression of epo mrna in the experimental samples . the cdna sequences for the genes of interest were obtained from genbank . all the primers sequences and rt - pcr parameters used in this study the pcr reaction mix contained 10x reaction buffer , 50 mm mgcl2 , dntp mix ( 10 mm each datp , dgtp , dctp and dttp at neutral ph ) , 1 m primers of epo or -actin , taq polymerase ( invitrogen , france ) , cdna and sterile water . pcr was carried out by using a dna thermal cycler ( r&d sytem , england ) as follows : 95c for 5 minutes ; then 35 cycles for -actin and 38 for epo of denaturation at 95c for 1 minute , annealing at 60c for 1 minute and extension at 72c for 1 minute each . a 10 l portion of the amplified pcr product was analysed by electrophoresis on a 2% agarose gel that was stained with ethidium bromide for visualization of dna bands by ultraviolet illumination . a 50base pair ( bp ) ladder dna molecular weight marker was used ( fermentas , france ) to provide a size reference for the test reactions . real - time rt - pcr technique was used to identify the distribution of vegf isoforms , vegfr1 vegfr2 and epor in the rna samples . the vegf common forward primer is located on exon 4 , the vegf120 reverse primer is located on the boundary of exon 5 and exon 7 , vegf164 reverse primer is located on the boundary of exon 5 and exon 8 , and vegf188 reverse primer is located on the boundary of exon 5 and exon 6 . the light cycler faststart dna master sybr green i ( roche biochemicals , stockholm , sweden ) was used for quantitative analyses of the generated cdna . the rt - pcr amplifications were performed in a total volume of 20 l , containing 5 l cdna sample , 4 l lightcycler - faststart dna master sybr green i , 0.5 l of each primer , and 2.5 mm mgcl2 . for each reaction , amplification was then performed for 45 cycles of switching between 95c for 10 s , gene dependant tm for 5 to 8 s , and 72c for 8 to 15 s depending on the length of the amplicon and followed by a melting point analysis from 65 to 95c . control cdna of normoxic mouse lung was diluted and amplified to create standard curves by plotting ct values versus cdna templates . relative amounts of mrna , normalised by -actin were calculated from ct values according to the manufacturer 's description . the relative quantification values were calculated according to the manufacturer 's protocol ( roche biochemicals , stockholm , sweden ) . the cp represents the pcr cycle at which an increase in fluorescence above a baseline signal can be detected . cp value was used to calculate the amount of rt - pcr product compared with the internal control , -actin . the cp value of -actin was subtracted from the gene cp value to evaluate the mean change in cp in each experimental group . gastrocnemius muscles from different groups ( nx , ahx and chx from wt and epo - tag mice ; n = 4 in each group ) were weighted and homogenized in 5-fold volume in ice cold buffer ( 10 mm tris , ph 8) . the protein content of the supernatant was determined by the bradford assay using bovine serum albumin ( bsa ) as standard . the quantity of total vegfa in the gastrocnemius muscles from wt and epo - tag mice in normoxia and following acute and chronic hypoxic exposure was determined by a commercial high - sensitivity elisa kit ( duoset kit ) according to the manufacturer 's instructions ( r&d systems europe , abingdon , uk ) . measurements were done in duplicate . for visualization of vascular endothelial cells we used the monoclonal antibody cd31 which recognizes platelet endothelial cell adhesion molecule ( pecam-1 ) , a glycoprotein strongly expressed in all endothelial cells . to that end , serial 10 m transverse sections from the midbelly region of frozen gastrocnemius muscles samples from nx wt , nx epo - tag , chx wt and chx epo - tag ( n = 5 in each group ) were cut in a cryostat ( leica , france ) at 20c and mounted in microscope slides ( superfrost plus , fisher scientific , france ) . the slides were fixed in cold acetone and incubated in 3% hydrogen peroxidase ( h2o2 ) to inhibit any endogenous peroxidase . then , they were blocked with 3% bsa before incubating with rat antimouse cd31/pecam-1 monoclonal antibody ( bd biosciences pharmingen , france ) diluted 1 : 100 in the protein blocking solution . tissue sections were incubated with a biotynilated antirat secondary antibody ( dako , france ) diluted 1 : 200 in blocking solution . staining was carried out using streptavidin - hrp ( dako , france ) and revelation was done by incubating slides in the dark in a 3 , 3-diaminobenzidine ( dab ) solution ( sigma , france ) . the sections were counterstained with haematoxylin ( sigma , france ) and mounted in aqueous mounting agent ( aquatex , darmstadt , germany ) . because the capillary tortuosity in muscles is a function of sarcomere length , the longitudinal 10 m cryo - sections were stained by using haematoxylin ( sigma , france ) and eosin ( sigma , france ) to make sure it was constant between the different conditions . the length occupied by 10 consecutive sarcomeres was measured per longitudinal section in 2 samples of nx and chx with the software described below . this technique of immunostaining does not allow to identify the various types of blood vessels ( arterioles , capillaries or venules ) nor the diameter of them . stained sections were studied under a light microscope connected to a digital camera ( coolpix 990 , nikon ) . image calibration was done by taking a photo of a micrometric glass coverslip at x40 magnification . explora nova image analysing software ( explora nova morpho , la rochelle , france ) was used to analyse the images . to determine the microvessel network we analysed two different sections . on each section , we have chosen two different areas for the deep region and for the superficial region . capillary density ( cd ) and capillary - to - fiber ratio ( c / f ) were quantified for each entire area . all transversely cut capillaries were counted and if a capillary was sectioned longitudinally , it was counted as one each time it crossed a junction between three or more muscle fibers . the individual fiber parameters were evaluated by counting an average of 70 fibers by area . for each individual fiber the following parameters were evaluated : ( a ) the number of capillaries around a single fiber ( capillary contacts , cc ) ; ( b ) the cc relative to the area of the fiber ( ccfa ) ; ( c ) the length of the contact between the microvessels and the fibers ( lc ) ; ( d ) the capillary - to - fiber ratio on an individual - fiber basis ( c / fi ) ; ( e ) fiber cross - sectional area ( fa ) ; and ( f ) fiber perimeter ( pf ) . obliquity in fiber sectioning was assessed by using the form factor ( ff ) that represents : ( 4 x fiber area)/(fiber perimeter) ( a perfect circle will have a ff of 1 , whereas a line 's ff will approach zero ) . the capillary - to - fiber perimeter exchange ( cfpe ) index was calculated to obtain an index of the size of the capillary - to - fiber interface and was determined from the equation : cfpe index = ( c / fi)/p . this index is expressed as a percentage of muscle fiber perimeters in contact with the capillary wall . two - way analysis of variance ( anova ; hypoxia versus epo deficiency ) was carried out and newman - keuls test was used for post hoc test . a nonparametric mann - whitney test was exceptionally used for plasma epo measurements since the nx wt group was only three mice making impossible to perform a two - way anova . the statistical analyses were performed using stastitica software ( startsoft , tulsa , ok ) . all values are expressed as mean sd . differences were considered to be significant at p .05 . body and muscle weights , hematocrits and haemoglobin concentrations of nx , ahx and chx of wt and epo - tag mice are presented in table 2 . body and muscle weights were similar in all groups whether submitted or not to hypoxia . hematocrit and haemoglobin concentrations were significantly 57% and 61% lower in nx epo - tag mice than in nx wt group demonstrating severe anaemia ( global effect , p < .001 ) . exposure to hypoxia for 14 days induced a polycythemia in wt ( p < .05 ) . epo - tag mice also responded to hypoxia exposure , and hematocrit values were 40% higher in chx than in nx mice ( p < .05 ) . however , haemoglobin concentration in epo - tag mice did not change when compared to normoxic values . in order to validate our model we chose to measure the plasma concentration in nx and ahx conditions for 24 hours because the hypoxia - induced erythropoiesis activation occurred during the early phase of exposure ( 59 days ) with a peak of expression of epo at 24 hours . the plasma and muscle epo measurements showed a strong reduction in epo - deficient mice ( epo - tag mice ) ( table 3 ) . in nx epo - tag mice , epo plasma concentration was 56% lower than in nx wt group ( p < .05 ) . acute hypoxia exposure increased epo plasma concentration in both groups , but remained 3-fold lower than in ahx wt animals ( p < .05 ) . similar results were reported for epo muscle concentration which was 2-fold lower in nx epo - tag mice than in nx wt group ( p < .05 ) . acute and chronic hypoxia exposure did not change epo concentration in the skeletal muscle in neither wt nor epo - tag mice . our results of classical rt - pcr did not demonstrate any expression of mrna encoding epo whatever the experimental condition ( figure 1(a ) ) . those results were confirmed by real - time rt - pcr ( data not shown ) . using the same transgenic model we demonstrated an upregulation of mrna encoding the epor in the brain which suggests an optimization of epo signalling to epo deficiency . in the skeletal muscle moreover , it can be noticed that the amount of epor is extremely weak when compared to -actin ( figure 1(b ) ) . we hypothesised that epo deficiency could induce changes in the microvessel network of the skeletal muscle as well as muscle fiber atrophy . immunostaining analysis of cd 31/pecam-1 was designed to visualise the distribution of vessels in gastrocnemius muscles ( superficial and deep region ) of wt and epo - tag mice after exposure to chronic hypoxia ( figure 2 ) . the morphometric parameters measured are reported in table 4 . the two - way anova showed a main global effect of epo deficiency in the microvessel network when expressed in c / f , c / fi and cc . these three parameters were higher in epo - tag mice when compared to wt ( p < .05 ; table 4 ) in both superficial and deep regions of the gastrocnemius muscle ( figure 2 ) . the lc / pf ratio was higher in epo - deficient mice compared with wt mice , but only in the superficial region of gastrocnemius ( + 34% and + 23% in nx epo - tag and chx epo - tag groups , resp . ; no statistical difference was shown in the deep regions , but the values of epo - tag mice tended to be higher than wt mice in normoxia and after chronic hypoxia ( table 4 ) . the absence of any change in ff showed that the cross - sectional morphology of muscle fibers remained unchanged ( table 4 ) . taken together , the results of ff and lc / pf suggest a change in microvessel tortuosity in the superficial region of the gastrocnemius muscle in epo - tag mice . the cd , fa and pf were not significantly altered in any examined groups and experimental conditions ( table 4 ) . equality , the cc expressed in relation to fiber area ( ccfa ) and the c / fi ratio expressed in relation to fiber perimeter ( cfpe ) were similar in all groups , whether submitted to hypoxia or not ( data not shown ) . the sarcomere length of the gastrocnemius was homogeneous among myofibrils of each group ( data not shown ) . because epo is required for oxygen delivery we suggested that epo deficiency could induce local hypoxia . the amount of active hif-1 was not changed between wt and epo - tag mice in both normoxia and hypoxia ( figure 3(a ) ) . mrna encoding hif-1 is unchanged in nx epo - tag mice compared to nx wt one ( figure 3(b ) ) . when exposed to acute hypoxia both groups exhibit an upregulation of hif-1 mrna ( p < .001 ; figure 3(b ) ) while no difference was observed in the chx groups . we analysed the amount of vegf protein in gastrocnemius muscle of wt and epo - tag mice both in normoxia and submitted to acute and chronic hypoxia by elisa . no significant difference was observed in vegf protein level between wt and epo - tag normoxic groups . in wt mice , the level of vegf protein increased by 29% ( p < .01 ) after acute exposure to hypoxia . in contrast , vegf protein content was reduced by 21% after chx , in comparison with normoxic values ( p < .05 ) . the vegf protein content in epo - tag mice failed to change with hypoxia exposure . in comparison with ahx wt , the amount of vegf protein was 30% lower in ahx epo - tag ( p < .01 ) whereas it was 28% higher in chx epo - tag than in chx wt ( p < .01 ) . using real - time rt - pcr , we quantified the expression of mrna spliced variant isoforms vegf120 , vegf164 and vegf188 as well as vegfr1 and vegfr2 of wt and epo - tag in the gastrocnemius muscle of nx , ahx and chx mice groups in order to examine the responses of vegf isoforms to epo deficiency and decreased oxygen transfer . our results showed that the expression of vegf120 and vegfr2 were modulated in hypoxia and/or epo deficiency only ( figures 4(a ) and 4(e ) ) . .001 ) in acute hypoxia in wt mice compared to nx groups . for epo - deficient mice , significant upregulation of vegf120 mrna vegfr2 mrna was upregulated in nx epo - tag group compared to nx wt ( p < .001 ) . in chronic hypoxia , vegfr2 mrna were upregulated in both epo and wt groups compared to corresponding normoxic ones ( figure 4(e ) ) . the rate of expression of vegf164 and vegf188 mrna remained unaffected by either hypoxia exposure or epo deficiency ( figures 4(b ) and 4(c ) ) . vegfr1 mrna was upregulated by acute hypoxia in epo - tag mice compared to nx epo - tag and ahx wt ( p < .01 ; figure 4(d ) ) . no difference was observed in chronic hypoxia and/or in normoxic epo - deficient mice compared to wt ones . in this work , we aimed to determine whether epo deficiency could be involved in the skeletal muscle fibers atrophy and the alteration of microvessel network . in fact , it has been suggested that epo could be an important growth factor for skeletal muscle development and repair . our principal findings are : ( 1 ) epo - deficient anemic muscles do not exhibit atrophy in both normoxia and hypoxia ; ( 2 ) microvessel network of epo - deficient mice is improved compared to wt mice ; ( 3 ) when exposed to hypoxia epo is not accumulated in hindlimb muscles and both mice linages exhibit an upregulation of vegf120 and vegfr2 in gastrocnemius muscle ; ( 4 ) epor expression is not upregulated by either hypoxia or epo deficiency . our transgenic model ( epo - tag ) is able to survive to 14 days in severe hypoxia ( 4500 m ) despite the weak expression of epo . in fact , in these mice , the expression of epo gene is reduced , but the production of epo still exists . the amount of epo is dramatically reduced in plasma resulting in a severe anaemia in these mice while a slight reduction is observed in hindlimb muscles . however , although epo availability was lower in epo - tag than in wt mice after acute exposure to hypoxia ( 24 hours ) , the epo protein expression remained responsive to the hypoxic stimulus as previously described . actually , in mice submitted to acute hypoxia , plasma epo is increased in both wt and epo - tag compared to normoxic mice even if the epo level is considerably lower in epo - tag than in wt mice . in hindlimb muscles , we did not find any difference between epo levels in normoxic and acute hypoxic mice . in rodent , it has been demonstrated an upregulation of mrna encoding epo in kidney as early as the first hour of exposure to hypoxia leading to a peak of plasma epo between the 12th and the 24th h of exposure . since we did not detect the presence of rna encoding epo at any experimental point , the weak amount of epo protein we measured in muscle probably corresponded to the epo present in capillaries or in the interstitial environment of myofibers . those results do not support the idea that epo is secreted by the skeletal muscle when exposed to both normoxia or hypoxia at rest as it has been shown in rats submitted to exercise . furthermore , the low amount of epor encoding mrna as well as the absence of regulation of its expression in epo - deficient or hypoxic gastrocnemius consolidates the hypothesis by which epo is not an important growth factor in the skeletal muscle in our experimental conditions . in order to explain the presence of mrna encoding epor in the gastrocnemius muscle we suggest that few nonmuscle cells could express epor lately , lebaron et al . demonstrated that cells dispersed in the fascia of rat quadriceps were able to respond to epo injections by stat5 phosporylation . taken together these results suggest that circulating nonresident cells originating from bone marrow could explain the expression of epor . moreover , the injection of rhuepo in musculocutaneous tissues induces the upregulation of endothelial nitric oxide synthase ( enos ) and prevents musculocutaneous tissues from ischemic damage . these results suggest that epo could have some undirect effects on the skeletal muscle such as increasing the perfusion of arteriolar or the mobilisation of nonresident stem cells in traumatizing conditions . in our work does not induce skeletal muscle atrophy even when mice are exposed to hypoxia , but we observe the improvement of microvessel network when determined through parameters evaluating angiogenesis such as c / f , c / fi , cc and lc / pf . the capillary number per area of muscle ( cd ) is an unreliable index of angiogenesis if the muscle fibers enlarge or atrophy [ 30 , 31 ] . indeed , some studies showed that body and muscle mass is reduced after exposure to hypoxia . these changes induce a decrease in muscle fiber size and an increase in cd not necessarily by the formation of new capillaries . in our work , we did not find a loss of body mass or muscle and the fa was not decreased in epo - deficient mice . the great variations of surface area measures can explain the nonsignificant change in cd despite the increase of c / f . therefore , we may conclude that the improvement of microvessel network can be attributed to angiogenesis in both studied regions . the improvement of both c / f and tortuosity ( lc / pf ) in the superficial region is probably due to the fact that glycolytic fibers , which are predominant in this region , are more sensitive to local hypoxia . indeed , in vitro , these factors were shown to stimulate endothelial cell migration and proliferation , which are key steps in the formation of new vessels [ 10 , 34 ] . suggesting that the lack of epo induces a default of oxygen supply to tissues which in turn activates a locally hif - vegf dependent angiogenic pathway . although we did not demonstrate any accumulation of either active hif-1 or vegf protein , we suggest that the improvement of capillary network occurred during muscle development . these results show that epo is dispensable first for microvessel formation and maintenance in the skeletal muscle . in chronic altitude hypoxia , nevertheless , there has been a number of discrepancies in the literature about physiological adaptations to altitude and angiogenesis in the skeletal muscle ( for review , see [ 36 , 37 ] ) . found an increased capillary density in the white part of the gastrocnemius muscle but no change in the number of capillaries around a single fiber in the red and white parts of gastrocnemius of mice submitted to 7 and 21 days of hypoxia ( ~7200 m ) . smith and marshall showed that an arteriolar remodelling within the skeletal muscle in rats during acclimatisation to chronic hypoxia occurs between the 7th and the 18th day of exposure to hypoxia ( 4500 m ) . olfert et al . did not find any hypoxia - induced angiogenesis in murin models . the deep region of this muscle is characterized as predominantly fast - twitch red fibers and the superficial region as predominantly fast - twitch white fibers . our results confirm the idea that chronic hypoxia does not provide a sufficient stimulus to induce capillary growth in mice skeletal muscles at least in the early phase of acclimatisation of hypoxia . actually , it has been described by some authors as a late acclimatisation ( up to 5 weeks ) to hypoxia which consists in skeletal muscle atrophy inducing in some cases the increase of capillary density ( for review ) . the real cause of this atrophy is not yet well known and factors such as intestinal malabsorption or inactivity have been suggested to be involved in this late acclimatisation to hypoxia . in this work we focused on the role of epo on the epo - dependent early phase of acclimatisation , so we may conclude that the lack of change in muscle capillary bed and the absence of skeletal muscle fiber atrophy in response to ambient hypoxia is not affected by epo deficiency . all studies dealing with hypoxia , vegf and skeletal muscles have addressed the vegf164 , which is the major isoform of vegf in the skeletal muscle . we did not find any changes in vegf164 and vegf188 expression in both epo - deficient or hypoxic gastrocnemius muscles . interestingly , both epo - deficient and wt mice exhibit a vegf120 response to hypoxia . this response is combined with the upregulation of vegfr2 in wt mice . among all vegf isoforms , vegf120 has been shown to have the weakest angiogenic effect . moreover , it has been demonstrated that vegf120 does not induce either the activation or the accumulation of endothelial cells in culture . lately , it has been proposed that vegf120 , when binding to vegfr2 receptor , may be involved in the control of dilatation and permeability of vessels especially on endothelial cells of the human umbilical cord . moreover , it has been demonstrated that vegf120 in brain melanoma was able to diffuse away from the tumour in order to recruit vasculature from surrounding tissues . taken together , these results may suggest that acute hypoxia induces the upregulation of vegf120 which acts through vegfr2 signalling and improves the recruitment of existing capillaries in order to ameliorate oxygen supply to the muscle . surprisingly , the upregulation of vegf120 in epo - deficient gastrocnemius is delayed when compared to wt . this phenomenon needs further investigation in order to understand if this delay is due to ( 1 ) epo deficiency ; ( 2 ) upregulation of vegfr1 , which is known to regulate the action of vegfr2 , or ( 3 ) the fact that vegfr2 is already over - expressed in epo - deficient mice . furthermore , these results suggest a nonangiogenic adaptation of microvessels when both groups of mice were exposed to hypoxia but this hypothesis needs to be verified through further investigation , using new imaging techniques , for instance . to conclude , this report showed that the gastrocnemius muscle possesses the remarkable feat to compensate epo deficiency - induced anaemia by developing its microvessel network . therefore epo deficiency alone is not a sufficient factor to explain muscle deconditioning in patients suffering from severe renal failure . moreover , we showed that the skeletal muscle exhibits an upregulation of vegf120 and vegfr2 when submitted to hypoxia , which suggests an improvement in the dilatation of pre - existing microvessel network in order to ameliorate oxygen supply .

erythropoietin ( epo ) and vascular growth factor ( vegf ) are known to be involved in the regulation of cellular activity when oxygen transport is reduced as in anaemia or hypoxic conditions . because it has been suggested that epo could play a role in skeletal muscle development , regeneration , and angiogenesis , we aimed to assess epo deficiency in both normoxia and hypoxia by using an epo - deficient transgenic mouse model ( epo - tagh ) . histoimmunology , elisa and real time rt - pcr did not show any muscle fiber atrophy or accumulation of active hif-1 but an improvement of microvessel network and an upregulation of vegfr2 mrna in epo - deficient gastrocnemius compared with wild - type one . in hypoxia , both models exhibit an upregulation of vegf120 and vegfr2 mrna but no accumulation of epo protein . epor mrna is not up - regulated in both epo - deficient and hypoxic gastrocnemius . these results suggest that muscle deconditioning observed in patients suffering from renal failure is not due to epo deficiency .

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

PMC4283748

intra - radicular posts are used to provide retention for restoration of the coronal structure of endodontically treated teeth [ 17 ] . in the recent years , the fiber reinforced composite ( frc ) posts have become more popular because of their suitable physical characteristics that are similar to tooth structure [ 8 , 9 ] . the overall retention of frc posts in the root canal depends on the bond strength between different parts of the combined many of the fiber post failures occur between the root canal wall and adhesive resin cement [ 14 , 15 ] . so , understanding the factors that interfere with the bonding between the root canal wall and resin cement is important . it has been claimed that this can decrease the bond strength between the frc post and root canal dentin [ 2 , 15 , 1820 ] . however , other researchers have reported that eugenol - containing sealers do not have any significant reduction in the adhesion between cemented frc post with resin cement to the root wall [ 18 , 2126 ] . the aim of this study was to evaluate the effect of the type of endodontic sealer on the bond strength of frc posts cemented with resin cement to the root wall dentin . the null hypothesis investigated was that the bond strength of frc posts cemented with resin cement in the presence of different endodontic sealers is not significantly different . in this in vitro study , 20 extracted human mandibular first premolars were selected . a total sample size of 20 ( at least 10 per group ) was required to detect a difference of at least 80% between any two groups with a power of 84% at a 5% significance level . the selected teeth did not have any caries , cracks or fractures , resorption , open apex or previous root canal treatments . the teeth were cleaned of calculus or soft tissue remnants and were placed for two hours in 2.5% naocl ( golrang , golrang co. , tehran , iran ) and then stored in 0.1% nan3 solution . diamond discs ( ref.070 , d&z , berlin , germany ) mounted in a dental lathe machine ( kavo polishing unit . ewl 80 , germany ) were used at low speed under constant water irrigation for removing teeth crowns to achieve a 15-mm root length . barbed broaches ( dentsply / maillefer , ballaigues , switzerland ) were used to remove pulp tissues . the same operator performed canal instrumentation using the step - back technique ( 1-mm short of the apical foramen ) . the master apical file was the number 35 k - file ( dentsply / maillefer , switzerland ) . during canal preparation stages , finally , paper points were used to dry the root canals ( aria dent , asia chemi teb co , tehran , iran ) . the roots were randomly divided into two groups of 10 specimens each ( table 1 ) . in each group , obturation was performed by the vertical condensation method using one type of root canal sealers ( table 1 ) and gutta - percha ( aria dent , tehran , iran ) . tokyo , japan ) was used to fill the coronal root canal orifices and the teeth were stored for one week in 100% humidity at 37c to ensure the setting of the used root canal sealers . then , coronal gutta - percha of each root was removed to a depth of 10-mm with a gates glidden drill # 3 ( dentsply / maillefer , switzerland ) and 4-mm gutta - percha was preserved in their apices . the canals were irrigated with distilled water and dried with paper points ( aria dent , tehran , iran ) . for post length and diameter similarity , a glass reinforced fiber post size # 3 ( hetco fiber post , hakim toos , mashhad , iran ) was tried in the post spaces and then all posts were cut to the length of 10-mm from their apex with diamond discs ( ref.070 , d&z ) mounted in a dental lathe machine ( kavo polishing unit , kavo ewl ) under water irrigation . in all groups , the shortened posts were cleaned with 70% ethanol for 60s , rinsed with distilled water and air - dried . according to a previous study ( 6 ) , the posts were immersed in 20% h2o2 for 20 minutes at room temperature . after air - drying , the post surfaces were painted with a single layer of a silane coupling agent ( ultradent porcelain etch and silane , ultradent products inc . , ut , usa ) for 60s and dried for 60s with gentle air stream . in all groups , the root canal walls were conditioned with an autopolymerizing primer ( ed - primer , kuraray medical inc . , then , the post spaces were air - dried and the excess primer was removed with paper points ( aria dent , iran ) . in the cementation stage , equal amounts of base and catalyst pastes of an adhesive composite resin cement ( panavia f2.0 , kuraray medical inc . , tokyo , japan ) were mixed and applied on the prepared post surfaces and into the post spaces with a lentulo spiral instrument ( dentsply / maillefer , switzerland ) . the fiber posts were inserted into the root canals using gentle finger pressure . after removing the excess cement around the post , oxygen - inhibiting gel ( oxyguard ii , kuraray medical inc . the resin cement was light cured for 60s by a halogen light unit with 500-mw / cm2 intensity ( coltolux50 , coltene , altstatten , switzerland ) . before each light exposure , coltolux light meter ( coltene , altstatten , switzerland ) was used for monitoring accurate light intensity of the light output . after storing all specimens in 37c saline for one week , all the specimens were subjected to thermocycling treatment for 5000 cycles at temperatures alternating between 5 and 55c for 30s each with an intermediate pause of 15 seconds . each dental root was sectioned perpendicular to its long axis using a diamond disc ( ref.070 , d&z , germany ) mounted in a cutting machine ( tl-3000 , vafaei industrial , tehran , iran ) at low speed under water irrigation to create three post / dentin slices ( coronal , middle and apical ) with 3-mm thickness for each slice . due to using tapered fiber posts , the post diameters and each slice size were measured using a digital caliper ( mitutoyo digital caliper 500 - 714 - 10 , mitutoyo co , tokyo , japan ) with 0.01 mm accuracy . push out test was performed with a universal testing machine ( walt+bai ag testing machines industries trass 4 , lhningen , switzerland ) at across head speed of 1 mm / min . the push - out pin was placed on the center of the apical end of the sliced fiber post and in an apico - coronal direction without inserting extra forces on the surrounding root canal walls . therefore , it was necessary to make three push - out pins in three diameters ( 0.7 , 0.9 and 1.0 mm ) that were used for each three - root section , respectively ( apical , middle and coronal parts ) . the peak force ( n ) required to extrude the fiber post from each root slice was recorded for all specimens . the bond strength in mpa was calculated with the following formula : a=(r1+r2)(r1r2)2+h2 in this formula , is the constant 3.14 , r1 is the coronal post radius , r2 is the apical post radius and h is the slice thickness in mm . , chicago , ill ) using two - way anova and post hoc tukey test at p<0.05 level of significance . table 2 shows the mean tensile bond strength ( tbs ) and standard deviation ( sd ) values for all experimental groups in different root canal regions . the two - way anova showed that root canal sealer as a main factor ( p=0.037 ) had significant effect on bs , but root canal dentin regions ( p=0.811 ) and the interaction between root canal sealers and root canal regions ( p=0.258 ) had no significant effects on bs ( table 3 ) . the lowest tbs mean value was seen in the endofill group and in the apical region . post hoc tukey test revealed that there were no significant differences between different root canal regions in both groups ( p>0.05 ) ( table 4 ) . the results of this investigation rejected the null hypothesis that different endodontic sealers do not have any effect on the bond strength of frc posts cemented with resin cement . according to the present study , the mean tensile bond strength ( tbs ) of fiber posts that were luted in the root canals with resin based sealer ( ah26 ) was significantly higher than those luted with eugenol containing sealer ( endofill ) . other studies have also showed that eugenol containing root canal sealers can decrease the bond strength of resin cements [ 13 , 16 , 1820 , 27 ] . it has been mentioned that eugenol ( 2- methoxy-4-allyphenol ) can spread in dentinal tubules and its phenolic components can deactivate the molecules in the growing polymer chains and jeopardize resin cement setting and decrease the bond strength [ 1 , 3 ] . however , other studies did not find any statistically significant difference between different types of root canal sealer on the retention of glass fiber posts using resin cements [ 21 , 22 , 24 , 25 , 28 ] . on the other hand , a greater amount of eugenol is released during the first two weeks of using eugenol containing sealer . in the present study so the remnants of the eugenol containing sealer could interfere with the polymerization reaction of resin cement and decrease its bond strength . however , some authors reported that there are no differences between effects of various root canal sealers on the tensile bond strength of fiber posts [ 2126 , 28 ] . it is stated that during post space preparation , most of the root canal sealer remnants are removed and the amount of debris and free eugenol available to inhibit the polymerization reaction of the resin cement is decreased . therefore , creating a clean post space is one of the most important factors in achieving bonding with resin cements . another technique for cleaning the post space wall is phosphoric acid etching followed by water rinsing [ 11 , 29 ] . self - etching systems that eliminate etching , rinsing , and drying steps may decrease the bond strength of self - etching systems due to incomplete cleaning of the root canal space [ 30 , 31 ] . the resin cement system used in this study has a self - etching bonding system ( ed primer ) that is less aggressive than etching with 37% phosphoric acid [ 29 , 32 ] . so the created thick smear layer in the post space might decrease the bond strength of the used resin cement in this study . bond strength of resin cement can be at the highest level if the orifices of dentinal tubules are kept open , but eugenol can spread in these tubules and modify the surface of the cement resin and decrease bond strength . it has been showed that using alcohol or acid conditioning in the post space can increase bond strength . the self - etching system ( ed - primer ) that was used in this study did not condition the post space wall and the lower bond strength of the endofill group and decreasing the bond strength from the coronal to apical region in this group can be explained by this fact . it is interesting that in ah26 group ( resin based sealer ) the bond strength was increased in more apical regions ( figure 1 ) . this can be due to lower penetration of the resin sealer into dentinal tubules or the presence of residues of resin based sealer in the apical portion of post space . these residues can bond with post space walls and may help better bonding with resin cement . this finding is in accordance with studies [ 33 , 34 ] that used resin cements and resin based sealers and found highest push out strengths in the apical sections . therefore , resin based sealers can be used in the inaccessible apical site within the root canal space ; thereby creating strong bonding strength as well as a fluid - tight seal . jha imputed a greater bond strength in the apical third to factors such as the greater wear of this region by endodontic reamers and creating a clean dentin that comes in better contact with the bonding agents . in addition , , he stated that there is an intimate contact between the post and root canal wall in this area , while the misfit between post and root canal space in the coronal region results in a greater volume of resin cement that can increase stress in the cement layer due to polymerization shrinkage . moreover , because the cement layer is the weakest area between the post and the post space wall , the greater thickness of the cement may be the cause of the reduced bonding strength observed in the coronal third . although these argumentations can explain the higher bonding strength in the apical third of specimens in the ah26 group , they can not clarify the opposite condition in the endofill group . in the endofill group , our results are in accordance with studies that have reported higher bonding strength in the more coronal thirds [ 5 , 11 , 35 ] . the higher tubular density and diameter of the coronal third of the post space are more than the other regions . furthermore , the dentinal hybridization is not uniform and lateral branches of resin tags are not seen in the apical thirds of post space . furthermore , more access to the coronal third of the canal makes etching and applying the adhesive agents [ 36 , 37 ] and direct light transmission to canal walls more effective in this area . however , aksornmuang et al . found no significant differences in the bond strengths among different regions of the root canal walls . they related this result to this fact that their used light source had a light intensity of about 903 mw / cm2 , which was not enough to completely cure composite resin . for example , the specimens had no coronal tooth structure , only one type of fiber post and adhesive were evaluated and the influences of fatigue loading on the push out tbs of specimens were not investigated . in vitro tensile tests for measuring bond strength may not be exactly representative of the clinical conditions . therefore , clinical studies should be performed to validate the results of the present study . within the limitation of this in vitro study it was concluded that : the type of root canal sealer had significant effect on the bond strength of cemented fiber posts.eugenol-based sealers ( endofill ) significantly reduced the bond strength of fiber posts luted with resin cement.there were no significant differences between different root canal regions in both groups . the type of root canal sealer had significant effect on the bond strength of cemented fiber posts . eugenol - based sealers ( endofill ) significantly reduced the bond strength of fiber posts luted with resin cement .

objectives : an important factor that interferes with the bonding between the root canal wall and resin cement is the root canal sealer remnant . there is controversy about the effect of eugenol - containing sealers on the bond strength between resin cements and fiber post . the aim of this study was to evaluate the effect of the type of endodontic sealer on the bond strength of frc posts cemented with resin cement to the root canal wall.materials and methods : in this in vitro study , 20 extracted mandibular first premolars were endodontically treated and divided into two groups according to the endodontic sealer used ( n=10 ) : g1 : ah26 ( resin based ) ; and g2 : endofill ( eugenol - based ) . after preparing post space , adhesive resin cement ( panavia f 2.0 ) was used for cementation of the fiber post to the root canal dentin . three 3 mm thick slices were obtained from each root.the push - out test was performed with a cross - head speed of 1 mm / minute . two - way anova and tukey post hoc tests were used for analyzing data ( =0.05).results : the two - way anova showed that different root canal sealers ( p=0.037 ) had significant effects on bond strength ( bs ) , but root canal regions ( p=0.811 ) and interaction between root canal sealers and root canal regions ( p=0.258 ) had no significant effects on bs . maximum and minimum mean values were observed in the ah26 group , the apical region and the endofill group in the apical region , respectively . post hoc tukey test revealed that there were no significant differences between different root canal regions in both cements ( p>0.05).conclusion : the region of root canal had no effect on the bond strength of cemented fiber posts to the root canal . eugenol - based sealers ( endofill ) significantly reduced the bond strength between fiber posts luted with resin cement to the root canal .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC3246378

let us remind ourselves of the unique range of properties this phenomenon possesses : 1 . the pattern of ball lightning movement proves that it is a self - contained object with a density approximate to the density of the air ( about 1.54.0 an ability to restore a ball shape after a smoke - like penetration through narrow openings and an ability to retain its shape under conditions of strong atmospheric turbulence are evidence of the existence of a substantial surface tension ( a mutual attraction between elements of the ball lightning substance ) ; 3 . the luminescence of ball lightning is mostly red - orange - yellow ( about 60% ) , and is white in about 25% of observations ; 4 . both low - temperature ( not burning ) and high - temperature ball lightning has been described by eyewitnesses who have had direct physical contact with ball lightning ; 5 . ball lightning with diameters in the range of 1030 cm has been observed most frequently , but objects of a much larger size have been described as well ; 6 . a globe - shaped non - luminous cloudlet is observed sometimes within several seconds at the site of the ball lightning s disappearance ; 9 . the energy content of ball lightning with a diameter of about 20 cm has been estimated in the range of several tens to 200 kj ; 10 . ball lightning is sometimes able to emit infrared radiation ( the sensation of thermal radiation ) , as well as emit strong radio noise , which has frequently been registered by radio receivers nearby ; 11 . powerful electromagnetic impulses can be generated when ball lightning explodes ( strong induced overvoltage and currents are demonstrated by both the breakdown of remote electrical equipment , and people far from the ball lightning explosion receiving electric shocks ) . an adequate model of ball lightning should guarantee an explanation all of the aforesaid characteristics . such a model should also explain the great variety and external dissimilarities of the conditions described by direct eyewitnesses of the process of ball lightning formation . in particular , the process of ball lightning formation has been repeatedly and directly observable : ( a ) when lightning strikes trees or ( b ) when lightning strikes lattice steel pylons or ( c ) when lightning strikes open copper wires or ( d ) when lightning strikes brick flues or ( e ) in short circuits in electrical equipment or ( f ) in powerful corona discharges of both natural and technological origin . models of ball lightning as a filamentary network of chain aggregates of nanoparticles slowly oxidizing in the air [ 3 - 6 ] allow us to explain the high energy content of this enigmatic object , though these models do not actually explain its ability to retain and easily restore its ball shape . unfortunately the mentioned models do not also give an explanation for the various electromagnetic effects of ball lightning . here we suggest an alternative aerosol , but not aerogel , model for ball lightning , which is able to explain all the aforementioned properties , as well as a diversity of observable conditions and processes of ball lightning formation . to facilitate discussion , we should briefly remind ourselves of the simplest design of electrochemical power sources . to make an electrochemical power source ( battery , accumulator , fuel cell , etc ) , it is necessary to use at least three components : an electrode reductant , an electrode oxidizer , and electrolyte , separating these electrodes ( a substance aiding the interelectrode transport of ions , but not electrons ) . any pair of substances , each having a different electron affinity and contacting through a suitable electrolyte , is inevitably involved in a reduction oxidation reaction and forms a certain electrochemical cell . such a cell is generally able to generate a voltage in the range of a few tenths of a volt to 5 volts . if one tries to mentally reduce the size of a standard battery , generating a voltage of about 1.5 volts , to the size of 100 nanometers or less ( the characteristic size of a smoke particle ) , it can be seen that the electrostatic intensity inside such a nanobattery , between the spatially divided components of the reductant and the oxidizer , will considerably exceed the sparkover electrostatic intensity ( at normal air temperature and pressure about 30,000 volts / cm ) . thus , a galvanic cell made in the form of a composite submicron or nanoparticle suspended in the air , will be spontaneously shorted by an electric discharge , arising initially on the particle surface ( then also in the adjacent air , in the immediate proximity to this surface ) because the electrostatic intensity is too high . we suggest a model where ball lightning is a cloud of composite particles , with sizes ranging from 5 to 1,000 nanometers , with each particle being a spontaneously formed nanobattery , short - circuited by a surface discharge . aerosol nanobatteries containing at least two key components of any galvanic cell reductant and electrolyte can be formed as a result of very different processes , including for example : ( 1 ) the volume co - condensation of the mixed evaporated products of a spark - arc erosion of composite condensed substances , or ( 2 ) an electrolysis of salt solutions ( or salt melts ) with following high - voltage electrospraying electrolysis - generated composite nanoparticles , or ( 3 ) a high - voltage or plasma electrospraying composition of molten metals , their mixed oxides , and electrolytes . as a result of these or similar processes , aerosol nanobatteries can apparently be spontaneously formed in at least two of their principal types either in the form of composite nanoaggregates ( fig . mixed sintered nanoaggregates of condensed smoke particles , containing solid reductant , electrolyte and oxidizer , inevitably form short - circuited aerosol nanobatteries nanocapsules , containing a core reducing agent and surface electrolyte layer , form short - circuited aerosol nanobatteries although such separate particles - nanobatteries are capable of generating only a standard voltage of tenths of a volt to a few volts , the super - sparkover electrostatic intensity inevitably arises on their surface . this leads to the development of an initial surface breakdown and to the excitation of microscopic contracted arc or arc - like discharges , running on the surface as well as in immediate proximity to the surface of each particle between the spatially divided areas , containing a reductant fuel and oxidizer . apparently , it is important to note that the certain additional conditions are necessary to facilitate the initial development of the breakdown on the surface of the nanobatteries . one of these conditions can be the increased initial surface electroconductivity of nanoparticles , for example due to an initial surface hydration or surface carbonization of nanoparticles . an alternative additional condition to facilitate the initiation of the breakdown on the surface of the nanobatteries can be their high temperature . in this case , the thermoionic and field emission can be major pre - ionization processes generating free seed electrons and initiating the surface breakdown in such white - hot nanobatteries immediately after their spontaneous air synthesis . at the same time , the strong photoionization and/or local production of the seed gaseous ions from a preceding corona , preceding normal lightning , or from a preceding electric arc appear also to be high - performance potential pre - ionization processes facilitating the initiation of microscopic arc discharges on the surface of both high - temperature and relatively cold nanobatteries . thus , apparently , there are two major functions of the electric discharge prior to the formation of ball lightning : ( a ) the synthesis of aerosol nanobatteries ; ( b ) the pre - ionization and ignition of the initial breakdown on the surface of the nanobatteries . generally speaking , aerosol nanobatteries can use both a condensed oxidizer ( a third possible component contained in the nanoparticle ) and external atmospheric oxidizers : first of all , atmospheric oxygen or water vapour , contacting with a core reductant of nanoparticles through a layer of electrolyte . as the electrostatic intensity , generated in the nanobattery , and the surface - to - volume ratio are very high the arc or arc - like discharges , irregularly migrating on a surface of each particle , provide an uninterrupted neutralization of the generated electrochemically charge disbalancement between the heterogeneous areas of the particle , including the areas with a low conductivity . apparently , these arc or arc - like discharges are the main reason for luminescence of low - temperature ball lightning . this attraction is caused by magnetic fields , which are generated around of each particle by closed loops of the galvanic and discharge currents . separate , at first distant from each other , sparkling aerosol particles - nanobatteries approach together and form a luminous ball cloud under the influence of mutual magnetic attraction . at the same time , since the galvanic currents flowing inside the aerosol particles and the surface discharge currents flowing mainly outside the particles form closed current loops , the galvanic and discharge currents inside such current loops are exposed to a mutual repulsion which in turn can result in the displacement of the initial surface discharges into the air space in proximity to the surface of the aerosol particles . gaseous products , for example , hydrogen , carbon monoxide , carbon dioxide , and the like , as well as the thermal energy , produced by each nanobattery as a result of intra - particle self - sustaining electrochemical reactions , excite a mutual repulsion of nanoparticles over short distances due to strong thermo- and diffusiophoresis . because of the powerful local generation of thermal energy and owing to surface discharges , the ionized gas layers develop around each aerosol particle . over short distances , these plasma layers are able to overlap each other , forming branched interparticle series - parallel circuits and connecting separate aerosol nanobatteries in a united aerosol electrochemical generator . accordingly , in these circumstances the substance of ball lightning is conducting and current - carrying low - temperature plasma with a condensed disperse phase aerosol nanobatteries continuously supporting a high ionization of the air disperse medium due to the surface and interparticle discharges . it is important that a collectivization of the current loops of separate aerosol particles , due to the electric arc overlapping between adjacent particles , substantially weakens their mutual magnetic attraction over short distances . it prevents an aggregation of particle - nanobatteries , and so they form a stable ball - shaped cloud with a density , slightly exceeding the air density ( fig . 3 ) . powerful interparticle magnetic attraction forms a stable cloud ball of short - circuited aerosol nanobatteries with total electric overlapping the surface discharges of separate particles various combinations of different reductants , electrolytes and oxidizers are able to form a great number of galvanic cells , including aerosol nanobatteries and clouds of them . apparently , one of the most widespread atmospheric reductants , which are frequently included in composition of the natural aerosol electrochemical power sources , is a soot carbon . in such cases , created for example after lightning strikes a tree , a thin layer of potassium carbonate ( an essential component of wood ash ) , co - condensed on the surface of black carbon nanoparticles , can play the role of a high - performance molten electrolyte in the spontaneous formation of high - temperature molten - carbonate aerosol fuel cells . a volume condensation of evaporated carbon with the production of black carbon nanoparticles is immediately followed by the condensation of molten carbonate layers on the surface of the hot carbon particles ( in these circumstances , the charged black carbon particles are condensation nuclei for evaporated carbonates ) . such a process of high - temperature co - condensation of the carbon fuel and carbonate electrolyte can result in the spontaneous creation of aerosol electrochemical generators with separate core - shell nanobatteries ( nanocapsules ) , suspended in an atmosphere containing oxidizer . the internal allocation of fuel ( the core carbon ) , and the surface position of molten carbonate electrolyte on the carbon particles , enables it to practically completely protect the carbon from normal high - temperature oxidation , and simultaneously allow its efficacious electrochemical oxidation by the atmospheric oxygen ( fig . 2 ) . in black carbon nanoparticles encapsulated in the molten carbonate electrolyte , at a temperature of nearly 900 c , electrochemical ( ion - mediated ) oxidation of the carbon should arise spontaneously and then be thermally self - sustained . the gaseous products of electrochemical oxidation of the core carbon , i.e. , co2and co , perforate the surface molten carbonate layers continuously , forming dynamic self - healed pores in these layers . as a result of the reaction carbon with carbonate ions , the carbon core acquires negative charge , while external surface of molten potassium carbonate shell acquires positive charge due to residual uncompensated ( surplus ) potassium kions . thus , electrochemical potential difference arises , and the co- and co2- generated dynamic pores in molten carbonate shell are initial channels for the arc discharges starting from carbon core of the nanobatteries to their external surface . thus , each separate battery - nanocapsule of this aerosol electrochemical generator contains the black carbon nanoparticle as the core carbon anode , while external surfaces of molten potassium carbonate shells of the batteries - nanocapsules are oxygen - depolarized cathodes of such aerosol nanobatteries supplied with air and co2 . it is worth mentioning that the core carbon electrode in these nanobatteries is an anode only within the framework of electrochemical interpretation . this carbon electrode is charged negatively , and in this case the carbon electrode simultaneously can be named as an electron - emitting cathode ( within the framework of electrophysical or electronic interpretation ) . apparently , high - temperature cathode spots can arise on the surface of the white - hot core carbon nanoparticle . these cathode spots emit the seed electrons for arc discharges due to powerful local thermoionic and field emission . the current density within such arc cathode spots can be extremely high , and high - power electron avalanche breakdown develops from cathode spots inside the co- and co2- generated dynamic pores . as soon as the electron avalanches reach an external surface of the core - shell nanobattery , the gas phase electrons are captured by the surface excess potassium kions and electronegative gas molecules . on the external surface of molten potassium carbonate shell , the cathode reaction , involving electrons , o2 , co2 , and metallic potassium ( the primary product of the k / electron recombination ) , regenerates new potassium k and carbonate ions . further the carbonate ions again repeat process of the oxygen transport through molten potassium carbonate shells to the core carbon anode probably , enormous reaction surface inherent in the nanobatteries and aerosol electrochemical generators , high - energy plasma chemical reagents ( similar to gas phase electrons and ions ) involved in electrode reactions , as well as high work temperature specifically inherent in carbon / air aerosol electrochemical generators cause very high effective power of electrochemical processes even without involving any additional cathode catalysts . interestingly , non - aerosol pilot plants of high temperature , molten electrolyte electrochemical cell devices , able to direct converting carbon black fuel to electrical energy with a voltage of 0.8 v and efficiency 80% , were recently developed and investigated . such carbon fuel cells , chemically similar to described here hypothetic aerosol carbon / air electrochemical power sources , generate electric power from an electrochemical reaction similar to the combustion reaction of carbon:(1 ) the net reaction ( 1 ) can be written as the sum of two half - cell reactions , involving the carbonate ions(2)(3 ) the carbon anode may also partially oxidize to co in a competitive reaction:(4 ) taking into account the experimentally obtained parameters of the voltage and the efficiency of high temperature carbon / air fuel cells with molten - carbonate electrolytes , let us try to estimate the potential characteristics of analogous aerosol electrochemical power source , i.e. , the potential characteristics of the carbon / air ball lightning . let the fuel of this ball lightning aerosol electrochemical generator be black carbon , with the electrolyte being a dynamically porous layer of molten potassium carbonate , condensed on the surface of black carbon aerosol nanoparticles . the volume of such a ball lightning is about 4 l , and the mass of the carbon fuel is 4 g at least . as the heat of the carbon combustion is about 33 kj per gramme , the energy content of this ball lightning can be about 130 kj . at the direct electrochemical conversion of this energy , the total electromagnetic energy of internal discharge currents of this ball lightning accordingly , a density of magnetic energy(5 ) where b is magnetic flux density ( tesla ) , 0 = 4 10is permeability constant ( h / m ) , and 1 is the black carbon aerosol s magnetic permeability , will reach about 20 kj / l , while the magnetic pressure p , maintaining the ball lightning sphericity and being equivalent to , will reach approximately 200 atmospheres . accordingly , in this particular case , the value of the interparticle local magnetic fields b will reach about 7 tesla . it explains , for example , why a powerful air drag is not able to tear the ball lightning substance apart , when the ball lightning escorts aircraft . so , let the lifetime of such a high - temperature aerosol electrochemical power source be about 50 s , during which it gradually spends its energy and then quietly fades . consequently , the average electromagnetic power of this aerosol power source should be about 2 kw ( i.e. , 100 kj/50 s ) . the visible luminescence of this ball lightning is caused by the plasma radiation of the surface particle and interparticle arc - like discharges , a high - temperature luminescence of the hot particles , as well as additional luminescence from the direct oxidation of carbon and carbon monoxide ( a competitive process , proceeding simultaneously with electrochemical oxidation and connected with the partial intracarbonate diffusion of molecular oxygen ) . according to the calculated value of average electromagnetic power above , and according to the above - mentioned value of the voltage of the non - aerosol prototype of the carbon black electrochemical power source , the average total value of internal discharge currents of this ball lightning should be about 2,500 amp ( i.e. , 2 kw/0.8 v ) . the value of these currents , distributed between all the nanoparticles , explain the existence of a powerful mutual magnetic attraction between the particles , a high surface tension value of the ball lightning substance , as well as the strong radio - interferences effects of ball lightning . let the ball lightning exist quietly for only 10 s , and then it explodes as a result of the self - propagation of local thermal fluctuations . at the moment of explosion , the temperature , ion conductivity of the electrolyte , rate of electrochemical reactions , discharge currents , and energy - release strongly increase . assuming that the explosion time is about 0.1 seconds and that the main part of the residual chemical energy of this ball lightning is converted into electromagnetic energy during this time , then obviously the total strength of the pre - explosive discharge currents can reach about 1,000,000 amp . assuming that the ball lightning explosion time is shorter , the total strength of the pre - explosive discharge currents can reach even greater values . a fast increase of discharge currents to such high values during the explosion and following fast droop of current to zero can explain the origin of powerful electromagnetic pulses and various strong distant induction effects , observed by eyewitnesses of ball lightning explosions . the principle of potential energy minimization allows us to expect magnetic ordering effects and the internal dynamic self - compensation of powerful local magnetic fields inside this system of dipole dipole magnetically interacting free aerosol current loops . therefore under conditions of weak external magnetic fields and at sufficient distances from the ferromagnetics , ball lightning as a whole can have only a minimal uncompensated magnetic moment . otherwise , and also , apparently , at the moment of the explosion , the uncompensated magnetic moment of the ball lightning can be substantial . it can lead to effects of attraction of ball lightning towards ferromagnetics , permanent magnets or current sources of external magnetic fields . the magnetic fields , measured inside the clouds of nanobatteries , should , apparently , strongly fluctuate . the large clouds or very large clouds ( e.g. , fog ) of nanobatteries are , probably , also able to interfere with radio communication owing to both the shielding and the radio noise generation . the widespread atmospheric aerosol fuel black carbon nanoparticles nevertheless is only one of numerous potential contenders to work as a reductant in the aerosol electrochemical power sources . in addition to the black carbon , many unoxidized substances ( e.g. , similar to si , zr , fe , cu , al , sn , pb , b , ca , w , s etc . ) or suboxidized substances ( e.g. , similar to feo , cu2o , sio etc . ) , hydrides , carbides , sulphides , silicides as well as fuel gases absorbed by nanoparticles , could apparently work as other probable reductants in the natural aerosol nanobatteries . probably , even some salts with a deficiency of oxygen , e.g. , similar to nitrites ( or sulphites ) being electrosprayed or condensed from vapour in the local atmosphere of the nitric oxide and nitrogen dioxide ( or correspondingly in the sulphur dioxide atmosphere ) in the form of submicron or nanoparticles could also work in the natural aerosol low - power nanobatteries - capsules as high - performance salt reductants . the subsequent air oxidation of these aerosol salt particles could cause the growth of nitrate or sulphate electrolyte layers on their surface . in these circumstances , the external oxidizer oxygen can react with the reductant only through the growing shell of a hydrated or molten electrolyte , in particular , a nitrate or sulphate electrolyte . such a concentric relative position of a reductant , electrolyte and oxidizer is one of the important conditions for the promotion of the preferred electrochemical , ion - mediated oxidation of the core salt reductant instead of its molecular oxidation . the potential natural sources of the electrolyte nanocomponents for low - temperature ball lightning could be , probably , atmospheric hygroscopic substances , in particular , salt ( first of all , chloride or sulfate ) cloud condensation nuclei , or the aerosol products of the erosion of nitrate , carbonate or phosphate minerals as well as atmospheric aerosol products of the volcanic origin ( fig low - temperature clouds of nanobatteries can also be spontaneously created on the basis of composite atmospheric particles , containing for example a mixture of hygroscopic condensation nuclei , metal or metal - oxide mineral particles , and organic nanoparticles at the same time , potential electrolytes for high - temperature ball lightning , i.e. , for ball lightning with temperatures of aerosol nanobatteries in an interval of 1002,000 c , could be substances similar to phosphoric acid , sulphuric acid , molten salts , molten or softened natural silicates , oxide and oxynitride glasses , solid metal - oxide electrolytes similar to clay beta - alumina with a wide range of the potential high - mobile ions at 250300 c which may be e.g. , na , k , li , ag , h , pb , sror ba . high - temperature batteries - nanocapsules apparently could also be the solid electrolytes on the basis of the zirconium oxide solid solutions , such as yttria - stabilized zirconias , calcia - stabilized zirconias , magnesia - stabilized zirconias etc . , or some nitride solid electrolytes , e.g. , similar to li3n , or the solid electrolytes on the basis of some composite oxides , e.g. , similar to li2+xc1xbxo3 , li4+xsi1xpxo4and li5+xag1xsixo4as well as the solid electrolytes on the basis of oxynitrides , e.g. , similar to lithium phosphorous oxynitride electrolytes . generally speaking , it is necessary to note that the processes of oxidation of aerosol reductant particles can often result in the formation of electrolyte layers on the surface of these particles in the form of electrolyte hydrates , electrolyte melts , or solid electrolyte layers instead of the ordinary dielectric oxide layers . such surface electrolyte layers can significantly reduce the standard rates of oxidation of these aerosol particles by molecular oxygen . at the same time , such electrolyte layers can naturally incite the subsequent competitive process of electrochemical oxidation of the core reductant of these particles . the capsules - nanobatteries with such surface electrolyte layers can be spontaneously formed for example , during the oxidation of primary aerosol particles in a damp atmosphere , in an atmosphere containing carbon dioxide , as well as in an atmosphere containing an acid or a free halogen . in similar circumstances , e.g. , the alkali or alkali - earth metal aerosol particles will be covered with growing hydroxide , carbonate , or with halogenide electrolyte shells instead of growing oxide layers . it is worth mentioning here that the growth of the surface hydrated layers of hydroxides or hydroxocarbonates , instead of the expected oxide layers , are a common outcome of open - air oxidation for many metals ( in particular : iron , copper , aluminium , brass , bronze , tungsten etc . ) . such ordinary surface layers , for example the patinas layers in the form of hydrated cu(oh)2cuco3and cu(oh)2co3 , or layers of hydrated aluminium oxide hydroxide , alo(oh ) , or layers of hydrated aluminium hydroxide , al(oh)3 , or layers of rust in the form of hydrated feo(oh ) and fe(oh)3 , are electrolyte substances which are thermostable enough to form both low - temperature and intermediate - temperature aerosol nanobatteries . alo(oh ) is converted into al2o3at a temperature of 420 c , the melting point of al(oh)3is 300 c , fe(oh)3is converted into fe2o3at a temperature of 500 c . at the same time , the capsules - nanobatteries with gradually developing surface layers of the oxide , nitride and oxynitride solid electrolytes can also be spontaneously formed at the high - temperature oxidation of many aerosol metal particles ( e.g. , zirconium - calcium , zirconium - yttrium alloys , lithium or lithium alloys , aluminium alloys etc . ) in a dry air or a pure nitrogen atmosphere . one can see that a lot of the oxidation processes in the various local atmospheres , also including the ordinary processes of open - air oxidation , can cause the growth of hydrated , molten , or solid electrolyte layers on the surface of the aerosol particles - reductants instead of the dielectric oxide layers . the formation of the similar electrolyte shells on the surface of aerosol particles , due either to their atmospheric oxidation or to the other above - mentioned processes , e.g. , similar to the volume co - condensation of the mixed vapour of reductants and electrolytes , can initiate an alternative electrochemical ion - mediated oxidation of the core particles reductants and , under pre - ionization conditions , convert such particles into aerosol discharge - shorted batteries - nanocapsules . it is possible that in addition to external gaseous oxidizers , some metals , their higher oxides , superoxides , ozonides , sulphides , chlorates as well as sulfur could , apparently , act as condensed oxidizing components , included in the composition of aerosol nanobatteries during their spontaneous air synthesis . certainly , it is clear that the fuel for ball lightning in the form of aerosol submicron and nanoparticles can only be made inside the local air volume with an initial deficiency of oxidizers . as is well known , such a local temporary deficiency of oxidizers can be spontaneously achieved by various means . oxidizers inside a confined space due to the substantial excess vapour of the future nanoparticle ball lightning fuel . another probable means for the local temporary neutralization of the influence of atmospheric oxidizers during ball lightning formation is the accidental simultaneous process of the generation of a reducing atmosphere of additional gas reductants hydrogen , carbon monoxide , hydrogen sulphide etc . this essential requirement of nanotechnology of metals the presence of an inert or reducing atmosphere at the manufacture of oxidable nanoparticles is completely applicable to the technology of nanoparticle ball lightning . therefore , the initial stage of the formation of ball lightning the process with a local deficiency of oxidizers enables the spontaneous self - assembly of the aerosol nanobatteries without a premature oxidative inactivation of the reductant components . however , normal atmospheric oxidation can also compete with the process of electrochemical oxidation after an initial formation of the batteries - nanocapsules is completed , and ball lightning is now in the oxidizer - enriched air . in this case , layers of dielectric oxides can theoretically appear on the surface of the core reductant , e.g. , due to the partial diffusion of molecular oxygen through the protective shell of an electrolyte , and complicate further charge transfer and the normal work of nanobatteries . but such an evolution of the nanobatteries work process requires the following : ( 1 ) the oxides , growing inside the nanobattery , should be condensed substances , but not gases , e.g. , similar to 2or so2 ; ( 2 ) these oxides should form very dense , compact , non - nanoporous dielectric layers directly contacting with the hydrated or molten electrolyte ; ( 3 ) at that , such oxide layers should be electrolyte - insoluble ; ( 4 ) besides this , these layers should not react with the electrolyte , water vapour , or oxides of nitrogen , as the similar chemical reactions will convert the dielectric oxides to electrolyte - salts , e.g. , silicates , hydroxides , nitrites , nitrates etc . ; ( 5 ) the oxide , nitride , or oxinitride layers generated should not be the solid electrolytes in themselves ; ( 6 ) the oxide , nitride , or oxinitride layers generated should not be the electronic semiconductor or the electronic conductor , including at the high temperatures , e.g. , similar to sn2,in2o3,al - doped zno , y ba cu o , pb bi sr ca ( 7 ) such oxide dielectric layers should also be proof against an influence of reductants similar to carbon , carbon monoxide , or hydrogen , i.e. , to an influence of the reducing substances which are either included into the nanobattery composition during the spontaneous synthesis of nanobatteries , or these reducing substances are synthesized during the work of the nanobatteries . as is obvious , the list of necessary requirements for the properties of the substance of the dielectric oxide layers , which can potentially complicate the work of the nanobatteries while growing on the surface of the core reductants , strongly limits the possible choice of such a detrimental ( for our model ) substance . the collective effects obviously should be of great importance in the life of aerosol clouds of nanobatteries . each nanobattery is indeed capable of generating a voltage only of about 1 v. but complexes aggregates containing thousands and millions of separate galvanic nanocells can be formed due to accidental statistic processes of aggregation in a high - density hot aerosol during ball lightning formation . repeated chaotic connections of a great number of nanobatteries in such dynamic complexes provide occurrence of plural series - parallel gas - discharge electric circuits inside the ball lightning . statistic formation of such macro - aggregates of nanobatteries with accidental series connections between them can provide arising dynamic voltaic piles of nanobatteries with enormous resulting voltage . these spontaneous multiple high - voltage dynamic nanoparticle generators will promote further electric collectivization of the current loops of separate aerosol nanobatteries through interparticle high - voltage discharges . apparently , not only local processes of the initial mechanical agglomeration of nanobatteries , but also the high initial electroconductivity of the seed low - temperature plasma of the preceding spark or corona discharge can contribute to the spontaneous electrical connection of separate nanobatteries into plasma - united series - parallel circuits . many billions of nanobatteries , electrically united by low - temperature plasma and electrically feeding this plasma , are contained inside a ball lightning cloud , which thus can be both a high - voltage and a high - current electrochemical aerosol generator . probably , the very long sparks from ball lightning to earthed objects , sometimes observable by eyewitnesses , even inside shielded rooms , can be a product of the super high voltage generated by the plural voltaic piles of nanobatteries , arising on the surface of ball lightning . apparently , a degree of electric collectivization of separate nanobatteries into plasma - united aerosol electrochemical generator can depend on a lot of linked conditions ( e.g. , on the temperature of nanobatteries , their form and concentration , a degree of the plasma ionization and a presence of ionized alkaline impurity , on magnetic permeability of nanobatteries , their gassing , on the currents of nanobatteries etc . ) . as the degree of electrical connection of the nanobatteries inside different aerosol electrochemical generators can be very various , electric power of such aerosol generators can also be alternatively redistributed either to maximal currents of separate nanobatteries , or to maximal voltage of dynamic voltaic piles of plasma - connected nanobatteries , or to intermediate values of currents and voltages adequate to complete electric power of the aerosol electrochemical generator . above , we have also mentioned the possibility of the generation of high and even extremely high currents inside the ball lightning electrochemical aerosol generators . at first sight if either the ion conduction of a nanobattery electrolyte or the electron conduction of a nanobattery reductant is very low , the galvanic and discharge currents inside a cloud of such nanobatteries , as well as the luminosity of such a cloud can also be very low , so that such ball lightning is hardly visible in bright sunlight , while the lifetime of this low - power ball lightning can be , on the contrary , increased . if the substance of the nanobattery reductant ( e.g. , similar to carbon , an extrinsic semiconductor , or metal ) and the substance of the nanobattery electrolyte ( e.g. , similar to salt / acid / alkaline melts or salt / acid / alkaline hydrates ) is moderately or highly conductive under the given conditions of temperature and air moisture , the discharge currents inside such a cloud of the aerosol nanobatteries can be moderate , high , or even extremely high . apparently , the currents in the range of several amperes to several thousand amperes distributed between all the aerosol particles of the cloud are the currents of a quiet state of ball lightning . discharge currents in the range of several thousand to several million amperes distributed between all the aerosol particles , are apparently pre - explosive and explosive currents of ball lightning . at the same time , we believe that the generation of such extremely high discharge currents inside ball lightning is also absolutely realistic . let black carbon , i.e. , a substance with quite a low conductivity , be the core reductant of the nanobattery . let an average diameter of the carbon core reductant particle be about 100 nanometers ( fig . so , the average number of nanoparticles in a 20 cm diameter ball lightning is 3.310 nanoparticles . the average cross - section area of carbon nanoparticle is of the order of the area where the galvanic current flows through the nanoparticle , i.e. , about 7.810 cm . the total area where the total current flows through all the aerosol nanoparticles will accordingly be about 2.610 cm . so , the total area is 26 m. a total electrical current of 10 amp ( distributed between all the aerosol particles ) through total area 2.610 cmwill result in a current density of about 3.8 amp / cm . for example , the standard recommended current density through carbon electrodes in a continuous furnace process is 27 amp / cm . thus , an extremely large total area , where intraparticle galvanic currents flow simultaneously through all the aerosol submicron or nanoparticles , causes a low average current density , which is absolutely acceptable for the normal work of hot carbon and the overwhelming majority of hot extrinsic semiconductors and metals as possible reductants in the aerosol electrochemical generators . as one can see , enormous reaction surfaces inherent in aerosol electrochemical generators readily enable to generate mega ampere currents and , consequently , mega watt electric power . therefore too high conductivity of the nanobatteries components ( in particular , electronic conduction of reductants and ionic conduction of electrolytes ) can considerably shorten a lifetime of ball lightning . as is well known [ 1,2,9 - 11 ] manmade ball - lightning - like objects can be generated with the aid of a great number of various methods . the superficial resemblance between the properties of these objects and the properties of the natural thunderstorm related ball lightning can be very significant . sometimes , only small formal distinctions raise doubts about the identity of the manmade and natural objects , e.g. , the lifetime distinctions , or the electromagnetic properties distinctions , or the density distinctions etc . ball - lightning - like objects , generated by electrical arc discharges from p - type doped silicon wafers in recent experiments , are undoubtedly the most impressive experimental advances in ball lightning science . really , mentioned luminous objects are very similar to natural ball lightning . however , the authors of these extremely interesting experiments correctly discuss two important limitations : first , the production of the luminous balls is not under complete control . second , free - floating balls were not observed . indeed , these limitations unfortunately leave a question open concerning the identity of the mentioned ball - lightning - like objects and natural thunderstorm related ball lightning . here , we would like to consider only two different models as possible interpretations of the experiments . the first model assumes that the ball - lightning - like luminous balls described in are clouds of nanobatteries . p - type doped silicon evaporated by arc discharge is condensed in the form of aerosol submicron or nanoparticles of amorphous silicon with boron trioxide microaddings . thus , a condensation cloud of burning hot nanoparticles of amorphous silicon is a cloud of potential silicon core reductants for aerosol nanobatteries . at 29 c room temperature and a relative humidity of 70% mentioned in , i.e. , under conditions of very high absolute humidity of ambient air and consequently under conditions of extremely high vapour pressure of the water in immediate proximity to high - temperature ball - lightning - like objects , the oxidation of the aerosol nanoparticles of amorphous silicon will result in the formation of electrolyte layers of silicic acids ( by reaction sio2with water vapour ) on their surface , instead of the expected sio2 dielectric surface layers . as is well known , at high temperatures silica scale layers are readily converted in the presence of water or water vapour to form silicic acids . although these electrolyte silicon species are volatile at such temperatures , their ablation is compensated for by the persistent formation of new silicic acids layers on the surface of the silicon aerosol particles under conditions of high air moisture , i.e. , in this case the processes of thermal ablation and water - mediated growth of such silicic acids layers are in dynamic equilibrium . thus , ball - lightning - like luminous clouds of discharge - shorted nanobatteries with silicon core reductants , dynamic electrolyte shells of hot silicic acids and external air oxidizers , can theoretically be created in experiments similar to due to high air moisture . the indirect verification of this model can easily be realized by a variation of the absolute humidity during experiments similar to . if the model works , an increase of the absolute humidity will increase the reproducibility of experiments , while lowering the absolute air humidity will decrease the reproducibility of the experiments ( i.e. , the probability of generating ball - lightning - like luminous balls ) . as the second model , it is possible to assume that ball - lightning - like objects generated in are only small pieces of semiliquid silica - silicon foam , which is formed on the surface and in the volume of the boiling silicon at the arc discharge in a local atmosphere with an initial deficiency of available oxygen . thus , it is possible to assume that a glowing silica - silicon foam material consists mainly of a mixture of : si ( melting point 1414 c , boiling point 3265 c ) + sio ( melting point > 1700 c , initial sublimation temperature 1250 c ) + sio2(melting point 1650 ( 75 ) c , boiling point 2230 c ) + si3n4(melting point 1900 c , with subsequent decomposition ) + sioxny , various highly thermostable silicon oxynitrides . in these circumstances , the gas frothing agent , foaming the silica - silicon sparks , is apparently gaseous silicon monoxide , sio . theoretically , carbon oxides and tungsten trioxide , wo3(melting point 1470 c , boiling point 1700 c ) , produced by the top tungsten or carbon electrodes , could also be suspected as potential frothing agents for the silica - silicon foam . however within the framework of the experimental geometry described , silicon monoxide appears to be the most probable foaming gas for the silica - silicon sparks . these burning hot pieces of semiliquid foam are thrown out from the electric arc and then gradually subjected to oxidation keeping a semiliquid state thanks to the thermal flux from the oxidation of suboxidized silicon . probably , the heat - resistant crust of these semiliquid foam pieces consists of dioxide , nitride and oxinitride of silicon . the slowed diffusion of oxygen into hot pieces of the silica - silicon foam through this superficial gradually hardening dioxide - oxynitride film guarantees the substantial lifetime of this ball - lightning - like phenomenon . pieces of gradually oxidable semiliquid foam are slightly similar to the soap froth in your bath , but their superficial hardening film and extremely high temperature easily guarantee an opportunity for their elastic bouncing on a cold firm surface . at the same time , such pieces of oxidable silica - silicon foam , holding internal heat due to a very low thermal conductivity , can be mechanically disintegrated and then again reunited as long as the internal walls of the foam pieces remain semiliquid . periodic casual breaks of oxygen through superficial passivating mixed layers of dioxide , nitride and oxinitride of silicon cause local bursts of power flux accompanied with jets and pulsations on the surface of hot liquid foam silicon pieces keeping their sphericity because of the high surface tension of the liquid silicon . the temperature and pressure of the foaming gas decrease at the end of the process and the hot foam pieces collapse with the formation of aerosol mixture of silicon dioxide , silicon nitride and silicon oxinitride . it is possible that the next candidates for analogous ball - lightning - like luminous objects could be , for example , plasmatrone - produced pieces of silicon - filled , or carbon - filled , or , for example , the silver powder ( frothing agent with boiling point 2162 c)aluminium powder ( oxidable heat source)filled alumina ( melting point 2054 c , boiling point 3000 c ) foam heat - insulating material . possibly , burning foam is the simplest explanation for this extremely interesting experimental phenomenon . moreover , it seems that the lightning synthesis of the similar hot pieces of slowly oxidable semiliquid foam can also be realized during a thunderstorm , and a part of ball lightning observations can apparently be attributed to observations of such burning foam phenomena . it is even possible to assume that the processes of oxidation in such natural lightning generated pieces of silicon- , metal- , or carbon - filled silicate or aluminosilicate hot foam can be spontaneously converted to the aforesaid alternative electrochemical oxidation of the impregnated reductants inside the hot molten electrolyte foam . nevertheless , taking into account some details of the movement and smoke - like behaviour of ball lightning , and especially taking into account some features of the process of initial self - assembly of the ball lightning substance of separate , significantly distant from each other , sparkling elements repeatedly mentioned by the eyewitnesses , we believe that it is impossible to explain all the observable characteristics of natural ball lightning within the framework of either burning foam or burning filamentary aerogel models , without the application of an aerosol model with long - range interacting particles . it seems that the proposed model allows us to explain all the observable characteristics of ball lightning , in particular a smoke - like behavior , an ability to keep the form of a ball under conditions of strong atmospheric turbulence , as well as the electromagnetic effects of ball lightning . this model also explains the great diversity of observable conditions and processes of ball lightning formation . in fact , any reduction - oxidation reaction inside the composite aerosol particles can be proceeded by an electrochemical mechanism under suitable conditions , and a lot of the intra - particle combinations of the three various substances , reductant - electrolyte - oxidizer , are capable of spontaneously forming short - circuited aerosol nanobatteries and the self - assembled ball clouds of these nanobatteries . the concrete instructions to experimentally simulate the ball lightning phenomenon are strongly dependent on the chosen fuel - reductant and on the method of its atomization , and so they require separate discussion . in particular , not only the black carbon aerosols , but seemingly also the carbon aerogels , coated with a surface film of molten carbonate electrolytes and heated in an oxidizing atmosphere could be good primary experimental targets to make high - temperature electrochemical aerogel power sources short - circuited by plural surface arc discharges and slightly similar to the aerosol electrochemical generators ball lightning described above . john f. cooper ( energy systems , materials science and technology division chemistry and materials science directorate , lawrence livermore national laboratory ) , dr . graham k. hubler ( materials & sensors branch , naval research laboratory ) for their useful comments .

despite numerous attempts , an adequate theoretical and experimental simulation of ball lightning still remains incomplete . according to the model proposed here , the processes of electrochemical oxidation within separate aerosol particles are the basis for this phenomenon , and ball lightning is a cloud of composite nano or submicron particles , where each particle is a spontaneously formed nanobattery which is short - circuited by the surface discharge because it is of such a small size . as free discharge - shorted current loops , aerosol nanobatteries are exposed to a powerful mutual magnetic dipole dipole attraction . the gaseous products and thermal energy produced by each nanobattery as a result of the intra - particle self - sustaining electrochemical reactions , cause a mutual repulsion of these particles over short distances and prevent their aggregation , while a collectivization of the current loops of separate particles , due to the electric arc overlapping between adjacent particles , weakens their mutual magnetic attraction over short distances . discharge currents in the range of several amperes to several thousand amperes as well as the pre - explosive mega ampere currents , generated in the reduction oxidation reactions and distributed between all the aerosol particles , explain both the magnetic attraction between the elements of the ball lightning substance and the impressive electromagnetic effects of ball lightning .

Introduction Model and Discussion Other Possible Components of Nanobatteries Competition Between the Processes of Normal and Electrochemical Oxidation Inside Nanobatteries Some Collective Effects Inside a Cloud of Nanobatteries Ball-lightning-like Objects and Natural Thunderstorm Related Ball Lightning Summary Acknowledgements

PMC2396421

quantitative reverse transcriptase polymerase chain reaction ( qrt pcr ) is a technique by which expression levels of individual genes can be quantified by measuring the abundance of their mrna products relative to a standard ( such as 18s rrna ) . it represents a robust means of gene expression quantification on its own , and is an ideal companion technique for confirmation or further inspection of results obtained by methods such as sage ( 1 ) and microarray analysis strategies ( 2 ) . pcr is traditional rt pcr , cycling temperatures to allow repeated steps of double - stranded nucleic acid melting , primer annealing , polymerase - driven synthesis of new double - stranded nucleic acid . in qrt pcr , oligonucleotide primers are designed to span intronic sequences , the portions of the genome not represented in mature mrna products . when these primers bracket an intronic splice site , a small product will be produced when the primers anneal to mrna during the reaction . an easily recognizable larger product ( or in the case of an extraordinarily long intron , no product at all ) would be produced should any contaminant genomic dna persist in the sample used for the reaction ( figure 1 ) . one such detection stratagem , referred to here as sybr green , employs a chemical agent that binds to synthesized double - stranded nucleic acid products generated during the course of the qrt pcr ( 3 ) . this associated molecule then fluoresces at 520 nm when excited with a wavelength of 497 nm ; this emitted wavelength is detected by the qrt pcr apparatus and recorded over the course of the reaction . combining information regarding increased fluorescent emission , temperature cycling , initial amounts of rna used in the qrt pcr reaction and fluorescence emission of an included standard allows a calculation to be made determining the levels of the targeted rna species , quantifying its expression in the original sample . figure 1.rtpcr primers are chosen in adjacent exons , with the predicted product spanning an intronic splice site . in this way , should any genomic dna persist in the rna preparation , any amplification from genomic dna will produce a long product ( a ) , containing both the targeted sequence and intervening intron . amplification from mrna ( b ) will produce only a short product as the intronic sequence has been spliced out of the mrna . pcr primers are chosen in adjacent exons , with the predicted product spanning an intronic splice site . in this way , should any genomic dna persist in the rna preparation , any amplification from genomic dna will produce a long product ( a ) , containing both the targeted sequence and intervening intron . amplification from mrna ( b ) will produce only a short product as the intronic sequence has been spliced out of the mrna . determining appropriate intron - spanning target sites and developing oligonucleotide primers against them involves several steps , starting with identifying target genes , determining their genomic sequence , their mrna coding sequence ( cds ) and the location of intervening intronic sequences . oligonucleotide primer development can be done by manually identifying ideal primer sites , but the majority of primer design is currently conducted with the aid of computational tools , such as muplex ( 4 ) , prifi ( 5 ) , the widely adopted primer3 utility ( 6 ) or its alternate interface , primer3plus ( 7 ) . primer3 is freely available as a standalone package and is available online through a simple web interface . however , for those users disinclined to run primer3 locally through a command line interface , designing primers against identified targets for each chosen gene individually through a web interface is potentially time - consuming and requires careful management of retrieved information . in addition to the management of these aforementioned data , there is also the issue of developing targets and primers , and selecting reaction conditions that will be most optimally successful . the difficulty of coordinating these data increases with the size of the qrt pcr project undertaken . we elected to address these issues of target site identification , primer development and optimization , and data organization to ameliorate the process for molecular biology laboratories . to this end , we have developed a set of optimized qrt pcr primer design conditions and a script that incorporates these conditions into an automated design procedure . multipride is designed to streamline and standardize the process starting with target gene identification through to primer design . after user identification of targets , multipride handles the entire procedure independent of additional user input , and produces organized , easily accessible primer design information . moreover , as all primer sets are designed with the same optimized reaction conditions , all qrt pcr reactions should be performable simultaneously or with the same settings from run to run . the steps that multipride follows are illustrated in figure 2 : starting with initial input , the script identifies and retrieves the required information from ncbi resources ( 8) . it then determines intronic splice sites , which are ideal qrt pcr target sequences . these ideal targets are then directed to a local install of the primer3 package that uses our empirically optimized hybridization conditions to develop sets of primers amplifying each identified target . the identified cds , intronic splice sites and primer design results are available to the user upon completion . we have chosen to employ a locally installed version of primer3 for use with multipride chiefly to allow users to exploit the power of their own laboratory computers rather than further taxing existing publicly available primer3 servers . figure 2.the process multipride follows , starting with inputted geneids and concluding with identified cds and primers for each intronic splice site . the processes described in the central gray box are those that are performed by the user 's computer . the process multipride follows , starting with inputted geneids and concluding with identified cds and primers for each intronic splice site . the processes described in the central gray box are those that are performed by the user 's computer . we developed a specific set of parameters for oligonucleotides designed by multipride , specifically deviating at key points from those default parameters specified by the primer3 package . first , we chose an annealing temperature range of 55.0c ( minimum ) , 57.5 ( optimum ) and 60.0c ( maximum ) instead of the default 60.0 , 63.0 and 65.0c range specified in the primer3 defaults . pcr reactions when working with mouse and rat rna samples , and because oligonucleotides designed with our temperature range typically exhibited a narrower range of ideal primer concentrations in reaction ( unpublished results ) . we also adjusted the maximum target oligonucleotide size to 25 nt from 27 nt to compensate for the reduction in annealing temperature . we altered the default settings for maximum self - complementarity and maximum 3 self - complementarity from values of 8.00 and 3.00 , respectively , to 5.00 and 2.00 , respectively , to reduce primer dimer formation . this repeat library is a merger of two of the base libraries offered with the primer3 package ( the rodent and simple and human libraries ) . we use this merged library for ease of primer development in our own cross - species experiments and it is not intended to confer any special benefit with the exception of simplicity for multi - species batch primer design . multipride was written entirely in perl , and uses the lwp module for certain functions . local installations of standard perl 5.8.0 , the lwp module [ available through the comprehensive perl archive network ( cpan ) ] and the primer3 package ( available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html ) are required for functionality . the user supplies an individual or a batch list of geneid numbers via command line interface . the script then collects the genomic sequence entry and first join data entry for that record . this information is used to identify exons and intronic splice sites in the genomic sequence , and prints the compiled cds to the screen , along with cds nucleotide positions for intronic splice sites . these splice sites will be specified as targets for primer3 oligonucleotide design in subsequent steps . in the case of genes that are composed of a single exon , no target is specified ; the entire monoexonic cds is a potential target site for oligonucleotide design . next , multipride invokes a locally installed version of primer3 , and using our hybridization conditions coupled with the intronic splice site positions , develops a maximum of 100 primer pairs spanning each splice site in the cds . multipride creates directories named for each geneid queried , and writes into these the primer3 output files named according to the convention geneid_splicesite.pr3 . these .pr3 files are readable in any text editor and are equivalent to the output a user may receive when performing an equivalent analysis using a primer3 server . those geneids that lack any of the requisite information ( an entry , genomic sequence or join data ) return an error message describing the nature of the error and an output file containing fasta - formatted cdna sequence if available . cell cultures were prepared of human weri - rb1 ( gift of dr j. boatright , emory university , atlanta , georgia ) or rat nrk-52e cells [ american type culture collection ( atcc ) , manassas , va , usa ] for subsequent rna extraction . dishes ( 60 mm ) of cells were grown to monolayer , media was removed and 1 ml of trizol ( invitrogen , carlsbad , ca , usa ) was added to each dish . then 900 l of lysed cells were removed from the dish . for mouse rna , a mixture of rna extracted from multiple tissues was used , including brain , heart , mammary gland , muscle , liver , lung , ovaries , spleen , submandibular gland and uterus . rna was prepared from both cultured cells and mouse tissue using trizol according to the manufacturer 's recommended protocol with the inclusion of an additional chloroform wash following organic phase separation , quantified using fluorescence methods and then diluted to 100 ng/l . rna samples weighing 5 g each were treated with turbo dna - free dnase ( ambion , austin , tx , usa ) to remove any contaminating dna . dnase - treated rna was then purified and reconcentrated using qiagen rneasy minelute columns ( qiagen , valencia , ca , usa ) and rna was eluted in 100 l of nuclease - free water ( 2 ng/l ) . oligonucleotides designed using multipride were ordered from ( mwg biotech , high point , nc , usa ) as high - purity salt free in 0.01 mol size . two forward and two reverse primers from the list of primers identified from our multipride analysis were ordered for a single splice site for each gene , generating four unique pairs of primers for each gene . for a given multiexonic gene , primer pairs were chosen from multipride 's generated results with priority placed on ( i ) splice site proximity to 5 end of transcript , ( ii ) splice site at least 100 bp from either end of transcript and ( iii ) at least two pairs of primers were generated for the specific chosen splice site . individual oligonucleotides were aliquoted into pairs at 1 m concentration ( per primer ) with the pairs set up as follows : pair 1f1/r1 ; pair 2f1/r2 ; pair 3f2/r1 ; pair 4f2/r2 . this concentration was considered a 5 primer pair stock for subsequent testing of primers at an initial 200 nm concentration of each primer in qrt pcr . for reactions that showed a single dissociation peak in the reactions when template rna was added but showed primer dimers in the absence of template rna , primers were retested at 100 nm concentration . for reactions that showed no amplification of fluorescent signal in the presence of template the primers were retested at 400 nm . pcrs were performed using a quantitect sybr green one - step rt pcr kit ( qiagen ) . reactions ( 25 l ) were set up with 5 ng of template rna or an equivalent volume of nuclease - free water as a control and 100 , 200 or 400 nm concentration of each of the primers in a pair to be tested . pcrs were performed in an abi-7500 real - time thermocycler ( applied biosystems , foster city , ca , usa ) . the reverse - transcriptase reaction began with 15 min at 95c to activate the polymerase , 50c for 30 min and then followed with 40 cycles of 95c for 15 s ( strand dissociation ) , 55 to 60c for 30 s ( primer annealing , temperature dependent on the primer pair ) , and 72c for 40 s ( dna synthesis ) . annealing temperature was 55c for primers designed to have a melting temperature of 57.5c , and incremental steps of + 0.1c were done with fluorescence data collected at each step and the derivative of the change in fluorescence was plotted by the sds software . those that showed high efficiency amplification of signal with template added and no amplification of signal in the absence of template were considered good amplification plots ( figure 3 ) . a primer pair was considered to have failed if the amplification plot showed amplification of signal from the reaction that contained no template or if the efficiency of the amplification was weak in the presence of template . those that showed a single peak for the reaction containing template and no peaks for the reaction containing no template were considered to be good dissociation curves ; we consider our threshold for success to be quite stringent , and may be more stringent than is required for some purposes ( figure 3 ) . those that showed multiple peaks for the reaction containing template or any peak including low melting temperature peaks ( primer dimers ) for the no template reaction were considered to be bad dissociation curves . each primer pair was graded as passed if both the amplification plot and the dissociation curves were good , or failed if either the amplification plot or the dissociation curve was bad , according to our aforedescribed criteria . ( a ) demonstrates an amplification curve for these primers , which is a plot of fluorescence intensity on the ordinate axis and the number of cycles on the abscissa axis . the increase in fluorescence is the result of free sybr green binding to double - stranded dna ( initially at a very low concentration ) , which increases roughly 2-fold per amplification cycle until primers and other reagents begin to run out . a smooth exponential increase in the initial stages of the pcr suggests the amplification of only one product , consistent with the primers being specific . ( b ) shows a representative dissociation curve for this reaction , plotting change in fluorescence against change in temperature . as double - stranded dna product is melted with increasing temperature , it dissociates into two single strands and releases complexed sybr green . the release of sybr green results in diminished fluorescence . the central sharp peak seen indicates the homogenous melting behavior of the double - stranded dna produced by the qrt a sample rt pcr using multipride - developed primers . ( a ) demonstrates an amplification curve for these primers , which is a plot of fluorescence intensity on the ordinate axis and the number of cycles on the abscissa axis . the increase in fluorescence is the result of free sybr green binding to double - stranded dna ( initially at a very low concentration ) , which increases roughly 2-fold per amplification cycle until primers and other reagents begin to run out . a smooth exponential increase in the initial stages of the pcr suggests the amplification of only one product , consistent with the primers being specific . ( b ) shows a representative dissociation curve for this reaction , plotting change in fluorescence against change in temperature . as double - stranded dna product is melted with increasing temperature , it dissociates into two single strands and releases complexed sybr green . the release of sybr green results in diminished fluorescence . the central sharp peak seen indicates the homogenous melting behavior of the double - stranded dna produced by the qrt multipride then displays the gene 's cds with a fasta - formatted header , along with the nucleotide positions of the splice sites relative to the cds . as described earlier , multipride then makes a directory for each geneid and places the primer3 output files in that folder , as shown in figure 4c . the output files can be read using any text editor ; in figure 4d the emacs terminal utility is used , showing the familiar primer3 output format . ( a ) the program is invoked using perl , and asks for geneid numbers as input . ( b ) multipride prints a fasta - formatted cds to the screen , along with the locations of splice sites . ( c ) the directory produced for a given geneid , containing the primer design files for each splice site . ( a ) the program is invoked using perl , and asks for geneid numbers as input . ( b ) multipride prints a fasta - formatted cds to the screen , along with the locations of splice sites . ( c ) the directory produced for a given geneid , containing the primer design files for each splice site . ( d ) the text file primer3 output for an individual splice site . a comparison between traditional , manual primer design and primer design conducted using multipride was undertaken . to manually design a single pair of primers for a single splice site of a single gene from our gene set ( human cadherin 13 , geneid 1012 ) we needed 12 min , 45 s to retrieve the genomic sequence , identify and confirm a single splice site , run primer3 for that single location and save all relevant files . the same gene was submitted to our script ; 1 min 26 s was required to identify and develop 100 pairs of primers for each of this gene 's 13 splice sites . this is 7 s per splice site , or roughly 110 times faster than the manual identification and primer development . multipride designed primers for all splice sites of the same 270 genes in slightly < 2 h , running on a 2.0 ghz dual processor power mac g5 ( apple computer , cupertino , ca , usa ) , factory condition with the exception of 6 gb of ram , using a 100 mbit / s internet connection for retrieving information from ncbi . of the 270 genes tested , 19 lacked sequence associated with their geneid , and therefore primers could not be designed . pcr conditions led to an immediate success rate of 75% among the 251 genes that we tested experimentally ; that is , for each targeted gene , 75% of the first batch of selected primers generated acceptable qrt table 1 describes the breakdown of first round success for each of the three species considered . rachlin and colleagues ( 4 ) describe a 39% chance for primers designed using their muplex utility ( which relies on default parameters for oligonucleotide and salt concentrations identical to primer3 's default parameters ) occurring in more than 10 genomic locations . should multiple locations for a given primer occur across multiple exons , that primer would give confounding results when used in qrt pcr . a number of failures are also due to double peaks that could result from amplification across an alternative splice site ; this has not been quantified . we feel these phenomena may account for much of our first - round failures . table 1.summary of results for qrt pcr amplifications using the first two selected pairs of multipride - developed primersspeciessuccessful qrt pcr reactions at ( 200 nm)total successful qrt pcr reactionstotal genes analyzedhuman ( % ) 39 ( 53)55 ( 74)74mouse ( % ) 61 ( 75)71 ( 88)81rat ( % ) 43 ( 45)63 ( 66)96all species143 ( 57)189 ( 75)251successful qrt pcr reactions at ( 200 nm) includes only those reactions that were successful at 200 nm primer concentration . total successful qrt pcr reactions includes those successful at 200 nm , as well as those that were not successful at 200 nm , but were subsequently successful when tested at 100 nm or 400 nm . summary of results for qrt pcr amplifications using the first two selected pairs of multipride - developed primers successful qrt pcr reactions at ( 200 nm) includes only those reactions that were successful at 200 nm primer concentration . total successful qrt pcr reactions includes those successful at 200 nm , as well as those that were not successful at 200 nm , but were subsequently successful when tested at 100 nm or 400 nm . pcr primer design for experiments of even modest scope can quickly become complicated and time - consuming . manual identification of ideal target sites is tedious and prone to user - introduced error ; individual queries directed against a public primer3 server are equally tedious . we do not attempt to quantify error rate for manual management of sequence and primer design data in this study . earlier findings described an error rate of 3033% for multiple - step tasks involving text editors , even amongst highly experienced users ( 9 ) . multipride greatly reduces the possibility of user - introduced error as all steps short of the initial geneid entry are managed entirely by the script . additionally , the individual files a user may generate when undertaking these tasks manually quickly accumulate and may further impact organization . we are confident that multipride will be of use to molecular biologists undertaking qrt pcr experiments of any magnitude .

quantitative reverse transcriptase polymerase chain reaction ( qrt pcr ) is a commonly employed gene expression quantification technique . this requires the development of appropriately targeted oligonucleotide primers , which necessitates the identification of ideal amplicons , development of optimized oligonucleotide sequences under most favorable pre - determined reaction conditions , and management of the resultant target - oligonucleotide pair information for each gene to be studied . the primer3 utility exists for development of oligonucleotide primers and fills that role effectively . however , the manual process of identifying target sites and individually generating primers is inefficient and prone to user - introduced error , especially when a large number of genes are to be examined . we have developed multipride ( multiple primer design ) , a perl utility that accepts batch lists of gene database identifiers , collects available intron and exon position data critical to qrt pcr primer development , and supplies these sites as identified targets for the primer3 utility . this automated gene to primer procedure is coupled with a set of optimized hybridization conditions used by the primer3 utility to maximize successful primer design . multipride and assembled repeat libraries are available upon request . please direct requests to aziesel@emory.edu .

INTRODUCTION METHODS RESULTS AND DISCUSSION

PMC4766328

the technological advancement in communication network has facilitated healthcare professionals across the world in accessing electronic medical records , such as medical images , and obtaining second opinions for high - quality diagnosis . as a consequence , medical images are exposed to security threat such as tampering of images , which may lead to wrong diagnosis and treatment ; thus a digital watermarking on medical image is introduced . so far most of the watermarking research has been done on single frame medical image which is impractical in the real environment , where most of the ultrasound medical images consist of multiframes ; thus a digital watermarking on multiframes medical images is proposed . watermarking could be applied on ultrasound medical images frame by frame sequentially but it would be time consuming for a large dataset ; thus a parallel environment is necessary for speeding up multiframes watermarking process by utilizing multicore technology . the performance improvements gained by the use of a multicore processor largely depended on the software algorithms used and their implementation . according to amdahl 's law , the possible gains are only limited on software portion that can be run in parallel simultaneously on multiple cores . digital watermarking is the technology that imperceptibly modifies original data and embeds them directly into image . digital watermarking in general is comprised of three major components : watermark generator : a desired watermark(s ) is generated for particular applications , which are optionally dependent on some keys.watermark embedder : watermark(s ) are embedded into the object , sometimes based on an embedding key.watermark detector : detecting the existence of some predefined watermark in the object . watermark generator : a desired watermark(s ) is generated for particular applications , which are optionally dependent on some keys . watermark embedder : watermark(s ) are embedded into the object , sometimes based on an embedding key . the purpose of medical image security is to maintain privacy of the patient information in the image and to assure data integrity that prevents the image from tampering . watermarking can be used in medical images to prevent unauthorized modification by authenticating the content of the image . tamper localization capable watermarking scheme can detect and locate modification of pixel values on the image . the tampered area can be recovered by retrieving the original pixel values that were stored on the image itself as a watermark . tamper localization is useful for deducing the motive of the tampering and whether any modification is legitimate . liew and zain proposed a reversible watermarking scheme ( tallor watermarking scheme ) by dividing image into roi ( region of interest ) and roni ( region of noninterest ) . roi is the significant part of the medical images that is used by doctors to diagnose the patient , and roni is the area outside the roi . watermarking for tamper detection and recovery is done in the roi area based on jasni 's scheme . the original least significant digits ( lsbs ) that are removed in watermark embedding process are stored in roni after compression . the stored lsbs later can be used to restore the image to its original bits value so the watermarking scheme can be reversible . the research was conducted on a single frame of ultrasound which is impractical in the real world ; thus a digital watermarking on multiframes medical images is proposed . in order to speed up multiframes watermarking processing time , in recent years there has been a surge of interest in running application in parallel to take advantage of multiprocessor and multicore systems . developments in microprocessor technologies have resulted in most processors having multiple computing cores in a single chip . parallel computing is a concept of performing tasks simultaneously by partitioning a large and complex problem into smaller tasks and solving each of them concurrently . there are two forms of parallelism : task parallelism and data parallelism ( as shown in table 1 ) . data parallelism emphasizes the distributed ( parallelized ) nature of the data , as opposed to the processing ( task parallelism ) . data parallelism is adopted in this experiment since each processor performs the same code ( watermarking code ) on different pieces of distributed ultrasound frames . optimal speedup from parallelization should be linear if the number of processing elements is inversely proportional to its run time . most of them have a near - linear speedup for small numbers of processing elements , which flattens out into a constant value for large numbers of processing elements . according to amdahl 's law , the overall speedup from parallelization would be restricted by a small portion of the program which can not be parallelized . a large and complex program if is the fraction of running time a program spends on nonparallelizable parts , then(1)limp11/p+=1. it is the maximum speedup with parallelization of the program , with p being the number of processors used . if the sequential portion of a program accounts for 10% of the runtime ( = 0.1 ) , then a 10x speedup will be the maximum , regardless of how many processors are added . gustafson 's law is another law in computing , closely related to amdahl 's law . it states that the speedup with p processors is(2)sp = pp1=+p1.amdahl 's law assumes that the total amount of work to be done in parallel is also independent of the number of processors , whereas gustafson 's law assumes that the total amount of work to be done in parallel varies linearly with the number of processors . applications are often classified based on the frequency of synchronization and communication needs between their subtasks . fine - grained parallelism is where an application has a high rate of communication among subtasks ; coarse - grained parallelism is where an application does not communicate many times per second , and it is embarrassingly parallel if an application seldom or never has to communicate . embarrassingly parallel applications are considered the easiest to parallelize . in the embarrassingly parallel problems , speedup factors could be achieved near the number of cores , or even more if the problem is partitioned enough to fit within each core 's cache(s ) , avoiding use of much slower main system memory . the matlab job scheduler ( mjs ) is the process that coordinates the jobs execution and their tasks evaluation . the mjs runs the submitted jobs in queue order , unless any jobs in its queue are promoted , demoted , cancelled , or deleted . mjs assigns task from the running job to each worker for execution and fetches result from workers upon the task completion . the cycle is repeated with another task . when all tasks for a running job have been assigned to workers , tasks were executed simultaneously by all workers in order to speed up execution of large matlab jobs . the mjs then returns the results of all the tasks in the job to the client session ( as shown in figure 1 ) . in this research , client and mjs are located in a single computer where a client ( end user ) has sent a job ( a stack of ultrasound medical images ) into mjs where it segregates the ultrasound medical images into multiple tasks ( subset of ultrasound medical images frames ) into workers ( cores ) and executes watermarking process in parallel . for example , in quad cores computer , mjs will segregate 15 frames of ultrasound medical images into 4,4,4,3 frames to each core / worker , respectively . job progresses through a number of stages upon its creation . in the mjs ( or other schedulers ) , each stage of a job is categorized by their state , such as pending , queued , running , or finished ( refer to table 2 ) . in watermarking process on a single frame of ultrasound medical image , watermark is embedded into ultrasound medical images and becomes an input file for watermarking authentication process ( as shown in figure 3 ) . the purpose of authentication process is to localize and recover the tamper region in medical images . in other words , the prerequisite of watermarking authentication process is watermarked ultrasound medical images , in which it is the output file generated by watermarking embedding process . process watermarking frame by frame sequentially was time consuming ; thus parallel computing on multicores was introduced to solve the problem concern . this is accomplished by dividing ultrasounds frames into tasks to each core , respectively , and performing watermarking process simultaneously with others . two modes of watermarking process in multiframes will be developed and compared ; there is sequential ( by using for loop ) versus parallel watermarking process ( watermarking on multicores ) ; the purpose is to prove that the parallel watermarking will have a significant improvement on elapsed time . in both sequential watermarking embedding and authentication process , therefore the elapsed time is proportional to the number of frames processed . the elapsed time could be reduced by using parallel computing on multicore processing technique and this technique enables ultrasound frames to be divided and distributed to multicore for parallel watermarking processing . sequential watermarking embedding and authentication process are sharing a common framework , where ultrasound medical images in dicom format are read and perform watermarking process frame by frame sequentially by using a for loop . processed frames will then concatenate into a variable named as a which will convert into dicom format at the end of the watermarking process . the relationship between both processes is that the output file of watermarking embedding process is the input file for watermarking authentication process . the difference between them is that authentication process has an additional step in identifying the tampered frames ; if it is 0 , it means nontampered ; else it is tampered and then image recovery is performed and tampered frame number is recorded and will be displayed upon the completion of watermarking process ( as demonstrated in figure 4 ) . the main algorithms of parallel watermarking process are dividing volumetric ultrasound medical images and distributing them into a number of cores and executing sequential watermarking processes on each core in parallel ; therefore a successful sequential watermarking process is a prerequisite in parallel watermarking process . the details of parallel watermarking process will be discussed in section 5.2 . in parallel watermarking process ( as illustrated in figures 5 and 6 ) , ultrasound multiframes medical images were loaded into a quad core microprocessor / cluster and create a job on the scheduler ; the job is then divided into tasks according to the number of cores in the microprocessor . the code implemented enables cluster to autodetect the number of cores available in the processor ; if the processor used is a quad core , then the job is divided into 4 tasks , where ultrasound frames are equally divided by 4 ; for example , if the total number of ultrasound frames is 30 , then it will be divided into 8,8,7,7 frames , and if the total number of frames is 15 , then it will divided into 4,4,4,3 frames . , watermarking process is carried out sequentially on the divided frames and at the same time it runs concurrently with other cores ( as illustrated in figure 7 ) . it is important to ensure that the generated frames output is in order after parallel watermarking process ; therefore the frame number has to be assigned and keep track before the frames are sent to the cores , respectively . upon the tasks completion , tasks were reassembled into a job , whereby all the frames will concatenate into an array and submit back to the cluster . the result is retrieved from all the tasks in the job with the function fetch output . all the frames will concatenate according to the frame number order and write into a dicom file . a job is deleted on two circumstances : when the scheduler encounters an error.when the job is finished . both parallel watermarking embedding and authentication process have a similar process flow as described above , except the process applied on each task , input and output files as listed in table 3 . tallor watermarking scheme , developed by liew and zain , will be executed in sequential and parallel modes . the elapsed time obtained from both modes will be compared and speedup factor of parallel relative to sequential watermarking process will be measured . it is to verify the efficiency of parallel framework . these areimperceptibility : testing the quality of medical images in terms of invisibility of watermarking in multiframes environment;elapsed time : the time taken to perform watermarking embedding and authentication process on medical images in multiframes environment;robustness to tampering : testing the effectiveness and efficiency of the tamper detection , localization , and recovery function in multiframes environment . imperceptibility : testing the quality of medical images in terms of invisibility of watermarking in multiframes environment ; elapsed time : the time taken to perform watermarking embedding and authentication process on medical images in multiframes environment ; robustness to tampering : testing the effectiveness and efficiency of the tamper detection , localization , and recovery function in multiframes environment . natick , ma , usa ) program on a laptop with quad core cpu of intel core i7 - 3630qm cpu @ 2.4 ghz , 2401 mhz , 4 core(s ) , 8 logical processor(s ) , and ram of 8 gb . three samples of ultrasound medical images in dicom format were used to test the system ( as shown in table 4 ) . the perceptibility of a watermarked image can be judged according to its fidelity and quality . the mean - squared - error ( mse ) and peak signal - to - noise ratio ( psnr ) were calculated by comparing the watermarked image and original image . if ii is a vector of n predictions , and ii is the vector of observed values corresponding to the inputs to the function which generated the predictions , then the mse of the predictor can be estimated by(3)mse=1niniiii2 . mse is the average term by term difference between the original image , i , and the watermarked image , i. if i and i are identical , then mse(i , i ) = 0 . a related distortion measure is the peak signal - to - noise ratio ( psnr ) , measured in decibels ( db ) . the problem with mean - square error is that it depends strongly on the image intensity scaling while psnr rectifies this problem by scaling the mean - square error according to the image range . psnr is defined as follows:(4)psnrdb=10 log10maxi2mse,where maxi is the peak value of the original image . if the signals are identical , then psnr is equal to infinity . a high psnr represents a high fidelity of a watermarked image . in this thesis , a high - quality watermarked image does not have any obvious noticeable distortion caused by the watermark embedding process . the assessment of quality is usually evaluated by human observers and is influenced by personal preferences which are subjective in nature . three different sets of ultrasound medical images which contain thirty and fifteen frames have been watermarked in two different ways : ( 1 ) sequentially and ( 2 ) parallel . it is important to ensure that the quality and fidelity of images were not affected by the way watermarking embedding process performed . both sequential and parallel watermarking embedding processes have produced the same mse and psnr result for each frame as indicated in table 5 except some negligible differences in the highlighted areas . this means that the operation either in sequential or in parallel mode does not affect the image quality and its fidelity . the psnr values reflect the medical image integrity and high psnr values indicate lesser distortion on medical image after watermarking process . psnr value reflected medical image fidelity ; ideally the watermarked medical image should be visually indistinguishable as original image . the psnr values are calculated for all images ranging within 48.29~48.74 db , which are within the acceptable range for diagnosis purposes and it has achieved imperceptibility as shown in figure 9 where the images before and after watermarking embedding process are visually indistinguishable as the original images . elapsed time is the time taken to perform watermarking embedding ( figure 8) and authentication process ( figure 12 ) on medical images in multiframes environment . this section is to test the speedup factor in parallel mode as compared to sequential mode in watermarking process . the formula of speedup factor is defined as follows:(5)speed up = elapsed time in sequnetial modeelapsed time in parallel mode . speedup factor is to measure the speed of parallel mode by factors relative to sequential mode . for example , if the speedup is 3 , this means parallel process is three times faster than sequential mode . table 6 shows that watermarking embedding process in parallel has achieved a significant speedup ( 14.13~19.29 ) relative to sequential process . in figure 10 , the elapsed time in sequential watermarking embedding process on sample_2 and sample_3 is similar but increases by double in sample_1 ; this means that the elapsed time in sequential process is proportional to the number of frames , whereas in parallel watermarking embedding process , the elapsed time results are consistent despite the number of frames processed . in conclusion , the number of frames does not have much impact on the parallel process as compared to sequential process . the watermarking embedding scheme is pixel oriented , which means different ultrasound sample with the same frame size will produce a similar result . the elapsed time is actually depending on many factors such as processor type , clock rate , memory speeds and use of memory catches , location of code in memory , compiler efficiency , and compiler optimization technique . different to watermarking embedding process , watermarking authentication process is to verify whether there is any tampering that occurred in the watermarked ultrasound medical images and then recovered the tampered frame to its original state . in table 7 , three watermarked ultrasound medical images have been fully tampered and the speedup factor ranges within 2.92~3.64 . this is because , firstly , watermarking authentication algorithm has more if - else branches as compared to watermarking embedding algorithm and , secondly , watermarking authentication process needs to return two results ( a string of tampered frames and concatenated ultrasound frames ) , whereas watermarking embedding process just returns one result , that is , concatenated frames of watermarked ultrasound medical images . in watermarking authentication process also is pixel oriented ; therefore the different sources have shown a little impact on elapsed time . it could be observed in figure 11 that watermarking authentication process in sequential mode is proportional to frame size but it is not the case in parallel mode ; the elapsed time does not have many changes in parallel mode at different frame size . the whole package of watermarking process is involving two steps : ( 1 ) watermarking embedding process and ( 2 ) watermarking authentication process . the high speedup in watermarking embedding process is compromised by the low speedup in watermarking authentication process . table 8 has shown that the overall speedup factor for three ultrasound medical images samples ranges within 5.21~6.60 . in order to demonstrate the tamper localization function in detecting forgery , counterfeited images were created by manually modifying the pixel values in the watermarked images using image processing software figure 13 shows an example of tampering on three frames ( frames numbers 2 , 4 , and 6 ) in ultrasound watermarked medical images . different set of tampered watermarked ultrasound medical images has been used to test the effectiveness and efficiency of the tamper detection , localization , and recovery function in multiframes environment ( as shown in table 9 ) . the function 's effectiveness was measured by checking whether it could detect the tampered frame number in multiframes environment ( as shown in figures 12 and 13 ) and is able to recover to its original form ( as shown in figure 14 ) . the function 's efficiency was measured by comparing the elapsed time taken for both sequential and parallel watermarking authentication process ( as shown in figure 15 ) . both effectiveness and efficiency testing were performed while testing the function 's robustness to tampering . for tampered free watermarked ultrasound medical images , the initial setup of parallel authentication process such as determining number of cores and dividing and distributing frames to each core was time consuming ; therefore it takes longer processing time than sequential version . for both sequential and parallel watermarking authentication process , the elapsed time is proportional to the total number of tampered frames . relative to sequential watermarking authentication , the efficiency of parallel watermarking authentication showed remarkably when there are more tampered frames . in summary , the elapsed time for parallel watermarking authentication process is varied based on the total number of tampered frames in ultrasound medical images . with 100% tampered watermarked ultrasound medical images , the efficiency of parallel version can be calculated based on elapsed time , such as 259.1/71 = 3.65 , which means parallel version can perform 3.65 times faster than sequential version . in other words , sequential watermarking authentication has performed a job all by itself , whereas parallel watermarking authentication has delegated a job to four workers / cores , in which parallel version has performed approximately 4 times faster than sequential version . in conclusion , the speedup factor was approximate to the number of cores since there is little communication between subtasks . the frame order of tampered frame also is a factor that affects the performance in parallel process ( as shown in figure 16 ) . both ( a ) and ( b ) have distributed 15 frames ( containing 9 tampered frames ) of watermarked ultrasound medical images to 4 cores , respectively . the main difference between ( a ) and the elapsed time for ( a ) is 5 seconds lesser than ( b ) . it is because ( a ) has an even distribution of tampered frames and hence fairer workloads among 4 cores as compared to ( b ) . the approach used to overcome the performance constraint in sequential watermarking process is by distributing ultrasound frames over multiple cores . the performance constraint for sequential watermarking process the capacity problem occurred when the existing hardware and software are unable to perform the anticipated computations in an estimated time . for example , it may not be feasible to conduct watermarking process on a large data size of dicom files in any reasonable manner . therefore , to run the entire computation may become impractical even though the existing hardware and software are capable of performing the watermarking process as required . the actual physical constraint , such as the processor speeds or total memory on a system , may cause the problem of capability , in which it will restrict the amount of watermarking processes performed . system upgrades may be a solution for the problem concern , but it is bounded by technology and cost constraint . in this case , the sequential watermarking problem may be partitioned into smaller and manageable parts that can be performed in parallel . however , the maximum number of parallel threads can not exceed the number of cores available on the system . the performance gain obtained by using multiple cores on a single system the proposed parallel watermarking scheme required little effort to separate the ultrasound medical images into a number of parallel tasks due to the little dependency ( or communication ) between those parallel tasks and achieved a speedup factor that is almost equivalent to the number of cores ; for example , quad cores microprocessor will result in a speedup factor of approximately four , which means parallel watermarking process is approximately four times faster than sequential mode in quad cores microprocessor . embarrassingly parallel problems , a phrase that comments on the ease of parallelizing such applications and the fact that it would be embarrassing for the programmer or compiler to not take advantage of such an obvious opportunity to improve performance . the performance of proposed parallel digital watermarking scheme could be further improved by using graphics processing unit ( gpu ) . both cpu and gpu could run thousands of threads concurrently , but gpu will have a better performance than cpu due to its larger number of cores possessed relative to cpu ; the difference in theoretical performance can differ by a factor ten in favour of the gpu ; therefore a parallel digital watermarking operation on gpu is recommended . in many cases a good example is image registration algorithms , where the gpu can be used to calculate a chosen similarity measure in parallel , while the cpu can run a serial optimization algorithm . so et al . made a comparison between cpus and gpus for ultrasound systems , in terms of power efficiency and cost effectiveness . the adoption of either gpu or hybrid cpu - gpu is largely dependent on the parallel adaption in an algorithm ; an algorithm that exhibited embarrassingly parallel problem will be suitably used in gpu whereas hybrid cpu - gpu is suitably applied in an algorithm that exhibited fine - grained parallelism . for future work , the proposed method could be applied on magnetic resonance images ( mri ) where the roi could be classified by using weighted - type fractional fourier transform approach prior to watermarking process . since the watermarking is pixel oriented , thus it could be also applied on nature images with lesser restriction on image fidelity requirement as compared to medical images .

with the advancement of technology in communication network , it facilitated digital medical images transmitted to healthcare professionals via internal network or public network ( e.g. , internet ) , but it also exposes the transmitted digital medical images to the security threats , such as images tampering or inserting false data in the images , which may cause an inaccurate diagnosis and treatment . medical image distortion is not to be tolerated for diagnosis purposes ; thus a digital watermarking on medical image is introduced . so far most of the watermarking research has been done on single frame medical image which is impractical in the real environment . in this paper , a digital watermarking on multiframes medical images is proposed . in order to speed up multiframes watermarking processing time , a parallel watermarking processing on medical images processing by utilizing multicores technology is introduced . an experiment result has shown that elapsed time on parallel watermarking processing is much shorter than sequential watermarking processing .

1. Introduction 2. Theory: Concept of Digital Watermarking 3. Theory: Parallel Computing in Multicores 4. Parallel Computing with MATLAB 5. Research Methodology: Sequential and Parallel Watermarking Embedding and Authentication Process 6. Experimental Design and Set-Up 7. Experimental Result 8. Conclusion and Future Work

PMC3399486

in the relationship between healthcare professionals and patients , there is an imbalance in knowledge regarding disease , prognosis , and treatment . the knowledge , competence , and expertise of healthcare professionals have been assumed to be strong and with the capacity to define patients ' best interests . based on their position and professional competence , the authority of healthcare professionals has regulated the degree to which patients are allowed to contribute to their treatment situation . the traditional view of the relationship between professionals and patients has recently been challenged from a different number of perspectives . formally , regulations [ 25 ] and developing views on health services management , patients have increasingly been encouraged to be more active in their treatment and care . furthermore , management - oriented leadership reforms have promoted the patient - as - partner view , maintaining that the patient is a participating producer of healthcare , and new social trends , including increased public access to information and knowledge via the internet . this has opened for some balancing of the information gap between professionals and patients : patients can use the internet to access information about disease progression , treatment alternatives , rights and choices , medical results , and unique qualities of hospitals or clinics . the emerging view is that healthcare professionals now meet patients as costumers who buy services and want choices , quality , and service . from a customer perspective the customer may want information about the service being purchased and a guarantee that the service is of high quality . all of these trends imply that communication and dialogue between healthcare professionals and patients have become important . however , recent research seems to indicate that the patient perspective is not given sufficient attention . in their danish study freil and knudsen found that one out of five patients reports insufficient involvement in his or her course of treatment . they assert that there is a general lack of user representation and surveys in the processes that shape health services organization and cultural issues . research on shared decision making is young , and new measurement instruments for decision making processes , outcomes and surrounding elements are constantly being developed [ 8 , 9 ] . few measurement instruments are widely used , and the extent of validation of scales differs . most instruments are self - reported scales which were tested for factorial validity without investigation of convergent and discriminant aspects of validity , and few of them were compared with each other . the objective of this paper is to develop and investigate the psychometric properties of a self - reported instrument that measures patient participation in decision making in surgical treatment from the patients ' perspective . we investigated the scope of patient participation through an analysis of norwegian surgical patients ' and healthcare professionals ' beliefs and practices related to patient participation in the decision making processes in surgical treatment ( i.e. , operations or surgical exploration ) . first , a literature review was conducted on concepts and models of patient participation in decision making . we reviewed the literature using the words patient participation , patient involvement , and user involvement in surgical treatment in the academic search elite , cinahl , medline , cochrane , psycinfo and isi databases , and in norwegian public documents on the subject of patient participation and user involvement . according to cahill an analysis of patient participation showed that the meaning of the concept is comparable to patient partnership , patient collaboration , and patient involvement . the literature review indicated that particularly important aspects of patient participation are empowerment , participation in treatment decision - making processes [ 1315 ] and active versus passive patient roles in treatment [ 1618 ] . these three general theoretical constructs were then included in an interview guide that focused on patients and healthcare professionals ' respective views on patient participation . the interviews were structured around the following questions : what does patient participation in surgical treatment imply ? , what preconditions facilitate patient participation in surgical treatment ? , what may hinder and promote patient participation in surgical treatment ? , and what are the possibilities to strengthen patient participation in surgical treatment ? secondly , qualitative interviews were conducted with four physicians and seven nurses from six surgical wards and seven patients who had undergone surgical treatment . the interviews identified domain dimensions to measure patient participation in decision making for surgical treatment . the interviews were conducted during two weeks in 2008 . applying purposive sampling [ 1921 ] , the eighteen participants were selected to ensure a broad representative sample , with experts of varying ages and sexes from medicine and nursing . the healthcare professionals were questioned about their general perceptions about medical / surgical decision making . the two patients identified in table 1 as back operation and hip replacement were interviewed two days before surgery ( these two patients were moved from the orthopedic ward to rehabilitation at another institution after surgery and could not be tracked thereafter because of anonymity . ) and the five remaining patients were interviewed 27 days after surgery . the aim of the interviews was to identify domain dimensions for measuring patient participation in decision making in surgical treatment . applying qualitative interview criteria we concluded that 18 interviews were sufficient to achieve saturation . third , qualitative content analysis was conducted using the methodological logic suggested by graneheim and lundman . based on a frequency count , four theoretical categories were defined as important for measuring patient participation in decision making in surgical treatment : information dissemination , formulation of options , integration of information , and control . the first category , information dissemination , contains patients ' opportunity to talk with answerable doctors and nurses upon admission . nurses give the patients general information about the hospital stay , the anaesthetics , and the preparations before surgery , and the doctors give the patients information about surgical procedures , treatment options , and consequences . the second category , formulation of options , is the patient 's possibilities to choose among options . the third category , integration of information , is about making the information understandable to the patients . patients describe a lack of possibilities to talk with physicians and that nurses were unable to take responsibility about treatment decisions . category four pertains to control which means rejection versus acceptance of paternalism and indicates patients ' role in decision making in treatment . the fourth step of the instrument development was to generate measurement ( questionnaire ) items . to ensure that the domain of the concept had been explored according to the four theoretical categories , we generated 12 items per category , resulting in a pool of 48 items . following the recommendations from churchill , validity and item generation included an evaluation of face and content validation of the items by an expert panel . the members of the expert panel consisted of four individuals with formal qualifications in health and social sciences . the panel gave feedback about the face validity of the items in terms of wording and clarity , and they assessed whether the items belonged together in the four theoretical categories . another basic requirement for content validity was the items ' ability to capture the meaning of the four categories . the members were instructed to associate items with each of the four categories , to comment on unclear or overlapping items and to give comment about the items ' and categories ' ability to capture patient participation in decision making in surgical treatment . this process resulted in 20 items that were further developed into questions in a questionnaire . the empirical testing of the instrument was conducted by distributing a questionnaire and written information about the research to patients ( n = 4,000 ) . the 4,000 patients were selected randomly from nine surgical wards at a norwegian university hospital . the patients had all undergone surgical treatment in the three months prior to receiving a questionnaire , from february to june 2009 . the first 2,000 questionnaires were sent in april 2009 , 1,000 questionnaires were sent in may 2009 , and the last 1,000 were sent in june 2009 . the hospital management consented to the participation , and the study was approved by the hospital research director , the head of the clinic at stavanger university hospital , the norwegian ethical committee ( institutional review board ) ( no . all potential respondents were provided with a full explanation of the study and were invited to participate . prior to obtaining consent , potential respondents were reassured that their decision to participate was voluntary and that they were free to withdraw from participation at any time . all items on the questionnaire were structured with a likert scale from 1 ( strongly disagree ) to 7 ( completely agree ) except for the background variables . empirical validation of the instrument followed the approach advocated for psychometric analysis by churchill and pett et al . . first the univariate distribution of items was assessed by calculating items ' mean , standard deviation , skewness , and kurtosis . then , an initial assessment of convergent validity was done by a principal component factor analysis for each of the four theoretical dimensions of customer participation followed by reliability evaluation by calculations of cronbach 's alpha . to further investigate convergent and discriminant validity , all the 20 items were entered in a factor analysis [ 24 , 26 ] . finally , a further assessment of discriminant validity was done by calculating the correlations between the sumscores of each of the four dimensions . the suitability of data for factor analysis was assessed by inspection of the correlation matrix , by computing the the kaiser - meyer olkin value ( kmo ) and by running bartlett 's test of sphericity . kmo values of .60 or greater and a significant bartlett 's test ( p < .05 ) for factor analysis were considered to be appropriate . for the factor analysis , at least three items per component were recommended , and for the correlation matrices the presence of coefficients greater than .30 was recommended . finally , the nomological validity of the measurement scale was assessed by evaluating the relationship between the four factors and the variables : i had no influence on treatment , and a norwegian version of the utrecht coping list ( ucl ) . the first variable focuses on subjective perceptions of fairness in treatment and justice in organizational settings and was expected to have a negative relationship to the patient participation in decision - making in surgical treatment scale . the ucl coping scale ( 9 items ) relates to the belief that one is able to respond to , and control , external situations . mean scores , standard deviation , skewness , and kurtosis values of 20 items are presented in table 3 . the skewness values ranged between .11 to 1.64 and kurtosis values ranged between .03 to 2.24 , indicating normally distributed items . as the cronbach 's alpha value of each factor was greater than .60 , the internal consistency and reliability were considered to be adequate . the first factor of each analysis captured a considerable amount of variance of each scale ( 50%61% ) , while each of the items ' lowest loadings on the first factor ranged from .62.77 , all indicating convergent validity . convergent and discriminant validity was further investigated by an exploratory factor analysis of all 20 items . the kaiser - meyer - olkin ( kmo ) measure of sampling adequacy increased to .90 , indicating a factorable correlation matrix and bartlett 's test of spherity reached statistical significance ( p = .005 ) for the 20 items . these empirical factors correspond closely to the theoretical dimensions ( 1 ) information dissemination , ( 2 ) formulation of options , ( 3 ) integration of information , and ( 4 ) control , but some cross - loadings and low factor loadings indicated that there was still room for improvement . after deleting four items , all items loaded over .30 on one factor and had low loadings on other factors . the result of a final analysis of the four - factor scale with 16 items is shown in table 5 . cronbach 's alpha was used to assess factor reliability and ranged from .63 to .80 . the two lowest alpha scores were estimated for the factors formulation of options ( .65 ) and control ( .68 ) . the low alpha scores for these factors may be considered in relation to the factors being measured using four and three items , respectively ; the number of items is a very important statistical parameter that influences the estimation of cronbach 's alpha [ 30 , p. 230 ] . nomological validity was assessed with a bivariate correlations analysis between the four dimensions of patient participation and the variable the correlation between first variable and the four factors varied between .10 and .21 ( p < .001 ) . the ucl coping scale correlated .53 , .29 , .66 , .39 ( p < .001 ) with the four participation dimensions , respectively ( table 6 ) . the aim of this study was to develop , empirically test , and validate an instrument specifically designed to understand patient participation in decision making in surgical treatment . empirical validation of the instrument followed the approach advocated for psychometric analysis by churchill and pett et al . . items were developed , validated , and placed in a questionnaire which was tested on 1,048 patients that had undergone surgical treatment . measuring patient participation in treatment is important to improve health services being provided , the dialogue between patients , and healthcare professionals and to identify what areas that could strengthen patient participation . the empirical testing of the instrument reveals a reasonable level of construct validity and reliability and suggests that patient participation is a multidimensional construct that can be measured by 16 items and the following four factors : information dissemination , formulation of options , integration of information , and control . the first factor , information dissemination , explains how information is given and what information the patients receive prior to their operations . the four items relate to general information on the surgical procedure prior to the operation , tests and examinations patients underwent , and benefits and ill effects from surgical treatment [ 3436 ] . the second factor is the formulation of options , in which healthcare professionals explain patients ' options , predict the outcome of treatments , and generate lists of possible treatment alternatives . integration of information , considers patients ' opportunities to speak to the physician and nurse before surgery , their opportunities to convey their needs as patients undergoing surgical treatment , and that their needs in connection with the surgical procedure were taken in consideration [ 1315 ] . the last factor is control , which refers to whether the surgical treatment is decided by the healthcare professionals , the patients , or both parties . the four dimensions had acceptable convergent and discriminant validity , and cronbach 's alphas ( ranging from .65 to .80 ) were more than adequate for this stage of scale development . compelling evidence was also found for nomological validity , the four dimensions of patient participation were , as expected , significantly correlated to ( negative correlations ) and the ucl coping scale . to evaluate the four factors for clinical practice , mean scores for each factor and a total score for the whole instrument have been calculated . the mean scores obtained for the factors information dissemination and integration of information were 5.4 and 5.3 , respectively ( scale range : 17 ) . patients had opportunities to speak to their responsible physicians and nurses after admission , which supported the patients regarding treatment and made the information understandable . the factor formulation of options explored to what extent initiatives regarding surgical treatment could be developed in cooperation with patients . the mean score of 2.6 for this factor , which was the lowest among the four factors , showed that involving patients in the formulation of options is given lower emphasis . thus , initiatives in connection with surgical treatment were negotiated to varying degrees with patient cooperation , and patients were not encouraged to participate in decisions regarding surgical treatment ( this item had the lowest mean score , 2.9 , for the component ) . in this paper we showed that patient participation in decision making in surgical treatment could be measured using a 16-item measurement instrument . analyzing from the healthcare professionals ' perspective may yield different results and seems suitable for future research . the purpose of the project is to increase the level of knowledge of patient participation in decision making and to identify to which degree patient participation in regard to surgical treatment exists . this knowledge is pivotal in order to further develop the user perspective for surgical patients and to ensure daily routines that consider the patients wishes to participate in the decision making in a plan of treatment . with our sample size of 1,048 patients , we more than satisfy the recommendation of having at least 1015 subjects per item to determine a stable factor structure . the results can only be generalized if the analysis of different samples reveals the same factor structure . norway is a rather homogeneous country , but some of the characteristics of the norwegian population might also be found in other countries with a western life style so it might be possible to generalize the results to other areas similar cultures . the instrument was intended for patients who have undergone surgical treatment , and it is also , in other studies , adapted to healthcare professionals . however , a response rate of 26% and the external validity problems related to the use of a single sample to modify a measure are potential biases related to external validity . future research may validate the instrument with a new and independent sample . patient participation in decision making can be difficult to achieve because of incongruence between patient and provider perceptions , knowledge , attitudes , and beliefs . contrasting the patients ' assessments of patient participation with the perspective of the healthcare professionals ' assessments can control for the bias of reporting more positive attitudes about patient participation than what can be justified by reality . the low degrees of patient participation and high level of paternalism experienced by patients may be interpreted as a need for facilitating patient participation in surgical treatment . this may be more important and beneficial for patients , which may also prove to be economically beneficial for society . the instrument is applicable to patients in surgical wards and is designed to be short and easy to administer . the further establishment of construct validity calls for numerous studies and different approaches over time to generate evidence of the relationships between measures .

the aim of this paper is to describe the development of a new , brief , easy - to - administer self - reported instrument designed to assess patient participation in decision making in surgical treatment . we describe item generation , psychometric testing , and validity of the instrument . the final scale consisted of four factors : information dissemination ( 5 items ) , formulation of options ( 4 items ) , integration of information ( 4 items ) , and control ( 3 items ) . the analysis demonstrated a reasonable level of construct validity and reliability . the instrument applies to patients in surgical wards and can be used to identify the health services that are being provided and the areas that could strengthen patient participation .

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

PMC3665018

it is the most common chronic respiratory disease in the world and one of the most common chronic diseases among children . this disease is characterized by variable and recurring symptoms , reversible airflow obstruction , and bronchospasm . asthma is clinically classified according to the frequency of symptoms , forced expiratory volume in 1 second ( fev1 ) , and peak expiratory flow rate . but , especially in iran , the general opinion is not so certain about the benefits of exercise for asthmatic patients . the incidences of eia among the asthmatic patients were differed according to season ( 48% in summer , 73% in spring / fall , and 91% in winter . this phenomenon is widespread among allergic children and can not be accurately predicted from the history . because of the risk of eia , some people generally believe exercise might be harmful for asthmatic patients . it is contrary to a medically accepted claim that states asthmatic patients need exercise as much as healthy people do , provided they consider some precautions to secure safety and avoid eia in exercise . with appropriate medical intervention and knowledge of asthma triggers , this problem is so controlled that many olympic champions have come from asthma sufferers . in the contemporary world , exercise rehabilitation is known as a complementary and necessary treatment for asthmatic patients.[810 ] therefore , today 's question asks about the type of exercise modality that leads to more benefits for asthmatic patients . until now it is showed that about 8 to 12 weeks aerobic exercise is effective in controling asthma symptoms , and improves pulmonary indices and quality of life.[1113 ] recently , some evidences emerged that verified rebound training as another beneficial method of exercise . it creates a safe and efficient framework for people of all age and ability groups . trampoline is also introduced as a safe and useful equipment for exercising all different parts of body . trampoline allows a person to bounce up and down on one or two legs with various upper and lower extremities movements . a national aeronautics and space administration ( nasa ) study demonstrated that at a certain level of oxygen intake , the pulse rate is lower on trampoline compared to running on treadmill . there is an evidence that suggests 12 week rebound exercises on rebounder shoes also improve the aerobic capacity.[17 spirometry indices such as forced vital capacity ( fvc ) , fev1 , and fev1/fvc ratio along with peak aerobic capacity ( vo2peak ) are traditionally used to document the progression in asthmatic patients . fvc is the volume of air that can forcibly be blown out after full inspiration , measured in liters . fev1 is the volume of air that can forcibly be blown out in one second , after full inspiration . in respiratory inflammatory diseases , the required time for forced expiratory increases and , as a result , fev1/fvc ratio ( fev1% ) is the ratio of fev1 to fvc . in young healthy people , this ratio is around 85% which gradually decreases to 75% by aging . in obstructive diseases ( asthma , chronic obstructive pulmonary disease ( copd ) , chronic bronchitis , emphysema ) , both values of fev1 and fvc is diminished , but the drop in fev1 is higher and this will cause the value of fev1% to drop down to below threshold values . the average values of fvc , fev1 , and fev1% for untrained healthy 30-year males are respectively 5.25 , 4.5 liter , and 85% . vo2max is the maximum capacity of body to transport and use oxygen during incremental exercise . it is widely accepted as the single best measure of cardiovascular fitness and maximal aerobic power . the average values for untrained healthy male and female are respectively 35 - 40 and 27 - 31 ml / kg / min . would be too risky , as any problem with respiratory and cardiovascular systems may be greatly exacerbated when a person exercise at his / her highest capacity . thus , some cautious protocols have been developed to measure the peak of vo2 at sub - maximal workloads . the procedure of sub - maximal tests is generally similar to maximal tests , but a certain percent ( usually 75 - 80% of maximum capacity ) of cardio - respiratory system is attained . values measured during sub - maximal tests are called vo2peak . aerobic exercise , alone or along with strength training , increases spirometry indices . it is indicated that exercise improves the pulmonary performance and reduces symptoms such as wheezing , coughing , chest tightness , and shortness of breath in asthmatic patients . in this study , we document the effects of aerobic and rebound exercises on fev1 , fvc , fev1% , and vo2peak of asthmatic patients . this study was approved by isfahan university research board . it was a quasi - experimental study conducted on one control and two experimental groups , namely aerobic and rebound training . as random group assignment had confliction with obtaining informed consent , group formation was voluntarily . experimental group , further to routine medical treatment , participated in their specific exercise programs . both exercise programs were supervised , consisted of two sessions of 45 to 60 minutes per week , and continued for 8 weeks . sample included asthmatic patients from the respiratory clinic of alzahra hospital in isfahan , iran . inclusion / exclusion criteria were being male aged 20 to 40 years , suffering intermittent to mild asthma for at least one year , being under routine medical treatment of the same respiratory clinic , ability of performing offered exercises , and suffering from no other disease complications . subjects , who experienced any radical changes in their routine medical treatment during the study , were excluded . every exercise session consisted of three parts : 15 minutes warm - up exercises , main exercises , and 15 minutes cool down exercises . main exercises of the first session lasted for 15 minutes and consisted of low intensity movements . session by session , the length and intensity of the main exercises were gradually increased , so that the final session lasted for 30 minutes and consisted of moderate to high intensity movements . the main exercise of aerobic group was jogging and was performed on a soccer pitch . time and intensity of exercises of aerobic group were matched to rebound group . in both groups , the intensity of exercises was controlled by interval self - monitoring of hr on radial pulse . exercise testing guidelines ( published by the american college of sports medicine ) were observed . before exercise , each subject was familiarized with the testing procedures . subjects were then encouraged to exercise to the limit of their tolerance on an automated treadmill ( power jack , england ) . vo2peak and peak heart rate ( hr ) were defined as the highest values achieved during exercise . all subjects were monitored during exercise recovery until hr returned to within 20% of baseline values . as measurement of vo2peak , fev1 , and fvc are influenced by asthma medications , for every subject the time and dosage of medication were fixed for pre- and post tests . other measurements : in this study , we also measured plasma endothelin level ( by enzyme - linked immunosorbent assays ( elisa ) method ) and quality of life ( by asthma quality of life questionnaire ) . this study was approved by isfahan university research board . it was a quasi - experimental study conducted on one control and two experimental groups , namely aerobic and rebound training . as random group assignment had confliction with obtaining informed consent , group formation was voluntarily . experimental group , further to routine medical treatment , participated in their specific exercise programs . both exercise programs were supervised , consisted of two sessions of 45 to 60 minutes per week , and continued for 8 weeks . sample included asthmatic patients from the respiratory clinic of alzahra hospital in isfahan , iran . inclusion / exclusion criteria were being male aged 20 to 40 years , suffering intermittent to mild asthma for at least one year , being under routine medical treatment of the same respiratory clinic , ability of performing offered exercises , and suffering from no other disease complications . subjects , who experienced any radical changes in their routine medical treatment during the study , were excluded . for every group , in both groups , every exercise session consisted of three parts : 15 minutes warm - up exercises , main exercises , and 15 minutes cool down exercises . main exercises of the first session lasted for 15 minutes and consisted of low intensity movements . session by session , the length and intensity of the main exercises were gradually increased , so that the final session lasted for 30 minutes and consisted of moderate to high intensity movements . the main exercise of aerobic group was jogging and was performed on a soccer pitch . time and intensity of exercises of aerobic group were matched to rebound group . in both groups , the intensity of exercises was controlled by interval self - monitoring of hr on radial pulse . exercise testing guidelines ( published by the american college of sports medicine ) were observed . before exercise , each subject was familiarized with the testing procedures . subjects were then encouraged to exercise to the limit of their tolerance on an automated treadmill ( power jack , england ) . vo2peak and peak heart rate ( hr ) were defined as the highest values achieved during exercise . all subjects were monitored during exercise recovery until hr returned to within 20% of baseline values . fev1 and fvc were measured by spirometer ( jager , germany ) . as measurement of vo2peak , fev1 , and fvc are influenced by asthma medications , for every subject the time and dosage of medication were fixed for pre- and post tests . other measurements : in this study , we also measured plasma endothelin level ( by enzyme - linked immunosorbent assays ( elisa ) method ) and quality of life ( by asthma quality of life questionnaire ) . descriptive information about fev1 , fvc , fev1% , and vo2peak of asthmatic patients are shown in table 2 . compared to values reported in introduction section for healthy untrained male adults , the data shows that fev1 , fvc , fev1% , and vo2peak of asthmatic patients are dramatically dropped . the interaction comparison is the most important one and when it is significant , it would be enough to focus on it . they demonstrate that , during the study , control group had no progress , while experimental groups made significant improvement . patients demographics ( meanstandard deviation ) patients descriptive statistics and result of repeated measure analysis of variance chartline of vo2 peak the present study suggests that exercise is better than no exercise . to understand which kind of exercise is more beneficial , it is necessary to pay attention to the slope of improvement lines in figures 1 to 4 . present study has shown that fev1 , fvc , fev1% , and vo2peak of asthmatic participants were much lower than healthy adults . it seems that there is still much room for more improvement with longer periods of exercise . it is well - documented that exercise improves work tolerance by decreasing ventilation rate for a given workload . improvement in respiratory muscles strength , cellular respiration , and cardio - vascular systems along with re - vascularization of skeletal muscles are among the direct physiologic reasons suggested for this claim . also , it seems that exercise rehab indirectly raises the knowledge and experience of patients for better care and management of the disease , and it subsequently alleviate the symptoms and reduces the consumption of bronchodilators . although benefits of aerobic exercises such as jogging and walking are evident , some patients still are not interested in such activities and would withdraw from them sooner or later . we decided to examine the effects of a totally different exercise such as rebound training . present study showed that by good supervision , patients could achieve self - management in rebound exercise without any risk of injury . therefore , it is not sensible to limit the type of aerobic exercise for asthmatic patients . it was shown that effects of rebound exercise not only are equivalent but even better than traditional aerobic exercises . it is the fact that the participants of the study were volunteers , and it might have intensified the results . however , it is just the point : exercise demands a lot of effort and , therefore , needs interest and should be accompanied with fun and enjoyment . we suggest for unskilled exercises such as rebound training , a period of supervised exercise is attempted before independence . supervisors need to address the skill acquisition as well as the volume ( frequency , intensity , and time ) of training . how effects of rebound therapy are justified ? we noted that rebound exercises are fun and they encourage a longer period of workout . we also noted that keeping balance and position on trampoline burden a lot of pressure on anti - gravity muscles and increases the blood flow to active muscles , and subsequently improves the functional capacity.[2527 ] movement on trampoline has a vibration effect on muscle spindles that fine tune the muscle tension . in another words , any hop or jump on a hard surface such as land activates the stretch reflex and increase the muscle tension . but , hop and jumps on trampoline are anticipated and flexibility of the surface cushions the contact and increases its time . it in turn controls the stretch reflex and leads to less muscle tone . it is why at a certain level of oxygen intake , the pulse rate is lower on trampoline compared to running on treadmill and oxygen efficiency is higher in trampoline exercises . last but not least , sedentary life style is one of the major disadvantages of the modern life . chronic diseases , including asthma , complicate and worsen the situation . in today 's iran , subsequently , the general fitness and specially the aerobic fitness fall lower than average of the society . on the other hand , when no supervised exercise is practiced , patients would find no opportunity to raise their awareness for preventing symptoms and asthma attacks . further to these disadvantages , lack of physical activity lowers the quality of psychosocial life as well . present study demonstrated that both aerobic and rebound exercises were worthwhile for mild to moderate male asthmatic patients . we demonstrated that the use of rebound exercises resulted in some advantages in vo2peak and respiratory indices , provided patients would be interested and enjoy the exercises . thus , the use of different exercise modalities in accordance to patients ' interests seems advisable . present study demonstrated that both aerobic and rebound exercises were worthwhile for mild to moderate male asthmatic patients . we demonstrated that the use of rebound exercises resulted in some advantages in vo2peak and respiratory indices , provided patients would be interested and enjoy the exercises . thus , the use of different exercise modalities in accordance to patients ' interests seems advisable .

background : there are some auspicious records on applying aerobic exercise for asthmatic patients . recently , it is suggested that rebound exercise might even increase the gains . this study was designed to compare the effects of rebound therapy to aerobic training in male asthmatic patients.methods:sample included 37 male asthmatic patients ( 20 - 40 years ) from the same respiratory clinic . after signing the informed consent , subjects volunteered to take part in control , rebound , or aerobic groups . there was no change in the routine medical treatment of patients . supervised exercise programs continued for 8 weeks , consisting of two sessions of 45 to 60 minutes per week . criteria measures were assessed pre- and post exercise program . peak exercise capacity ( vo2peak ) was estimated by modified bruce protocol , forced vital capacity ( fvc ) , forced expiratory volume in 1 second ( fev1 ) , and fev1% were measured by spirometer . data were analyzed by repeated measure analysis of variance ( anova).results : significant interactions were observed for all 4 criteria measures ( p < 0.01 ) , meaning that both the exercise programs were effective in improving fvc , fev1 , fev1% , and vo2peak . rebound exercise produced more improvement in fev1 , fev1% , and vo2peak.conclusions:regular exercise strengthens the respiratory muscles and improves the cellular respiration . at the same time , it improves the muscular , respiratory , and cardio - vascular systems . effects of rebound exercise seem to be promising . findings suggest that rebound exercise is a useful complementary means for asthmatic male patients .

INTRODUCTION METHODS Study design Selection criteria Exercise training Measurements Data analysis RESULTS CONCLUSION DISCUSSION Practical applications

PMC1533268

the central nervous , immune , and endocrine systems communicate through multiple common messengers . over evolutionary time , what may be termed integrated defense system(s ) ( ids ) have developed to coordinate these communications for specific contexts ; these include the stress response , acute - phase response , nonspecific immune response , immune response to antigen , kindling , tolerance , time - dependent sensitization , neurogenic switching , and traumatic dissociation ( td ) . these idss are described and their overlap is examined . three models of disease production are generated : damage , in which idss function incorrectly ; inadequate / inappropriate , in which ids response is outstripped by a changing context ; and evolving / learning , in which the ids learned response to a context is deemed pathologic . mechanisms of multiple chemical sensitivity ( mcs ) are developed from several ids disease models . model 1a is pesticide damage to the central nervous system , overlapping with body chemical burdens , td , and chronic zinc deficiency ; model 1b is benzene disruption of interleukin-1 , overlapping with childhood developmental windows and hapten - antigenic spreading ; and model 1c is autoimmunity to immunoglobulin - g ( igg ) , overlapping with spreading to other igg - inducers , sudden spreading of inciters , and food - contaminating chemicals . model 2a is chemical and stress overload , including comparison with the susceptibility / sensitization / triggering / spreading model ; model 2b is genetic mercury allergy , overlapping with : heavy metals / zinc displacement and childhood / gestational mercury exposures ; and model 3 is mcs as evolution and learning . remarks are offered on current mcs research . problems with clinical measurement are suggested on the basis of ids models . large - sample patient self - report epidemiology is described as an alternative or addition to clinical biomarker and animal testing.imagesfigure 1figure 2figure 3figure 1figure 2figure 3figure 4figure 5

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PMC4899594

fangji huangqi tang ( fht ) is a classical formula of traditional chinese medicine ( tcm ) . it was firstly recorded in jing gui yao lue by zhongjing zhang in the han dynasty . the formula consisted of four herbs , including stephania tetrandra s. moore , astragalus membranaceus ( fisch . ) bge . , atractylodes macrocephala koidz . , and glycyrrhiza uralensis fisch . lots of doctors recommended fht for the treatment of chronic glomerulonephritis , cardiac edema , and rheumatic arthritis in chinese clinical application based on their actual experience . also , experts began to do their research work to reveal the potential effective compounds in fht . for safe and effective use of fht in the clinic , quality control and evaluation of this formula with multiple compounds ' identification and determination became essential and important . it has already been found out that fht contains different kinds of components , such as flavonoids ( liquiritigenin , isoliquiritigenin , liquiritin , formononetin , and calycosin ) , alkaloids ( fangchinoline and tetrandrine ) , lactones ( atractylenolide i , atractylenolide ii , atractylenolide iii , and atractylon ) , and saponins ( astragaloside i , astragaloside ii , astragaloside iii , and astragaloside iv ) . among these compositions , fangchinoline , tetrandrine , and liquiritigenin , as well as calycosin , all showed good effects on anti - inflammatory and improving immunity [ 47 ] . the critical strategy for quality control and evaluation of fht should be applied to study as much of these effective components as possible . several common analytical techniques were usually used for multiple chemical compounds ' determination in tcm , including hplc - dad , uv , hplc - elsd , and lc - icpms [ 814 ] . however , these methods showed lots of drawbacks and technical shortcomings , such as time - consuming sample running , poor sensitivity , peak interferences , and limitation of detectors on components identification . as it is widely accepted , the uhplc - qqq - ms technique has a special advantage of high resolution and acute sensitivity . and it is now a very powerful tool for the determination of complex chemical compounds in tcm prescriptions , by which the identification of each analyte is also accomplished at the same time . for this reason , a simple , rapid , and sensitive method for simultaneous determination of 10 components in fht was developed based on uhplc - qqq - ms technique in this paper , and the feasibility of this technique on tcm quality control and evaluation was strongly demonstrated . stephania tetrandra s. moore and atractylodes macrocephala koidz . were obtained from hebei chinese medicine group corporation ( hebei , china ) . fangchinoline , tetrandrine , calycosin-7-o--d - glucoside , atractylenolide i , atractylenolide iii , liquiritin , isoliquiritin , liquiritigenin , isoliquiritigenin , and glycyrrhizin with purity of 99% or higher were purchased from the sichuan victor biological technology co. ( chengdu , china ) . hplc - grade methanol and acetonitrile were purchased from e. merck ( merck , darmstadt , germany ) . purified water was from a milli - q system ( millipore corporation , bedford , ma , usa ) . formic acid was of analytical grade and was obtained from nanjing chemical reagent company ( nanjing , china ) . the uhplc system ( shimadzu , kyoto , japan ) consisted of lc-30ad binary pump , an autosampler ( model sil-30sd ) , an online degasser ( dgu-20a5r ) , and a column temperature controller compartment ( cto-30a ) . an extend - c18 column ( agilent , usa , 2.1 mm 100 mm , 1.8 m ) at a temperature of 30c was used for separation . mass spectrometric detection was performed using triple quadrupole 5500 instrument ( ab sciex , concord , ontario , canada ) equipped with an electrospray ionization ( esi ) source . the separation of these ten compounds was achieved by linear gradient elution using a mobile phase consisting of 0.1% ( v / v ) aqueous formic acid ( a ) and acetonitrile ( b ) . the gradient program was as follows : 0.10.8 min , 5% b50% b ; 0.83 min , 50% b90% b ; 33.1 min , 90% b5% b. the flow rate was set at 0.4 ml / min . the samples were placed in an autosampler at 4c prior to injection and the injection volume was 10 l . both positive and negative ionization mass spectrometric analyses were conducted due to the physicochemical properties of the ten analytes . the scan mode was set as multiple reactions monitoring ( mrm ) and the selected monitor ions were m / z 623.2/174.1 for tetrandrine , m / z 609.3/367.1 for fangchinoline , m / z 233.1/187.2 for atractylenolide i , m / z 249.1/231.0 for atractylenolide iii , m / z 447.2/285.1 for calycosin-7-o--d - glucoside , m / z 821.3/351.2 for glycyrrhizin , m / z 417.1/255.1 for liquiritin , m / z 417.1/255.1 for isoliquiritin , m / z 256.2/135.0 for liquiritigenin , and m / z 256.2/135.0 for isoliquiritigenin . also , another selected monitor ion pair was selected for the identification purpose for each analyte . they were m / z 623.2/181.3 for tetrandrine , m / z 609.3/192.2 for fangchinoline , m / z 233.1/215.1 for atractylenolide i , m / z 249.1/163.0 for atractylenolide iii , m / z 447.2/270.0 for calycosin-7-o--d - glucoside , m / z 821.3/193.1 for glycyrrhizin , m / z 417.1/135.0 for liquiritin , m / z 417.1/135.0 for isoliquiritin , m / z 256.2/119.1 for liquiritigenin , and m / z 256.2/119.1 for isoliquiritigenin . source condition parameters were as follows : curtain gas set at 35 , ion source temperature adjusted to 550c , and ion source gas 1 ( gs 1 ) and ion source gas 2 ( gs 2 ) both at 55 . according to the original composition and preparation process of fht recorded in jin - gui - yao - lue , fht was obtained in the following procedure : pieces of stephania tetrandra s. moore ( 12 g ) , astragalus membranaceus ( fisch . ) bge . ( 6 g ) were mixed together in a ratio ( 4 : 5 : 3 : 2 , w / w / w / w ) and macerated in deionized water for 30 min before being decocted twice with boiling water ( 1 : 10 , w / v and 1 : 8 , w / v , resp . ) , 20 min for each time . the solution was then filtered through a four - layer mesh and was diluted with water to 1000 ml . 2 ml of the above solution was added to 50 ml vessel , and 15 ml of 60% methanol solution was added for ultrasonic extraction . then , moderate volume of 60% methanol solution was added to the scale of the 50 ml vessel . the solution was centrifuged at 12,000 rpm for 5 min and was filtered through 0.22 m membrane . stock solutions of the 10 analytes were separately prepared by diluting the reference standards with methanol . all the standards were soluble in methanol and completely dissolved in the mixed working solution . and the mixed working solution containing 10 compounds was diluted to appropriate concentration ranges for the construction of calibration curves . the calibration curve of each compound was performed with at least seven appropriate concentrations levels . the concentrations of the 10 components in fangji huangqi tang should be included in the range of the standard curve . the limit of detection ( lod ) and limit of quantification ( loq ) under the present chromatographic conditions were determined at a signal - to - noise ( s / n ) ratio of 3 and 10 , respectively . intraday variations within one day were all taken into consideration to determine the precision of the developed method . the relative standard deviation ( rsd ) certain amounts of 10 standards were accurately added to a fht sample and then processed and analyzed as described in sections 2.2 and 2.3 . the total amount of each analyte was calculated from the corresponding calibration curve , and the recovery of each analyte was calculated by the following equation : recovery ( % ) = ( amountdetermined amountoriginal)/amountspiked 100% , where amountdetermined is the determined total amount of each analyte , amountoriginal is the original amount of each analyte in samples determined , and amountspiked is the spiked amount of each analyte with 80% , 100% , and 120% ratio of the original amount . in order to investigate the stability of the processed sample solution , the same sample solution was stored at 4c and analyzed every 12 h for 1 day . in addition , the 10 stock solutions were all determined every week to investigate whether obvious degradation had happened under their preservation condition . according to the published papers , many different kinds of components were identified in fht including alkaloids , flavonoid , and lactones . also , these chemical compounds were proved to be the main effective components in fht . the ten selected compounds for simultaneous determination belonged to different chemical classes and covered a wide polarity range . so , it is of great significance to screen out an appropriate sample preparation method based on the effective extraction for various effective compounds . therefore , a novel sample method has been developed to collect different polar components on a solid phase extraction column . this simple and convenient technique was suitable for traditional chinese medicine prescriptions detection by enhancing the sample polarity scope , as well as reducing the pollution of the instrument . it was difficult for researchers to perform good separation for the chemical constituents in fht because of the complexity of the composition and the existence of isomers . in this paper , the uhplc conditions were well optimized , including the type of column , column temperature , mobile phase system , and flow rate . after a comparison of different brands of columns including agilent c18 , waters c18 , and thermo c18 , agilent column finally gave out sound separation for the ten compounds to be determined . in addition , different kinds of mobile phase systems such as acetonitrile and methanol with a variety of modifiers ( including formic acid and acetic acid ) were tested . as a result , a solution of 0.1% ( v / v ) aqueous formic acid and acetonitrile at 0.4 ml / min was used as the optimal chromatographic elution system . typical source conditions were used as follows : curtain gas was set at 35 ; ion source temperature was adjusted to 500c ; ion source gas 1 ( gs 1 ) and ion source gas 2 ( gs 2 ) were both set at 55 . independent parameters for the detection of each compound were optimized by injecting standard solution into ms system under mrm mode , including the declustering potential ( dp ) , the collision energy ( ce ) , the entrance potential ( ep ) , and the collision cell exit potential ( cxp ) . chromatographic peaks of fht were identified by comparing the retention time with that of each reference compound . the representative extract ions chromatograms of the standard mixture solution were shown in figure 2 . ( a ) stands for fangchinoline ( 0.98 min ) ; ( b ) stands for atractylenolide i ( 2.28 min ) ; ( c ) stands for tetrandrine ( 1.01 min ) ; ( d ) stands for atractylenolide iii ( 1.83 min ) ; ( e ) stands for calycosin-7-o--d - glucoside ( 1.04 min ) ; ( f ) stands for liquiritigenin ( 1.47 min ) and isoliquiritigenin ( 1.72 min ) ; ( g ) stands for liquiritin ( 1.17 min ) and isoliquiritin ( 1.31 min ) ; ( h ) stands for glycyrrhizin ( 1.60 min ) . according to the mass - to - electric charge ratio of each compound , table 2 shows the regression equation for each reference compound , as well as the lod and loq values and the mass spectrometry information . all the components showed good linearity ( r > 0.9991 ) in a relatively wide concentration range . tables 3 and 4 provide precision and recovery results of the 10 components , respectively . the intraday variations of the compounds were < 2.53% , the relative standard deviation ( rsd ) of repeatability test was below 2.68% for all the analytes of interest , and the extraction recoveries of the components ranged from 98.51% to 101.10% . stability was evaluated by analyzing all the analytes in samples kept at 4c for 24 h. they were all found to be stable within 24 h ( rsd < 3% ) . as a result , the developed method was proved to be precise , accurate , and sensitive for the simultaneous quantification of the major compounds of fht . the developed uhplc - ms / ms method was subsequently applied to analysis and quality evaluation of fht . a rapid uhplc - ms / ms method was established for the comprehensive analysis of fht . furthermore , ms / ms proposed high sensitivity which is helpful for the quantification of the low - content compounds . the method was successfully applied to the simultaneous determination of 10 bioactive compounds in fht . this available , rapid , and reliable method is fit for routine analysis and effective quality control of this chinese herbal prescription .

the aim of this study is to develop an ultrahigh performance liquid chromatography method coupled with triple quadrupole mass spectrometry for simultaneous determination of tetrandrine , fangchinoline , atractylenolide i , atractylenolide iii , calycosin-7-o--d - glucoside , glycyrrhizin , liquiritin , isoliquiritin , liquiritigenin , and isoliquiritigenin in fangji huangqi tang ( fht ) . the chromatographic separation was performed on a reversed - c18 column , eluted with a mixture of 0.1% acetic acid and acetonitrile at 0.4 ml / min . the separation of these ten compounds was achieved by linear gradient elution . the method was strictly validated with respect to specificity , precision , accuracy , and repeatability . all the compounds showed good linearities ( r 0.999 ) . the loqs of the ten components were 0.36 , 0.18 , 0.09 , 0.43 , 0.02 , 1.89 , 0.26 , 0.18 , 0.61 , and 0.48 ng / ml for tetrandrine , fangchinoline , atractylenolide i , atractylenolide iii , calycosin-7-o--d - glucoside , glycyrrhizin , liquiritin , isoliquiritin , liquiritigenin , and isoliquiritigenin , respectively . the lods of the ten components were 0.11 , 0.05 , 0.03 , 0.13 , 0.01 , 0.57 , 0.08 , 0.05 , 0.18 , and 0.14 ng / ml for tetrandrine , fangchinoline , atractylenolide i , atractylenolide iii , calycosin-7-o--d - glucoside , glycyrrhizin , liquiritin , isoliquiritin , liquiritigenin , and isoliquiritigenin , respectively . the method was proven to be specific and reliable , which would provide a meaningful basis for the quality control and evaluation of fht during its clinical application .

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

PMC4341315

most poor people seek help at public run hospitals where services are free , and those from families below the poverty line ( bpl ) are exempted from payments for medicines and investigations . while this is helpful , there are other hidden costs which the family has to bear . patients often need to buy medicines which are unavailable in the hospital , meet the expenses for food , transport , etc . the total costs usually workout to be well beyond the means of those coming from bpl households . in fact , health care costs often lead to further impoverishment and sets off a vicious cycle of ill - health and poverty , one fuelling the other . this had been investigated in maternal health and it is relevant for scaling up of mental health care too . the financial impact of hospitalization for psychoses needs to be evaluated , especially on the poorer sections of the society . this study aims to assess the treatment related expenses of patients admitted to a government general hospital with psychotic illness . the present study was conducted after getting approval from institutional research board of government medical college , thrissur . patients who meet the international classification of diseases ( icd)-10 criteria for schizophrenia , bipolar mood disorders , and other psychotic conditions were prospectively identified from the psychiatry wards of government medical college , thrissur . they were interviewed after getting informed consent ( written ) and we used specially designed study proforma for data collection . these in - depth interviews were aimed to explore the financial burden experienced by the family during hospitalization . the major components of expenses during the hospital stay were categorized as cost of medicines , investigations , food , travel , and other incidental expenses . the details of medication and investigations were collected from the case records on the last day of hospitalization . we also analyzed the different categories of expenses and calculated the total expenses incurred during hospitalization . all those who had a bpl card issued by the revenue department of kerala were considered as poor . we used descriptive statistics such as mean , median , range , and standard deviation to explain the results . mann whitney u - test was used to compare costs among bpl and above poverty line ( apl ) patients . spearman 's rank correlation coefficient was used for finding out the correlation between study variables . there were 104 cases who received an icd-10 diagnosis of psychosis during the 6-months study period . four cases had to be excluded due to lack of consent and inability to schedule the interviews on the day of discharge . only 3% of the subjects were illiterate while 34% had primary level education , and the rest had education up to high school or more . the number of earning members in each family ranged from 1 to 4 while the average family size was four . pension benefits were available to 29% of the families , but the average pension was only inr 300/month . about a half of all patients ( 49% ) were engaged in skilled , semiskilled or unskilled work , often for daily wages , before the onset of illness . about 21 of them stopped working less than a month prior to admission whereas 13 patients were not working for the last 1 - 6 months . during the hospitalization , about 49% of the caregivers had to cut back work resulting in an average loss inr 1500 from the caregiver income during the hospitalization period alone . nearly half ( 45% ) of the families had availed loans for different purposes , and a significant proportion ( 22.2% ) of them took loans to meet the expenses of hospitalization for psychosis . the expenses incurred during hospitalization included cost of medicines , laboratory investigations , food , travel and other incidental costs . though most patients ( 76% ) received free medicines from the hospital , some drugs had to be purchased . almost a quarter ( 24% ) had to purchase all the necessary medicines from elsewhere . both bpl and apl patients spent almost same amount for purchase of medicines ( z = 0.469 , p = 0.639 ) . more hospital days meant more money spent on medication ( r = 0.442 , p = 0.00 ) . while most patients ( 94% ) needed laboratory investigations , only a minority ( 19.2% ) could get all the investigations done in the hospital itself . many ( 41.5% ) patients had to pay and use other facilities for some investigations . among 94 patients who needed investigations , patients from bpl households spent significantly less amount of money for investigations ( z = 2.682 , p = 0.007 ) and this expense did not depend on the duration of hospitalization ( r = 0.101 , p = 0.320 ) . even though , food was available at free of cost for inpatients from bpl families , the quantity was insufficient . the average cost of food during the hospitalization was inr 1200 , and this increased with the duration of hospital stay ( r = 0.619 , p = 0.00 ) . among the 100 patients admitted in the hospital , 65% hailed from thrissur district and the remaining 35% were from the neighboring districts such as palakkad , malappuram , and ernakulam . majority of patients ( 60% ) made use of vehicles such as car , auto or ambulance for conveyance . the number of persons accompanying the patient on their journey to the hospital was 1 - 5 while 1 - 3 persons accompanied the patient after getting discharged from the hospital . an average cost of travel per patient was inr 396 for their journey to and fro the hospital . the total direct expense of hospital stay was calculated as the sum of cost of medicine , laboratory test , food , travel , and other miscellaneous expenses . the average total expenses including indirect expenses was inr 3975 and this increased with the duration of hospital stay ( r = 0.515 , p = 0.00 ) . almost half of the families ( 46% ) had received financial support from relatives outside their household and this averaged inr 500 . majority ( 67% ) of patients treated for psychosis in a government - run medical college came from low - income households living below the poverty line . the average out of pocket expense was more than 1200 rupees and averaged inr 100/day . the average cost of hospitalization rose to about inr 4000 when indirect expenses are added on . we did not find a significant difference between apl and bpl families in the expenses related to hospitalization except that the patients from bpl families spent less on investigations that were free for them . many families had to rely on hired vehicles for bringing the patient to the hospital . there are schemes that allow reimbursement of the expenses for transport during emergency obstetric care . there is no provision to reimburse the cost of transport of a person who needs hospital admission and care for psychosis . government should extend such schemes to include those who need hospitalization for a psychotic illness . almost half ( 45% ) of the families had availed loans , a clear indication of their strained financial status . about a fifth of these families borrowed money to meet the expenses for the current hospitalization and treatment . this is a matter of great concern as health care costs , especially out of pocket expenses and is a major cause for further impoverishment of poor households . lack of support would lead these families to take more loans and get into a debt trap . our study shows that health care cost , especially the costs associated with management of psychosis is well beyond the financial capability of low - income families . government should recognize this and offer financial support to meet the treatment related expenses of people with psychosis . the model of janani suraksha yojana can be adopted for financing expenses of psychiatric hospitalization . many families are laboring under serious financial strain while they are engaged in the treatment of psychosis . the governments should seriously consider financial support for treatment of poor people affected by major health conditions like psychoses . support should extend well beyond free services and should include strategies to finance the direct and indirect costs of treatment . to the best of our knowledge , this is the first study from india to report the costs associated with hospitalization for people with psychotic illness in a general hospital setting . the treatment of mental illness involves significant expenditure , frequent , and considerable use of health care resources . health care financing in india is vastly different from western countries and some developed economies where in health insurance plays a very large role . even there , the situation is bad , if the provision for third party payment is unavailable . there are schemes like rashtriya swasthya bima yojana ( rsby ) and other health insurance policies under government of india that are running essentially for inpatient service for people from bpl families . people with psychiatric illness were kept out of the benefits of rsby scheme till recently . . a special scheme to support management of psychotic disorders like schizophrenia and bipolar disorders would be helpful to many families . good access to effective treatment and adherence to long - term prophylactic treatment is important in the management of schizophrenia and recurrent bipolar disorder . poverty is a barrier that denies access to good quality mental health care for them . governments and policy makers should design and implement schemes to finance treatment of psychosis that eventually will help to improve outcomes . the cost of care leading to further impoverishment is a real problem , especially in low - income settings . we need to adopt an intervention which goes well beyond the conventional management of illness to prevent this .

background : combination of ill health and poverty poses special challenges to health care providers . mental illness and costs are linked in terms of long - term treatment and lost productivity , and it affects social development . the purpose of the present study is to assess the economic burden of poor families when a family member needs hospitalization due to psychosis.materials and methods : the information was gathered from caregivers of 100 psychotic inpatients of medical college hospital of kerala during a period of 6 months . data regarding components of expenses such as cost of medicine , laboratory investigations , food , travel , and other miscellaneous expenses during their inpatient period were collected by direct personal interview using specially designed proforma . the data were analyzed using epi - info software . the patients below the poverty line ( bpl ) were compared with those above poverty line ( apl).results : there was no significant difference between patients from bpl and apl in respect of amounts spent on the studied variables except for laboratory investigations during the hospital stay.conclusions:the results showed that the studied subjects are facing financial difficulties not only due to hospitalization , but also due to the recurrent expense of their ongoing medication . the study recommends the need of financial support from the government for the treatment of psychotic patients .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC4629954

diabetes is a major global health problem , which currently affects millions of people worldwide with high and escalating costs to societies.1,2 we have established the diabetes impact study 2013 with the aim to provide an updated account of the current and future epidemiological profile of diabetes in denmark together with an appraisal of the costs to patients and society that is attributable to diabetes . the study takes advantage of the establishment of the danish national diabetes register ( ndr ) in 20063 which , together with the high quality of danish health registers , offers unique opportunities for epidemiological and public health research in diabetes.4 here , we present the diabetes impact study 2013 and the first epidemiological results , with focus on the trends in the incidence , the mortality , and the resulting prevalence of diabetes in denmark from 2000 to 2011 . as a novelty , we also introduce the framework of using register data to classify diabetes by complications status to investigate the epidemiological characteristics of chronic complications of diabetes . ndr is established on the basis of information contained in already existing danish health registers,3 including the danish national patient register ( dnpar),5 the danish national prescription registry,6 and the danish national health service register.7 linkage of person - specific data between the registers is possible using the personal identification number that is assigned to each danish citizen and is used for administrative purposes throughout the public and private sectors in denmark . as previously summarized,3,8 inclusion in ndr takes place for a given person when the first of several possible ascertainment criteria are met : the first date of either a contact registered in the dnpar with a diagnosis relevant to diabetes mellitus , fulfillment of specific criteria of purchasing prescriptions with either insulin or peroral antidiabetic drugs , fulfillment of one of two specific patterns of measurement of blood sugar , or a registration of a chiropodist service specifically provided to a person with diabetes . all data on moves , migrations , and deaths recorded for the population of ndr registrants were obtained from the danish civil registration system , which , since 1968 , has kept electronic records on all demographic data for all danish inhabitants.9 ndr is supposed to cover all persons fulfilling inclusion criteria and being alive at the end of 1996 plus all persons with inclusion date since the start of 1997 . for the present analysis , a data set was extracted with all patients cumulatively registered with ndr on july 3 , 2013 ( n=497,232 ) . for these patients , all contacts ( regardless of the date of contact ) in dnpar were extracted , including coded data in all diagnostic and therapeutic interventions performed , together with information on primary and secondary diagnoses underlying the contact , the hospital , and all relevant dates related to the contact . dnpar contains information on all activities in the danish hospitals back to 1977 for inpatient contacts and 1995 for outpatient contacts , including visits to emergency rooms.5 ndr is established on the basis of information contained in already existing danish health registers,3 including the danish national patient register ( dnpar),5 the danish national prescription registry,6 and the danish national health service register.7 linkage of person - specific data between the registers is possible using the personal identification number that is assigned to each danish citizen and is used for administrative purposes throughout the public and private sectors in denmark . as previously summarized,3,8 inclusion in ndr takes place for a given person when the first of several possible ascertainment criteria are met : the first date of either a contact registered in the dnpar with a diagnosis relevant to diabetes mellitus , fulfillment of specific criteria of purchasing prescriptions with either insulin or peroral antidiabetic drugs , fulfillment of one of two specific patterns of measurement of blood sugar , or a registration of a chiropodist service specifically provided to a person with diabetes . all data on moves , migrations , and deaths recorded for the population of ndr registrants were obtained from the danish civil registration system , which , since 1968 , has kept electronic records on all demographic data for all danish inhabitants.9 ndr is supposed to cover all persons fulfilling inclusion criteria and being alive at the end of 1996 plus all persons with inclusion date since the start of 1997 . for the present analysis , a data set was extracted with all patients cumulatively registered with ndr on july 3 , 2013 ( n=497,232 ) . for these patients , all contacts ( regardless of the date of contact ) in dnpar were extracted , including coded data in all diagnostic and therapeutic interventions performed , together with information on primary and secondary diagnoses underlying the contact , the hospital , and all relevant dates related to the contact . dnpar contains information on all activities in the danish hospitals back to 1977 for inpatient contacts and 1995 for outpatient contacts , including visits to emergency rooms.5 by means of the personal identification number , data for each of the registrants in ndr were linked with dnpar to establish relevant clinical milestones for each patient . these milestones included the date of first occurrence of the diagnosis of diabetes , and the date of first occurrence of codes classifying for chronic complications related to diabetes ( see establishing the diagnosis of diabetes section ) . information from danish civil registration system was used to establish the date of death ( regardless of the cause of death ) and the date of emigration as relevant . the date of the clinical diagnosis of diabetes is not available in ndr and has been operationally established as the first date of fulfillment of a criterion for inclusion in ndr.8 if the first registration of a diagnosis or an intervention code specific for diabetes occurred in dnpar for a patient before the date of inclusion registered with ndr , this date has been used as a proxy for the date of diagnosis . the right column of table 1 shows how groups of codes of diagnoses and interventions have been used in this process . the complete list of all the codes of diagnoses and interventions which qualify for the diagnosis of diabetes and related complications may be accessed from http://www.appliedeconomics.dk/index.php/publications/reports-for-download . we have attempted to assess when a patient has developed chronic diabetes complications based on coded information from the dnpar . in the absence of published guidelines , we have relied on consultations with clinical experts combined with decisions made on the basis of the actual distribution of relevant codes . this classification scheme comprises three complication groups on a tentative basis : without evidence of complications from available data ( complication group 0 , cg0 ) ; with evidence of at least one complication of intermediate impact ( complication group 1 , cg1 ) ; and with evidence of at least one complication of major impact ( complication group 2 , cg2 ) . we have assumed irreversibility so that once qualified for cg1 , the patient can not revert to cg0 and can only remain in cg1 until death or progression to cg2 . for patients with transition directly from cg0 to cg2 , it has been assumed , for computational reasons , that the patient first progresses to cg1 , but instantaneously progresses further to cg2 without producing person - time in cg1 . the date of progression to a given complication group has been taken as the first occurrence of a code of diagnosis or procedure registered in dnpar qualifying for a cg . table 1 ( middle column ) provides a summary of this information and the complete list of all the codes of diagnoses and interventions , which qualify for a complication group and may be accessed from http://www.appliedeconomics.dk/index.php/publications/reports-for-download . codes not qualifying for any complication are assigned to cg0 by default . some codes of diagnoses and procedures are specific for diabetes ( eg , diabetic retinopathy ) , while others are unspecific ( eg , myocardial infarction and amputations ) . for patients having developed such complication(s ) before the operational date of the diagnosis of diabetes , the patients will be assigned to the concerned cg at the date of diagnosis of diabetes and the patient can only contribute with person - time at risk for progression to a more advanced cg . the epidemiological analyses of transition rates and prevalence proportions have been performed within the framework of an illness - stage - death model outline in figure 1 . the number of persons present in each of the stages , represented in the flow chart in figure 1 , on the last date of a given calendar year represents the prevalence of patients with the complication status for that year . these numbers provide for the annual complication - specific prevalence proportions by division with the total population size . annual transition rates ( incidence rate of the disease ; morbidity rates reflecting rates of progression from cg0 to cg1 and from cg1 to cg2 , respectively ; and mortality rates from each of the complication groups ) have been estimated from the numbers of transitions divided with the appropriate numbers of person - years at risk during the calendar year concerned . for each calendar year , we calculated individual person - time , in terms of 1 ) the exact number of days experienced in any combination of 5-year age - groups ; and 2 ) complication status based on the transition dates between age - groups and complication groups . age groups were further collapsed to the groups : 024 , 2549 , 5074 , and 75 + years . the analyses of morbidity and mortality were done on the basis of age standardization within each sex using the age composition of the population of persons with diabetes 2006 ultimo . for the estimation of the incidence rate of diabetes , population time at risk of developing diabetes for each calendar year was obtained from statistics denmark,10 with subtraction of the person - time experienced with diabetes during that year in order to estimate true person - time at risk in the denominator . the analysis of incidence rate of diabetes was done on the basis of age standardization within each sex , using the age composition of the danish population 2006 ultimo in four groups : 024 , 2549 , 5074 , and 75 + years.10 -based analyses were employed to test for heterogeneity over calendar time . an approximated poisson distribution and the binomial distribution for rates and proportions were used to calculate 95% confidence intervals . because we consider the first operational years of the ndr ( 19971999 ) as a running - in phase , the present analysis focuses on the period from 2000 to 2011 . the diabetes impact study 2013 is an observational register - based investigation with no contact to registered persons . permission to extract and analyze data was obtained from the danish data protection agency ( jnr 2013 - 21 - 1749 ) and the danish national board of health ( fseid-00000440 ) . as of july 3 , 2013 , a total of 497,232 persons were registered in ndr . after exclusion of 688 persons with invalid or inconsistent data , 496,514 persons ( 99.9% of all registrants ) were included in the present analysis.8 table 2 contains a summary of all events observed and inferred from the registry data by complication group for the total population , with stratification for men and women . the trend in standardized incidence rate is shown in figure 2 , demonstrating an increasing incidence , with fluctuations . there is strong evidence of heterogeneity between calendar years ( p<0.001 ) . in absolute numbers , the incidence increased from approximately 18,000 new cases in year 2000 to almost 32,000 new cases in year 2011 , corresponding to an average annual increase at approximately 5% . the men : women standardized rate ratio over the period was 1.25 ( p<0.001 ) . approximately 75% of the persons with diabetes , slightly higher for women than for men , were without complications at diagnosis , and this proportion remained largely constant through the period of analysis ( table 2 ) . an increasing number of persons progressed to cg1 and cg2 , respectively ( table 2 ) , over the period . however , when taking into consideration the person - time at risk of developing these complications , the rate of developing complications decreased for both complication groups ( figure 3 ) , mostly pronounced for the category of severe complications ( cg2 ) . these trends were statistically significant for men as well as for women ( p<0.001 ) . the rate of progression to cg1 was higher for men than for women , with a men : women standardized rate ratio at 1.37 ( p<0.001 ) , whereas the rate of progression to cg2 was more similar , yet statistically significantly different , for men and women with men : women standardized rate ratio at 1.03 ( p<0.001 ) . the number of deaths ( regardless of cause of death ) increased slightly from approximately 8,300 in year 2000 to almost 12,000 in year 2011 ( table 1 ) . however , when taking into consideration the person - time at risk of dying in the various complication groups , the mortality rate decreased for both men and women and for all complication groups ( figure 4 ) . overall , the standardized mortality rate declined from 5.7 deaths per 100 person - years to 3.9 deaths per 100 person - years , corresponding with a decrease of 3% annually . the mortality rate was lowest for cg0 , with a decline in the rate from 2.9 to 2.0 deaths per 100 person - years ( men : women standardized rate ratio : 1.27 , p<0.001 ) . the mortality rate was intermediate for cg1 , with a decline in the mortality rate from 5.1 to 3.3 deaths per 100 person - years ( men : women standardized rate ratio : 1.09 , p<0.001 ) . the mortality rate was highest for cg2 , with a decline in the mortality rate from 11.4 to 7.4 deaths per 100 person - years ( men : women standardized rate ratio : 1.15 , p<0.001 ) . the number of prevalent patients increased from approximately 140,000 patients at the end of the year 1999 to more than 300,000 at the end of the year 2011 ( table 2 ) . the standardized prevalence proportion increased from 28 per 1,000 population 1999 ultimo to 52 per 1,000 population 2011 ultimo ( figure 5 ) . for each of the years covered by the study , men had a statistically significant ( p<0.001 ) higher prevalence proportion than women , with the men : women standardized proportion ratio ranging from 1.1 to 1.4 . the relative prevalence of patients in cg0 fell slightly from approximately 58% at the end of 1999 to approximately 55% at the end of 2011 ( figure 6 ) . this was caused by an increase in the proportion of patients in cg2 from less than 23% at the end of 1999 to more than 25% at the end of 2011 , whereas the proportion of patients in cg1 was almost constant throughout the period of analysis . as compared with women , there were relatively fewer men in cg0 and relatively more men in both cg1 and cg2 ( data not shown ) . we have used already existing national health register data to establish a comprehensive characterization of the central epidemiological features of diabetes in denmark , with all advantages and limitations attached to this approach . data from the nationwide danish health registers are generally considered to have a very high level of completeness in terms of coverage of all the relevant persons and activities.4 furthermore , using already existing data collected for purposes independent of the study concerned reduces both bias and need to collect additional data . nevertheless , a series of limitations are involved when using ndr and the other danish health registers for research purposes.8 thus , ndr may include up to 20% registrants who do not have diabetes . a sensitivity analysis with exclusion of these persons yields a reduction of approximately 30% for all key figures presented in table 1 , but with similar trends in the proportions and rates as found for the total material . another concern is that for more than 10% of the registrants , the operationally established date of diagnosis of diabetes is systematically biased toward too late a date.8 we have addressed this issue by using the full information available in dnpar to establish the earliest possible date of registration with a diabetes - specific event . relevant data on diabetes and comorbidity captured in the primary health care sector are not available in ndr and dnpar , including values of glycosylated hemoglobin as well as measurements of microalbuminuria and results of screening for diabetes eye complications . the data extracted from the dnpar may suffer from misclassifications and errors in coding practice , and in addition , cause omissions of otherwise relevant registrations ; such errors may lead to misclassification of the date of onset of complications . compared with previous epidemiological analyses based on ndr,11 our approach is novel as we attempted to assess complication status and categorize it by level of impact . even though this may be questioned on clinical grounds , it seems to work at the group level as evidenced by the fact that the mortality level is higher for the group of patients with complications of moderate impact as compared with patients without complications , but is even higher in the group of patients with complications of severe impact . with these limitations in mind , we believe our results accurately reflect the current trends in diabetes epidemiology in denmark and extend previous epidemiological analyses based on ndr by confirming an increasing incidence level.11 because the diagnosis of diabetes is established by means of the first occurrence of certain events , fluctuations in incidence occur as a consequence of changes in administrative practice , like reimbursement for services specific for patients with diabetes , but since the majority of patients are ascertained by hospital diagnoses or prescriptions with antidiabetic drugs , such explanation seems less likely.8 alternatively , the increasing incidence level might be explained by increased awareness , leading to earlier diagnosis than in previous times . if so , it would be expected that the proportion of patients having signs of complications already at the time of diagnosis should decrease over time , but this is not the case , as evidenced in table 2 . currently , no data are available to assess the impact of changes in overweight and obesity on the incidence rate of diabetes , thus making further investigations highly needed . we find that the prevalence of diabetes is increasing exponentially as evidenced by the net influx to the population of patients with diabetes ( ie , the difference between the annual number of new cases and the annual number of deaths ) , which is steadily increasing . our study demonstrates that this trend is driven not only by an increasing incidence , but also by a decreasing trend in the risk of developing complications as well as a decreasing mortality level regardless of the presence of complications , but with most reduction among patients with severe complications . these trends have been observed also in subsets of patients attending the steno diabetes center in denmark and have been ascribed to intensified glycemic control in the patient population.12 in this perspective , the emerging so - called diabetes epidemic is partly a result of improved care . we believe that the trends seen in the danish population may be generalized to other nations with similar socioeconomic characteristics . our results have demonstrated that for incidence , complication , and mortality rates as well as prevalence proportions , men have higher levels than do females . concerning incidence rates and prevalence proportions , this pattern was also observed from the first epidemiological analyses on the basis of ndr data,11 but so far these observations remain unexplained . together with our previously published study on the validation of the ndr,8 the results of the present study as part of the diabetes impact study 2013 , we will perform in - depth analysis of the relative importance of those factors that drive the prevalence and use such models to establish scenarios for the future evolution of the prevalence of diabetes in denmark , in line with similar approaches in other communities.13 new insight into the future societal burden of diabetes will be obtained by combining epidemiological data with information on patients demographic , ethnic , and socioeconomic characteristics as well as estimates of costs attributable to diabetes within categories characterized by age , sex , and complication status . the prevalence of patients with diabetes registered in denmark is rising exponentially due to the synergistic effect of increasing incidence combined with decreasing rate of developing complications as well as decreasing mortality in the patient population . further studies will seek to quantify the relative importance of these factors , and this will enable for establishment of scenarios for estimating the future burden of diabetes .

purposeas part of the danish diabetes impact study 2013 , we present trends in the incidence , morbidity , mortality , and prevalence of diabetes in denmark for the period 2000 through 2011.patients and methodsthe danish national diabetes register was established in 2006 and is assumed to cover all patients with diabetes , alive as of the end of 1996 , and all subsequent new cases . the present study is based on the content of the register as of july 3 , 2013 ( n=497,232 patients ) . using the personal identification code assigned to all danish inhabitants , all available supplementary information from the danish national patient register and the danish civil registration service was used to define the date of diagnosis of diabetes and the first date of experiencing complications ( grouped according to impact and severity).resultsduring the period of 2000 to 2011 , the incidence rate of diabetes increased approximately 5% annually . during the same period , decreasing trends were observed for both the rates of progression in complications and of the complication - specific mortality . during the same period , the prevalence of diabetes doubled.conclusionthe increasing prevalence of diabetes in denmark is driven by increasing incidence combined with decreasing morbidity and mortality in the population of patients with diabetes . these mechanisms will be explored further as part of the diabetes impact study 2013 , together with investigations into the socioeconomic and health economic aspects of diabetes .

Introduction Patients and methods Data sources Establishing milestones in the lifetime cause of diabetes Establishing the diagnosis of diabetes Occurrence and classification of complications relevant for diabetes Framework of epidemiological analysis Ethical aspects Results Incidence Complications Mortality Prevalence Discussion Conclusion

PMC4819234

deep brain stimulation ( dbs ) has demonstrated significant therapeutic benefits in ( i ) treating symptoms relating to neurological motor disorders including parkinson 's disease , essential tremor , and dystonia , ( ii ) controlling epilepsy , and ( iii ) providing relief from chronic pain . dbs has been also employed for treatment refractory neuropsychiatric indications including tourette 's syndrome , obsessive compulsive disorder , and for treatment resistant depression . more recently , dbs has been has been proposed as a potential treatment of severe drug and alcohol addiction . considerable research involving laboratory animals has been conducted in recent years to study the underlying principles of dbs and the mechanisms through which its therapeutic effects are mediated . however , a bottleneck in establishing the therapeutic mechanisms and benefits of dbs has been the lack of portable dbs systems that enable long term brain stimulation in freely moving laboratory animals . as a consequence of this , there remains a gap in the existing experimental data investigating mechanisms of chronic dbs action in preclinical animal studies . most of the existing dbs devices employ complex circuitry and are , as a consequence , quite bulky . these devices are connected to the implanted electrode through long insulated wires that run from the device to the animal 's head . the stimulator often needs to be disconnected from the electrode over the course of the study ( i.e. , overnight as the animal is returned to its home cage ) . thus , in order to better reflect the longer duration stimulation applied to the brain in clinical trials , the laboratory animal would ideally receive this brain stimulation continuously without interruption for days or even weeks . in order to improve the quality of preclinical work , the animal needs to be able to freely move , eat , sleep , swim in water , and carry out several other activities . when connected to the dbs device through long wires , behavioral tests and normal behaviors the progress of understanding the therapeutic mechanisms and benefits of dbs will benefit from the reduction of the size of power requirement of dbs devices . this paper presents a novel design for a low power micro deep brain stimulation device for laboratory animals . three different configurations of the device are presented : single piece head mountable , single piece back mountable , and two piece back mountable . an investigation of the literature reveals that thus far only a few dbs devices have been reported that are suitable for long term brain stimulation of laboratory animals . millard and shepherd developed an implantable , single channel stimulator that generates charge balanced biphasic current pulses delivered to a bipolar electrode array for chronic stimulation of neural tissue ( see fig . the subject is placed in a small chamber surrounded by three orthogonal coils of wire which are driven to generate a magnetic field . currents are induced in wire coils in the implanted stimulator then regulated to produce biphasic current pulses with fixed amplitude of up to 500 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm a}$\end{document}. phase duration is adjustable from 25 to 250 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm s}$\end{document } per phase . stimulus rate can be continuously varied , and the temporal precision of the stimulus means that the stimulator can be used in behavioral experiments or for generating electrically evoked potentials . the application of the stimulator on chronic electrical stimulation of the auditory nerve was described . developed a lightweight implantable micro stimulator device for mice that delivers biphasic pulses to two individual electrode pairs ( see fig . it consists of an implantable stimulator , two stimulation electrodes , an infrared camera system including a monitor and a programming and test fixture to program the stimulus current . the stimulator housing contained a polycarbonate cylinder with a screw top , and was 30 mm in length with a diameter of 8 mm . the total length of the lead system from the stimulator to the connector pins was 45 mm . the microcontroller then had the highest power consumption of all the components of the circuit . to maximize battery life , the microcontroller was put in standby mode in between two consecutive biphasic pulse patterns . the bench top validation and in - vivo implementation of the device was presented . the results indicate that the wireless implantable stimulator in mice delivered continuous bilateral stimulation without restricting the animal mobility and hygiene . in - vivo testing showed that stimulation of the mice ventral striatum yields similar results as previously shown by others in rats where conventional deep brain stimulation techniques were used . 1.micro dbs devices developed for laboratory animals by ( a ) millard and shepherd , ( b ) haas et al . , ( c ) liu et al . , ( d ) kouzani et al . , and ( e ) forni et al . . micro dbs devices developed for laboratory animals by ( a ) millard and shepherd , ( b ) haas et al . , ( c ) liu et al . , ( d ) kouzani et al . , and ( e ) forni et al . . liu et al . reported a head mountable apparatus and implant method suitable for chronic dbs in rats ( see fig . they evaluated the effect of chronic dbs of the rat nucleus accumbens on morphine reinforcement using their dbs apparatus . an electrode was stereotaxically implanted into the core of unilateral nucleus accumbens and connected to the dbs apparatus fixed to the rat skull . a 900-second conditioned place preference paradigm was used for determining the effect of electrical stimulation on morphine reinforcement . kouzani et al . developed a head mountable dbs device for laboratory animals ( see fig . it produces continuous monophonic current pulses of 90 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm s}$\end{document } width , with frequency of 130 hz , and amplitude of 200 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm a}$\end{document}. to produce timing pulses , a general purpose timer chip was used in its astable mode . to deliver electric current pulses , the device was able to produce non - stop stimulating current pulses for about nine days using a 280 mah coin battery . they did not carry out experiments with laboratory animals , but used a 1 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm k}\omega$\end{document } resistor to model the animal 's brain tissue . ewing and grace reported a dbs device for the continuous delivery of brain stimulation in rats . their device was configured to stimulate with square , monophasic , anodic , constant current 100 ms pulses at frequency of 130 hz and amplitude of 100 ma . the device was carried by the animals via rodent jackets and connected to the electrodes via an external cable . they used the device to examine how five days of stimulation affected rhythmic brain activity in freely moving rats . synchronized video and local field potential data was collected from animals in their home cages before , during and after stimulation . they reported that the initial changes in power observed with short term stimulation were replaced by altered coherence , which may reflect the functional action of dbs . this easy removal property enables removing or switching the microstimulator during the experiments without having to anaesthetize or operate on the animal , thereby minimizing stress , and maximising the quality of the behavioural and biological data . the dbs system consists of three components : a stimulating electrode , a stimulator support and an electrical portable microstimulator . the microstimulator is made up of two main blocks : one adjustable pulse generator and one voltage to current converter . the pulse generator block is based on a series of four cmos invertors ' chains with a feedback loop allowing to regulate the frequency and a buffer stage on the two last ones , both being coupled through a variable network allowing to vary the duty cycle . the voltage to current converter is based on a classical bipolar transistor wilson current mirror directly supplied by the buffer output to minimize consumption during off phases of the pulse . its weight is around 5.6 g with the resin protective coat , and 7.4 g with the two watch batteries . the initial setting of the current parameters is done before mounting the microstimulator on the head of the rat using an oscilloscope and a 15 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm k}\omega$\end{document } output reference resistance . to validate the device , they performed continuous dbs of the subthalamic nucleus in a classical rat model of pd . they showed that the long duration stimulation reduces significantly pd - induced akinesia without inducing animal discomfort and tissue damage . the millard and shepherd 's device requires a magnetic field that is generated by applying a high voltage to the excitation coil which may have undesirable impact on biological cells . 's device requires new data to be uploaded to the microcontroller and some fixed resistors be changed for changing the characteristics of the stimulation current . the size of the device is large , and the device can only stimulate for several hours . considering the characteristics of the current devices , there is still a need to reduce their power consumption , size , weight , cost , and simplify their maintenance and mounting procedures . a low power , lightweight portable micro dbs device that can be carried by the animal during the course of a clinical trial is presented in this section . based on our preclinical research requirements , the duration of the cathodic pulse is set to 90 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm s}$\end{document } , the frequency of stimulation is set to 130 hz , and the amplitude of current pulses is set to 200 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm a}$\end{document}. however , the stimulation parameters of the device are adjustable as described in the programming subsection . the device employs the passive charge balancing method to minimize undesirable effects including tissue damage . the passive charge balancing method is based on an ac - coupling capacitor reducing the demanding electronic circuit . the device consists of the following components : microcontroller , current source , stimulation electrode , power source , and printed circuit board . three different configurations of the micro dbs device are constructed : single piece head mountable , single piece back mountable , and two piece back mountable . attiny 24a is a low power 8-bit microcontroller based on the avr enhanced reduced instruction set computing architecture . it provides 2 kb of flash , 128 bytes of eeprom and sram , 12 i / o lines , 32 registers , an 8-bit timer / counter , a 16-bit timer / counter , internal and external interrupts , 8-channel 10-bit adc , a programmable watchdog timer , and internal calibrated oscillator . it offers four selectable power saving modes : ( i ) in idle mode , the cpu is stopped while allowing the sram , timer / counter , adc , analog comparator , and interrupt system to continue functioning , ( ii ) in adc noise reduction mode , switching noise during adc conversions is minimized by stopping the cpu and all i / o modules except the adc , ( iii ) in power down mode , registers keep their contents and all chip functions are disabled until the next interrupt or hardware reset , ( iv ) in standby mode , the crystal / resonator oscillator is running while the rest of the device is sleeping , allowing very fast start up combined with low power consumption . the pseudo code for the program that implements the deep brain stimulation operation is given in fig . pseudo code for the program that implements the deep brain stimulation operation . in the first step , this is done by initializing the two data direction registers of the microcontrollers , ddra and ddrb . the other i / o pins are configured as input pins . in the second step , this is achieved by initializing the power reduction register , prr . in the third step , the led is flashed to show the user that the device has started and is operating correctly , and that the battery is in good condition . in the fourth step , the 16-bit timer / counter 1 unit is programmed to generate the stimulation waveform . the unit facilitates precise event management , wave generation , as well as signal timing measurement . it has a number of control registers that can be programmed to determine the desired operation of the unit . in this work the timer can be programmed to operate in 15 different waveform generation modes . in this work , the timer is operated in fast pulse width modulation ( pwm ) mode 14 . it counts from a bottom value to a top value then restarts from the bottom value . for generating a stimulation waveform of width 90 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm s}$\end{document } , and frequency 130 hz , the icr1 and ocr1a registers are set to 7692 and 90 , respectively . however , by varying the content of the timer control registers , the characteristics of the generated waveform can be changed , resulting in the adjustment of the stimulation parameters . finally , in the fifth step , for power efficiency and to maximize the battery lifetime , the microcontroller is made to enter the idle mode . in this mode , accordingly , the cpu and the execution of the program is halted and no further instruction is run , but timer 1 continues operating to provide the desired waveform on its output pin without the intervention of the cpu . the produced waveform is used to switch on / off the transistor pdtc143te that controls the operation of a current source . to deliver electric current pulses , lm334 is an adjustable current source with good current regulation from 1 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm a}$\end{document } to 10 ma under a voltage range of 1 v to 40 v. the device has three terminals and offers true floating current feature . the current amplitude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm i}_{\rm set}$\end{document } is determined by an external resistor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm r}_{\rm set}$\end{document } as follows : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm i}_{\rm set}=(1.059\times{\rm v}_{\rm r})/r_{\rm set}$\end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm v}_{\rm r}$\end{document } is the voltage across \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm r}_{\rm set}$\end{document}. the average \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm v}_{\rm r}$\end{document } for the desired operating temperature range is about 62 to 66 mv . for the current amplitude of 200 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm a}$\end{document } , we therefore used \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm r}_{\rm set}=330 \omega$\end{document}. to achieve charge injection , the stimulation electrode is implanted into the target tissue . we used an electrode by plasticsone that has a stainless steel twisted wire electrode extend below a threaded plastic pedestal . to supply energy to the micro dbs device for the duration of a clinical trial , a battery is used . for the single piece head mountable configuration , two cell button silver oxide 1.55 v batteries the diameter and the height of each battery are 11.6 mm and 5.4 mm , respectively . the weight of the battery is 2.3 g. the capacity of each battery is 190 mah which is well adequate for operating the device for about ten days . the two batteries are stacked together using conductive glue to form a 3.1 v source . for the single piece back mountable and the two piece back mountable configurations , one cell button lithium manganese dioxide 3 v battery is used to power the device . the diameter and the height of the battery are 20 mm and 3.2 mm , respectively . the total weight of the battery is 2.8 g. the capacity of the battery is 235 mah which is also well adequate for operating the device for over eleven days . to accommodate the components of the micro dbs device , and connect them according to the circuit schematic shown in fig . 2 , two printed circuit boards are developed , one for the single piece head mountable as well as the two piece back mountable configurations , and another for the single piece back mountable configuration . after fabrication of the printed circuit board , the electronic components of the micro dbs device are assembled onto the boards . ( a ) the board for the single piece head mountable as well as the two piece back mountable configurations . ( b ) the board for the single piece back mountable configuration . fabricated boards . ( a ) the board for the single piece head mountable as well as the two piece back mountable configurations . this configuration incorporates both the stimulation board and the battery pack as a single piece device that can be attached directly into the stimulation electrode implanted into the target tissue . the board includes a two pin terminal for connection to the positive and negative sides of the battery pack , and another two pin terminal for connection to the electrode . a thin brass sheet is cut into small pieces and attached to the positive and negative sides of the battery pack using conductive glue . then , the power terminals are soldered onto the brass pieces on the positive and negative sides of the battery pack . a sample of the single piece head mountable micro dbs device is shown in fig . 5 . the weight of different components of different components of the single piece head mountable micro dbs device is measured using a laboratory scale . the weight of the stimulation board is 0.71 g , the weight the battery is 2.56 g , and the weight of the entire device is 3.27 g. during the implant procedure , the threaded plastic pedestal of the electrode is fixed into the skull . next , the stimulation device including the battery is mounted into the electrode via the two pin electrode terminal on the device . then , the device is sealed with dental cement on top of the animal 's head . this configuration incorporates the stimulation board , a 20 mm cell button battery holder , and the cell button lithium manganese dioxide 3 v battery in a single piece device . the board includes a two pin 2.54 mm terminal for connection to the electrode . a two way 2.54 mm receptacle is used to plug a pair of thin wires into the electrode terminal of the stimulation device . the other end of the wires is connected to a two pin plug that is mounted into the electrode fixed into the skull . the cell button lithium manganese dioxide 3 v battery is placed inside the battery holder . the jacket is closed by using a velcro fastener , and a hook gives a secure fit . the advantage of this configuration is the fact that the battery can be easily replaced extending the operation time of the device unlimitedly . 6 shows samples of the single piece back mountable micro dbs device . fig . the weight of different components of different components of the single piece back mountable micro dbs device is measured using a laboratory scale . the weight of the stimulation board is 1.25 g , the weight of the battery is 2.83 g , the total weight the battery and the battery holder is 3.83 g , and the overall weight of the entire device is 5.08 g. this configuration is similar to the single piece back mountable formation . the difference is that in this configuration the stimulation board and the battery - holder / battery are separate . the stimulation board is connected to the battery holder using a pair of thin wires instead of being secured underneath the battery holder . the stimulation device can be sealed using hot glue and then placed under the animal skin . alternatively , it can be placed inside the rat jacket near the battery holder . a sample of the two piece back mountable device is shown in fig . the overall weight of the device is similar to that of the single piece back mountable device which is around 5.1 g. fig . after construction of the micro dbs devices , their performance was evaluated in terms of the lifetime of the battery and also the operation of the device . three tests were conducted : bench test , in - vitro test , and in - vivo test . the performance of the micro dbs was tested using a 1 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm k}\omega$\end{document } resistor that modeled the animal 's brain tissue . the bench testing of the micro dbs device with a 1 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm k}\omega$\end{document } resistor . the steady state current consumption of the device after 10 , 20 , and 30 hours of continuous operation was measured using a keithley 2401 sourcemeter . the sourcemeter has precision current measurement capability within the range 10 pa-1 a. the average steady state current consumption of the device was found to be around 870 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm a}$\end{document}. next , to practically determine the number of days the device can be operated by using the 235 mah cell button battery , the sourcemeter was programmed to read the battery voltage and store it every five minutes . the variation of the battery voltage for the continuous operation of the micro dbs device using a 235 mah cell button battery is shown in fig . as can be seen from the figure , the battery voltage remained steady over the course of the first eleven days . theoretically , the operating voltage of the attiny 24a microcontroller is within the range 1.85.5 v. however , the system continued producing stimulating current pulses below 1.8 v in day 12 . once the battery voltage fell below 1.5 v , the microcontroller stopped working , and thus the device stopped producing the stimulation pulses . the red arrow in fig . 8 shows when the device ceased operation due to the low battery voltage after over twelve days of continuous operation . 8.variation of the battery voltage under continuous operation of the micro dbs device using a 235 mah cell button battery . variation of the battery voltage under continuous operation of the micro dbs device using a 235 mah cell button battery . a comparison of the head - mountable configuration against similar current devices is presented in table i. in comparison of the lifetime of each device , the strength of the stimulation current as well as the use of an external source should be taken into account . the head - mountable configuration offers a much smaller size , and yet longer term higher strength stimulation current pulses . table icomparison of micro dbs devicesref.sizesourceweighttermparameters14mm \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \times$\end{document } 12mmmagnetic coupling2.5gun- limited10050o \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a$\end{document } 2525o \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu { \rm s}$\end{document } 505000hz8 mm \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \times$\end{document } 30mmbattery ( 4.65v)2.1g10 hours201 oc \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu { \rm a}$\end{document } 6i \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 0\mu { \rm s}$\end{document } 131hz20 mm diameterbattery ( 3v/ 200mah)--0.20.5ma 0.031.4ms 1200hz25 mm diameterbattery ( 3v/ 280mah)7.8g9 days200 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu { \rm a}$\end{document } 9o \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm s}$\end{document } 130hz15 mm \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \times$\end{document } 28mmbattery ( 6v1 36mah)7.4g7 days50121 - \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 0\mu { \rm a}$\end{document } 08o \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu { \rm s}$\end{document } 0130hzproposed12 mm diameterbattery ( 3.1v/ 190mah)3.2g10 days200 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu { \rm a}$\end{document } 9o \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu { \rm s}$\end{document } 130hz comparison of micro dbs devices the micro dbs device was tested in - vitro at mayo clinic by dr abulseoud 's team . the test was conducted by placing the stainless steel twisted wire part of the stimulation into a tank of physiologic saline solution . the cathodic pulses of 90 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm s}$\end{document } duration , 130 hz frequency , and 200 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm a}$\end{document } amplitude were delivered to the electrode by the device which was powered by the 235 mah cell button battery . an oscilloscope was used to confirm the continuous delivery of current to the stimulating electrode . after the successful bench test and in - vitro test , a number of two piece back mountable micro dbs devices ( see fig . 7 ) were tested in - vivo at mayo clinic by dr abulseoud 's group . the devices were used in a study to examine the role of dbs in reducing ethanol preference in alcohol preferring rats . eleven adult male wistar rats ( charles rivers ) 1216 weeks at time of experiment were used . a pair of thin wires connected the stimulation board to the electrode . a second pair of wires connected the stimulation board to the battery holder . each device created pulses of 90 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm s}$\end{document } duration , 130 hz frequency , and 200 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu{\rm a}$\end{document } amplitude whilst being powered by a 235 mah cell button battery . the stimulation board , the battery holder and the battery were all placed in a rat jacket ( harvard apparatus ) and secured on the back of the animal . the pair of wires connecting the stimulation board to the stimulating electrode was tunneled under the skin at the back of the neck and externalized for a short distance above the stimulating electrode where it was connected ( see fig . the animals did not appear to be bothered by the device , the jacket , or the wires . animals were implanted with bipolar twisted electrode ( plasticsone ) into the right nucleus accumbens shell under isoflurane inhalation anesthesia ( 3.0% during induction and 1.5% during maintenance ) ( see fig . coordinates from bregma ( flat skull ) are : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm ap}=2.04$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm m}\hbox{-}{\rm l}=1.2$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm d}\hbox{-}{\rm v}=8.0$\end{document } ( from skull ) tilt : 10 degrees . animals were allowed to recover for three days post - surgery then were divided into two groups : continuous stimulation for 7 days using the head mounted device in a jacket \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( { \rm n}=5)$\end{document } and sham group did not receive stimulation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( { \rm n}=6)$\end{document}. data on body weight and water consumption as well as locomotor activity counts was collected during the week before stimulation and during the week of stimulation ( for the continuous group ) . the weight and water consumption of animals are shown in fig . 10 . as can be seen from the figure , the sham group had less weight at start of experiment , but no significant effect for stimulation on weight as well as total water consumption 10.body weight and water consumption of the animals before stimulation week and during stimulation week . body weight and water consumption of the animals before stimulation week and during stimulation week . animals \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( { \rm n}=5)$\end{document } were housed under 12:12 hour dark : light cycle ( lights on at 6.00 am ) circadian locomotor activity counts in home cage were recorded using an infrared motion detector interfaced with a computerized data acquisition system ( clocklab ) and later analyzed using matlab software . no significant difference in total locomotor activity counts pre , during , or after stimulation was observed ( one way repeated measure anova \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \rm p}=0.2$\end{document } ) ( see fig . the stimulating device was well tolerated and the stimulation at the nac did not alter weight , water consumption or locomotor activity , however it resulted in changes in alcohol consumption and preference in alcohol preferring rats ( data not shown here , and will be published by dr . the paper presented the development and testing of a low power lightweight micro deep brain stimulation device for laboratory animals . it can be easily carried by the animal during the course of a clinical trial . after construction , the performance of the device was evaluated through three tests : bench test , in - vitro test , and in - vivo test . during the tests , the device was able to produce non - stop stimulating current pulses for over twelve days using a 235 mah cell button battery . the evaluation of the device in a study to examine the role of dbs in reducing ethanol preference in alcohol preferring rats was successful . whilst it did not alter weight , water consumption or locomotor activity of the animals , it resulted in changes in alcohol consumption and preference in alcohol preferring rats . in order to minimize the power consumption , size , and weight of the device , however , such feature can be implemented by incorporating a recording electrode and a signal conditioning interface circuit . the microcontroller used in the device has 10-bit analog - to - digital converter channels that can be used to convert the conditioned output of the recording electrode to a high resolution digital signal . in addition , similar to the current stimulation devices involving any stimulation electrode , wire , and/or electronic component , performing mri / fmri with this device can also cause tissue damage from component heating particularly around the electrode . the advantages offered by the device include : ( i ) ultra - low cost when mass produced . it is expected that the entire device will cost less than$5 ; ( ii ) ultra - low maintenance and ease of use ; ( iii ) quick and easy battery replacement enabling ultra - long term stimulation .

deep brain stimulation has emerged as an effective medical procedure that has therapeutic efficacy in a number of neuropsychiatric disorders . preclinical research involving laboratory animals is being conducted to study the principles , mechanisms , and therapeutic effects of deep brain stimulation . a bottleneck is , however , the lack of deep brain stimulation devices that enable long term brain stimulation in freely moving laboratory animals . most of the existing devices employ complex circuitry , and are thus bulky . these devices are usually connected to the electrode that is implanted into the animal brain using long fixed wires . in long term behavioral trials , however , laboratory animals often need to continuously receive brain stimulation for days without interruption , which is difficult with existing technology . this paper presents a low power and lightweight portable microdeep brain stimulation device for laboratory animals . three different configurations of the device are presented as follows : 1 ) single piece head mountable ; 2 ) single piece back mountable ; and 3 ) two piece back mountable . the device can be easily carried by the animal during the course of a clinical trial , and that it can produce non - stop stimulation current pulses of desired characteristics for over 12 days on a single battery . it employs passive charge balancing to minimize undesirable effects on the target tissue . the results of bench , in - vitro , and in - vivo tests to evaluate the performance of the device are presented .

Introduction Existing Micro Brain Stimulation Devices for Laboratory Animals Proposed Micro Brain Stimulation Device Evaluation Conclusion

PMC4579733

partial anomalous pulmonary venous drainage ( papvd ) is a developmental anomaly of the cardiovascular system , the cause of which can be sought both in the abnormal gene structure , as well as in environmental factors . the essence of the defect seems to be the persistence of the early - stage embryo connections between the systemic venous circulation ( major celiac venous plexus ) and pulmonary circulation in the absence of a proper connection between the pulmonary veins and the left atrium of the heart , probably as a result of abnormal growth process of mesenchymal tissue located behind the heart ( dorsal mesocardial protrusion , dmp ) , in the area of so - called second heart field ( rear field ) toward the venous / alluvial pole of the heart . the lack of blood inflow from one or more pulmonary veins into the short , single common pulmonary vein which leads blood to the left atrium is visible at approximately the 8 week of the embr yo 's age ; this results in excessive angiogenesis of the celiac plexus and the emergence of their numerous connections with the cardinal venous system and lung parenchyma . as a result , during the diagnosis of papvd a correct connection of two or three pulmonary veins with the left atrium is found , while the other pulmonary veins lack internal lumen ( no lumenization ) or undergo atresia . the part of pulmonary parenchyma affected by the vascular anomaly drains venous blood to the venous system drainage area via the system of main and vertical veins or through the coronary sinus . the aim of this paper is to trace the applied strategy and diagnostic test technique on the example of a patient with turner syndrome , concomitant aortic valve anomaly and a very rare form of mixed partial abnormal pulmonary venous connection . a 10-year - old girl with turner syndrome , remaining under observation since infancy due to moderate insufficiency and mild stenosis of bicuspid aortic valve , was admitted to the clinic of gastroenterology owing to recurrent gastrointestinal bleeding , and then referred to a follow - up echocardiography examination to the echocardiographic laboratory at the department of children 's cardiac surgery of the medical university of warsaw before a scheduled colonoscopy under general anesthesia . transthoracic echocardiography confirmed the aortic pathology described above and it was stated at the same time that the size of the cavities of the left atrium and the left ventricle as well as the thickness and contractility of the left ventricular free wall were within the normal range appropriate for the child 's age . however , what attracted the examiner 's ( wm ) attention was paradoxical motion of the interventricular septum and enlargement of the right ventricle . it 's diameter measured in lv long axis view exceeded upper normal limits ; the dimensions of the right atrium and the pulmonary trunk were also borderline , what prompted the examiner to attempt to identify the cause of this condition ( fig . 1 ) . presentation m reveals the enlargement of the right ventricle ( rv ) and an abnormal motion of the ventricular septum ( ) a phenomenon that indicated the possibility of a low - pressure systemic - pulmonary shunt despite the absence of the atrial septal defect . additional marking : lv left ventricle right ventricular enlargement accompanied by paradoxical motion of the inter ventricular septum is characteristic feature of the right ventricular volume and/or pressure overload but not the left ventricular one in the course of aortic defect . today , in spite of the widespread echocardiography , the detection of right heart enlargement caused by atrial septal defects and/or partially abnor mal pulmonary venous drainage still may be delayed . less common reasons include : hemodynamically significant tricuspid and/or pulmonary valve insufficiency ( e.g. , in milder forms of ebstein 's anomaly ) , pulmonary hypertension , etc . structural abnormalities of the right heart valves and the presence of atrial septal defects were excluded in the described case , including those located marginally ( e.g. of sinus venosus type ) . also no echocardiographic features of pulmonary hypertension were found ( low tricuspid and pulmonary valve regurgitant flow velocities ) . the image analysis of a part of the atrial septum located in the vicinity of the mouth of the superior vena cava ( normal , without a sinus venosus defect ) revealed a widening of the paracardiac section of the vein and clearly turbulent flow of blood into the right atrium . no entry of the right upper pulmonary vein into the left atrium was visualized , but lower right and both left pulmonary veins joining the left atrium were shown ( fig . the diameters of the left pulmonary veins were smaller than that of the right lower pulmonary vein . precise visualization of pulmonary and systemic veins was hampered by adverse anatomical conditions in a child with turner syndrome ( short neck restricting suprasternal access , inspiratory position of the thorax , abdominal discomfort ) who reluctantly engaged in the examination . visible left atrium ( la ) and three tributaries of the pulmonary veins : lower right vein ( 3 ) and two left veins ( 1 , 2 ) . the bloodstream flowing into the left atrium from the left upper pulmonary vein ( 1 ) is clearly narrower than the others . superior vena cava satisfactory visualization of the superior vena cava was achieved by using right parasternal projections with the patient positioned laterally on the right side , especially during maximal exhalation . about 3 cm above the right atrium a rather wide , horizontally extending vein was revealed in the transverse plane . it emerged from the lung tissue and ran to the superior vena cava which expanded visibly at that level . located in its close proximity was the opening of the azygos vein which ran in the sagittal plane ( figs . the examining physician 's attention was drawn to the image of the left brachiocephalic vein , also enlarged and showing increased flow . during the search for the cause with the use of high left parasternal projections with the patient positioned laterally on the left side , a vertical vein of ca . 8 mm diameter and with a turbulent flow , running from the bottom to the left brachiocephalic vein was observed . the initial segment of the vessel not revealed due to being obstructed by pulmonary tissue ( figs . visible opening of a wide vein ( rupv ) running to the enlarged superior vena cava ( svc ) over the branch of the right pulmonary artery ( rpa ) . right brachiocephalic vein color doppler facilitates the visualization of an abnormal , horizontally oriented ( in the transverse plane ) superior vena cava ( svc ) tributary . markings : rupv right upper pulmonary vein , rpa right pulmonary artery , aoasc ascending aorta high right longitudinal parasternal projection . superior vena cava visualized in the long axis . at a distance of about 3 cm from the exit to the right atrium ( ra ) superior vena cava ( svc ) is significantly widened ( # ) , and the section in the immediate vicinity of the exit to the right atrium has a normal diameter . additional markings : la left atrium , rpa right pulmonary artery high left parasternal projection . blue color is used to mark the flow in the descending aorta ( aod ) , red color to mark the upward movement in the vein located leftward from the aorta which drains into the left venous angle ( v ) . additional markings : lb left brachiocephalic vein a slight deviation of the transducer to the left reveals a more peripheral section of the vertical vein ( v ) which runs from the depths of the lung tissue , above the left pulmonary artery ( lpa ) . additional marking : la left atrium the presumptive diagnosis of a rare variety of mixed partial abnormal pulmonary venous drainage was made : the right upper pulmonary vein into the superior vena cava , and the left upper pulmonary vein ( lupv ) through persistent vertical vein to the left brachiocephalic vein . however , what remained vague were anatomical details of left pulmonary veins , as in the first phase of the examination the opening of two veins to the left atrium was revealed , which suggested that the image of the left pulmonary veins was correct . the fact that the additional venous channel on the left side could be explained by an unusual drainage from the left lung ( a greater number of pulmonary veins from which the upper ones exit incorrectly ) , the presence of an abnormally running and larger than usual systemic vein ( for example , accessory azygos vein , top left intercostal vein ) or the existence of a connection between the venous and pulmonary venous systems ( levoatrial cardinal vein or venous vessel connecting lupv with the left brachiocephalic vein ) . it was essential to dispel the doubts in order to determine further course of procedure , and , above all , to schedule surgical correction . partial anomalous pulmonary venous drainage , comprising a relatively small part of the lung parenchyma ( usually one incorrectly connecting vein or a larger number of small veins ) does not cause significant hemodynamic consequences that require surgery . in case of anomalous drainage from more extensive areas , the shunt becomes hemodynamically significant and requires a corrective surgery . due to the abnormal function of the aortic valve it was impossible to carry out echocardiographic calculation of qp : qs flow ratio which is important in making indications for cardiac surgery . it should be emphasized that the implications of right ventricular volume overload may be partially masked by aortic regurgitation , resulting in an additional volume burden on the left ventricle . thus , the severity of kinetic disorders of the interventricular septum could not be a sufficient indicator of the size of the leak . minimally invasive computed tomography angiography ( angio - ct ) and magnetic resonance ( mr ) of the cardiovascular system are free from the constraints associated with the presence of aerated lung tissue and bone structures reflecting ultrasound . moreover , they allow precise three - dimensional reconstruction of the heart and vascular structures , and the calculation of the mutual relationship of pulmonary and systemic flows . in the described case , the angio - ct examination ( figs . 8 , 9 ) confirmed the abnormal connection of the right upper pulmonary vein to the superior vena cava . it turned out that the venous drainage from the left lung occurred through three veins : the bottom and middle ones running correctly into the left atrium while the upper , considerably smaller than the others , drained via a vertical vein into the left venous angle . it is worth noting that the review of the chest x - ray taken for the purpose of correct determination of the area to be examined , revealed both abnormal vascularity of the right lung , which suggested that the right upper pulmonary vein was connected to the superior vena cava . it also revealed a shade corresponding to the vertically extending vein that connected to the left brachiocephalic vein . the left upper pulmonary vein drains via the vertical vein ( 3 ) to the enlarged brachiocephalic vein ( 1 ) . highly situated right upper pulmonary vein ( 4 ) returns the blood to the wide superior vena cava ( 2 ) . rear view showing the enlarged superior vena cava ( 1 ) with the exit to the right upper pulmonary vein ( 2 ) . three pulmonary veins connect with the left atrium : one on the left side ( 3 ) and two right ones ( 4 , 5 ) . the upper left pulmonary vein drains via the vertical vein ( 8) to the brachiocephalic vein . additional markings : 7 ascending aorta , 6 right pulmonary artery a simplified diagram of the pulmonary veins anatomy ( author : mak ) . markings : la left atrium , ra right upper pulmonary vein , lapv left additional pulmonary vein , v vertical vein , vbc brachiocephalic vein , svc superior vena cava upper , lupv left upper pulmonary vein , lipv left inferior pulmonary vein it was estimated that the small size of the abnormally connecting left pulmonary vein classified the systemic - pulmonary shunt as hemodynamically insignificant and justified the adoption of the watchful waiting approach . important arguments for relinquishing the surgery included also the awareness of major technical difficulties that would occur in any attempt to correct the defect , as well as a possibility of the deterioration of the aortic valve function in the future . a potential necessity for surgical treatment of the valve would be associated with reopening the chest in the future if the patient was operated on at present . in the absence of significant symptoms that could be attributed to abnormal drainage of pulmonary veins , partial anomalous pulmonary venous drainage is among rare congenital anomalies found in 0.20.7% of autopsy studies . it affects the right lung ( 8588% ) rather than the left one ( 910% ) , and occasionally affects both lungs ( 22.5% ) . in the majority of cases the defect is accompanied by a sinus venosus defect of the septum ( 6065% ) or asd2/ pfo ( 20% ) . papvd is diagnosed in up to 13% of patients with turner syndrome most commonly it takes a mixed form without the atrial septal defect . the presented case is another good example of many difficulties which may arise both in diagnostics and the management of patients diagnosed with pulmonary venous anomalies . in a situation where a relatively small part of the pulmonary connection drains abnormally , the circulatory symptoms are not intensified and therefore are hard to detect in a clinical examination . typical auscultatory phenomena , such as overactive , deformed precordium , ejection murmur above the pulmonary artery , rigid split of the second heart sound above the pulmonary artery , are present only in case of the large shunt . in the examined patient the ejection murmur was diagnosed at the base of the heart and it was preceded by a protosystolic click and quiet diastolic murmur radiating towards the apex . the presence of these symptoms has been explained so far with the bicuspid aortic valve , diagnosed by echocardiography already in early childhood . abnormalities revealed in the chest x - ray and electrocardiogram , which are associated with the right ventricular volume overload and increased pulmonary blood flow , also depend on the severity of the shunt and thus are not perceptible in benign forms . no information on a conducted radiological examination of the chest or electrocardiogram was found during the analysis of medical records . transthoracic echocardiography is a primary diagnostic method of structural heart defects , but the diagnosis of pulmonary vein anomalies in a young , uncooperative , often crying and agitated child is very difficult . the main obstacle is the presence of exceedingly aerated lung tissue , usually making it impossible to trace the superior vena cava throughout its entire course and impeding the visibility of the entries of the pulmonary veins into the left atrium . moreover , in a child with turner syndrome and a short , webbed neck , there is limited suprasternal access which normally enables a good insight into the vascular structures of the upper mediastinum . what is very useful in the assessment of the atrial septum , the superior vena cava and right pulmonary vein is the right parasternal access with the patient positioned on the right side and exhaling deeply , but such conditions can be obtained only in older patients , able to follow commands and accepting the inconvenience of the long duration of the examination . in the described case , it was difficult to establish the optimal cooperation with the suffering ( because of abdominal pain ) child , tired with successive burdensome examinations . in most cases the abnormal flow of right upper pulmonary vein coexists with sinus venosus atrial septal defect . an isolated connection of the upper right pulmonary vein to the superior vena cava occurs much more rarely . in the absence of significant features of right ventricular volume overload , a visualization of the normal morphology of the atrial septum and particularly its upper part may encourage the abandonment of direct visualization of the course and connection of each pulmonary vein . in the described case , an additional diagnostic trap was the presence of two left pulmonary veins running into the left atrium . coming across such an image is usually an indication to identify a correct drainage of left pulmonary veins . this observation of a vertical venous channel directly adjacent to the left lung and running into the left venous angle usually requires an extremely careful analysis of the images obtained from the zygomatic indentation and/or high left parasternal accesses . in the described case , because of the patient 's neck anatomy and her negative attitude towards the examination , the suprasternal access was greatly limited , which significantly reduced the chance to visualize abnormal veins . a not her factor which may influence the delay in the papvd diagnosis is a gradual , slow build - up of the right ventricle volume overload symptoms . the medical history revealed that no size abnormalities of the right atrium , right ventricle or pulmonary artery had been reported to date , and there were also no anomalies of the ventricular septum kinetics . it is highly probable that in the earlier period the severity of overload irregularities was much smaller , so they were not perceptible in echocardiography . it is worth noting that it was the finding of the features of anatomically unexplained volume overload of the pulmonary circulation that became the reason for a more indepth image analysis which determined the cause of this condition . finally , it should be noted that the correct interpretation of the echocardiographic image could be hindered by the examiner 's focus on the previously identified irregularities associated with the bicuspid , non - closing aortic valve . some features of this anomaly , such as the left ventricular volume overload associated with aortic regurgitation , which can prevent the paradoxical motion of the ventricular septum , and the widening of the ascending aorta , causing the displacement of the superior vena cava , could have a masking effect on the papvd symptoms . in conclusion , the authors would like to emphasize that the currently widely available ultrasound diagnostics combined with the examining physician 's thoroughness may be considered a valuable tool for the initial detection of systemic and pulmonary venous anomalies in the mediastinal area in children . authors do not report any financial or personal links with other persons or organizations , which might affect negatively the content of this publication and/or claim authorship rights to this publication .

the authors present a case of echocardiographic diagnosis of a rare congenital cardiovascular anomaly in the form of mixed partial anomalous pulmonary veins connection in a 10-year - old girl with turner syndrome and congenital mild stenosis of insufficient bicuspid aortic valve , made while diagnosing the causes of intestinal tract bleeding . the article presents various diagnostic difficulties leading to the delayed determination of a correct diagnosis , resulting from the absence of symptoms of circulatory failure in the early stage of the disease and the occurrence of severe and dominant auscultatory phenomena typical for congenital aortic valve defect which effectively masked the syndromes of increased pulmonary flow . the authors discuss the role of the impact of phenotypic characteristics of the turner syndrome , in particular a short webbed neck restricting the suprasternal echocardiographic access and the presence of psychological factors associated with a long - term illness . the importance of indirect echocardiographic symptoms suggesting partial anomalous pulmonary veins connection in the presence of bicuspid aortic valve , e.g. enlargement of the right atrium and right ventricle , and paradoxical interventricular septum motion were emphasized in patients lacking asd , pulmonary hypertension or tricupid and pulmonary valve abnormalities . the methodology of echocardiographic examination enabling direct visualization of the abnormal vascular structures was presented . special attention was paid to the significance of highly sensitive echocardiographic projections : high right and left parasternal views in sagittal and transverse planes with patient lying on the side , with the use of two - dimensional imaging and color doppler . finally , the limitations of echocardiography resulting from the visualization and tracking of abnormal vascular structures hidden behind ultrasound non - conductive tissues were indicated , as was the role of other diagnostic modalities , such as angio - ct and/or nuclear magnetic resonance .

Introduction Case report Discussion Conflict of interest

PMC4635833

human schistosomiasis is a disease caused by any of 5 species of trematodes , the blood flukes , in the genus schistosoma , and infections are acquired by contact with freshwater containing parasite larvae . world health organization ( who ) estimates that over 237 million people required treatment for schistosomiasis in 2010 with estimates of up to an additional 779 million at risk globally . about 85% of the people at risk of schistosomiasis are from africa with more than 90% of the cases occurring in sub - saharan africa . king estimated that schistosome infections accounted for 24 - 56 million disability - adjusted life years ( dalys ) lost in 2010 . two major schistosome species are prevalent in sub - saharan region , schistosoma mansoni and schistosoma haematobium causing intestinal and urogenital schistosomiasis , respectively . both types of schistosomiasis are hyper - endemic in the great lake regions of east africa , owing to the favorable habitat for snails of biomphalaria and bulinus genera , which are the intermediate host for s. mansoni and s. haematobium , respectively . surveys have revealed the presence of the disease in other parts of the country , including lake victoria . lakeshore communities are typically dependent on water from these lakes , rivers , and ponds for various daily activities , including cooking , bathing , and washing clothes . fishing is usually a major occupation of men of these communities , further bringing them into contact with water . historical studies have revealed that both schistosomiasis mansoni and schistosomiasis haematobium have been endemic for a long time in tanzania . through a full consideration of the amount of end - organ pathologies to the liver ( in the case of s. mansoni and s. japonicum infections ) and bladder and kidneys ( in the case of s. haematobium infection ) , together with chronic morbidities associated with impaired child growth and development , chronic inflammation , anemia , and other nutritional deficiencies , some new disease burden assessments estimate that schistosmiasis accounts for up to 70 million dalys lost annually . who estimates that more than 1 billion people are chronically infected with soil - transmitted helminths ( sths ) . sths persist exclusively in the poorest populations often living in remote , rural areas , urban slums , or in conflict zones . ascaris lumbricoides , hookworms ( ancylostoma duodenale and necator americanus ) , and trichuris trichiura are the most common sth species with global prevalence of about 1,000 , 900 - 1,300 , and 500 million cases , respectively [ 16 - 18 ] . of the sth species , hookworms are the most widely distributed species occurring throughout much of east africa . both schistosomiasis and sths have long been known to be co - endemic in the lake victoria basin in tanzania . a comaparable situation has been reported in the kenya and uganda sides of the basin . a. lumbricoides and t. trichuria have the highest prevalences in areas close to lake victoria of tanzania , western kenya , and southeastern uganda . the morbidity caused by sths several studies have revealed the impact of sth infections as significant predictors of protein - energy malnutrition , iron - deficiency anemia ( ida ) , vitamin a deficiency ( vad ) , and poor academic performance among schoolchildren in different countries [ 26 - 29 ] . moreover , these consequences may continue into adulthood with effects on the economic productivity which trap the communities at risk of infections in a cycle of poverty , underdevelopment , and disease . hence , benefits of successful sth control programmes extend well beyond eliminating sth as they improve the nutritional and health status of children as well as contribute to higher educational attainment , labor force participation , productivity , and income among the most vulnerable populations [ 31 - 33 ] . schistosomes and sth - immunosuppressive features have possible impact on promoting susceptibility to hiv / aids , tuberculosis , and malaria , and there is evidence that female genital schistosomiasis caused by s. haematobium may significantly increase the likelihood of contracting hiv / aids . moreover , there are adverse effects on pregnancy outcome and agricultural worker productivity [ 2,6,36 - 39 ] ; helminths are strongly associated with poverty in sub - saharan africa . therefore , new and ongoing efforts to control and eliminate helminths and neglected tropical diseases ( ntds ) generally represent key elements for achieving africa 's millennium development goals ( mdgs ) . as such , the successful sth control programmes have enormous benefits of improved nutritional and health status of children for higher educational attainment , labor force participation , productivity , and income among the most vulnerable populations . mass drug administration ( mda ) has been a major approach to control human helminthiases in developing countries . in order to control schistosomiasis and sths , who recommends mda if the prevalence exceeds 10% and has the target of deworming at least 75% of school - aged children and other high - risk groups by administration with praziquantel for schistosomes and albendazole or mebendazole for sths . conversely , control of helminth infections has been suggested as the means to facilitate control of the big 3 species , especially by reducing the frequency of malaria fevers , the frequency of severe and cerebral malaria , and the prevalence of anemia [ 45 - 47 ] . furthermore , new evidence points to substantial geographic overlap between ntds and the big 3 helminthic diseases , with emerging data suggesting that control of ntds could actually become a powerful tool for combating hiv / aids , tuberculosis , and malaria . the main objective of this study was to carry out a survey on schistosomiasis and sths in order to suggest feasible and effective intervention strategies in the lake victoria basin , tanzania . the study area is the basin of lake victoria located on the northwest of tanzania ( fig . the area stretches from the west through south to the eastern side of the lake . it is comprised with kagera , mwanza , and mara on the lake shore and shinyanga about 60 km away from the lake . the study area is bordered by uganda on the north , and rwanda and burundi on the west . the main ethnic groups include wahaya , wasukuma , wazinza , wakerewe , wajita , wakurya , and waluo . much of the activities have a bearing to schistosomiasis and sth infections as they are performed without protective gear . the level of sanitation and hygiene in the area is as low as in any poor resource settings . collection of stool and urine specimens was done at the selected schools where inhabitants visited . each village person was given 1 stool container and 1 urine container on the first day and asked to bring the urine and stool specimens . the stool samples collected were processed to make duplicate kato - katz thick smears covered with cellophane soaked in glycerine and malachite green . the smears were examined for hookworms and other helminth eggs or larvae within 1 hr after preparation . the egg counts on each of the 2 slides were added together and divided by 2 to get the mean number of egg counts . urine samples were collected after 10.00 a.m. to diagnose s. haematobium based on detection of eggs in urine using the filtration technique with nuclepore membranes according to who . urine samples were mixed thoroughly and 10 ml were drawn using a syringe and passed through the membrane where eggs were logged . examination was done on site where detected eggs were counted and expressed as the number of eggs per 10 ml of urine . data entry was done using dbase iv ( borland international , scotts valley , california , usa ) and a double entry system was used for quality control . data was transferred to stata version 8 software ( statacorp 2000 , college station , texas , usa ) for analysis . analysis was done by generation of some frequency tables , cross tabulations , and calculation of prevalence . the ethical and scientific clearance was obtained from the medical research coordinating committee of the national institute for medical research before the implementation of the study . the study team visited villages where community leaders were met and the objectives , procedure , potential harm , and benefits of the study were explained to them . the leaders in turn convened meetings of the community members and the same was explained to them before the consent to participate in the study was sought . any participant would be free to withdraw from the study at any time of the study period when he or she felt to do so . the decision to refuse or withdraw from the study would have no negative effect on benefits provided during and after the study . all subjects who would be found infected with schistosomiasis or intestinal helminths would be treated following standard treatment guidelines . participants were informed that information will be confidentially kept only using code numbers instead of names of participants . the study area is the basin of lake victoria located on the northwest of tanzania ( fig . the area stretches from the west through south to the eastern side of the lake . it is comprised with kagera , mwanza , and mara on the lake shore and shinyanga about 60 km away from the lake . the study area is bordered by uganda on the north , and rwanda and burundi on the west . the main ethnic groups include wahaya , wasukuma , wazinza , wakerewe , wajita , wakurya , and waluo . much of the activities have a bearing to schistosomiasis and sth infections as they are performed without protective gear . the level of sanitation and hygiene in the area is as low as in any poor resource settings . collection of stool and urine specimens was done at the selected schools where inhabitants visited . each village person was given 1 stool container and 1 urine container on the first day and asked to bring the urine and stool specimens . the stool samples collected were processed to make duplicate kato - katz thick smears covered with cellophane soaked in glycerine and malachite green . the smears were examined for hookworms and other helminth eggs or larvae within 1 hr after preparation . examination for s. mansoni eggs the egg counts on each of the 2 slides were added together and divided by 2 to get the mean number of egg counts . urine samples were collected after 10.00 a.m. to diagnose s. haematobium based on detection of eggs in urine using the filtration technique with nuclepore membranes according to who . urine samples were mixed thoroughly and 10 ml were drawn using a syringe and passed through the membrane where eggs were logged . examination was done on site where detected eggs were counted and expressed as the number of eggs per 10 ml of urine . data entry was done using dbase iv ( borland international , scotts valley , california , usa ) and a double entry system was used for quality control . data was transferred to stata version 8 software ( statacorp 2000 , college station , texas , usa ) for analysis . analysis was done by generation of some frequency tables , cross tabulations , and calculation of prevalence . the ethical and scientific clearance was obtained from the medical research coordinating committee of the national institute for medical research before the implementation of the study . the study team visited villages where community leaders were met and the objectives , procedure , potential harm , and benefits of the study were explained to them . the leaders in turn convened meetings of the community members and the same was explained to them before the consent to participate in the study was sought . any participant would be free to withdraw from the study at any time of the study period when he or she felt to do so . the decision to refuse or withdraw from the study would have no negative effect on benefits provided during and after the study . all subjects who would be found infected with schistosomiasis or intestinal helminths would be treated following standard treatment guidelines . participants were informed that information will be confidentially kept only using code numbers instead of names of participants . a total of 1,606 from 36 selected communities , mostly from the shoreline of the lake victoria basin in tanzania , were recruited for the study ( table 1 ) . out of 1,606 adults who provided stool specimens , 199 ( 12.4% ) were positive for s. mansoni , 349 ( 21.7 ) for hookworms , 133 ( 8.3% ) for a. lumbricoides , and 33 ( 2.0% ) for t. trichiura . a total of 1,400 adults provided urine for s. haematobium diagnosis out of whom 25 ( 1.8% ) from mwanza and mara were positive for eggs ( table 1 ) . the highest prevalence ( 18.9% ) of intestinal schistosomiasis were recorded in mwanza region followed by kagera with 17.8% . it was only in kagera region that all the 3 common sths at various prevalence rates were found . interestingly , no schistosomiasis haematobium patient was recorded in shinyanga region despite its being located in the hinterland . shinyanga is naturally the driest of the 4 regions , and the study was done in mid - july during draught when s. haematobium transmission is low . the few adult members with urogenital infections observed in mwanza and mara were from wet areas with paddy fields . such areas are good habitats for snail hosts of the genus bulinus . by that time bulamba locality was dry besides being close to the lake . the study team visited a total of 9 localities in 5 out of 8 districts in kagera region . in this region , ( 17.8% ) were positive for intestinal schistosomiasis , 182 ( 37.3% ) for hookworms , 113 ( 23.2% ) for ascariasis , and 33 ( 6.8% ) for trichuriasis ( table 2 ) . the highest prevalence ( 77.1% ) of intestinal schistosomiasis was recorded at bwina locality in chato district followed by bwanga ( 30.3% ) and kiziramuyaga ( 18.5% ) in chato and muleba districts , respectively . hookworm infections were the highest at runazi ( 69.6% ) , bwina ( 68.8% ) , and kiziramuyaga ( 66.2% ) in biharamulo , chato , and muleba districts , respectively . the prevalence of ascariasis was the highest at kyenshama ( 66.0% ) in muleba district , followed by an urban community of bunena ( 42.9% ) , then bwina ( 31.3% ) and bwanga ( 24.2% ) , both in chato district . trichuriasis was highly prevalent at bunena ( 33.3% ) in bukoba urban district ( table 2 ) . the study team visited a total of 12 communities from 7 of the 8 districts of mwanza region . out of 471 adult participants who provided stool specimens , 89 ( 18.9% ) had intestinal schistosomiasis and 42 ( 8.9% ) had hookworms . no one was found to be infected with ascaris , trichuris , or other intestinal helminths ( table 3 ) . out of 265 adults who had their urine specimen examined , only 3 ( 1.1% ) from 2 communities of geita had urogenital schistosomiasis . about a half ( 51.1% ) of those who presented stools at bungonya in sengerema had intestinal schistosomiasis . six of 14 individuals ( 42.9% ) at tumaini in ilemala and 21 out of 50 examinees ( 42.0% ) at nyamikoma in magu district were found infected with intestinal schistosomiasis . mwaginghi in kwimba district recorded the highest prevalence of hookworms ( 50.0% ) followed by tumaini ( 35.7% ) and lumeji ( 21.6% ) in ilemela and magu districts , respectively ( table 3 ) . we recruited 344 adult members from mara region who provided both stool and urine specimens . the region had relatively low prevalence ( 6.7% ) of s. mansoni and even lower prevalence ( 0.9% ) of hookworms . the highest recorded prevalence of intestinal schistosomiasis in the region was from nyamitwebili where out of 44 members who provided stool specimens , 8 ( 18.2% ) were positive for the disease . mwisenge locality was the second by having 7 ( 13.7% ) positive cases for the disease . only hookworms were recorded at minigo in rorya district and mwisenge in musoma urban districts . with the exception of bulamba in bunda district , all communities in mara region had cases infected with s. haematobium . shinyanga region had totally different findings from the other 3 regions . in this region , out of 303 members who provided both stool and urine specimens , only 33 ( 10.9% ) were positive for hookworm eggs ( table 5 ) . the highest prevalence for hookworms was found in kahama district at bukomela and mseki localities with 40.4% and 33.3% prevalence , respectively ( table 5 ) . the study results showed that generally the prevalence of s. mansoni , s. haematobium , and sths is widespread in the study area . however , the prevalence of s. mansoni in kagera region was very low with the exception of a few pockets in chato district . this could be attributed to the fact that lake shores in kagera region are open ones instead of bays such that when the wind blows makes the waves splash the shores making the place an unconduncive habitat for snail intermediate hosts for schistosomes . moreover , some of the shores in kagera region are formed by escarpments thus deep and less habitable by the snails . the situation is quite different from shores in chato district in the southwest of lake victoria which are mainly shallow and in bays thus not susceptible to strong winds and can habor the snails . the relatively higher prevalence of ascariasis and trichuriasis in kagera region than in other regions could be attributed to the difference in soil temperature , altitude , soil type , and rainfall [ 51 - 53 ] . kagera region is wet almost throughout the year as compared to the rest of the regions in the area . the highest prevalence of s. mansoni in kagera region that was recorded at bwina community could be attributed to the location of the study site . bwina ward is a thin peninsular with an average width of less than 2 km . as such , most of the area is surrounded by lake victoria water that is inhabited by the snail intermediate host biomphararia species for s. mansoni . bwanga and kiziramuyaga localities are close to the lake area that has small bays that are conduncive areas for the snail hosts . this has an implication of the region having the best breeding ground of the sths as well as more people consuming more vegetables that may be carrying the helminth eggs than those from other areas . the absence of urogenital schistosomiasis in kagera could be due to an absence of ponds and streams during the time of the study , and the fact that no paddy cultivation is practiced in the area . the high prevalence of intestinal and urogenital schistosomisiasis in the study area was a function of the distance from lake victoria ; the former being more prevalent at localities close to the lake , whilist the latter is more so away from it . the high prevalence of intestinal schistosomiasis at nyamitwebili and mwisenge localities could be associated with their being at the lake shore . minigo had a lot of paddy fields that are good habitats for snail intermediate hosts for s. haematobium . the distribution of s. haematobium and s. mansoni along lake victoria is largely related to the distribution of the intermediate hosts . along the shore of the lake , members of the genus biomphalaria are common with populations living along the lake shores and islands being highly affected by s. mansoni , as the risk of infection increases . in mara region , the distribution of s. mansoni was similar to that of mwanza with more s. mansoni close to the lake and s. haematobium on the hinterland . moreover , in areas where s. haematobium was high , there was a significant prevalence of hookworms . the findings are in line with the ones from a previous study in magu district in mwanza region . the spatial distribution of s. haematobium and hookworm is in accordance with the presence of ponds and streams as well as wetness and warmth of the soil in the reference area that are prerequisite for proliferation of the 2 helminths . we could not easily establish why there were no infections due to a. lumbricoides and t. trichiura in mwanza region or intestinal schistosomiasis in shinyanga region . a major reason could be geographical as stated above or there were treatment programmes prior to this study . the study results showed that schistosomiasis and 3 sths infections ( hookworms , ascaris , and trichuris ) were co - endemic in lake victoria basin in tanzania with a high probability of polyparasitism in the study participants . the prevalence of s. mansoni , s. haematobium , and sths ranged from low to moderate in most parts of the study area . intestinal schistosomiasis was prevalent along the lake victoria shores and decreased with distance from it . the distributions of both schistosome and geohelminth infections have important implications for integrated intervention including periodic community deworming programmes , provision of safe water supply , and proper health education relevant to good personal hygiene and good sanitary practices . these activities will facilitate the reduction of both prevalence and intensity of infections of schistosomiasis and sths in such endemic communities . pre- and post - intervention studies to assess knowledge , attitude , and practices of the concerned population can not be overemphasized .

the objective of this study was to carry out a community survey on schistosomiais and soil - transmitted helminth ( sth ) infections in order to suggest feasible and effective intervention strategies in lake victoria basin , tanzania . a total of 37 communities selected from 23 districts of the 4 regions in the lake victoria basin of tanzania were involved in the study . from each of the selected locality , 50 adult community members , 25 males and 25 females , were recruited for the study . each study participant was requested to submit stool and urine specimens . from each stool specimen , duplicate kato - katz thick smears were prepared and microscopically examined for schistosoma mansoni and sth eggs . urine specimens were processed by the filtration technique and microscopically examined for schistosoma haematobium eggs . ultrasound examination for morbidity due to schistosomiasis was performed . mass treatment was done using praziquantel and albendazole for schistosome and sths infections , respectively . out of 1,606 adults who provided stool specimens , 199 ( 12.4% ) were positive for s. mansoni , 349 ( 21.7% ) for hookworms , 133 ( 8.3% ) for ascaris lumbricoides , and 33 ( 2.0% ) for trichuris trichiura . out of 1,400 participants who provided urine specimens , 25 ( 1.8% ) were positive for s. haematobium eggs . because of the co - endemicity of these afflictions and their impact on vulnerable population groups , the helminthiasis could be simultaneously treated with 2 drugs , praziquantel for schistosomiasis and albendazole for sths .

INTRODUCTION MATERIALS AND METHODS Study area and population Specimen collection and examinations Data management and analysis Ethical considerations RESULTS DISCUSSION

PMC4458553

group a streptococcus ( gas , otherwise known as streptococcus pyogenes ) is one of the most important causes of infectious disease morbidity and mortality , with an estimated prevalence of severe disease of at least 18.1 million cases leading to more than 500,000 deaths annually . much of the burden exists in less developed countries where control strategies are difficult to implement and ensure efficacy . a vaccine is therefore widely acknowledged as an important strategy to reduce the burden of gas disease globally . several vaccine candidates are presently in various stages of preclinical development , and a limited number have also reached early phase clinical trials [ 3 , 4 ] . a key element in vaccine development is the availability of a validated and standardized immunoassay that correlates with immune protection . however , despite decades of research , there is no single standardized immunoassay and certainly no human correlate of protection ( cop ) established for gas . in the case of other bacterial pathogens , such as streptococcus pneumoniae and group b streptococcus ( gbs ) , for which vaccines are widely available or development is underway , cops provide a means of assessing the true efficacy and immunogenicity of potential vaccines [ 6 , 7 ] . a solid understanding of bacterial pathogenesis and host immune response lays the foundations for robust cops . it is widely agreed that the m protein is an important virulence factor of gas , conferring both adhesion and antiphagocytic properties through binding of various host proteins and interacting with the complement pathway [ 8 , 9 ] . the m protein is an alpha - helical coiled - coil dimer extending from the surface of the bacteria as a fibril . its structure is divided into conserved , central variable , and n - terminal hypervariable regions . some m proteins may have a nonhelical portion at the distal end of the n - terminal region , but the significance of this is unknown . there are a number of a , b , c , and d repeats that vary between the different m proteins , with increasing sequence conservation downstream of the hypervariable region . the current gas classification system , known as emm - typing , is based on sequencing of 1015% of the emm gene ( which encodes the m protein ) with 223 emm - types identified worldwide [ 12 , 13 ] . recently , a new adjunct method of classification , known as the emm - cluster system has been developed , which organizes the 223 emm - types into 48 distinct emm - clusters based on the phylogenetic analysis of the whole m protein and its structural and binding properties [ 1416 ] . studies in mice have demonstrated that mucosal iga antibodies against the m protein prevent colonization and adherence of gas [ 17 , 18 ] . binding of type - specific igg antibodies to the n - terminus of the m protein in the host activates the complement pathway , leading to deposition of c3b and facilitating phagocytosis and killing . limited evidence suggests that these type - specific antibodies provide long - lasting protection against homologous strains . whilst the conserved region of the m protein has not been as extensively studied as the n - terminus , it has also been shown to elicit osonophagocytic antibodies [ 2023 ] . antibodies produced against the b repeat region of the m protein are not thought to be opsonic and thus unlikely to be protective . memory b cells and t cells also appear to play a role in propagating immunity , and several immunodominant t cell epitopes have been mapped , although the exact mechanisms of cell - mediated immunity in gas are still unclear [ 21 , 23 ] . non - m protein virulence factors such as c5a peptidase , lipoteichoic acid , protein f , and streptococcal fibronectin - binding protein have been shown in animal studies to raise protective antibodies that contribute to host immunity , particularly in preventing colonization [ 18 , 25 ] . antibodies to streptococcal toxins such as streptococcal pyrogenic exotoxins a , b , and c and streptococcal erythrogenic exotoxin b have also been proposed to play a role in in vivo immunity . other potentially immunogenic antigens that have been investigated as vaccine candidates include streptococcal protective antigen , serum opacity factor , streptococcal pili , s. pyogenes cell envelope protease , and gas carbohydrate . whilst all of the above may play a role in gas immunity , it is believed that opsonic type - specific antibodies remain responsible for clearing infection . the exact natural history of immunity against gas infections also remains unclear , although there are distinct peaks of increased incidence at different age groups for each gas disease ( figure 1 ) . for instance , the incidence of gas superficial infections is high in children but decreases in adulthood . anti - gas antibody levels in adults are much higher compared with children , suggesting that this natural acquisition of antibodies due to exposure over time provides protective immunity against gas diseases later in life [ 20 , 26 ] . similarly for severe and invasive gas diseases , there is decreased incidence in adulthood ; the significant peak in the elderly is likely to be due to comorbid illness and immunosenescence [ 2729 ] . a correlate is an attribute that is statistically associated with an endpoint ( without the association necessarily being causal ) , whilst a surrogate or mechanistic cop ( mcop ) has the added criterion of being part of the causal pathway and the mechanism by which a vaccine induces protection ( figure 2 ) . in 2011 , it was agreed that a roadmap for gas vaccine development was needed to harness the efforts of the international community and two key components were identified as ( 1 ) definition of human cops for gas and ( 2 ) development of high throughput standardized assays that accurately represent in vivo immunity [ 5 , 25 , 35 ] . a key long - term advantage of cops is in enabling vaccine licensure by obviating the need for demonstrating field efficacy in the scenario where efficacy trials are logistically or ethically challenging . establishing the efficacy and safety of vaccine candidates can be a lengthy and expensive endeavour involving large - scale phase iii trials . whilst it is likely that the first gas vaccine candidate to be licensed will be approved based on phase iii trials using pharyngitis as an end - point , cops may provide an alternative for second generation vaccines . if an immunological marker is established as a cop and it is demonstrated that a vaccine candidate meets or exceeds an immune threshold , then it may be approved based on serological data alone . successful precedents include vaccines for meningococcal serogroups b and c [ 3840 ] , influenza , s. pneumonia , and haemophilus influenzae type b ( hib ) . not only can cops fast - track the licensure process and potentially reduce costs , but they can also be useful when placebo randomized controlled trials ( rct ) are no longer possible or ethical ( as in the case of hib ) . this may be the case if a successful gas vaccine is developed ; it may be unethical to allow cases of rhd and potentially fatal invasive diseases to occur in subsequent vaccine development trials when a vaccine would prevent them . correlates of protection may also be used to inform seroepidemiology studies and surveillance of populations postvaccination . this could be relevant to gas because of high global diversity of strains that may evoke different patterns of immune responses and to evaluate whether a vaccine will introduce pressure that leads to the emergence of new strains . further applications that could be explored include passive protection , where the cop is used to determine the amount of antibody to administer in passive immunizations and as screening or diagnostic tools , for instance , in testing antibody levels against rubella for pregnant women . thus , establishing a cop would provide an adjunct tool of great benefit in gas vaccine development . firstly , there are technical difficulties in developing a high quality , standardized immunoassay for gas . further , the utilization of the assay to determine a cop for gas raises its own set of challenges in terms of study design and use of animal or human models . it should be reproducible across different laboratories with a common standard operating protocol and equivalent data sets . for gas , one of the key points will be ensuring that antigens and gas strains used by different laboratories are comparable . the methodology needs to be based on a thorough understanding of gas pathogenesis , host interactions , and immune response in order to be clinically relevant . it is worth noting that these criteria for the establishment of a robust standardized immunoassay in gas are important not only in terms of defining a cop , but also in securing reliable immunological data for the conventional aspects of vaccine development to enable proof - of - concept studies , successful progression through trial phases , and ensuring ongoing funding from the relevant sources . a robust immunoassay will also be particularly critical in helping to overcome the specific challenges in gas vaccine development such as ensuring coverage across the many different strains and variation in geographical distribution . there are currently two main types of immunoassays utilized in gas research : assays that give a purely quantitative measurement of antibody such as the enzyme - linked immunosorbent assay ( elisa ) and functional bactericidal assays that demonstrate immunity through evidence of phagocytic killing of gas ( table 1 ) . assays that have been established for other pathogens that have potential application to gas include those for s. pneumoniae and gbs . the opsonophagocytic assays proposed as cops may not necessarily apply to all gas protective antigens . the elisa has been used for quantification of antibodies in gas in the development of all the major gas vaccine candidates in testing of both rabbit and mice antisera , as well as human antisera in two phase i trials [ 4 , 17 , 21 , 23 , 4449 ] . the elisa has been used successfully in s. pneumoniae research to establish cops and in gbs to propose potential cops values , suggesting that the elisa may be a good candidate for determining gas cops . one issue is the choice of antigen to use , such as recombinant m protein , or synthetic peptides [ 4 , 44 , 47 ] . the peptide method is generally favoured in preclinical studies as it allows for the testing of antibodies specific to the vaccine epitope only and minimizes the amount of non - specific antibodies detected [ 10 , 44 , 47 ] . however , there is a theoretical concern that the folding structure of peptides may be altered from the normal structure when within a whole m protein and consequently give results that may not be clinically relevant . for example , in s. pneumoniae research , initial elisas were found to correlate poorly with in vivo reactions due to measurement of nonfunctional antibodies to pneumococcal cell wall polysaccharide ( c - ps ) in addition to anti - capsular antibodies . this problem was overcome by preabsorption of serum with highly purified c - ps before use in the elisa , to remove non - functional antibodies . similarly , preabsorption with pneumococcal type 22f capsular polysaccharide neutralized antibodies cross - reactive to common epitopes , leaving only serotype - specific antibodies to be measured in the elisa . for gas , the addition of a preabsorption step with an unrelated m peptide or full m protein could increase the specificity and detection of functional antibodies . a slightly different approach was taken in gbs , where alterations to the antigen were made [ 53 , 54 ] . the gbs iii polysaccharide was mixed with methylated human serum albumin ( hsa ) to increase adherence of the antigen to the microtitre plates and coupled with biotin or nonmethylated has , and a subsequent study found using nonmethylated hsa produced the highest specificity and sensitivity . in gas , mixing the full m protein with fibrinogen or hsa to bind the b repeat and c repeat portions of the m protein , respectively , could reduce detection of non - functional antibodies . there is currently no standardized protocol between gas laboratories , making it difficult to confidently compare results between studies . two approaches could potentially be used to ameliorate this issue : ( 1 ) attempting to regulate all steps and reagents used or ( 2 ) providing a standard serum for laboratories to use as a reference . the latter is utilized for s. pneumoniae , where elisas are standardized against the reference serum , 007 sp , created from volunteers immunized with the 23-valent vaccine . martins et al . took a novel approach by developing a multiplexed fluorescent immunoassay using luminex multi - analyte profiling technology ( luminex corp . igg antibody responses against nine common gas antigens ( including the m protein ) conjugated to fluorescent microspheres were simultaneously measured using either human sera or animal antibodies . whilst the assay quantified antibody responses to antigens in a high throughput fashion , a high degree of nonspecific binding of igg to microspheres was reported and no comparison with elisa or bactericidal assays was performed . the principle of gas bactericidal assays is to incubate whole blood with bacterial inoculum and then determine bactericidal activity by measuring colony - forming units ( cfu ) . there are two variations , the direct bactericidal test ( dbt ) and the indirect bactericidal test ( ibt ) . the dbt utilizes the test subject 's whole blood as a source for serum and phagocytes . the ibt only uses serum from the test subject and nonimmune human blood from a separate donor as the source of phagocytes , with the aim of reducing variability as donors are screened to ensure that there are no type - specific or cross - reactive antibodies present . the ibt has lower sensitivity with low antibody titres , leading to the development of an optional preopsonization step where antisera is first incubated with gas to increase antibody binding before adding the source of phagocytes . currently , the ibt is the most commonly used method to assess functional immunity to gas [ 4 , 4448 ] . despite this , there are still many variations in methodology between laboratories ( table 2 ) , and even in the world health organization established protocols , two main methods of performing the ibt are described . inter - assay variability is high because of the different methodologies and because of the unstandardized element of donor blood . the need for fresh human blood is problematic as it means that assays need to be performed within two hours of venepuncture . both the donor and test subject must have the same blood group , or only group o blood can be used , to prevent an abo incompatibility reaction . the ibt is labour intensive and low throughput , making it a less suitable choice for determining a cop for gas . a variation of the ibt using light microscopy of wright smear stains to define opsonization rather than counting cfu has also been used to test gas vaccine candidates [ 46 , 60 ] . however , in the phase i trial of the hexavalent gas vaccine , less of a response was seen in the ibt when compared to this version of the opsonophagocytic assay , suggesting that in some cases association of bacteria with the cell wall of leukocytes may not equate to killing . the hl-60 opsonophagocytic assay ( opa ) is an alternative functional opsonic assay that could potentially be utilized in gas , given its prior success with s. pneumonia and gbs . the hl-60 cells used as the source of phagocytes are human promyelocytic leukaemia cells capable of being cultured and differentiated , thus eliminating the need for human donors and potentially decreasing inter - assay variability . the assay tests the bactericidal activity and ability of sera to opsonize viable bacteria in the presence of differentiated hl-60 neutrophils and complement . romero - steiner et al . first developed the hl-60 opa to measure functional antibodies against s. pneumoniae . they demonstrated for seven serotypes that the hl-60 opa correlated highly with opas using peripheral blood leukocytes ( pbl ) , the previous standard for effector cells . over time , there have been a number of variations trialled and modifications made to the assay ( table 3 ) . the hl-60 opa was tested in various multilaboratory trials using multiplexing and automated colony counting , showing robust inter - assay and interlaboratory reproducibility and validation [ 63 , 64 ] , and a standardized protocol is now available online . however , it was found that the execution of the opa is not always tightly regulated across all laboratories , and whilst overall agreement is sufficient , there remains higher variability compared to elisa . further , the correlation between elisa , hl-60 , and pbl opas is poor for some specific pneumococcal serotypes . for example , five serum samples tested for serotype 19f immunity had elevated elisa igg titres but no opsonophagocytic activity . this may in fact be a problem with the elisa ; a study comparing elisa and opa in children with human immunodeficiency virus ( hiv ) in south africa found raised elisa antibody titres in both infected and noninfected groups , but lower opa titres in hiv - infected individuals , suggesting that the elisa could be detecting non - protective antibodies . nevertheless , evidence of bactericidal activity of functional antibodies is desired when considering licensure of a new vaccine candidate , and thus the who has determined for s. pneumoniae conjugate vaccines that an opa titre of 1 : 8 in conjunction with an elisa antibody concentration of at least 0.35 g / ml demonstrates that a candidate provides sufficient protection to be considered for licensure . it has been proposed that the use of the 007sp reference serum could also increase standardization . the development of a reference serum for gas would create a degree of uniformity across laboratories and help facilitate immunoassay and vaccine development . furthermore , the establishment of a standard collection of gas isolates for laboratories to reference would increase standardization in the performance of immunoassays used in vaccine studies . attempts to increase throughput of the assay have focussed on developing a fluorescent opa ( flopa ) using fluorescence activated cell sorting ( facs ) to measure the uptake of fluorescently labelled , whole killed gbs cells , or antigen - coated fluorescent microspheres . the flopa has multiple advantages including the ability to be multiplexed , the ability to standardize complement and effector cells , and a faster running time . a criticism of previous flopas has been the inability to distinguish between adhered and internalized bacteria . fabbrini et al . have overcome this for gbs with phrodo , a ph - sensitive fluorogenic dye that only emits a bright red signal in an acidic environment . this ph - dependent reaction means that phrodo - labelled bacteria are only detected when they are in phagolysosomes . bacteria that are bound to the hl-60 cell wall show only a very low signal on facs , and thus the actual bactericidal antibodies can be measured rather than just measuring adhered antibodies . this method was shown to be reproducible and as sensitive and specific as a traditional opa . the potential applications of this technique are promising ; however no studies using human sera have been carried out to date , and facs remains an expensive method requiring skilled technicians . once a standardized immunoassay is established , studies can be carefully designed to determine what levels of immune markers correlate with protection . a key challenge in establishing a cop is the inherent heterogeneity present within populations and environments . there is a distinct variance in distribution of strains in developed countries when compared with resource poor regions such as africa and the pacific [ 70 , 71 ] . the highest rates of gas diseases are in these low socioeconomic areas [ 1 , 72 ] , where crowding , poor hygiene , and tropical climate are likely to result in greater exposure and infection pressures . early studies suggested that different gas strains require different levels of antibodies to be considered immune . if that is the case , it may be difficult to accurately determine specific cops due to the challenges of obtaining comprehensive data from developing regions and the apparently constantly evolving epidemiological landscape [ 1 , 70 ] . individual host factors can also alter cops , as a person 's immunity changes with age and between individuals . the level of antibody that is protective in a young healthy adult may not protect against infection in an elderly individual . gas bactericidal antibodies have been shown to persist for up to 30 years but to varying degrees between persons , making it potentially difficult to predict cops in the long - term . cell - mediated immunity involving b and t cells has also been shown to play a role in long - term memory . in particular , memory b cells are capable of mounting a rapid response to gas infection in animal studies , potentially meaning that long - term immunity may be more dependent on an individual 's cellular response and that low circulating antibody titres do not necessarily correlate with a lack of protection . for instance , the hib conjugate vaccine was licensed based on a cop of serum antibody concentration of 0.041.0 g / ml but variations from this range have been shown in different studies to be protective , and it is thought that immunologic memory and population differences have not been accounted for . the choice of antigen(s ) for a vaccine candidate , route of administration , and immune marker targeted can also result in different cops . for instance , the protective titre required for type - specific antibodies raised by the 30-valent vaccine may be different from that raised by the j8-dt vaccine against the conserved region of the m protein . secretory iga antibodies are part of mucosal protection and inhibit gas colonization but may not always necessarily correlate with serum igg levels or protection against invasive disease . there is some evidence that mucosal iga antibodies may be able to contribute to immunity in certain cases , with intranasal immunisation of mice shown to induce salivary antibodies and protect against subsequent intranasal challenge [ 82 , 83 ] . these studies raise the possibility that mucosally delivered vaccines ( e.g. , intranasal ) would require separate development of mucosal assays and cops . it is also unclear whether serum opsonophagocytic igg antibodies involved in clearing infection are protective against recurrent skin infections , as most studies of immune protection against gas have traditionally focused on pharyngitis . animal models provide a way of studying in vivo immunity and pathogenesis , and pathogens that have used animal models to determine cops include clostridium tetani and clostridium botulinum . however , there have been difficulties in developing accurate disease models and bridging the gap from animal to human immune response because gas is a human - only pathogen . there have been numerous animal models of gas infections developed , spanning from murine models for virulence and vaccine studies to rhd in rats ; invasive disease in rabbits or pigs ; and pharyngitis in primates . murine models have been used in studies of several vaccine candidates [ 47 , 86 ] . however , there are several limitations to these models : only a select number of gas strains are virulent in mice ; repeated passage of strains may be needed but result in mutations altering virulence ; colonization is often difficult to achieve ; and true pharyngitis does not occur . as such , murine models are unlikely to give an accurate indication of human cops for gas . nonhuman primate models have been established in an attempt to provide a more clinically relevant model of gas pharyngitis . although there is variable reproducibility of the severity of clinical disease seen in monkeys , they have been shown to produce type - specific opsonic antibodies ( measured by bactericidal assays ) and could be utilized in challenge or vaccine studies to evaluate cops . traditionally , cops have been established through a rct phase iii vaccine trial as has been the case for hib , tuberculosis , and s. pneumoniae vaccine candidates [ 34 , 88 ] . other studies nested within rcts may also be used to inform cops , as demonstrated by a case - control study of the acellular pertussis vaccine . the lack of phase iii trials has hindered the establishment of cops for gas as rcts ideally allow for an understanding of the relationships between the vaccine , immune markers , and clinical endpoint ( figure 2 ) for cops to be determined with appropriate statistical power . similar challenges face the gbs vaccine field where there is no vaccine beyond phase ii trials currently available . lin et al . used a case - control study to estimate a cop in order to facilitate vaccine licensure . maternal and cord serum samples from infants across 14 hospitals were compared by elisa to establish a relationship between igg levels against gbs and early - onset disease in neonates . in this study , an antibody titre 5 g / ml was protective against neonatal disease . a case - control study conducted by baker et al . also evaluated maternal antibody levels required to protect against gbs early - onset disease , finding that an overall titre 1 g / ml conferred a 70% risk reduction . more specifically , it was identified that different antibody levels were found to be protective for the different gbs types ia , iii , and v . there are limited data from human natural infection studies for a number of gas vaccine candidates . for example , a study of serum igg antibodies against p145 , the parent peptide of j8 , showed an age - related increase in antibody titres suggesting that these antibodies may correlate with protection over the life course ( figure 1 ) . similarly , anti - c5a peptidase titres were found to be in significantly higher concentrations in adults than in children in minnesota . further , a study of children aged 514 years in mexico observed higher titres of anti - gas carbohydrate antibodies that correlated with reduced colonization of the pharynx suggesting that there may be a threshold protective antibody titre . these studies suggest that defining a cop without data from phase iii clinical trials will be challenging , although an important consideration in the future remains . additionally , whilst this evidence is insufficient to establish a cop , it may help to inform clinical proof - of - concept , that is , determination of whether an intervention is biologically active or inactive . proof - of - concept is an important bridging step in ensuring that government bodies , investors , and pharmaceutical companies continue to support the vaccine development process and allow for larger scale efficacy studies where a cop may then be established . data from phase i and ii clinical trials are also relevant to the current status of gas vaccine research as has been the case for phase ii trials of the botulinum f toxoid vaccine and the meningococcal c conjugate vaccine in the united kingdom . in the case of meningococcal c , protective titres extrapolated from animal studies were used to inform human cops , and vaccine licensure was approved by comparing cops with the preexisting vaccine . given the limited clinical applicability of animal models and the potential drawbacks of phase iii clinical trials , human challenge models would provide a controlled method for testing of new vaccines . many pathogens including , but not limited to s. pneumoniae , cholera , malaria , and dengue virus , utilize human challenge models in their fields . three pharyngeal challenge studies of gas were carried out in the 1970s with a total of 178 volunteers ( table 4 ) [ 9496 ] . importantly , the trials were safe , with penicillin administered within the therapeutic timeframe and patients closely monitored to prevent progression into invasive disease or autoimmune sequelae . in a potential gas human challenge model , individuals would be infected with a carefully predetermined virulent strain of gas known to cause pharyngitis and followed through to see if they develop clinical disease . by analyzing blood samples taken at different time points and comparing immune markers with clinical outcomes , one

group a streptococcus ( gas ) is known to cause a broad spectrum of illness , from pharyngitis and impetigo , to autoimmune sequelae such as rheumatic heart disease , and invasive diseases . it is a significant cause of infectious disease morbidity and mortality worldwide , but no efficacious vaccine is currently available . progress in gas vaccine development has been hindered by a number of obstacles , including a lack of standardization in immunoassays and the need to define human correlates of protection . in this review , we have examined the current immunoassays used in both gas and other organisms , and explored the various challenges in their implementation in order to propose potential future directions to identify a correlate of protection and facilitate the development of m protein - based vaccines , which are currently the main gas vaccine candidates .

1. Introduction 2. Immune Response to GAS Infection 3. Immune Correlates of Protection 4. Development of a Standardized Immunoassay for GAS 5. Implementing Immunoassays to Determine a Correlate of Protection

PMC2686428

haematopoiesis is the process whereby haematopoietic stem cells ( hscs ) differentiate into at least 14 types of mature blood cells , all of which have distinct microscopic appearance and perform different essential functions . like other stem cells , hscs possess the capacity for multi - lineage differentiation and are unique among blood cells , in that they have the ability to produce identical copies of themselves for the entire lifetime of the organism ( self - renewal ) . hscs are extremely rare ( 110 hscs per 100 000 cells in adult murine bone marrow ) and during ontogeny derive from mesoderm , with sequential sites of haematopoiesis including the yolk sac , the aorta - gonad mesonephros region ( agm , an area surrounding the dorsal aorta ) , the placenta , fetal liver , thymus and finally the bone marrow in the adult animal . a major strength of the mouse blood system is the ability to isolate and intravenously transplant distinct subpopulations and assess the functional readout in vivo . as a consequence , many paradigms of the wider field of stem cell biology progress in the development of monoclonal antibodies directed at cell surface antigens , followed by fluorescence - activated cell sorting ( facs ) , has permitted the isolation of many haematopoietic subpopulations with differing lineage capabilities . in adult haematopoiesis , long - term hscs ( lt - hscs ) give rise to short - term hscs ( st - hscs ) , which in turn produce common myeloid progenitors ( cmps ) , common lymphoid progenitors ( clps ) and lymphoid primed multipotent progenitors ( lmpps ) . clps eventually give rise to b- and t cells , and also to natural killer ( nk ) and dendritic cells . cmps give rise to megakaryocyte / erythroid ( meps ) , granulocyte / macrophage ( gmps ) , and eosinophil ( cfu - eo ) and basophil ( cfu - ba ) progenitors . gmps give rise to the committed precursors of neutrophils and macrophages ( figure 1 ) . the classic scheme of haematopoiesis features a tree that splits into the myeloid and lymphoid lineages . the progressive restriction of developmental fates is carried out by lineage - specific transcription factors that control lineage - specific expression profiles . of note , the previously recognized distinction between the myeloid and lymphoid lineages has recently been disputed by the finding that t - cells precursors retain the ability to ultimately give rise to macrophages ( 31,32 ) . the haemangioblast is a multipotent cell and a common precursor to both haematopoietic and endothelial cells ( 33 ) . the haemangioblast has been isolated from gastrulating mouse ( 34 ) and zebrafish ( 35 ) embryos as mesodermal subpopulations , although their in vivo functionality awaits demonstration . progenitors : lt - hsc ( long - term haematopoietic stem cell ) , st - hsc ( short - term haematopoietic stem cell ) , lmpp ( lymphoid primed multipotent progenitor ) ; lymphoid : clp ( common lymphoid progenitor ) , ctnkp ( common t and natural killer progenitor ) , dn - x ( double negative x ) , dpl ( double positive large ) , dps ( double positive small ) ; myeloid : cmp ( common myeloid progenitor ) , mep ( megakaryocyte and erythrocyte progenitor ) , bfu - e ( blast - forming unit erythrocyte ) , cfu - e ( colony - forming unit erythrocyte ) , mk - p ( megakaryocyte progenitor ) , cfu - mc ( colony - forming unit mast cell ) , cfu - eo ( colony - forming unit eosinophil ) , cfu - ba ( colony - forming unit basophil ) , gmp ( granulocyte and macrophage progenitor ) , cfu - g ( colony - forming unit granulocyte ) , cfu - m ( colony - forming unit macrophage ) ; dendritic : cdp ( common dendritic progenitor ) . the coloured cell types are those included in bloodexpress . overview of adult haematopoiesis in the mouse . the classic scheme of haematopoiesis features a tree that splits into the myeloid and lymphoid lineages . the progressive restriction of developmental fates is carried out by lineage - specific transcription factors that control lineage - specific expression profiles . of note , the previously recognized distinction between the myeloid and lymphoid lineages has recently been disputed by the finding that t - cells precursors retain the ability to ultimately give rise to macrophages ( 31,32 ) . the haemangioblast is a multipotent cell and a common precursor to both haematopoietic and endothelial cells ( 33 ) . the haemangioblast has been isolated from gastrulating mouse ( 34 ) and zebrafish ( 35 ) embryos as mesodermal subpopulations , although their in vivo functionality awaits demonstration . progenitors : lt - hsc ( long - term haematopoietic stem cell ) , st - hsc ( short - term haematopoietic stem cell ) , lmpp ( lymphoid primed multipotent progenitor ) ; lymphoid : clp ( common lymphoid progenitor ) , ctnkp ( common t and natural killer progenitor ) , dn - x ( double negative x ) , dpl ( double positive large ) , dps ( double positive small ) ; myeloid : cmp ( common myeloid progenitor ) , mep ( megakaryocyte and erythrocyte progenitor ) , bfu - e ( blast - forming unit erythrocyte ) , cfu - e ( colony - forming unit erythrocyte ) , mk - p ( megakaryocyte progenitor ) , cfu - mc ( colony - forming unit mast cell ) , cfu - eo ( colony - forming unit eosinophil ) , cfu - ba ( colony - forming unit basophil ) , gmp ( granulocyte and macrophage progenitor ) , cfu - g ( colony - forming unit granulocyte ) , cfu - m ( colony - forming unit macrophage ) ; dendritic : cdp ( common dendritic progenitor ) . the coloured cell types are those included in bloodexpress . haematopoiesis involves a progressive restriction of differentiation potential , mirrored at the molecular level by the establishment of lineage - specific expression profiles which are controlled by combinatorial interactions of transcription factors ( tfs ) . conventional gene knock - out experiments have been used to illustrate the stages at which haematopoiesis is blocked in the absence of specific tfs . this has the effect of deregulating the expression of the locus ( e.g. scl / tal1 and lmo2 in t - cell acute leukaemia ) or generating chimeric fusion proteins , as in myeloid and lymphoid leukaemias ( mll , runx1 and tel / etv6 ) . public databases that address the need to integrate disparate information on blood development have typically focused on a specific subset of blood cells , or on a specific aspect of gene behaviour . for example , hemopdb ( 4 ) focuses on promoters and tfs that are active during haematopoiesis in human , mouse and rat . epodb ( 5 ) is built upon a controlled vocabulary of keywords important for erythropoiesis ( the development of a red blood cell from a bfu - e cell ) to mine public databases . those genes eventually considered to be relevant to vertebrate erythropoiesis are integrated into the database together with their structural , functional , regulatory and gene expression information . hembase ( 6 ) harbours ests and genes from mrna gene libraries from developmentally staged , primary human erythroblasts . finally , lymphtf - db ( 7 ) holds information on murine tfs and their specific targets at various points in b- and t - cell development . at present , no publicly available database has integrated expression profiles across all major branches of the haematopoietic hierarchy . reconstruction of the regulatory networks that govern the differentiation of haematopoietic stem cells towards the multiple mature lineages will depend on ready access to comprehensive expression datasets . here we present bloodexpress , an integrated platform and database of mouse blood expression with flexible search capability that covers the most immature stem cells , intermediate multipotent progenitors and mature blood cell types . bloodexpress integrates expression data from 271 individual array experiments derived from 15 distinct studies : akashi et al . ( 9 ) , chen et al . ( 10 ) , dudziak et al . ( 11 ) , ficara et al . these datasets represent 37 distinct blood cell types , including all major precursors and mature cell types ( figure 1 ) . tables s1 and s2 summarize the technical details of the expression studies and the purification strategies for the above cell types , respectively . the expression data of the above studies were obtained via the gene expression omnibus ( 23 ) and stembase ( 24 ) , or were kindly supplied by the authors . microarray probes were mapped to the mouse ensembl gene database ( ncbi m37 assembly ) ( 25 ) and discretized to present ( p ) , absent ( a ) , or unknown ( u ) values ( if the gene was not represented on the array ) . to do this , all 271 distinct arrays were processed with the mas5calls method of the affy library ( 26 ) , which performs a wilcoxon signed rank test on every probe set . the p 0.04 as recommended by affymetrix is indicative of specific signal , a p 0.06 indicates lack of specific signal , and a p - value of between 0.040.06 is typically flagged as marginal. however , in order to diminish the number of false positives , all probe sets with p > 0.04 were annotated as a. for those studies that included biological replicates , the following strategy was adopted : if the p - values of a given probe were 0.04 in > 50% of biological replicates , then the probe in question was flagged as p in that particular cell type . most genes on the affymetrix arrays present in the database are represented by a single probe . as a second step for those genes represented by multiple probes , only if > 50% probes were labelled as p. we compared the results of our p - value - based discretization strategy with the published dataset by chambers et al . ( 9 ) , where a normalization score 5 for a given affymetrix probe is reported by the authors as indicative of specific signal . the chambers et al . ( 9 ) dataset covers eight cell types and contains two biological replicates per cell type . in order to compare the specific signal as provided by the wilcoxon signed rank test ( p - value ) and the normalized scores as provided in chambers et al . ( 9 ) , the two datasets were discretized as described above : only if > 50% of probes across biological replicates had a p 0.04 or normalized score 5.0 , would the probe be annotated as p. also , for those genes with multiple probes , only if > 50% of probes from the previous probe - level annotation step were flagged as p , would the gene ultimately be annotated as dataset ( 9 ) , both with the p - value and normalized score cutoffs , the overlap between the two matrices was found to be 91% . no significant improvement in overlap was observed by varying the recommended p - value cutoff of 0.04 or the cutoff for specific signal from the normalized scores . it is likely that a larger number of biological replicates in the chambers et al . 's study ( 9 ) would have resulted in a greater overlap . moreover , the expression patterns of some genes known to be involved in haematopoietic processes were inspected . these included the b - cell - specific factor pax5 , the transcriptional regulators bcl11a / bcl11b , which control b- and t - cell development , respectively , and the gfi1/gfi1b transcriptional repressors involved in lymphoid versus erythroid development . presence / absence calls for these genes across the haematopoietic hierarchy were found to be in good agreement with published experimental data . annotation of transcription factors was carried out by mapping all mouse ensembl genes to the dna - binding domain ( dbd ) database ( 27 ) . the dbd is a database of predicted sequence - specific dna - binding domains based on a hidden markov model library of the superfamily and pfam databases . many potential users of bloodexpress may want to be able to focus their analyses on transcription factors because transcriptional regulation is a key factor controlling haematopoiesis , a fact underlined by the large number of transcription factor genes that play key roles in normal haematopoiesis and/or the development of leukaemia ( 13 ) . information stored for each mouse ensembl gene includes its annotated name in the ensembl database , its description , alternative gene names according to the mouse genome database ( 28 ) , and its chromosomal location . moreover , the panther database ( 29 ) was used to retrieve the ontology annotation for the ensembl genes . the bloodexpress database can be accessed through a user - friendly web interface ( http://hscl.cimr.cam.ac.uk/bloodexpress/ ) with two principal modes of searching the database : gene - centric and cell type - centric . for gene - centric searches , the search::genes interface ( figure 2 ) allows the user to input a gene i d in ensembl or mgi formats , and thus retrieve the blood cell types where the gene in question is expressed . in those cases where an mgi gene name is shared by more than one gene ( e.g. sly ) , keyword searches are also allowed , in which case the input is matched against the ensembl gene name and description fields . as before , a number of candidates are returned for the user to choose from . the information ultimately returned on every gene includes the ensembl gene i d ( hyperlinked to the ensembl database for further exploratory analysis ) , the ensembl gene name , its chromosomal coordinates , the ensembl gene description and whether it is a predicted tf according to the dbd database . if the gene in question is a predicted tf , the search::genes interface also allows the user to optionally retrieve detailed superfamily / pfam protein family annotation . this is followed by a detailed list of cell types where the gene has been found to be expressed , with reference to the microarray paper by means of a hyperlink to pubmed ( http://www.pubmed.org/ ) . the user can search a gene 's expression pattern by entering its mgi or ensembl i d , or by specifying a keyword that is searched against a list of gene names and gene descriptions before a list of candidates is returned to the user . the user can also specify a similarity threshold to retrieve genes with similar expression patterns . the user can search a gene 's expression pattern by entering its mgi or ensembl i d , or by specifying a keyword that is searched against a list of gene names and gene descriptions before a list of candidates is returned to the user . the user can also specify a similarity threshold to retrieve genes with similar expression patterns . while it is useful to know the cell type(s ) where a particular gene of interest is expressed , it is also valuable to ascertain what other genes follow a similar pattern of expression across the haematopoietic hierarchy . since a gene 's expression pattern in our database is laid out as a vector where the columns are the distinct cell types , vector comparisons can be employed to identify identical and similar patterns of expression . the metric we have used to compare vectors relies on the hamming distance ( hd ; 30 ) . in information theory , the hd between two vectors of equal length is the number of positions for which the corresponding symbols are different . the percentage similarity between the genes in our database is calculated as follows in what we call the expression similarity score ( ess ) : ess = [ ( l hd)/l ] 100 , where l is the number of cell types being compared and hd is the number of cell types where the expression calls for the two genes being compared are different . the search::genes interface allows the user to specify a level of similarity between the query gene and other genes in the database . the query gene is compared on - the - fly against the rest of the genes in the database , and those genes with an ess above the cutoff specified by the user are returned . in those cases where the gene of interest has been covered in a limited number of cell types in the original studies , an additional score , the coverage score , is provided and which divides the ess described above by the fraction of cell types upon which the comparison has been performed out of all the distinct cell types in the database . the coverage score thus provides an estimate of how general the expression pattern in question is across the haematopoietic hierarchy . for cell - type - centric searches , the user can select a set of cell types in the search::cells interface ( figure 3 ) . a list of all the ensembl genes expressed in the specified cell types is returned with their detailed annotation and hyperlinked to the mouse ensembl database . moreover , the user can further refine queries by excluding those genes that are expressed in a second list of cell types . the resulting gene list can be further filtered by considering only predicted transcription factors as annotated in the dbd database ( 27 ) and/or those genes with specific ontology terms based on the annotation provided by the panther database ( 29 ) . each of the three main levels of annotation of the panther database ( biological process , pathway and molecular function ) contains a number of second - level categories ( 31 , 152 and 29 categories , respectively ) . the user is thus able to filter those genes with a specific ontology annotation such as defense / immunity protein ( mf00173) , t - cell activation ( p00053) or oncogenesis ( bp00281). figure 3.snapshot of the cell type - centric search::cells web interface . the user can select a cell type to return a list of genes expressed therein . queries can be refined further by excluding those genes that are expressed in the second list of cell types . the resulting gene lists can be filtered further by considering only predicted transcription factors , or genes with specific ontology terms . the user can select a cell type to return a list of genes expressed therein . queries can be refined further by excluding those genes that are expressed in the second list of cell types . the resulting gene lists can be filtered further by considering only predicted transcription factors , or genes with specific ontology terms . the server is implemented as a set of perl cgi scripts running under apache ( http://www.apache.org/ ) . bloodexpress integrates expression data from 271 individual array experiments derived from 15 distinct studies : akashi et al . ( 9 ) , chen et al . ( 10 ) , dudziak et al . ( 11 ) , ficara et al . these datasets represent 37 distinct blood cell types , including all major precursors and mature cell types ( figure 1 ) . tables s1 and s2 summarize the technical details of the expression studies and the purification strategies for the above cell types , respectively . the expression data of the above studies were obtained via the gene expression omnibus ( 23 ) and stembase ( 24 ) , or were kindly supplied by the authors . microarray probes were mapped to the mouse ensembl gene database ( ncbi m37 assembly ) ( 25 ) and discretized to present ( p ) , absent ( a ) , or unknown ( u ) values ( if the gene was not represented on the array ) . to do this , all 271 distinct arrays were processed with the mas5calls method of the affy library ( 26 ) , which performs a wilcoxon signed rank test on every probe set . the p 0.04 as recommended by affymetrix is indicative of specific signal , a p 0.06 indicates lack of specific signal , and a p - value of between 0.040.06 is typically flagged as marginal. however , in order to diminish the number of false positives , all probe sets with p > 0.04 were annotated as a. for those studies that included biological replicates , the following strategy was adopted : if the p - values of a given probe were 0.04 in > 50% of biological replicates , then the probe in question was flagged as p in that particular cell type . most genes on the affymetrix arrays present in the database are represented by a single probe . as a second step for those genes represented by multiple probes , only if > 50% probes were labelled as p. we compared the results of our p - value - based discretization strategy with the published dataset by chambers et al . ( 9 ) , where a normalization score 5 for a given affymetrix probe is reported by the authors as indicative of specific signal . the chambers et al . ( 9 ) dataset covers eight cell types and contains two biological replicates per cell type . in order to compare the specific signal as provided by the wilcoxon signed rank test ( p - value ) and the normalized scores as provided in chambers et al . ( 9 ) , the two datasets were discretized as described above : only if > 50% of probes across biological replicates had a p 0.04 or normalized score 5.0 , would the probe be annotated as p. also , for those genes with multiple probes , only if > 50% of probes from the previous probe - level annotation step were flagged as p , would the gene ultimately be annotated as dataset ( 9 ) , both with the p - value and normalized score cutoffs , the overlap between the two matrices was found to be 91% . no significant improvement in overlap was observed by varying the recommended p - value cutoff of 0.04 or the cutoff for specific signal from the normalized scores . it is likely that a larger number of biological replicates in the chambers et al . 's study ( 9 ) would have resulted in a greater overlap . moreover , the expression patterns of some genes known to be involved in haematopoietic processes were inspected . these included the b - cell - specific factor pax5 , the transcriptional regulators bcl11a / bcl11b , which control b- and t - cell development , respectively , and the gfi1/gfi1b transcriptional repressors involved in lymphoid versus erythroid development . presence / absence calls for these genes across the haematopoietic hierarchy were found to be in good agreement with published experimental data . annotation of transcription factors was carried out by mapping all mouse ensembl genes to the dna - binding domain ( dbd ) database ( 27 ) . the dbd is a database of predicted sequence - specific dna - binding domains based on a hidden markov model library of the superfamily and pfam databases . many potential users of bloodexpress may want to be able to focus their analyses on transcription factors because transcriptional regulation is a key factor controlling haematopoiesis , a fact underlined by the large number of transcription factor genes that play key roles in normal haematopoiesis and/or the development of leukaemia ( 13 ) . information stored for each mouse ensembl gene includes its annotated name in the ensembl database , its description , alternative gene names according to the mouse genome database ( 28 ) , and its chromosomal location . moreover , the panther database ( 29 ) was used to retrieve the ontology annotation for the ensembl genes . the bloodexpress database can be accessed through a user - friendly web interface ( http://hscl.cimr.cam.ac.uk/bloodexpress/ ) with two principal modes of searching the database : gene - centric and cell type - centric . for gene - centric searches , the search::genes interface ( figure 2 ) allows the user to input a gene i d in ensembl or mgi formats , and thus retrieve the blood cell types where the gene in question is expressed . in those cases where an mgi gene name is shared by more than one gene ( e.g. sly ) , keyword searches are also allowed , in which case the input is matched against the ensembl gene name and description fields . as before , a number of candidates are returned for the user to choose from . the information ultimately returned on every gene includes the ensembl gene i d ( hyperlinked to the ensembl database for further exploratory analysis ) , the ensembl gene name , its chromosomal coordinates , the ensembl gene description and whether it is a predicted tf according to the dbd database . if the gene in question is a predicted tf , the search::genes interface also allows the user to optionally retrieve detailed superfamily / pfam protein family annotation . this is followed by a detailed list of cell types where the gene has been found to be expressed , with reference to the microarray paper by means of a hyperlink to pubmed ( http://www.pubmed.org/ ) . the user can search a gene 's expression pattern by entering its mgi or ensembl i d , or by specifying a keyword that is searched against a list of gene names and gene descriptions before a list of candidates is returned to the user . the user can also specify a similarity threshold to retrieve genes with similar expression patterns . the user can search a gene 's expression pattern by entering its mgi or ensembl i d , or by specifying a keyword that is searched against a list of gene names and gene descriptions before a list of candidates is returned to the user . the user can also specify a similarity threshold to retrieve genes with similar expression patterns . while it is useful to know the cell type(s ) where a particular gene of interest is expressed , it is also valuable to ascertain what other genes follow a similar pattern of expression across the haematopoietic hierarchy . since a gene 's expression pattern in our database is laid out as a vector where the columns are the distinct cell types , vector comparisons can be employed to identify identical and similar patterns of expression . the metric we have used to compare vectors relies on the hamming distance ( hd ; 30 ) . in information theory , the hd between two vectors of equal length is the number of positions for which the corresponding symbols are different . the percentage similarity between the genes in our database is calculated as follows in what we call the expression similarity score ( ess ) : ess = [ ( l hd)/l ] 100 , where l is the number of cell types being compared and hd is the number of cell types where the expression calls for the two genes being compared are different . the search::genes interface allows the user to specify a level of similarity between the query gene and other genes in the database . the query gene is compared on - the - fly against the rest of the genes in the database , and those genes with an ess above the cutoff specified by the user are returned . in those cases where the gene of interest has been covered in a limited number of cell types in the original studies , the ess will only be based on a limited number of comparisons . an additional score , the coverage score , is provided and which divides the ess described above by the fraction of cell types upon which the comparison has been performed out of all the distinct cell types in the database . the coverage score thus provides an estimate of how general the expression pattern in question is across the haematopoietic hierarchy . for cell - type - centric searches , the user can select a set of cell types in the search::cells interface ( figure 3 ) . a list of all the ensembl genes expressed in the specified cell types is returned with their detailed annotation and hyperlinked to the mouse ensembl database . moreover , the user can further refine queries by excluding those genes that are expressed in a second list of cell types . the resulting gene list can be further filtered by considering only predicted transcription factors as annotated in the dbd database ( 27 ) and/or those genes with specific ontology terms based on the annotation provided by the panther database ( 29 ) . each of the three main levels of annotation of the panther database ( biological process , pathway and molecular function ) contains a number of second - level categories ( 31 , 152 and 29 categories , respectively ) . the user is thus able to filter those genes with a specific ontology annotation such as defense / immunity protein ( mf00173) , t - cell activation ( p00053) or oncogenesis ( bp00281). figure 3.snapshot of the cell type - centric search::cells web interface . the user can select a cell type to return a list of genes expressed therein . queries can be refined further by excluding those genes that are expressed in the second list of cell types . the resulting gene lists can be filtered further by considering only predicted transcription factors , or genes with specific ontology terms the user can select a cell type to return a list of genes expressed therein . queries can be refined further by excluding those genes that are expressed in the second list of cell types . the resulting gene lists can be filtered further by considering only predicted transcription factors , or genes with specific ontology terms . the server is implemented as a set of perl cgi scripts running under apache ( http://www.apache.org/ ) . bloodexpress provides the first comprehensive integration of published expression datasets , covering the majority of mouse blood cell types and accessible through a user - friendly web interface ( http://hscl.cimr.cam.ac.uk/bloodexpress/ ) . the gene - centric and cell - type - centric interfaces allow the distinctive and important advantage of identifying gene expression patterns across the well - defined haematopoietic hierarchy . the flexibility of filtering by gene functional category enhances the capability of the bloodexpress platform for discovering previously uncharacterized genes with expression patterns similar to those of genes already known to be important in blood development . transcriptional regulation is key in controlling haematopoiesis as many tfs , or their regulation , are mutated in leukaemias . the reconstruction of regulatory networks that govern the differentiation of haematopoietic cells towards the multiple mature cell lineages , in both normal and pathological states , will be facilitated by the large - scale exploration of gene expression patterns as provided in bloodexpress . the datasets included in bloodexpress can aid in the reconstruction and/or analysis of regulatory networks by providing gene lists for gene set enrichment analysis as well as the analysis of consistency of inferred networks across multiple lineages . moreover , the underlying database structure and web server implementation is such that it could be readily adapted for studying regulatory networks in other biological contexts and stem cell systems . future developments of bloodexpress include the integration of additional mouse blood cell expression datasets as they become available and , most importantly , those of mouse models of various types of leukaemia and other haematopoietic diseases . the leukaemia research fund , medical research council and the leukemia and lymphoma society . funding for open access charge : leukemia and lymphoma society .

haematopoiesis is the process whereby blood stem cells give rise to at least fourteen functionally distinct mature cell types , and represents the best characterized mammalian adult stem cell system . here we introduce the bloodexpress database , the first public resource integrating mouse blood cell expression profiles . bloodexpress enables the searching of data from individual studies in a single database accessible through a user - friendly web interface . microarray datasets have been processed uniformly to allow their comparison on the bloodexpress platform . bloodexpress covers the majority of murine blood cell types , including both progenitors and terminally differentiated cells . this allows for the identification of dynamic changes in gene expression as cells differentiate down the well - defined haematopoietic hierarchy . a gene - centric interface returns haematopoietic expression patterns together with functional annotation and a list of other genes with similar expression patterns . a cell type - centric interface allows the identification of genes expressed at specific points of blood development , with the additional and useful capability of filtering by specific gene functional categories . bloodexpress thus constitutes a platform for the discovery of novel gene functions across the haematopoietic tree . bloodexpress is freely accessible at http://hscl.cimr.cam.ac.uk / bloodexpress/.

INTRODUCTION MATERIALS AND METHODS Contents of BloodExpress The BloodExpress web interface Implementation SUMMARY AND FUTURE DEVELOPMENTS SUPPLEMENTARY DATA FUNDING

PMC156637

cross - cultural comparisons , case - control and prospective observational studies identified a relationship between diet , blood pressure and lipids levels [ 3 - 6 ] , but there is still considerable scientific uncertainty about the relationship between specific dietary components and cardiovascular risk , especially in mediterranean populations . in this work we quantified the public health benefit of fruit and vegetable consumption on the primary prevention of acute coronary syndromes ( acute myocardial infarction or unstable angina ) using a large sample of cardiac patients and frequency - matched controls from all greek regions . the cardio2000 is a multicentre case - control study that explores the association between several demographic , nutritional , lifestyle , clinical and biochemical risk factors with the risk of developing non - fatal acute coronary syndromes . from january 2000 to march 2002 , we randomly selected 848 coronary patients and 1078 cardiovascular symptom free subjects ( controls ) . the number of the enrolled subjects was decided through power analysis , in order to evaluate ( two sided ) differences in the coronary relative risk greater than 7% with statistical power greater than 80% and significant level less than 5% . according to the population distribution provided by the national statistical services ( ministry of economics , census 1991 ) , we stratified our sampling into all the greek regions . controls were mainly individuals who visited the outpatient departments of the same hospital at the same period with the coronary patients , for routine examinations or minor surgical operations . in a few cases ( in country hospitals ) where the available number of hospitalised controls was not sufficient for the matching procedure , we enrolled into the study friends or colleagues of the coronary patients . all participants were informed about the aims of the study and agreed to give the requested information . in order to eliminate recall bias we tried to retrieve precise information from cases and controls and medical history through hospital or the insurance records . the coronary patients were selected from the admission listing of the cardiological clinics . according to the study 's protocol the patients were hospitalised for an acute first event of coronary heart disease ( acute myocardial infarction or unstable angina ; stable angina was excluded from the analysis ) . the selection criteria for cardiac patients are : diagnosis of first acute myocardial infarction ( mi ) . mi was defined by any two features : electrocardiographic changes , compatible clinical symptoms , and specific diagnostic enzyme elevations of sgot , cpk , ldh and troponin t or i ( 49% of the patients had mi ) ; or diagnosis of unstable angina ( i.e. one or more angina episodes at rest within the preceding 48 hours ) corresponding to class iii of the braunwald classification ( 51% of the patients had unstable angina ) . afterwards , we randomly selected subjects without any clinical symptoms , signs or suspicions of cardiovascular disease in their medical history ( controls ) , frequently matched to the patients by age ( 3 years ) , sex , and region . the cardio2000 investigators performed the evaluation of clinical symptoms and signs in the series of cases and controls . the information regarding the investigated medical factors was retrieved from the subjects ' medical records , along with lifestyle characteristics , through a confidential , detailed questionnaire administered during physician interview after the second day of hospitalisation , for cases and at entry for controls . in particular , the educational level was classified into three groups : group i : education through high school ( 0 9 years of schooling ) ; group ii : high school , technical education up to 14 years of schooling ( but not academic ) and group iii : ( university ) . former smokers were those who had stopped smoking for at least one year , and never smokers those who never smoked a cigarette in their lives . those who stopped smoking for less than a year were classified as smokers . quantification of smoking status was based on the calculation of pack - years adjusted for nicotine content equal to 0.8 mg per cigarette . physical activity was defined as any type of non - occupational physical exercise , at least once / week during the past year , and was graded in qualitative terms such as light ( expended calories < 4 kcal / min , i.e. walking slowly , stationary cycling , light stretching etc . ) , moderate ( expended calories 47 kcal / min , i.e. walking briskly , outdoor cycling , swimming moderate effort etc . ) and vigorous ( expended calories > 7 kcal / min , i.e. walking briskly uphill , long distance running , cycling fast or racing , swimming fast crawl etc . ) . the rest of the subjects were defined as physically inactive . moreover , since physical activity is an important factor for cardiovascular disease , we took into account the occupational activity status of all participants . the evaluation of the nutritional habits was accomplished with a validated questionnaire based on the guidelines from the department of nutrition of the national school of public health . we measured the consumption of several food items as an average per week during the past year . the frequency of consumption was quantified approximately in terms of the number of times per month this food was consumed . thus , daily consumption multiplied by 30 and weekly consumption multiplied by 4 and a value of 0 was assigned to food items rarely or never consumed . in order to describe overall diet we used composite scores , which are necessary for the evaluation of epidemiological associations . specifically for fruit and vegetable intake the participants reported their average consumption of a specific portion size for these foods over the past year , including apples , grapes , pears , bananas , oranges , etc , as well as broccoli , carrots , spinach , lettuce salad , yellow squash , and tomatoes . vegetable intake was categorized into < 1 serving / day , 1 1.49 servings / day , 1.5 1.99 servings / day , 2 2.49 servings / day , and 2.5 + servings / day , based both on maintaining a natural gradient of exposure and including adequate participants in each category . the provided list of fruits and vegetables was comprehensive enough to capture most types of fruits and vegetables that our subjects consumed . although fruit juice was recorded it was not evaluated due to low consumption ( < 5% ) . alcohol , a potential confounder for total energy intake , was measured by daily ethanol consumption ( red or white wine , beer , and other beverages ) , in wineglasses of 100 ml and 12% ethanol concentration ) as done by other investigators . according to the collected medical information , the majority of the controls ( 86% ) and the patients ( 83% ) had at least one laboratory measurement during the past 12 months . in addition , we measured , both in patients and controls , arterial blood pressure levels in the right arm ( average of 3 measurements having the patient seated and rested ) , total cholesterol and fasting glucose levels . for the cardiac patients the measurements were collected during the first 12 h of hospitalization and at the end of the interview for the controls . the previous information as well as the patients ' and controls ' reports assisted us in characterizing the subjects as having hypertension , hypercholesterolemia , or diabetes . in keeping with the long - standing classification criteria used in several population - based studies , patients whose blood pressure , according to their medical records , were greater or equal to 140 / 90 mmhg or were taking antihypertensive medication were classified as hypertensives . hypercholesterolemia was defined as cholesterol levels greater than 220 mg / dl or greater than 200 mg / dl when two other risk factors for coronary heart disease were present or if hypo - lipidemic treatment was administered . diabetics were those with fasting blood glucose greater than 125 mg / dl or those who were under special diet or treatment . finally , we measured the height and the weight both in patients and controls and we calculated the body mass index ( bmi = weight / { height } ) . in addition , we asked for any significant changes in their body mass during the past years . obesity was defined as bmi > 29.9 kg / m. presence of premature coronary heart disease among first - degree relatives ( < 55 years for male relatives and < 65 for female relatives ) was also recorded . further details regarding the aims , design and methodology of the cardio2000 study have been previously presented [ 10 - 13 ] . continuous variables are presented as mean one standard deviation , while qualitative variables are presented as absolute and relative frequencies . in order to fit a multivariate risk model that evaluates the investigated parameters on the risk of developing acute coronary syndromes , stratified by sex contingency tables were conducted in order to investigate associations between categorical variables and groups of study , by the calculation of the overall chi - squared criterion . also , wilcoxon non - parametric criterion was applied in order to evaluate differences in continuous measurements ( i.e. body mass index ) between groups . estimations of the relative risks of developing acute coronary syndromes were performed by the calculation of the odds ratio ( or ) and the corresponding confidence intervals through multiple conditional logistic regression analysis , adjusting for age ( in years ) , body mass index ( bmi ) ( kg / m ) , smoking , alcohol intake , physical activity , history of high cholesterol or hypertension or diabetes or premature chd , and use of multivitamins . we then conducted a stratified analysis according to smoking status ( past , current , never ) to evaluate whether the relation between fruits and vegetable intakes and chd risk differed by these variables . tests of linear trend across increasing categories of vegetable consumption were conducted by assigning the medians of intakes in categories ( servings / day ) treated as a continuous variable . all reported p - values are from two - sided tests and compared to a significant level of 5% . continuous variables are presented as mean one standard deviation , while qualitative variables are presented as absolute and relative frequencies . in order to fit a multivariate risk model that evaluates the investigated parameters on the risk of developing acute coronary syndromes , stratified by sex contingency tables were conducted in order to investigate associations between categorical variables and groups of study , by the calculation of the overall chi - squared criterion . also , wilcoxon non - parametric criterion was applied in order to evaluate differences in continuous measurements ( i.e. body mass index ) between groups . estimations of the relative risks of developing acute coronary syndromes were performed by the calculation of the odds ratio ( or ) and the corresponding confidence intervals through multiple conditional logistic regression analysis , adjusting for age ( in years ) , body mass index ( bmi ) ( kg / m ) , smoking , alcohol intake , physical activity , history of high cholesterol or hypertension or diabetes or premature chd , and use of multivitamins . we then conducted a stratified analysis according to smoking status ( past , current , never ) to evaluate whether the relation between fruits and vegetable intakes and chd risk differed by these variables . tests of linear trend across increasing categories of vegetable consumption were conducted by assigning the medians of intakes in categories ( servings / day ) treated as a continuous variable . all reported p - values are from two - sided tests and compared to a significant level of 5% . seven hundred ( 83% ) of the coronary patients were males and 148 ( 17% ) were females , while 862 ( 80% ) of the controls were males and 216 ( 20% ) were females . the data analysis revealed that 365 ( 43% ) of the cardiac patients and 722 ( 67% ) of the controls consumed some quantity of fruit and vegetable daily . of these , 28 ( 3% ) patients and 15 ( 1% ) controls reported consuming < 1 serving / day ; 351 ( 41% ) patients and 377 ( 35% ) controls reported consuming 1 1.49 servings / day ; 344 ( 40% ) patients and 428 ( 40% ) controls reported 1.5 1.99 servings / day ; 100 ( 9% ) patients and 150 ( 14% ) controls reported 2 2.49 servings / day , and 33 ( 7% ) patients and 100 ( 10% ) controls consumed 2.5 + servings / day ( p for trend < 0.001 ) . we then examined the association between the consumption of fruit , or vegetable and the prevalence of hypertension , diabetes mellitus , hypercholesterolemia , as well as cigarette smoking , body mass index , and physical inactivity , since these factors are considered modifiers of the effect of fruits and vegetables on coronary risk . after stratifying our analysis by group of study ( patients vs. controls ) , an inverse association revealed between fruit and vegetable consumption and cigarette smoking ( p - value = 0.008 , p - value = 0.04 , respectively ) , prevalence of hypertension ( p - value = 0.038 , and p = 0.067 , respectively ) , and diabetes mellitus ( p = 0.044 , and p - value = 0.04 , respectively ) . furthermore , a positive association was found between fruit and vegetable consumption and education level both in patients and controls ( p - value = 0.042 , p - value = 0.001 , respectively ) . no significant associations were observed between fruit and vegetables intake and the prevalence of hypercholesterolemia ( p = 0.574 , p - value = 0.804 , respectively ) , obesity ( p - value = 0.579 , p - value = 0.565 , respectively ) , alcohol consumption ( p - value = 0.939 , p - value = 0.919 , respectively ) and physical inactivity status ( p - value = 0.384 , p - value = 0.067 , respectively ) . after adjusting for the aforementioned risk factors , those in the upper quintile of fruit consumption ( 5 or more items /day ) had 72% lower relative risk for chd ( or = 0.28 , 95% ci 0.11 0.54 ) , compared with those in the lowest quintile of intake ( < 1 item / day ) . similarly , consumption of vegetable more than 3 days / week was associated with 70% lower relative risk for chd ( odds ratio = 0.30 , 95% ci 0.22 0.40 ) , compared with those that they did not consume vegetables . of particular interest , a 10% reduction in coronary risk was observed for every additional piece of fruit consumed per day ( or = 0.90 , 95% ci 0.85 0.97 ) , after controlling for the conventional cardiovascular risk factors . adjusting for the same covariates in an analysis of the overall trend , considering vegetable intake as a continuous variable , we found an odds ratio of 0.81 ( 95% ci 0.67 0.93 ) for developing chd per each additional serving / day of vegetable consumed . the individual effect of fruit and vegetable consumption on coronary risk is presented in table 2 . moreover , green vegetable intake showed a 12% reduction on coronary risk per item / day ( odds ratio = 0.88 , 95% ci 0.77 0.97 ) , and vitamin c - rich fruits showed an 11% reduction per item / day ( odds ratio = 0.89 , 95% ci 0.84 0.95 ) . characteristics of the study 's population comparisons were based on the chi - squared test and the wilcoxon criterion . we then stratified the previous analysis by smoking status ( current vs. never / former smokers ) and found that the protective effect of fruit and vegetable consumption on coronary risk is diminished in the subgroup of heavy ( + 31 cigarettes per day ) current smokers . consumption of 2.5 + servings of vegetable per day is associated with a 27% lower risk of developing acute coronary syndromes compared to those who consumed < 1 serving per day ( odds ratio = 0.73 , 95% ci 0.27 1.96 ) . moreover , each 1-piece / day increase of fruit consumption is associated with 5% lower coronary risk in current smokers ( odds ratio = 0.95 , 95% ci 0.86 1.04 ) . the present study revealed that even a moderate consumption of fruit and vegetable 1 2 servings per day ) is associated with significantly lower risk of coronary events , even after controlling for several potential confounding risk factors ( table 2 ) . furthermore , the risk was progressively lower as the consumption of fruits or vegetables increased . those in the highest quintile of consumption ( 2.5 or more serving per day ) had about 70% lower relative risk when compared to those in the lowest quintile moreover , some protection appears to be provided even in the group of current smokers . results from the multivariate logistic model that was developed for the evaluation of the effect of fruits and vegetables consumption on coronary risk the odds ratios were estimated after taking into account the effect of hypertension , hypercholesterolemia , diabetes mellitus , family history of premature coronary heart disease , physical activity status , smoking habits , body mass index , alcohol consumption , and education status of the participants . our findings are in accordance with those of previous epidemiological studies , which concluded that the consumption of fruits and vegetables might reduce disease risk , particularly for cancer and cardiovascular disease . however , less is known about the direct association between fruits and vegetable intake and risk of acute coronary syndromes . nine of ten ecological studies two out of three case - control studies and six of sixteen cohort studies reported a significant protective association of chd with consumption of fruit and vegetables . moreover , a review of 250 observational studies reported that increased consumption of fruits and vegetables could reduce cardiovascular deaths from 6% to 22% ( 8,000 deaths annually ) . our findings support that even low consumption of fruits and vegetables ( 12 servings per week ) is associated with about 45% lower coronary risk ( table 2 ) . consumption of 2 or more servings per week is associated with about 70% reduction in relative risk . this translates to a considerable prevention of acute coronary events , carries a large public health potential , and emphasises the importance of following dietary recommendations as a whole . moreover , our findings on smokers and the consumption of fruits and vegetables are worth mentioning . although statistical significance was not achieved , a trend was evident , suggesting that the consumption of fruits and vegetables may offer some protection for current smokers . since smokers consume substantially less fruit and fruit juice then non - smokers with heavy smokers eating the least , special attention should be given to dietary interventions for smokers in order to improve their nutrition . a discussion of the mechanisms responsible for the protection fruit and vegetable consumption offers is beyond the scope of this study . however , several investigators reported that the antioxidant , potassium , fiber , and folate content of fruits and vegetables could at least partially explain their protective effect of chd [ 18 - 20 ] . other compounds such as flavonoids , phytates , lycopene , carotenoids , and other phytochemicals in vegetables may also have significant protective effects in reducing coronary risk . despite our attempt to control for several known confounding factors , it is still possible that the observed inverse association between fruits and vegetable intake and coronary risk could be at least partially explained by other factors associated with heart - healthy behaviours . also , in retrospective case - control studies two main sources of systematic errors may exist , the selection and the recall bias . in order to eliminate selection bias we set objective criteria both for patients and controls . however , insignificant misclassification may exist , since a small percentage of asymptomatic coronary patients may be wrongly assigned to controls , even they were evaluated by a cardiologist . moreover , in case - control studies it is usually observed that patients who had a recent adverse event are more likely to place greater emphasis on several factors related to the disease than the control group ( recall bias ) . to reduce this type of bias and analyse precise information , we obtained accurate information from the patients as well as from their relatives or their accompanying persons and compare their responses with kendall 's tau - criterion . concerning the medical information we avoided recall bias by obtaining detailed data from subjects ' medical records however , over / under estimation of the relative risk may exist , especially due to the measurement of nutritional and smoking habits and the onset of the investigated cardiovascular risk factors . moreover , the coronary patients who died at entry or the day after were not included into the study . this bias could influence our results . however , the proportion of deaths during the first two days was less than 4% . thus , it is unlikely that the exclusion of fatal events has significantly altered our findings . finally , we tried to reduce the potential effect of uncontrolled or unknown confounders using the same study base , for patients and controls . the presented scientific evidences are sufficient to give good reason for public health education and promotion aimed at a substantial increase in the consumption of fruits and vegetables . however , our data do not suggest evidences for causality and population trials are needed in order to provide scientific proof of their efficacy . dp : design of the study , statistical analysis and drafted the manuscript , cp : design of the study and drafted the manuscript , pk : editing , cc : drafted the manuscript , mv : editing , cs : conception of the study and editing , and pt : conception of the study and editing . this study is supported by research grants from the hellenic heart foundation ( 11/19992002 ) . the authors would like to thank the physicians and the specialists that coordinated the data collection of this study : dr k tzioumis ( athens , crete , pelloponisos ) , dr j skoumas ( athens ) , dr n papaioannou ( athens , thessalia ) , dr p starvopodis ( ionian islands ) , dr l karra ( aegean islands ) , dr d antoniades ( macedonia ) , dr g rembelos ( aegean islands ) , dr d markou ( athens ) , a moraiti ( athens ) , d evagelou ( crete ) , dr s vellas ( attica , hpeirous ) , dr . g skoumbourdis ( sterea hellas ) , b meidanis ( macedonia , sterea hellas , thessalia ) , dr s loggos ( attica ) , dr g zavitsanakis ( sterea hellas ) , dr i elefsiniotis ( athens ) , dr a theodorakis ( sterea hellas ) , dr n marinakis ( aegean islands ) , dr t kyratzoglou ( east macedonia ) and s zombolos ( peloponnese ) .

backgroundthe relation between diet and human health has long been investigated . the aim of this work is to evaluate the association between chd risk and the consumption of fruit and vegetable , in a large sample of cardiac patients and controls.methodsstratified sampling from all greek regions , consisted of 848 ( 700 males , 58 10 years old and 148 females , 65 9 years old ) randomly selected patients , admitted to the cardiology clinic for a first event of an acute coronary syndrome ( acs ) . in addition we selected 1078 frequency paired , by sex - age - region , controls in the same hospitals but without any clinical suspicion of chd . using validated food - frequency questionnaires we assessed total diet , including fruit and vegetable intake , on a weekly basis . multiple logistic regression analysis estimated the relative risk of developing acs by level of fruits and vegetables intake after taking into account the effect of several potential confounders.resultsdata analysis revealed that the benefit of fruit or vegetable consumption increases proportionally by the number of servings consumed ( p for trend < 0.001 ) . after adjusting for the conventional cardiovascular risk factors , those in the upper quintile of fruit consumption ( 5 or more items / day ) had 72% lower risk for chd ( odds ratio = 0.28 , 95% ci 0.11 0.54 , p < 0.001 ) , compared with those in the lowest quintile of intake ( < 1 items / day ) . similarly , consumption of vegetable more than 3 days / week was associated with 70% lower risk for chd ( odds ratio = 0.30 , 95% ci 0.22 0.40 , p < 0.001 ) , compared with those that they did not consume vegetables . of particular interest , a 10% reduction in coronary risk was observed for every one piece of fruit consumed per day ( odds ratio = 0.90 , 95% ci 0.85 0.97 , p = 0.004).conclusionsconsumption of fruits and vegetables seems to offer significant protection against chd .

Background Methods Statistical analysis Results Discussion Conclusion Competing interests Authors' contributions Acknowledgment

PMC5009051

an 18-year - old male visited our hospital complaining of right knee pain that had lasted for 7 months . the patient was 182.1-cm tall and weighed 103.4 kg ( 228 lb ) and was a wrestler who had been active in athletics for over 10 years . on physical examination , the joint was negative for locking , snapping , and giving way and showed a normal range of motion ; however , he had been experiencing a catching sensation for 1 year . dull pain was localized on the anterolateral side of the right knee , and there was tenderness in the same region . 1 ) . given the patient 's physique , athletic activity ( wrestling ) , and the size of the phlm tear , we thought that the radial tear of the phlm would become further aggravated and may cause additional injuries to surrounding cartilages , and decided to perform an arthroscopic examination . arthroscopy showed two ligament structures that arose from the anterior horn of the medial meniscus ( ahmm ) and ahlm , respectively and were attached to the intercondylar notch of the lateral condyle of the femur . these two ligaments were combined together , extended superiorly , ran parallel to the acl , and then inserted into the intercondylar notch in the 12 o'clock direction ( fig . describing this combined ligament separately , the anteromedial mfl , which started from the ahmm , appeared to have the typical pattern that surgeons can occasionally observe4 ) ( fig . 2b ) , and the anterolateral mfl , which had never been found or reported , started from the ahlm , ran parallel to the acl , and inserted into the superior portion of the intercondylar notch anterior to the acl ( fig . 2c ) . because the ahlm was not attached to the tibia , hypermobility occurred and led to degeneration of the ahlm . in addition , an outerbridge grade ii cartilage injury was visible on the anterior portion of the tibial plateau ( fig . a fibrous structure assumed to be the anterolateral mfl and the anteromedial mfl were identified ( fig . 4 ) . although the characteristics of the patient 's pain were inconsistent with phlm radial tears , such an injury can completely disable the load - bearing function of the meniscus and thus result in an articular cartilage injury , wear , and degeneration . thus , an arthroscopic partial meniscectomy for the radial tear of the phlm was performed . at 1-year short - term outpatient follow - up , appropriate anatomic placement of the tibial meniscus attachment is necessary to preserve critical knee joint functions7 ) . the anteromedial mfl that is an anomaly of the ahmm has been descried by some authors8 ) and they have contended that ahmm hypermobility can lead to wear and subsequent degenerative changes9 ) . however , there has been only 1 reported case of anterolateral mfl that was found in a patient with congenital absence of the acl , indicating how extremely rare the anomaly is6 ) . on review of the present case 's mri , ligamentous structures were identified anterolateral to the normal acl , which demonstrated low signal intensity on sagittal t1- and t2-weighted mri images ( fig . 4 ) . because the signal intensities were similar to those of normal ligaments around the knee , we suspected it could be the anterolateral mfl that had been observed during arthroscopy . we discovered the lateral meniscus ( lm ) that had a normal tibial attachment of the phlm but the ahlm lacked the tibial attachment that otherwise ran parallel to the acl and inserted into the superior portion of the intercondylar notch anterior to the acl . the present case also showed ahlm hypermobility , resulting in early meniscal degeneration at age 18 and an injury to the surrounding articular cartilage ( fig . 3 ) . since there had been no reports of this type of anomaly , we decided to administer rehabilitative treatments ( e.g. , strengthening exercises and activity modifications ) in the outpatient clinic . furthermore , the concomitant presence of anteromedial and anterolateral mfls that were combined as a single structure is a new anatomical anomaly that has never been described in the literature . the patient had been previously diagnosed with a radial tear of the lm in the contralateral knee and had undergone a partial menisectomy 10 months earlier performed by the same surgeon . no ligament abnormalities were observed in that knee on arthroscopy or mri , indicating that it was a unilateral anomaly . the present case report is significant in that it is the first description of an anterolateral mfl . orthopedic surgeons should be aware of this anatomical anomaly , and future description of more cases would add to our knowledge regarding anomalous meniscus insertion .

anatomical variations of the meniscus are a common anomaly that knee surgeons frequently encounter . however , anomalies of the anterior horn of the lateral meniscus ( ahlm ) are extremely rare . in this report , we present a newly discovered anomaly of the ahml : an anterolateral meniscofemoral ligament is described with clinical features and radiographic and arthroscopic findings .

Case Report Discussion

PMC2914339

understanding metabolic activity from the underlying genotype is one of the most addressed problems in computational biology . of particular interest is the issue of the identification of the reactions that are indispensable for an organism to survive , grow or perform a specific function in a given growth medium or , conversely , of the potentially most harmful knock - outs or enzymopathies . several experimental protocols are able to assess the essentiality of gene products ( and hence of the corresponding metabolic reactions ) , ranging from individual knock - outs to transposon mutagenesis and rna interference [ 15 ] . computational approaches on the other hand might provide important clues on the system - level organization by investigating genome - scale network reconstructions . the functional modularity of metabolic networks suggests that topological aspects may provide a key to identify a class of essential pathways [ 6 , 7 ] . however the metabolic genotype only constitutes the frame on the top of which the dynamic phenotype is built . the essentiality of a metabolic pathway will in general depend on both structural considerations based on the network reconstruction from genomic information , and on the model of metabolism defined on it , for example , on the corresponding steady state fluxes . in e.coli , phenotypical essentiality of metabolic genes has been associated with a reduced allowed variability of the corresponding fluxes , suggesting that dynamically stiff reactions may constitute an evolutionarily robust backbone of metabolism conserved over different species . here we attempt a more thorough integration of topological and dynamical views to obtain a more comprehensive insight into a metabolic network 's organization , efficiency , and ability to respond to perturbations . we will first associate the essentiality of a reaction with a measure of its topological replaceability by enumerating the alternative paths from substrate to product along the network edges , with the rationale that from a purely structural viewpoint more replaceable reactions are less likely to be crucial nodes of the network . then we will validate and compare the essentiality map thus obtained with the metabolic phenotype resulting from the definition of a general constraint - based model for metabolic flux prediction . we shall see that dynamical and structural measures of essentiality may offer complementary views of a reaction network 's robustness . we carry out our analysis on the metabolic network of the human red blood cell ( hrbc ) , one of the most studied complexes in systems biology , from the earliest mathematical models of single biochemical pathways [ 9 , 10 ] to the currently available genome - scale reconstructions . the reason for this choice lies essentially in its limited size . on the one hand , it allows to compute reaction replaceabilities exactly by a suitable modification of johnson 's algorithm for counting loops in a directed graph . on the other , it allows for the efficient application of various sampling methods to the space of viable flux states [ 8 , 13 ] . the latter is a vital ingredient to address many important properties of erythrocytes . indeed for some organisms under certain conditions it is reasonable to assume that the metabolic activity is aimed at maximizing a subset of the metabolic reactions ( or a function of them ) associated with a certain biological function . in such cases for example , e. coli 's metabolism has been shown to maximize biomass production under evolutionary pressure , but after a genetic knockout it responds with a minimum rearrangement of fluxes . while the production of the cofactors ensuring the maintenance of osmotic balance and the release of oxygen may be argued to be their metabolic goal information - rich directions in flux space must be retrieved by coupling the underlying constraints on fluxes with other types of analyses . much understanding has indeed been obtained from the uniform sampling of feasible states [ 13 , 16 , 17 ] and by functional studies , like the computation of extreme pathways , of metabolic regulatory structures [ 19 , 20 ] and of metabolic pools . these aspects combined make hrbcs a key benchmark for both theories of metabolism and computational tools . it is worth noting that the detailed structural information we derive ( i.e. , the full map of alternative paths for each substrate / product pair ) can not be retrieved by other methods . unluckily , computation times still prevent scaling the approach we employ up to networks larger than a few hundred nodes . given a reaction network , we want to compute , for any pair of metabolites a and b that are , respectively , substrate and product in a reaction i ( this situation will be indicated by aib ) , the number ab( ) of alternative pathways , excluding reaction i , of length allowing for the conversion a b , see figure 1 . the rationale is that a reaction performing a metabolite conversion aib for which ab( ) ( or , more properly , ab( ) ) is large will be more easily substituted , in case of an enzymopathy or a knockout , than one for which the above quantity is small . finding paths connecting two points of a directed network is a long - studied problem in computer science . the focus is usually on locating the shortest paths or the fastest way to find any path . enumerating all the distinct paths between two vertices is however a less confronted issue . in our case it is crucial to avoid overcounting , for example , due to self - intersecting paths . we will identify the substitutive paths using the following trick : for each pair ( a , b ) of metabolites such that aib , revert i fictitiously . this results in a new graph where an auxiliary edge bia replaced the edge aib , see again figure 1 . counting the number of alternative reaction chains producing b from a then comes down to computing the number of directed cycles , that is , non self - intersecting directed closed paths along the edges of the new graph , passing through the fictitious edge bia . thanks to the limited size of the hrbc network it is possible to solve this enumeration problem , will denote here the number of reactions in the alternative pathway ( = 4 in figure 1 ) . the space of viable fluxes will be defined through a constraint - based approach which relies on more general assumptions than flux - balance analysis ( fba , ) . fba is the standard method to model steady - state reaction networks where mass balance constraints are imposed to every metabolite . for a reaction network with n reactions and m metabolites , let us denote by a and b , respectively , the m n matrices of output and input stoichiometric coefficients . the stoichiometric matrix is given by s = a b. letting = ( i)i=1 denote a vector of fluxes ( with properly chosen bounds i i i ) , the concentrations c = ( c)a=1 of metabolites vary in time according to c=s-u , where u = ( u)a=1 stands for the net cellular uptake of metabolite a ( u > 0 if a is a global output of metabolism , u < 0 if a is consumed by the organism , u = 0 if a is mass - balanced ) . assuming a steady state , the concentrations are constant in time ( i.e. , c=0 ) and vectors satisfying s = u , or ( 1)(ab)=u , represent flux configurations ensuring that each metabolite meets its production or consumption constraints at fixed concentrations . as n is typically larger than m , the system is underdetermined and feasible flux states form a convex set of dimension n - rank(s ) embedded in the n - dimensional space of fluxes . in absence of a selection criterion that allows to pick one solution out of this set ( as e.g. , a maximum biomass principle ) , a uniform sampling of the solution space should be carried out . when n is sufficiently small ( as for hrbcs ) , this can be achieved effectively , albeit at a considerable computational cost , by monte carlo methods [ 13 , 16 ] or by message - passing procedures . here we will consider a different but related flux scheme based on von neumann 's ( vn ) model of reaction networks . in the vn framework , one fixes the environment through a small set of intakes on nutrients and defines a self - consistent flux problem where the network chooses , given a target growth rate , how much of the nutrients to use and which metabolites are globally produced . the equations describing the vn model have been studied by statistical mechanics methods in [ 23 , 24 ] . for an intuitive derivation , note that the quantities a and b represent , respectively , the total output and the total input of each metabolite for a given flux vector . then a flux vector such that a b , with some constant > 0 , describes a network state where metabolites are being produced at a rate at least equal to , since for each of them the total output is at least times the total input . it is simple to see that as increases the volume of such flux vectors shrinks continuously ( for = 0 every flux vector is a solution ) . in particular , there exists a value of , representing the maximum metabolic production rate compatible with the stoichiometric constraints , above which no suitable flux vectors exist . the presence of conserved metabolic pools implies = 1 , so that in metabolic networks optimal steady state fluxes correspond to the solutions of the solutions of ( 2 ) do not coincide with those of ( 1 ) even for u = 0 . interestingly , a finite volume of ( optimal ) flux states turns out to satisfy the above constraints . this trait is at odds with both the behavior of the solutions of ( 2 ) for a random reaction network ( where a single solution survives at ) and with the optimization that is usually coupled to fba ( where typically a single flux state maximizes the objective function ) , and points to the robustness of metabolic phenotypes . for e.coli , in particular , the solutions of ( 2 ) have been shown to reproduce both the large - scale organization of fluxes and the individual measured rates . in addition , fluxes with the smallest solution - to - solution fluctuations , representing the most susceptible parts of the network , turn out to be strongly correlated with e.coli's phenomenologically essential genes . the main technical advantage in using the vn scheme lies in the fact that its solution space can be sampled uniformly at very modest computational costs even for genome - scale models . the algorithm allowing for this , which has been recently applied to sample e.coli's solution space , is detailed in the following section . we consider the hrbc metabolic network studied in , a map of which is shown in figure 2 ; table 1 lists reactions and the corresponding abbreviations . the network comprises three main pathways , namely , glycolysis ( reactions 113 ) , the pentose phosphate ( pp ) pathway ( 1421 ) and the adenosine metabolism , with a total of m = 39 metabolites linked by n = 59 reactions : 49 internal reactions ( 34 of which come from the splitting of 17 reversible processes ) , 3 auxiliary fluxes to maintain the osmotic equilibrium and the redox state of the cell ( atpase , nadhase , nadphase ) and 7 uptake reactions to guarantee the intake of the necessary nutrients ( glu , ade , ado , ino ) , and of the cytosol elements ( h2o , h , pi ) . the forward and backward parts of reversible reactions are treated separately throughout this study , both in the structural and in the flux analysis . structural vulnerabilities are identified by analyzing the loop structure of a modified metabolic reaction network , created from the original one by inverting in turn the direction of the single reaction for which we want to compute the replaceability , as explained in figure 1 . the fastest known exact algorithm ( for the worst case scenario ) of this cycle enumeration problem for a directed graph was introduced by johnson . we shall now shortly describe its key ideas , referring to for a pseudocode . given a directed graph with n vertices and e edges , the algorithm is designed to build non self - intersecting paths from a root vertex r to itself , loading them onto stacks . the main ingredients allowing for an optimal exploration of the graph are ( a ) a smart choice of the root vertex , and ( b ) an efficient method to avoid duplicating cycles and repeating searches on the same portions of the graph . to achieve this , vertices are initially ordered in a lexicographic sequence , and the algorithm only selects as roots those nodes that are the vertex ( in the initial ordering ) of at least one cycle . the algorithm described in guarantees to find such vertices in o(n + e ) operations . moreover , to avoid self - intersections , each time a node is loaded onto a stack it is also given a blocked it was proven by johnson that if a vertex v stays blocked as long as every path from v to the root vertex r intersects the current path at a vertex other than r , the algorithm outputs all cycles exactly once . by sufficiently delaying the unblocking of each of these vertices and by keeping track of the portions of the graph that have been searched holding the current stack , the maximum time that can elapse between two consecutive cycle outputs can be reduced to o(n + e ) . the same holds for the time window before the first cycle is delivered and for the one after the output of the last cycle . hence , the total time needed to list the , say , c cycles of the graph is o((n + e)(c + 1 ) ) . in our case , each fictitious reaction reversal generates a new graph , so that computing the complete substitutability map for a network of n reactions requires a time of the order o(n(n + e)(c + 1 ) ) . for practical reasons , we perform this analysis on the bipartite metabolic network ( as in figure 1 ) rather than the reduced network of figure 2 . this implies that in our case n = n + m. one can in principle consider different measures of replaceability of a metabolic conversion aib . the quantity ab = ab( ) , counting the total number of paths alternative to i from a to b of any length , is perhaps the most obvious option . taking into account the fact that , typically , longer detours can be less convenient than shorter ones from an energetic viewpoint one could instead consider -weighted functions like ab = exp ( )ab( ) , with the caveat that shorter pathways might require more atp than longer ones we limit ourselves to identifying three key reaction groups that are independent of the replaceability measure used : the group of reactions such that each substrate - product pair involved in them can be substituted ( this is putatively the part of the network that is most robust to enzymopathies ) ; the group of reactions that can not be substituted , corresponding to the most harmful enzymopathies ; the group of reversible reactions that are only replaceable in one direction , corresponding to the situation in which a conversion ab can only be substituted in one direction in case of a knockout . optimal flux vectors , that is , solutions of ( 2 ) , are computed by the algorithm introduced in based on . the idea is to modify fluxes iteratively until all inequalities in ( 2 ) are satisfied . ( with = 1 in our case ) define ( ) = a b and let ( ) denote the rows of ( ) , for a { 1 , , m}. let also , for each iteration step t , (t ) be the flux vector at step t and ( 3)m(t)=arg min a a()(t ) , at each t , the algorithm runs as follows . if ( ) (t ) < 0 , update fluxes according to ( 4)i(t+1)=max { 0,i(t)+im(t)( ) } , and iterate in t. else , if ( ) (t ) 0 stop , that is , (t ) is a solution . convergence to a solution is rigorously ensured for all 0 < , and can be approximated with the desired resolution by iterating the above process for increasing values of . to guarantee that solutions are well defined one can either resort to setting fixed upper bounds on i 's or , as we do , impose a linear constraint of the form ii(t ) = n on the solutions ( this is equivalent to singling out one flux as the reference unit for the other fluxes ) . different initial points generate trajectories to different solutions at and the sampling of the solution space thus obtained turns out to be uniform . contrary to fba , the solution space of vn 's model is generically not a polytope . indeed much useful information can be retrieved from its shape . as a means to characterize it we employ the average overlap between different optimal flux vectors , defined as follows . let and denote two distinct solution vectors of ( 2 ) and , for each flux i , let ( 5)q(i)=2iii2+i2 . and , equals 1 if flux i takes on the same value in solutions and and decreases as the values differ more and more . averaging q(i ) over different pairs of solutions provides a measure of the allowed variability of flux i ( smaller variability corresponds to larger average overlap ) , complementary to the standard deviation of the resulting flux distribution . the complexity of the solution space can then be roughly understood by distinguishing narrower directions with larger overlap or less variable fluxes from broader ones . it is reasonable to think that a cell will be more sensitive to perturbations ( e.g. , knockouts ) of fluxes with larger overlap . analyzing the susceptibility of the solution space to perturbations along the directions identified by different fluxes then allows to extract a list of the potentially more deleterious perturbations , in analogy with previous work on e.coli . in order to test the hrbc network against enzymopathies , one can first employ a structural criterion : the knockout of a metabolic conversion a b that is less easily substituted is more likely to be deleterious for the cell than the knockout of a highly replaceable conversion . as said above , we concentrate here on a coarse - grained view of replaceability based on classifying reactions into the groups ( a ) , ( b ) , and ( c ) defined above , with groups ( b ) and ( c ) containing potentially essential reactions . the second criterion is based on fluxes : fluxes with smaller allowed variability ( i.e. , larger overlap ) in the healthy cell are more likely to be essential links of the network than fluxes whose value can be changed over a larger range without losing optimality . as is to be expected , the essentiality maps produced in these ways have a large degree of similarity , and reactions in the group ( b ) discussed above coincide with the physiologically most critical parts of hrbc 's metabolism . the simplest way to simulate an enzymopathy on flux deficiencies can be partial , that is , of a smaller degree , the closer i is to the upper limit of the allowed range in the healthy cell , or total if i=0 . such constraints cause in principle a modification of the solution space along the direction i which in turn cascades on the entire volume , modifying the optimal states of the metabolic network . ( for the sake of simplicity we exclude the highly replaceable currency exchange fluxes from this discussion . ) the most replaceable core of the network lies in the pp pathway ( reactions 1721 ) , which constitutes the main source of nadph , the key metabolite that in erythrocytes limits the accumulation of peroxides protecting the cell from hemolysis . the high reliability coming with replaceability partly explains the reason why this group of reactions plays a central role not just as an auxiliary pathway for glycolysis , see the following analysis of fluxes . unreplaceable reactions are instead lined up along glycolysis ( numbers 1,6,813 ) , in the bridge between glycolysis and the pp - pathway ( 14 and 16 ) or in auxiliary modules ( 22 , 27 , 29 ; the adeamp conversion in 25 is also not replaceable being directly linked to the ade uptake ) . the physiologically most deleterious knockouts ( hk , pk , and g6pdh ) all belong to this group . for instance , deficiency in the level of g6pdh is the basis of different types of hemolytic anemias , including favism , and is also linked to malaria resistance . finally , there is a group of reversible reactions ( numbers 2 , 4 , 7 , 15 , 23 , 26 ) that can be replaced only in one direction . note however that the last three of these could still be replaced in case of an enzymopathy if a proper medium is selected . for instance , if reaction 15 is removed , it could be substituted by an alternative chain of reactions provided 6pgc is externally supplied . this is instead not possible for reaction 4 and possibly 23 ( depending on the directionality of reaction 26 ) , as a knockout in these cases would necessarily result in a net production of fdp and r1p . the flux distribution corresponding to optimal states in the healthy and enzyme deficient hrbc are displayed reaction by reaction in figure 4 , obtained by sampling 10000 solutions of ( 2 ) , while a pictorial representation of the optimal flux states is given in figure 5 . for the healthy cell ( black line in figure 4 and top left panel in figure 5 ) the large flux backbone is formed by the second part of glycolysis ( crucial for atp , nadh , and 23dpg production ) and the pp pathway ( nadph production ) . the latter gives a substantial contribution to the former , not just as salvage way . in addition to glu , which is the fundamental substrate for hrbcs , the ino uptake plays an important role as an alternative way to the pp pathway . it is worth stressing that these solutions imply a net production of 23dpg , the crucial regulator for oxygen release , which is obtained without any imposed constraint . this picture is strongly reminiscent of the first eigenpathway obtained by extreme pathways analysis in , though the thermodynamic constraints and production requirements used in , including one on 23dpg , are more strict than the self - consistent analysis presented here . comparing the distributions with fba studies on the same system , one notices instead a general rearrangement of fluxes in the network apart from glycolysis . a close inspection reveals that such a rearrangement is mostly quantitative , as preferred reaction directions are generically preserved , the noteworthy exception being the rpi flux , that in the vn solution strengthens the pp pathway with respect to the fba solution . this scenario is not surprising in view of the basic difference between fba and the vn approach . it should be kept in mind however that the a priori constraints on flux variability are quite more strict in fba than they are in the vn model , and the flux distribution appear to be particularly sensitive to the assumed upper and lower bounds for the fluxes . in figure 6 one sees that the large overlap backbone ( signaling dynamically stiff fluxes ) coincides to a large degree with the structurally most vulnerable parts of the network . note that the overlap of reactions 2 , 4 , 15 and 26 is larger in the direction that can not be replaced , further pointing to a higher susceptibility , and that currency reactions ( 3135 ) belong to the most constrained part of the network . revealingly , however , topological and dynamical characterizations prove to be complementary in some cases . this is seen , for example , from reaction 3 , which is flux - constrained but also highly replaceable , so that the damage due to removal is limited even in presence of a small allowed dynamical range . ( a similar picture holds for reaction 23 . ) to conclude , we remind that in our framework uptake fluxes are optimized variables not fixed by boundary conditions . in the optimal state five of the uptakes have a limited allowed variability , implying rather severe constraints on the cell 's environment . we have simulated the most studied enzymopathies by constraining the flux of the corresponding reaction . indeed , we have observed appreciable differences in relevant cellular functions compared to the nondeficient case only under full enzyme deficiencies , as also observed in within a standard fba optimization approach . even under the most serious enzyme deficiencies the network appears to be able to maintain the production of atp , nadh and nadph almost constant , see also . as shown in figure 4 , the alterations in the flux distributions are not particularly striking and indeed we do not observe global flux rearrangements on the network 's scale . the g6pdh enzymopathy appears to only cause local changes , confirming the structural predictions , the overlap calculations and also in agreement with clinical observations . the synoptic analysis of figure 5 shows that in general the response to the perturbation consisted in a drop of the glu uptake , and in a reduction of the glycolytic flux , while the rapoport - leubering shunt ( reactions 8 - 9 ) for the production of 23dpg remains particularly stable , as does the adenosine metabolism . for the glycolytic deficiencies pk and hk we further observe an increase of the ino uptake to sustain the pp pathway and allow for the second part of glycolysis , and with it the production of atp and nadh , to take place . detailed flux configurations corresponding to the next most severe enzymopathies ( hk , en , pgk and pgm ) are available from http://chimera.roma1.infn.it/sysbio . in this work we compare two robustness measures for biochemical networks , one based on structural properties ( the reaction replaceability ) , the other based on dynamical stiffness ( the overlaps ) . the former can be exactly assessed by enumerating the alternative paths joining substrates and products of a given reaction in a network . the latter depends on both the network topology and the model defined on it . within vn 's frame , we found that unreplaceable reactions mostly correspond to processes with a smaller allowed flux variability . in such directions , reaction removals as well as constraints on the fluxes are expected to be generically harmful . reactions with limited ( but non zero ) replaceability tend to have instead smaller overlap , so that while the reaction is difficult to substitute still its flux can be largely adjusted . in an evolutionary perspective , the former pathways appear as frozen , and perturbations at these nodes will require large - scale flux rearrangement , while a mutation affecting the latter group may be neutral and could be preserved across generations . these , albeit structurally robust , are dynamically constrained and should be considered as essential pathways of the metabolism as well . integrating dynamical and structural characterizations may thus provide a rather complete picture of the emerging network robustness . the fact that topological and dynamical essentiality may not coincide could also prove to be important in view of the present challenges to understand the dynamical basis of topological modularity . extended flux state sampling was achieved here by an algorithm that is easily scalable to larger networks , see . exhaustive structural analysis instead was made possible by the small size of hrbc 's metabolic network , which has served here as a model system to test basic concepts and algorithms . the use of the same procedure on a larger network , such as e. coli 's , is likely to be prevented by cpu time growth . however , message - passing algorithms , designed specifically to solve combinatorial optimization or counting problems albeit approximately on graphical models , may be a suitable replacement .

characterizing the capabilities , key dependencies , and response to perturbations of genome - scale metabolic networks is a basic problem with important applications . a key question concerns the identification of the potentially most harmful reaction knockouts . the integration of combinatorial methods with sampling techniques to explore the space of viable flux states may provide crucial insights on this issue . we assess the replaceability of every metabolic conversion in the human red blood cell by enumerating the alternative paths from substrate to product , obtaining a complete map of he potential damage of single enzymopathies . sampling the space of optimal steady state fluxes in the healthy and in the mutated cell reveals both correlations and complementarity between topologic and dynamical aspects .

1. Introduction 2. Approach 3. Methods 4. Results 5. Final Remarks

PMC3323857

the seminal discovery of adult brain plasticity in animals and humans has hugely influenced the theories and concepts applied in neurorehabilitation research and their translation into practice , in particular with regards to movement deficits in acquired brain injury [ 1 , 2 ] . this work directly translated into new and , in some cases , more efficient interventions for patients with mild to moderate hemiparesis ( e.g. , [ 35 ] ) . however , much less research has specifically investigated the reorganization of the motor system in patients with poor or minimal functional abilities and , most critically , the rehabilitation of these patients [ 6 , 7 ] . firstly , standard rehabilitation approaches are difficult to apply when patients have little voluntary movement . secondly , the lack of funding for regular one - to - one physical therapy sessions beyond the postacute phase , together with the common assumption that substantial improvements in functional motor ability are unlikely once the first 6 months of recovery have passed , primes the health care system and patients to accept the status quo . while the latter is true for the whole range of functional recovery , the impact is particularly grave for patients with poor functional recovery and also affects the research effort . thirdly , the motor system of patients with poor residual recovery can not easily be studied with the paradigms adopted from basic science research , such as finger opposition or grip movements . these methodological challenges can be overcome by excluding patients with high levels of spasm or by limiting the study group to those with relatively good levels of motor control . as a result , the motor system of patients with very poor recovery , in particular in chronic state , has been studied much less than that of patients with mild or moderate impairment . this translates directly into studies on treatment efficacy , and indeed , the availability of suitable treatments per se . based on the aforementioned considerations we argue that research on the mechanisms of long - term recovery , and their interaction with treatment efficacy , needs to widen its focus to the population of stroke survivors with severe long - term motor deficits . hereinafter we briefly summarise the present literature and further discuss the challenges involved in the study and treatment of patients with minimal motor recovery . upper limb paresis occurs in 85% of the patients and substantially impacts disability in the long term . animal models have tried to reproduce stroke lesions that occur in humans , with variable degrees of success . the majority of focal ischemia models involve the middle cerebral artery ( mca ) territory , the most commonly affected arterial territory in ischemic strokes in humans . rat focal ischemia models are frequently used because of low cost , similarities between vasculatures of rats and humans , fewer concerns from the general public compared to nonrodents , and availability of clear behavioral outcomes [ 1113 ] . severe mca infarcts in rodents leading to long - lasting sensorimotor and cognitive deficits can be produced by proximal mca occlusion induced by electrocoagulation . complete or partial mca occlusion can be also achieved by insertion of an intraluminal filament . in the filament model for example , if endothelin-1 , a vasoconstrictor drug , is injected topically or intracerebrally , the forelimb motor cortex is typically spared , but infarcts vary in location and extension depending on the sites and route of drug administration . in models of occlusion of the distal mca or its branches , cortical frontoparietal infarcts are associated with less severe deficits than in the cortico - subcortical infarcts produced by proximal mca occlusion . furthermore , multiple embolic infarcts can be produced in brain areas supplied by the mca , after injection of microspheres , macrospheres , a thrombotic clot , or purified thrombin into the internal carotid artery [ 19 , 20 ] . finally , light activation of photosensitive dyes such as rose bengal makes small cortical infarcts possible , by occlusion of cortical vessels . many of these techniques have not been applied exclusively to small animals such as rats , mice , gerbils , or rabbits , but also to cats , dogs , pigs , and monkeys . larger animals have proportions of gray / white matter that are more similar to those found in human brains , in contrast with lissencephalic brains of rats and mice . however , technical limitations and costs limit widespread use of focal ischemia models in larger animals . it is recommended , for instance , that new drugs be tested first in rodents , before efficacy is investigated in gyrencephalic species . they offer powerful opportunities : the possibility to objectively monitor behavioral outcomes , manipulate experimental conditions , and obtain images ( mri , micropet ) or intracortical recordings of neuronal activity in living animals ; to scrutinize molecular mechanisms of cell death and recovery ; to work with strains and transgenic animals that present hypertension , atherosclerosis and obesity , among other factors that are common in patients with stroke ; and to perform postmortem histological evaluations . still , conclusions based on animal models of focal ischemia must be examined with caution . for instance , small , selective cortical infarcts leading to mild sensorimotor deficits that tend to improve quickly are present in some of the models but are not common in humans , even though similar clinical features can occur in subcortical , lacunar infarcts . in addition , background pathophysiology is not shared between focal cerebral ischemia in rodents and humans . fast arterial recanalization is achieved in several animal models , while in humans recanalization can occur either after r - tpa administration or spontaneously but , unfortunately , does not happen at an early phase after ischemic injury in most patients [ 8 , 2427 ] . lack of arterial recanalization is strongly associated with more severe strokes and lower probabilities of recovery . furthermore , rodent models have often included young healthy animals while , in humans , the bulk of strokes is concentrated in the elderly [ 2832 ] . age is a potent predictor of poor outcome in humans [ 33 , 34 ] . interestingly , despite high mortality rates in old mice , levels of recovery at four weeks have been reported to be similar in aged and young animals . other factors may contribute to poor outcome in humans , ranging from different mechanisms underlying strokes in different age groups to self - fulfilling prophecies in stroke care in the aged , and importantly , to comorbidities that impact recovery . diabetes mellitus and atrial fibrillation , for instance , are significantly associated with more severe outcomes and lower chances of recovery [ 36 , 37 ] . hypertensive rats have poorer collateral blow flow and also worse outcomes after focal ischemia than nonhypertensive animals . the demand for stroke models in hypertensive , obese , and aged rodents has been underlined . contrasts between the compelling efficacy of a myriad of drugs in animal models of neuroprotection and the systematic failure of the same drugs when administered to patients in clinical trials underscore the requirement for animal models that more realistically approach human strokes [ 13 , 22 , 39 ] . however , bigger rates of complications and mortality in aged or unhealthy animals , technical difficulties ( anesthesia , complex surgeries in less flexible arteries , etc . ) , and hence the greater costs involved present challenges for advancements of research in the field . in particular , high mortality rates limit the opportunity to study recovery of animals with severe strokes in the chronic phase . in summary , there is a gap between pathogenesis and clinical features of severe strokes in humans and pathogenesis and clinical features of the most widely used models of focal ischemia in animals . high costs and lower expectations for substantial improvements in activity or quality of life are also major obstacles for rehabilitation of patients with severe strokes . still , as stroke mortality decreases in parallel with advances in health care , it is expected that more patients with severe strokes will survive the acute phase over the next decades . at the moment , thus , animal models of focal ischemia that match severe human strokes more closely are deeply needed . despite these limitations , animal models have provided unique insight on plastic mechanisms underlying sensorimotor recovery after focal ischemia , and on how to enhance beneficial patterns of reorganization to obtain behavioral gains ( e.g. , ) . constraint - induced movement therapy , for instance , shown to improve motor outcomes in humans with different types and sizes of strokes , was developed based on seminal studies that underscored the importance of the amount of use of the paretic limb to promote enhancement of motor function [ 41 , 42 ] . the phenomenon was observed in monkeys with small cortical infarcts submitted to intracortical microstimulation and behavioral testing . as mentioned before , such small infarcts are rarely observed in humans , but still , the information gained by the model was a major step forward in stroke rehabilitation . constraint - induced therapy has now been successfully applied to patients with various types of strokes , with more severe deficits and worse motor prognoses than the monkeys included in the model of forced use of the affected limb [ 1 , 3 , 43 , 44 ] . over the past decades , neuroimaging and neurophysiology techniques have emerged as paramount tools to study brain reorganization in humans [ 4550 ] . overall , functional neuroimaging studies in patients with stroke have shown that good performance is associated with augmented activity in preexisting networks during a motor task , rather than assignment of networks that are not overtly active during these tasks in healthy subjects . furthermore for instance , according to the model of interhemispheric inhibition , an imbalance in activity between the ipsilesional and the contralesional primary motor cortex ( m1 ) can occur after stroke [ 51 , 52 ] a number of studies have shown that this imbalance in activity between the two hemispheres can impair motor performance of the affected hand , at least in some patients in the chronic phase after stroke [ 51 , 52 ] . whether interhemispheric callosal fibers mediate this phenomenon or whether it depends on changes in activity of cerebellar or thalamic pathways , remains an open question . however , the vast majority of published studies that investigated effects of modulation of interhemispheric inhibition only included patients with mild to moderate motor impairments , likely due to difficulties in developing tasks and applying available neurophysiology and neuroimaging tools to more severely affected patients [ 5159 ] . in regard to recruitment of ipsilesional or contralesional secondary motor areas , striking differences in patterns of function were unveiled when paradigms of investigation were applied to patients with poor recovery , compared to those of less affected patients or healthy subjects . recruitment of secondary motor areas occurs when the outflow from m1 is disconnected from the spinal cord in large cortical , cortico - subcortical or subcortical strokes , as well as in strokes that strategically damage the corticospinal tract [ 60 , 61 ] . at least in part , more pronounced patterns of activity in secondary motor areas in the ipsilesional and/or contralesional areas are associated with more severe disruption of the corticospinal pathway , and excessive activation of secondary motor areas correlates with poorer motor behavior [ 48 , 50 , 62 ] . whether excessive activations of secondary motor areas or contralesional m1 are maladaptive , whether they represent the best possible remodeling option after severe injury , or whether they may play hero or villain roles depending on motor tasks / circumstances remains to be determined . there are indications that motor performance may actually rely on activity of contralesional areas in more severely affected patients . for instance , it has been shown that transient disruption of the contralesional dorsal premotor cortex by transcranial magnetic stimulation slows motor performance of the paretic hand to a greater extent in patients with worse motor performance compared to less impaired patients . in addition , activation of the contralesional dorsal premotor cortex has been demonstrated to be greater in patients with more severe hand motor impairment , compared to healthy subjects and to less affected patients . on the other hand , disruption of the ipsilesional dorsal premotor together , these studies provide evidence that ( 1 ) contralesional areas are positively , functionally relevant in at least some well - recovered patients in the chronic phase ; ( 2 ) in patients with severe motor deficits , behavioral gains , albeit small , may occur by augmented activity of networks that are normally either minimally active or not active at all in healthy brains . severity of motor impairment seems to be a key factor influencing patterns of rewiring after stroke , but age , brain status before stroke , intensity , and timing of rehabilitative interventions , among other factors , are also likely to play pivotal roles in the process [ 33 , 34 , 6567 ] . a key concept to develop effective rehabilitation interventions is heterogeneity of mechanisms underlying stroke as well as plastic processes that lead to recovery of function after neuronal injury . mechanisms underlying neurological impairment , recovery of activity , and participation are also distinct . until now , most proof - of - principle studies or clinical trials have excluded patients with severe sensorimotor impairments and the lack of evidence - based effective interventions has nurtured a nihilistic approach for rehabilitation of these patients . moreover , patients with severe sensorimotor impairments often present with cognitive impairments , depression and are faced with massive changes in psychosocial interactions [ 6870 ] . further research efforts must not only address restoration of sensorimotor function but also incorporate an integrative approach to target neuropsychiatric domains , personal experience / expectations , environmental conditions , and psychosocial factors . changes to the functional organization of neural representations and their behavioral concomitants have been described for a number of human study models such as amputates ( e.g. , [ 71 , 72 ] , musicians [ 73 , 74 ] , blind ( e.g. , [ 7578 ] ) and deaf persons ( e.g. , [ 79 , 80 ] ) , as well as learning paradigms ( e.g. , [ 8184 ] ) . together these studies suggest that sensory representations are sensitive to enhanced or altered sensory stimulation . this knowledge has the prospect to devise interventions that capitalize on the plastic capacities of the adult brain , mainly training- or practice - based interventions . the mechanisms of brain plasticity interact with psychological processes and behavior and together provide a number of considerations for the conceptualization of interventions . injury to the peripheral or central nervous system changes receptive field characteristics of neurons and neural representations through deprivation of the original afferent inputs these neurons receive [ 85 , 86 ] . the key mechanisms driving this change are the disinhibition of silent synapses , and the loss of inhibitory or excitatory inputs from lesioned neural populations connected to none - lesioned regions , including homologous areas in the two hemispheres . in both cases , it is important to appreciate the effects of functional changes in nonlesioned areas and their influence on the control of the impaired behavior . as a consequence , rehabilitation efforts should not only focus on the impaired function per se , for example , the affected upper - limb in the case of hemiplegia , but also consider the effects of use or nonuse of the lesser - affected extremities . this is particularly relevant for patients with poor recovery as they will most likely entirely rely on the less - affected extremity in everyday behavior . this might , at least in theory , increase the interhemispheric inhibition exerted by the nonlesioned hemisphere . sensory stimulation and these changes are most likely driven through hebbian mechanisms as well as dendritic and axonal processes . critically , use - related reorganization is not driven by increased neural activation alone but heavily depends on the behavioral relevance of the activity [ 9092 ] . this association of sensory stimulation and consequential changes in neural representations and receptive field parameters has been demonstrated most elegantly in monkeys who received auditory and tactile simulation within the same protocol . behaviorally , the animals were only rewarded for responses in one of the two stimulation modalities , and subsequent changes in the brain were only observed for the rewarded modality . thus this data indicates that neural representations are only susceptible to stimulation - induced reconfigurations if this stimulation is attended to and of behavioral significance . therefore , we argue that interventions aiming to enhance recovery through the induction of practice - induced plasticity not only need to focus on the actual practice element of the intervention but also consider how the intervention characteristics enforce attention and provide tangible and motivating feedback . motor control is a complex behavior that goes far beyond motor execution and the processes typically associated with primary motor cortex function . cognitive processes such as motor planning , error - monitoring and attention can heavily influence motor performance . motor rehabilitation research , however , typically conceptualizes motor function as the ability to execute a movement with little consideration being given to the cognitive processes that might influence this ability . for example , few studies have investigated how much patients with hemiplegia benefit from advanced movement preparation . the respective behavioral costs or benefits result from perceptual , cognitive , and motoric components of the stimulus - response cascade , such as stimulus - response mapping and response selection . few studies have investigated advanced movement preparation in patients . a study by verleger et al . suggests that well - recovered patients show little difference to controls in a motor priming task . using a similar paradigm in patients with poor recovery , our group found marked behavioural and electrophysiological difference between patients and matched control . most strikingly , the data suggests that patients are more sensitive to advance information ( manuscript submitted ) . thus , it appears that visual precues can facilitate or hinder apparent affected arm abilities , and that the magnitude of this effect is modulated by the severity of the motor deficit . while more research is needed to fully understand the interaction of cognitive processes , stroke severity , and motor performance , the findings summarised previously suggest that using advance movement information in a cognizant and explicit manner may be a beneficial addition to rehabilitation interventions . in order to translate improvements in motor ability into real - world benefits , treatments have to obtain not only better motor control over the affected arm , but also a transfer of these newly acquired abilities into the curriculum of everyday behaviors . behavior change can be facilitated through a number of measures falling under the cbt ( cognitive behavioral therapy ) umbrella . these measures use not only learning principles , which in themselves are likely to facilitate use - related plasticity processes , but also tools that enhance motivation , adherence , and coping . the latter , again , are likely to increase the patient 's engagement with the intervention , which in turn is likely to improve outcome . conceptualizing the rehabilitation of motor function as changing motor behavior rather than improving motor ability represents a significant shift in theoretical perspective . it implies that practice - based interventions should include treatment strategies that actively support sustained learning and behavior change , through the explicit use of learning principles and cbt elements . the latter facilitates not only real - world benefit but also enhances the processes of use - related functional reorganization that drive improvements in motor control . interview data ( unpublished ) from patients with severe chronic hemiplegia indicates that these patients experience disproportionate psychological and service - related barriers . i hate it ( the hand ) and want to make it invisible , and it ( the hand ) looks horrible and is no good ; i 'd cut it off if i could suggest that patients do not only face the actual physical impairment but also face psychological barriers to using any residual ability . this aspect is particularly important for interventions that rely on motor practice because the patient 's engagement with the intervention is directly linked to their ability to cope with their disability . however , more often than not physiotherapy is provided without considering the psychological barriers associated with the use of the hemiplegic hand . we therefore argue that in order to improve the prospects for patients with low - functioning hemiparesis it is necessary to better understand the psychology of poor recovery and build that knowledge into motor rehabilitation interventions . in addition to the psychological barriers low - functioning patients seem to experience , our interview data suggests a strong perception amongst these patients that the care system is presumptuous and does not provide for them . this is illustrated by comments such as my doctors gave up on me after 3 month , my physio tried to work with my hand initially but soon gave up , the ot only taught me how to manage things ( with my good hand ) , i guess this was because she knew that my ( paretic ) hand would be no good , and there is nothing they can do for me because i can not move . these comments highlight not only the need to understand the patient perspective and a holistic approach to long - term care of these patients , but also a critical need to develop motor rehabilitation treatments that help patients to enhance their residual motor ability and enhance its real world benefit . this affects not only the patients ' general levels of activity but , of course , also the level of engagement with the therapy process . not a lot can be gained in a therapy session with a tired patient ! moreover , tiredness and fatigue are linked to sleep , and sleep is likely to be a modulating factor of recovery . moreover , patients with chronic low - functioning hemiparesis seem to suffer from sleep difficulties at least as frequently as the general population . it is therefore likely to further aggravate the difficulties patients already have with activities of daily living and to reduce the benefit patients can get from therapy and other activities . finally , an increasing body of literature suggests that sleep enhances brain plasticity in general and procedural learning specifically ( e.g. , [ 97100 ] ) . assuming that brain plasticity is the main driver for the recovery of function , good and sufficient sleep is likely to facilitate and probably enhance the rehabilitation effort . support for this assumption is provided by a series of studies suggesting that motor learning can be positively influenced by sleep [ 101 , 102 ] . the issues raised previously are relevant to all patients but probably have greater significance in patients with poorer recovery . therefore , treatments for these patients in particular should incorporate measures to counter tiredness and fatigue and monitor the quality of nocturnal sleep as well as daytime sleepiness . jeannerod 's theory of neural simulation suggests a shared neural network for the control of overt and covert movement modalities such as movement observation and motor imagery . this theoretical framework provides a powerful tool for research in patients with poor recovery . generally , evidence from behavioral , neuroimaging , and psychophysiological studies confirms jeannerod 's notion of equivalence in healthy populations . for example , several fmri studies have shown comparable activations in the cortical motor regions when participants observe or imagine hand movements ( e.g. , [ 104107 ] ) . thereby , these activities are similar to the activations obtained when the same movements are actually performed in the scanner . typically , these studies use tasks that can be practiced prior to scanning and can be performed in the scanner . while this approach yields interesting and important insights , it lacks ecological validity , particularly with regard to the rehabilitation context . taking these considerations on board , szameitat and colleagues [ 108 , 109 ] studied motor imagery of complex actions , such as eating with knife and fork or running . using an fmri paradigm , they successfully demonstrated that imagery of such complex movements is feasible in the scanner environment and leads to meaningful activations in the motor system . these findings are not trivial because complex everyday actions can not be practiced prior to the scanning . the person will therefore rely on their motor memory rather than the experience obtained through practicing the actual task ( e.g. , finger tapping ) immediately before the scanning begins . in this sense , the imagery of complex movements , by default , affords less stringent experiment control . however , if motor imagery is to be used as a study tool for patients with poor recovery , their inability to move will prevent practice prior to scanning , and therefore , these patients will also perform the task by relying on their motor memory . evidence showing the feasibility and suitability of a paradigm that omits practice is therefore particularly relevant for the application in patients with poor residual recovery . moreover , the study of complex actions with fmri opens the door to the investigation of the representation of activities of daily living and the alteration of those representations throughout recovery and/or treatment . as mentioned previously , the cognitive processes involved in advance movement preparation are likely to play an important part in the person 's ability to function in everyday life but might also enhance or hinder the rehabilitation effort . understanding to what extend the principle of equivalence also holds for the higher cognitive processes involved in motor planning is therefore important . in a series of eeg experiments , kranczioch and colleagues [ 110 , 111 ] directly compared execution , imagination , and observation of finger movements in an advanced motor preparation paradigm . these studies firstly showed that the eeg - derived erps provide a good tool for the study of covert movements . this is important because the high temporal resolution of eeg typically requires precisely timed stimuli , which is a challenge for the imagery condition . at the same time , not all patients can part - take in mri scanning ( e.g. , because of metal in their body ) and eeg can therefore provide an alternative method to study motor processes . covert movements not only provide a good vehicle to study the reorganized motor system of those patients unable to execute the kind of controlled hand movements used in experimental paradigms requiring overt responses , but can also be employed to induce enhanced neural activation of motor circuitries which aids functional reorganization and recovery . coined by sharma and colleagues as a backdoor to the motor system after stroke , therapeutic approaches using covert movement modalities have recently been tested [ 7 , 113116 ] . while initial evidence is promising , there are a number of questions that need to be answered in due course . for example , literally all our knowledge on covert movement has been obtained in younger persons , typically university students . aging is known to affect the motor system ( e.g. , [ 117119 ] ) ; in fact , the way a person moves is quite indicative of older age and frailty . it is therefore not inconceivable that motor - specific mechanisms of covert movement , determined in younger populations , are differentially affected by age . similarly , the ability to imagine , or to focus on stimuli during observation , relies heavily on cognitive and perceptual processes and may therefore , again , be modulated by age . the latter may of course also be affected by the stroke [ 121 , 122 ] . more research that characterizes covert movement in healthy older persons is therefore needed to help tailor treatment development . in addition , it is presently unclear whether imagery and observation provide equally suitable treatment pathways , and how this interacts with lesion location . while several studies have explored the effects of mental practice on recovery , there is , to the best of our knowledge , no direct comparison between these methods . pilot data from our group suggests that motor imagery is best able to activate the reorganized motor system in patients with chronic sever hemiparesis . poor long - term recovery of motor function after stroke is a major public health issue and a big problem for patients and their families . but treatment provision is not satisfactory , research in this area is limited , and ( at least some ) patients feel abandoned by the health care system . a deeper understanding of the complexities involved in motor control and their interaction with the mechanisms of brain plasticity as well as psychological aspects of recovery is needed not only to maximize the treatment outcome for patients but also to tailor health service provisions and support infrastructures accordingly . despite the incredible advancements in brain imaging and rehabilitation research , and the growth of knowledge on brain plasticity over the last 20 years , there is little we can offer to patients with minimal recovery at present . a targeted and interdisciplinary research effort is required to meet the need for research and treatment development . this is necessary for the sake of the individuals affected as well as those who fund the health and welfare systems .

functional reorganization forms the critical mechanism for the recovery of function after brain damage . these processes are driven by inherent changes within the central nervous system ( cns ) triggered by the insult and further depend on the neural input the recovering system is processing . therefore these processes interact with not only the interventions a patient receives , but also the activities and behaviors a patient engages in . in recent years , a wide range of research programs has addressed the association between functional reorganization and the spontaneous and treatment - induced recovery . the bulk of this work has focused on upper - limb and hand function , and today there are new treatments available that capitalize on the neuroplasticity of the brain . however , this is only true for patients with mild to moderated impairments ; for those with very limited hand function , the basic understanding is much poorer and directly translates into limited treatment opportunities for these patients . the present paper aims to highlight the knowledge gap on severe stroke with a brief summary of the literature followed by a discussion of the challenges involved in the study and treatment of severe stroke and poor long - term outcome .

1. Background 2. Animal Models of Focal Ischemia versus Human Strokes 3. Capitalizing on Adult Brain Plasticity to Enhance Motor Recovery: Neural and Behavioral Considerations 4. Overt and Covert Movement: Alternative Ways of Stimulating the Motor System 5. Concluding Remarks

PMC3531036

health - care workers ( hcws ) are at increased risk of acquiring infections with blood borne pathogens because of occupational exposure to blood and other body fluids . the human immunodefienciency virus ( hiv ) the incidence of all injuries with potential to transmit these infections varies between occupational groups but is more prevalent in those providing primary care and performing invasive procedures.12 factors that determine risk of significant exposure to hiv include frequency of needle stick injuries ( nsi ) and the prevalence of hiv in the patient population , amongst others . the estimated yearly risk of hiv infection for anaesthetists in the united states , for example has been put at between 0.000130.032% depending on the sero - prevalence of hiv within the patient population.3 guidelines on the management of occupational exposure to hiv exist . despite these clear guidelines , hcws , especially those providing primary care generally take inadequate measures following occupational exposure to hiv.45 the first case of documented seroconversion after a specific occupational exposure to hiv was reported in 1984 and an approximate number of 1000 cases occur each year due to accidental exposure.67 nigeria ranks among the top three countries with the highest burden of hiv / aids ( unaids ) and as such family physicians ( fps ) who are involved in the provision of care at all levels of the health system are constantly at risk of acquiring hiv and other blood borne pathogens . the aim of this study therefore was to assess the awareness and knowledge of fps in nigeria regarding hiv post exposure prophylaxis . the society of family physicians of nigeria ( sofpon ) holds her scientific meetings annually . at the 13 annual national conference of the society , as well as at a training workshop organized by the national postgraduate medical college for fps in training , questionnaires were distributed to doctors to assess their level of awareness and knowledge regarding the use of postexposure prophylaxis to prevent the transmission of hiv . this was a cross - sectional questionnaire - based survey with nationwide representation of fps in nigeria . knowledge was assessed by asking if the respondents knew whether hiv pep was effective in reducing / preventing hiv transmission ; which body fluids were considered to have high - risk for hiv transmission ; the initial first - aid measures to employ in the event of accidental exposure ; the best timing for commencement of hiv pep following exposure and the ideal antiretroviral ( arv ) drug regimen for hiv pep . respondents were scored based on the number of correct responses to the 23 items listed on the questionnaire to assess knowledge of hiv pep ; with all questions equally weighted . for the purpose of this study , adequate knowledge was defined as correctly answering at least 70% of the 23 listed items . data was entered and analyzed using epi info statistical package version 3.3.5 ( cdc , atlanta , georgia ) . differences between those with adequate knowledge and those with inadequate knowledge were analyzed using chi - squared test . to determine independent predictors of adequate knowledge , we used multivariate logistic regression . the human research ethics committee ( hrec ) of the jos university teaching hospital approved the study . a total of 300 questionnaires were administered and 186 were returned . only 175 had adequate information and were included in the analysis . the characteristics and risk profile of responding physicians are presented in table 1 . there were 43 ( 24.6% ) female respondents with a mean age 38 7 years but ranged from 2764 years . ninety - seven ( 55.4% ) of the respondents were junior residents , 32 ( 18.3% ) were senior residents while 46 ( 26.3% ) were consultants . the majority ( 82.8% ) of the respondents worked in government facilities and 156 ( 90.2% ) had donor funded hiv / aids treatment programs in their hospitals . one hundred and seventy - one ( 97.7% ) of the respondents were aware of the concept of hiv postexposure prophylaxis . one hundred and forty two ( 82.1% ) had existing protocols for hiv pep at their workplaces , 13 ( 7.5% ) did not have hiv pep protocols at their workplaces , and 18 ( 10.4% ) were not aware of the existence of hiv pep protocols in their facilities . one hundred and twenty ( 69.8% ) were in medical specialties at the time of the survey . when asked if they considered themselves at risk of acquiring hiv from the workplace , 158 ( 91.3% ) indicated that they were at risk of acquiring hiv from the workplace while 15 ( 8.7% ) indicated otherwise . with regards to the frequency of contact with body fluids , 94 ( 54.7% ) respondents had daily contact with body fluids , 30 ( 17.4% ) had weekly contact while 48 ( 27.9% ) handled body fluids infrequently . one hundred and twenty ( 69.4% ) of the respondents reported history of needle stick injuries and majority ( 66.7% ) of those reporting needle stick injuries ( nsi ) had had multiple injuries . only 26 ( 21.6% ) of the 120 respondents that had history of nsi had received antiretroviral prophylaxis following such nsi . characteristics and risk profile of 175 family physicians in nigeria surveyed on their knowledge and awareness of hiv postexposure prophylaxis the level of awareness and knowledge of hiv pep is presented in table 2 . one hundred and seventy one ( 97.7% ) respondents were aware of the existence of the concept of hiv pep and an equal number of respondents ( 171 ) were aware that hiv pep was effective in reducing the transmission of hiv . seventy - four ( 51.4% ) respondents correctly identified the risk of transmission of hiv from a nsi to be around three per 1000 injuries . sixty - three ( 43.8% ) over estimated the risk while 7 ( 4.9% ) underestimated the risk of hiv transmission from needle stick injuries . regarding knowledge of various high risk body fluids for hiv transmission , the following proportions were obtained for correct identification of the following body fluids as high risk for hiv transmission : breast milk 88.6% , peritoneal fluid 85.7% , synovial fluid 77.0% , pleural fluid 87.0% , and cerebrospinal fluid 85.8% . however , 12.7% , 17.7% , and 11.5% incorrectly identified urine , saliva and feces respectively as high - risk fluids for hiv transmission . results of the responses of 175 family physicians in nigeria surveyed on their knowledge and awareness of hiv postexposure prophylaxis one hundred and thirteen ( 70.2% ) and 166 ( 97.1% ) correctly stated both measures as two first aid procedures to perform at the needle stick site . fifty - five ( 34.4% ) respondents erroneously thought that applying hypochlorite to the wound was an appropriate first aid measure at the needle stick site . with regards to types of exposures with high risk for hiv transmission , all ( 100% ) the respondents knew that exposure of broken skin to body fluids was high risk for hiv transmission . one hundred and thirty eight ( 81.7% ) and 137 ( 83.5% ) identified mucous membrane exposure and percutaneous injuries were high - risk exposures for hiv transmission respectively . one hundred and fifty - four ( 93.9% ) of the respondents knew that hiv pep should be commenced within one hour of a high - risk needle stick injury , while 10 ( 6.1% ) thought pep could still be effectively used after 72 h following a needle stick injury . in response to what the ideal drug regimen is for high - risk exposure , 94 ( 57% ) respondents identified an expanded 3-drug regimen as ideal while 54 ( 32.7% ) and 17 ( 10.3% ) incorrectly stated 2-drug and one - drug regimens , respectively . one hundred and forty ( 83.3% ) respondents said hiv pep should be used for 4 weeks , while 26 ( 15.5% ) respondents over estimated the duration of use and two ( 1.2% ) underestimated the duration of use of hiv pep . of 167 physicians responding to the question of the administration of hiv pep for nonoccupational exposures , 102 ( 61.1% ) indicated that it was appropriate to use hiv pep for nonoccupational exposure while 65 ( 38.9% ) did not support the use of hiv pep for nonoccupational exposures . for the purpose of this study , adequate knowledge was defined as correctly answering 16 ( 70% ) of the 23 listed items . one hundred and thirty - nine ( 79.4% ) of the 175 physicians surveyed had adequate knowledge while 36 ( 20.6% ) had inadequate knowledge . we compared respondents with adequate and inadequate knowledge by sex , work setting ( government or other ) , receipt of donor funds for hiv treatment and rank ( junior - senior house officers / registrar versus senior - senior registrar / consultant doctor ) as shown in table 3 . junior doctors had more adequate knowledge compared to senior doctors regarding hiv pep ( p=0.005 ) . being male was also associated with adequate knowledge ( p=0.05 ) . however , no single factor was independently predictive of adequate knowledge on multivariate analysis . results of comparison of 175 family physicians in nigeria with and without adequate knowledge regarding hiv postexposure prophylaxis one hundred and seventy one ( 97.7% ) respondents were aware of the existence of the concept of hiv pep and an equal number of respondents ( 171 ) were aware that hiv pep was effective in reducing the transmission of hiv . seventy - four ( 51.4% ) respondents correctly identified the risk of transmission of hiv from a nsi to be around three per 1000 injuries . sixty - three ( 43.8% ) over estimated the risk while 7 ( 4.9% ) underestimated the risk of hiv transmission from needle stick injuries . regarding knowledge of various high risk body fluids for hiv transmission , the following proportions were obtained for correct identification of the following body fluids as high risk for hiv transmission : breast milk 88.6% , peritoneal fluid 85.7% , synovial fluid 77.0% , pleural fluid 87.0% , and cerebrospinal fluid 85.8% . however , 12.7% , 17.7% , and 11.5% incorrectly identified urine , saliva and feces respectively as high - risk fluids for hiv transmission . results of the responses of 175 family physicians in nigeria surveyed on their knowledge and awareness of hiv postexposure prophylaxis one hundred and thirteen ( 70.2% ) and 166 ( 97.1% ) correctly stated both measures as two first aid procedures to perform at the needle stick site . fifty - five ( 34.4% ) respondents erroneously thought that applying hypochlorite to the wound was an appropriate first aid measure at the needle stick site . with regards to types of exposures with high risk for hiv transmission , all ( 100% ) the respondents knew that exposure of broken skin to body fluids was high risk for hiv transmission . one hundred and thirty eight ( 81.7% ) and 137 ( 83.5% ) identified mucous membrane exposure and percutaneous injuries were high - risk exposures for hiv transmission respectively . one hundred and fifty - four ( 93.9% ) of the respondents knew that hiv pep should be commenced within one hour of a high - risk needle stick injury , while 10 ( 6.1% ) thought pep could still be effectively used after 72 h following a needle stick injury . in response to what the ideal drug regimen is for high - risk exposure , 94 ( 57% ) respondents identified an expanded 3-drug regimen as ideal while 54 ( 32.7% ) and 17 ( 10.3% ) incorrectly stated 2-drug and one - drug regimens , respectively . one hundred and forty ( 83.3% ) respondents said hiv pep should be used for 4 weeks , while 26 ( 15.5% ) respondents over estimated the duration of use and two ( 1.2% ) underestimated the duration of use of hiv pep . of 167 physicians responding to the question of the administration of hiv pep for nonoccupational exposures , 102 ( 61.1% ) indicated that it was appropriate to use hiv pep for nonoccupational exposure while 65 ( 38.9% ) did not support the use of hiv pep for nonoccupational exposures . for the purpose of this study , adequate knowledge was defined as correctly answering 16 ( 70% ) of the 23 listed items . one hundred and thirty - nine ( 79.4% ) of the 175 physicians surveyed had adequate knowledge while 36 ( 20.6% ) had inadequate knowledge . we compared respondents with adequate and inadequate knowledge by sex , work setting ( government or other ) , receipt of donor funds for hiv treatment and rank ( junior - senior house officers / registrar versus senior - senior registrar / consultant doctor ) as shown in table 3 . junior doctors had more adequate knowledge compared to senior doctors regarding hiv pep ( p=0.005 ) . being male was also associated with adequate knowledge ( p=0.05 ) . however , no single factor was independently predictive of adequate knowledge on multivariate analysis . results of comparison of 175 family physicians in nigeria with and without adequate knowledge regarding hiv postexposure prophylaxis hiv / aids continues to be a serious public health concern and occupational exposure of hcws to this virus poses a threat to health care delivery systems in resource - limited settings . ensuring occupational health and workplace safety pose serious challenges in our clinical care settings ; therefore , studies relating to awareness , knowledge , and attitude / practices of hcws are vital as they help to inform policy on occupational postexposure prophylaxis against blood borne pathogens such as the hiv . this study showed that the greater majority ( 97.7% ) of family physicians in nigeria are aware of the concept of hiv pep and its effectiveness in reducing hiv transmission . those with existing hiv pep policies in their facilities accounted for 82.1% of the respondents . while only about half ( 51.4% ) of the respondents correctly identified the risk of hiv transmission from nsi , there was a high level of knowledge of high - risk body fluids and types of exposures to hiv infection . we demonstrated good knowledge of first aid measures to be instituted at exposure sites and also good knowledge of the best time to commence hiv pep , as well as the ideal arv regimen to use in the event of a high - risk exposure to hiv . however , when the level of knowledge was compared between senior and junior doctors , junior doctors were more knowledgeable about hiv pep than senior doctors . our respondents are similar in gender and age distribution when compared to previous reports from nigeria89 as well as in other middle and low - income countries,1012 but differed significantly in terms of awareness and knowledge of high - risk fluids for hiv transmission . the levels of awareness of hiv pep and its effectiveness in reducing hiv transmission were 97.7% and 99.4% , respectively , for our respondents . among hcws in a hospital in uganda,11 95% of the respondents had heard about hiv pep but only 61% believed it was able to reduce the risk of hiv transmission . similarly , in a study of hcw from ethiopia,10 100% of the doctors surveyed believed that hiv pep could reduce the risk of hiv transmission but only 50% had adequate knowledge of hiv pep . in a survey of anaesthetists in nigeria,13 92.1% of the respondents were aware of the concept of hiv pep , however , only 39.7% had existing hiv pep policies in their institutions . a recent survey among hcw in central nigeria9 reported that 99% of the doctors had ever heard of hiv pep but only 56.5% were aware of the existence of written policies in their institutions . in a survey among junior doctors in the united kingdom , 93% of those surveyed had heard of pep for hiv ; however , only76% were aware that hiv pep reduced the transmission of hiv.18 an earlier study among general practitioners in australia reported a lower level of awareness of hiv pep of 68.5% compared to our respondents.14 the high levels of awareness of hiv pep obtained in our cohort may be attributable to the fact that , there has been increased government and donor funds through the pepfar and global funds to fight aids and malaria to implement training of hcw and increase access to hiv treatment programs in health care settings in nigeria . the risk of a nsi from a patient with hiv resulting in seroconversion is estimated to be around 0.3%.15 overall , 51.4% of our respondents correctly estimated the risk of seroconversion from nsi . less than half ( 43.8% ) over estimated the risk and 4.9% underestimated the risk . this low level of risk estimation among fps in nigeria is similar to that reported from other medical specialties.13 similarly , among british anaesthetists , only 34% were aware of the true risk of hiv transmission from nsi.16 the need for adequate knowledge of nsi risk of hiv transmission can not be overemphasized among hcw especially primary care physician 's as this is essential in the risk assessment process of hiv pep administration . the level of knowledge on the correct identification of both high and low - risk body fluids for hiv transmission was high for our respondents . similar rates have been reported among hcw and surgery resident doctors in nigeria917 but much lower rates of knowledge of high and low risk body fluids were found among nigerian and uk anaeasthetists.1617 even though universal precaution is advocated for every patient , correct knowledge of high - risk body fluids for hiv transmission is essential so that extra precaution may be taken to further minimize the risk of hiv transmission . following a nsi , the first priority should be to promote active bleeding of the wound and to wash thoroughly with soap and running water . the majority of ( > 90% ) of our respondents knew the first - aid measures to be instituted at nsi site and 93.9% knew that pep following an hiv prone nsi should be commenced within one hour . other studies reported much lower rates of knowledge ( between 1538.5% ) about the optimal timing for pep in the event of a nsi.12131819 when our respondents were assessed for adequacy of knowledge of hiv pep , 79.4% had adequate knowledge . the proportion of our respondents with adequate knowledge is higher than the 41.0% reported among surgery residents in the eastern part of nigeria17 and the 50% obtained among doctors in ethiopia.10 among nigerian dentists , 64.4% were reported to have good knowledge.8 a survey that compared doctors based on adequate versus inadequate knowledge found younger age ( < 40 versus > 40 years ) , male gender and type of practice setting ( teaching hospital versus other settings ) and shorter length of practice ( < 10 versus > 10 years ) to be significantly associated with adequate knowledge.10 younger or junior doctors are more likely to be undergoing postgraduate training and this may explain why they had more adequate knowledge of hiv pep as hiv medicine is incorporated into their training curricula . the reason why male gender was associated with better knowledge may be due to the unequal distribution of respondents in our study and other studies because majority of our respondents were male . our study reports high level of awareness , knowledge of hiv risk transmission from nsi and adequate knowledge of hiv pep among fps in nigeria . these overall findings may be attributed to the fact that nigerian hcws have received increased training activities in hiv medicine because of increased government funding and commitment to fight hiv / aids . also , donor driven initiatives like the us governments presidents emergency plan for aids relief ( pepfar ) and the global fund to fight aids , tb and malaria ( gftam ) have contributed to capacity building in hiv / aids management especially in the health sector . despite the level of awareness and knowledge demonstrated by the family physicians in our study , majority of those that had been exposed to nsi did not access hiv pep , even though most of their institutions had existing hiv pep policies and protocols . this knowledge practice gap provides opportunities for targeted interventions to improve hcws access to existing policies and protocols for effective utilization of hiv pep which is needed to prevent seroconversions from nsi and other modes of occupational exposure , especially since recent reports from nigeria indicate that this knowledge practice gap is generalized among those at risk.92021 special programs to improve knowledge and increase access to hiv pep targeting senior doctors should also be encouraged and instituted . our study was cross - sectional in nature but the results are generalizable because it was a nationwide survey that included fps from all over the country . in conclusion , our study has revealed that despite the high levels of awareness and knowledge of hiv pep among nigerian family physicians , there exists a knowledge - practice gap necessitating the need for enhanced prevention education to improve or close this gap .

background : to determine the level of awareness and knowledge of hiv postexposure prophylaxis ( hiv pep ) and determinants of adequate knowledge among family physicians in nigeria.materials and methods : this was a cross - sectional questionnaire - based survey conducted among 175 family physicians at two national conferences.results:majority ( 97.7% ) of the respondents was aware of the concept of hiv pep and 99.4% believed it was effective in preventing hiv transmission . over two third of our respondents had been exposed to nsi ; however , less than 25% of those exposed received pep . there was high level of knowledge of the various high - risk body fluids as well as types of high - risk exposures . 93.9% of our respondents knew that hiv pep should commence within 1 h of exposure , 83.3% knew the correct duration of hiv pep , but only 57.0% knew the ideal pep regimen for high - risk exposures . the total mean score for our respondents was 17.82.9 with 79.4% having an adequate score . being a junior doctor and male sex were associated with adequate knowledge.conclusion:this study shows that despite high levels of awareness and knowledge of hiv pep , access to its use among family physicians in nigeria is still sub - optimal .

INTRODUCTION MATERIALS AND METHODS RESULTS Awareness and knowledge of HIV PEP Factors associated with adequate knowledge of HIV PEP DISCUSSION CONCLUSION

PMC3135088

it is well established that stroke survivors have low levels of physical fitness and muscle strength that impact on their ability to perform everyday activities and affect their independence and community participation . physical inactivity after stroke has many negative sequelae that may impact upon potential reorganization of brain function and on recovery of motor abilities . there is greater awareness now of the need for patients to practice everyday actions intensively in order to regain skill in motor performance . however , it is only recently that interest has turned to the physiological effects of inactivity in bringing about a reduction in aerobic capacity and physical endurance and to the effect of this deconditioning on an individual 's capacity to raise the energy levels in motor training that are necessary to improve functional performance and brain reorganization . physical activity is positively related to aerobic capacity , the capacity of the cardiorespiratory system to supply oxygen ( the cardiac output ) and of skeletal muscle to utilize oxygen . low aerobic capacity limits performance of activities of daily life and increases the risk of falls and dependence on others . it is also a significant determinant of poor bone health ( specifically osteoporosis ) in individuals with chronic stroke . reduced cardiorespiratory fitness and inactivity after stroke appear to be related to a combination of pathological ( comorbid cardiovascular disease , coronary artery disease ) , physiological ( decreased muscle activation and motor control , and changes in muscle length and stiffness ) , and environmental factors ( little opportunity or incentive for physical activity ) . for the elderly , these factors may be combined with an age - related decline in cardiorespiratory fitness reported to be approximately 10% or greater per decade . environmental factors contributing to declining fitness during rehabilitation may include excessive bed rest and physical and mental inactivity . observational studies in the usa and europe , carried out in the 1980s , found that the majority of a patient 's day in hospital rehabilitation may be spent being alone and inactive [ 3 , 4 ] . a similar situation was observed more recently in an australian institution where only a small proportion of the day was spent in physical activity [ 5 , 6 ] . one study of physical activity within the first 14 days of acute stroke found that although most patients , 84.5% , were free to move out of bed , more than 50% of the patients ' time was actually spent resting in bed . only 13% of individuals observed were engaged in activities that might have the potential to improve mobility and prevent secondary complications . a multicentre longitudinal project has compared inpatient care and recovery patterns between four european rehabilitation centres . patients in the uk centre spent less time in therapy and more time sitting inactive by their beds than those in the german or swiss centres , with uk therapists spending more time doing legally required administrative tasks with less time to care for their patients ( 1 hour per day ) [ 8 , 9 ] . clearly , if meaningful and challenging physical and mental activity play a critical part in driving optimal brain reorganization , there is an urgent need throughout the world for patients to be more active during their hospital rehabilitation than they currently tend to be . however , it is not only the time spent in active exercise that is significant but whether what the individual is doing in that time is sufficiently vigorous and relevant to induce a training effect not only on motor skill but also on level of fitness . a study of patients in physiotherapy and occupational therapy between 2 and 14 weeks after stroke , during which heart rate was monitored , indicated that therapy may be of insufficient intensity to produce a cardiorespiratory training effect . on average , in a physiotherapy session , 42% of the time was spent inactive in lying , 11% active in lying , 16% active in sitting , and 31% active in standing . if it was present , the aerobic component of a typical physiotherapy session lasted less than 3 minutes . although one might expect progressively higher exercise intensities over time as functional status improved , any increase in hr mean and hrpeak did not reach statistical significance . during occupational therapy a considerable percentage of time was spent in sitting , discussing issues related to discharge , equipment needs and home management . the reorganization of rehabilitation to enable specific task training and practice , with the progressively increasing intensity of exercise required not only for motor learning but also directed specifically at muscle strength and endurance , and aerobic cardiorespiratory fitness , is a major challenge . in this paper we argue for increased effort to be made to reorganize therapy delivery after stroke to take account of these needs , in particular specific cardiorespiratory training , and we give some examples of how this might be achieved [ 11 , 12 ] . it is well known that people respond negatively to prolonged physical inactivity . however , after stroke the consequences may be particularly severe , since the reorganization of the brain that follows the neural lesion is thought to be dependent on what the patient does and the meaningfulness of this , as well as the environment in which activity takes place . early onset of active and moderately vigorous rehabilitation , especially of weight - bearing activities , could be expected to influence not only the rate and extent of physical recovery , by preventing neuromuscular disuse changes related to long periods of rest , but also to have a role in minimizing cognitive deterioration , depression , and anxiety . early mobilization ( helping patients to get out of bed into sitting , standing up , and walking ) is now recommended in a number of clinical guidelines for acute stroke ( e.g. , ) . in general , if there is no progression of neurological deficits , active rehabilitation should commence within 2448 hours . the few existing studies provide limited but promising evidence of the benefits of early exercise on functional recovery , as well as on early depression , medical complications , health care costs , and harm minimization . a recent cochrane review concluded that although further research is needed in this field , the evidence suggests that stroke units practicing early mobilization as part of standard care should continue to do so . there is evidence that early , frequent , and active mobilization can influence recovery . a stroke unit in trondheim , norway , a physiotherapist and nurse take a staged approach , first sitting the patient up in bed , and then over the edge of the bed , then into a chair or into standing as appropriate . once this first mobilization has been successfully completed , the patient is then assisted frequently throughout the day to repeat out of bed activities , progressively increasing activity as able . a study in 2008 compared physical activity levels of rehabilitation patients in this early mobilization centre with stroke units in melbourne , australia . it was found that trondheim patients spent twice as long standing and walking and less time lying in bed . patients were observed to change position from lying to sitting to standing twice as often as patients in melbourne . these results are only meaningful if it can be shown that early physical activity actually does improve patient outcome , and there is some indirect evidence that it has a positive effect . a large , international multicentre randomised controlled trial , the australian very early rehabilitation trial ( avert ) , is currently underway aimed at testing whether early and frequent mobilization results in fewer deaths and less disability at 3 months after stroke compared with current stroke practice . the recognition of the specificity of neuromuscular processes and resultant development of a task - specific training and practice model of rehabilitation with emphasis on activating motor learning processes , provides a framework for functional rehabilitation , and biomechanics and exercise physiology provide the means to combine skill and aerobic training methods even in this early phase of rehabilitation [ 11 , 12 ] . for example , simple methods of providing increased intensity in the practice of significant actions in this early period include specific and repetitive practice of standing up and sitting down , aimed both at improving the effectiveness of performance of standing up itself but also the person 's ability to perform the action with sufficient repetitions to produce an efficient performance in terms of o2 uptake . the ability to stand up and sit down is critical to the rehabilitation process , and biomechanical principles provide guidelines to reducing the effort required to stand up in the very early stages of mobilization . for example , biomechanical investigations have shown that positioning the feet back to 75 ankle dorsiflexion and raising the seat height each reduces the muscle forces required . walking on a treadmill with body weight support via harness connected to an overhead support system may be the only means of practicing walking for more severely affected weak and dependent individuals early after their stroke , and it is reported to be well tolerated and effective . without the treadmill and harness , practice of walking will be delayed . treadmill walking also allows for independent and semi - supervised practice for those with more ability , as well as improving aerobic capacity , and increasing walking speed and endurance . the very early provision of assisted overground and harness - supported treadmill walking is probably critical to good postdischarge functional capacity in terms of both performance and energy levels . an early goal for walking overground and independently is to walk to the next appointment or to walk at least part of the way rather than being transported in a wheelchair . the goal can be to walk this distance 5 times a day , increasing distance as soon as possible , and keeping a record of progress which gives the patient a specific attentional focus . the results of several motor learning studies in which the person 's attention was focused on the outcome of an action rather than the action itself ( or the quality of the movement ) , have shown that , for example , walking to a prearranged place as quickly as possible can produce more effective performance than focusing attention on the quality of the movement . in early practice of overground walking , a sense of security is provided if the therapist or nurse assists by holding a belt with grab handles ( e.g. , handi - lift / walk belt , pelican manufacturing , osborne park , western australia 6017 ) . it is increasingly acknowledged that the rehabilitation team must adapt the delivery of interventions , increase the time spent in meaningful exercises and task practice , to meet the needs of brain reorganization , skill relearning , and improved physical and mental fitness . the low intensity , low duration , and low frequency of physiotherapy common in many rehabilitation centres , following outdated and insufficiently challenging therapy models of intervention , sometimes for as little as 30 minutes to one hour per day , severely restrict the optimal level of recovery required for participation in daily and social activities after stroke . therapists need to move away from reliance on one - to - one therapy to a model in which the patient practices not only in individualized training sessions with a therapist but also in groups , and in circuit training , where patients practice at work stations set up for weight - bearing strength training exercises , and to encourage practice of specific actions . patients are semisupervised and assisted as necessary by therapist and aide , with similar attitudes and methods to those who work in sports training . the modern rehabilitation workspace needs to provide an environment built to encourage and challenge physical activity . such an environment includes a suspended harness system that allows practice of balancing and walking tasks , feedback and computerized devices to provide information and incentive , exercise machines such as treadmills with harness suspension , stationary bicycles , including an electronically braked isokinetic ergometer , and stepping machines . some centres are developing and testing electromechanical training aides , including robotic devices and virtual reality systems . even simple technological aids can increase time spent in physical and mental activity by enabling patients to practice independently . assistive devices and the assistance of therapy aides can drive physical participation and increase intensity of practice , motivation , competition , and personal responsibility . patients can also work in a twosome , one assisting the practice of the other . for both therapist and patient , the focus of physiotherapy is always on the optimization of functional motor actions critical to everyday life . optimization in this context refers to training a person 's performance to be as efficient and as effective as possible in achieving a functional goal , effective referring to a goal achieved and efficient meaning at the least physiological cost . for this latter , the focus is particularly on exercising the large muscles of the lower limbs and trunk , a requirement of cardiorespiratory fitness training . mobility requires lower limb muscles that are strong enough and well enough coordinated to support , transport , and balance the body mass in a gravitational environment , with muscles capable of producing force quickly enough and with the appropriate timing ( i.e. , power ) to avoid unexpected trips and slips . all of this requires appropriate energy levels and aerobic fitness . as an example , take an action that is performed many times in daily life . the ability to stand up and sit down effectively without falling is essential to independent living as well as a critical prerequisite for upright mobility , yet it is one of the most physically demanding actions we perform regularly , requiring greater strength of lower limb extensors and range of motion of hips , knees , and ankles than walking or stair climbing . lack of independence in this action is a common source of falls [ 34 , 35 ] , and is one of the most likely factors associated with institutionalization . guidelines for training : standing up and sitting down based on biomechanical experimentation and clinical evidence , are provided in a recent textbook . in a randomized controlled study of a program for training standing up after stroke , britton and colleagues assessed the effects of a 30 min standardized training program of standing up and sitting down . the aim of each session was to increase the number of repetitions , and the number of stands and sits was recorded throughout each day using an activpal accelerometer activity monitor . the number of repetitions in the exercise group increased on average from as few as 18 per day to 50 stands . in addition , in this group there was a significant mean increase in percentage of body weight taken through the paretic ( 10% ) leg after 1 week of training . in contrast , in the control group , the weight taken on the paretic leg decreased during the period of the clinical trial . the strength of this study is in the prescribed protocol which included not only the number of repetitions per day but also a record of what the patient actually did and how many repetitions , that is , the dosage . this latter is notable , as dosage of therapy has not been an issue in rehabilitation until recently and has rarely been reported in clinical trials . the ability to regain functional motor skills and increase the intensity of exercise and practice in rehabilitation involves in large part the acquisition of balance . central to balance control is the need to keep the centre of mass within the perimeter of the base of support , or on track to a new base of support as in walking or running . it is becoming increasingly clear that although there are many different types of exercise available , some are more likely to improve balance and prevent falls than others , but overall it appears that balancing mechanisms are very specific to the action being performed , the purpose of the action ( the task ) and the environment in which it takes place . there is increasing evidence that challenging balance exercises in standing with the feet close together and exercises for the lower limb muscles performed in standing against body weight resistance [ 39 , 40 ] , for example , in standing up and sitting down , step ups , heels raise and lower , marching , stair walking , semisquats , and reaching to the floor sideways and forward to pick up an object , performed with increasing numbers of repetitions and without reliance on the upper limbs for support and balance are the optimal way to improve balance as well as flexibility , strength and endurance , and fitness . these exercises can be made more challenging by increasing the height of steps and chairs and by increasing and varying speed . despite the positive changes taking place in physical rehabilitation , it appears to be common for deconditioning to be evident in the stroke community at discharge from rehabilitation . we know that physical activity is positively related to aerobic capacity and improving aerobic capacity during rehabilitation must be addressed if individuals are to become part of their community again . further information on testing aerobic fitness , methods of increasing intensity of effort as a person improves , and the monitoring of physiological responses is provided in several publications ( see [ 11 , 42 , 44 ] ) . the rehabilitation period , often very short , provides an opportunity to ensure that patients are not only prepared for discharge but also understand the need to continue with physical activity after discharge in order to increase their motor skill and fitness levels . the promotion of a physically active lifestyle has become an important issue in health policy in many countries . the world health organization ( who ) has recognised this by modifying and revising the classification of impairments , disabilities , and handicaps . the most recent international classification of function ( icf ) provides greater focus on quality of life and integration of physical activity into daily life , emphasising limitation in activity rather than disability and restriction in participation rather than handicap . recently , who issued a warning that a sedentary lifestyle is one of the most serious , yet insufficiently , addressed public health problems of our time , and this is of course as relevant to people disabled through stroke as it is to anyone else . although many people affected by stroke regain the ability to walk by the time they are discharged from rehabilitation , it is well documented that many have sufficiently low physical endurance to limit their ability to perform household tasks or even to walk short distances . it has been found that walking may require a much higher level of energy expenditure than before the stroke , and that this can limit the ability to walk outside the house . minimum criteria for successful community walking include an independent walking velocity of 0.8 m / s or greater , the ability to negotiate uneven terrain and kerbs , and the physical endurance to walk 500 m or more . in a review of 109 people discharged from physiotherapy , cardiorespiratory fitness training can address both the efficiency with which people affected by stroke can walk and the distance they are able to achieve . the loss of independent ambulation outdoors has been identified as one of the most debilitating of stroke sequelae . among stroke survivors one year after stroke , the most striking area of difficulty was low endurance measured by the distance walked in a 6-minute walk test ( 6mwt ) . those subjects able to complete this test were able to walk on average only 250 m compared to the age - predicted distance of > 600 m , equivalent to 40% of their predicted ability and not far enough for a reasonable and active lifestyle . the detrimental effect of low exercise capacity and muscle endurance on functional mobility and on resistance to fatigue is likely to increase after discharge if follow - up physical activity and exercise programs are not available . a physically active lifestyle has several fitness and health benefits and is also critical for stroke survivors . exercise helps maintain fitness and participation , but also has a very real protective function , helping to prevent recurrent stroke and further cardiovascular disease , reducing the risk of falls , increasing physical independence and improving quality of life . in addition , a randomized controlled trial provides evidence of increased activity of cortico - subcortical networks produced by repetitive treadmill training of stroke - affected individuals . similar changes were not found in the control group who had a comparable program that provided neither aerobic stress nor required many active repetitions . exercises directed at maintaining or improving aerobic function , strength and endurance , flexibility , and neuromuscular control are all strongly recommended for stroke survivors in order to further the recovery process , increase confidence and improve well - being . currently , the challenge for many survivors is to find in their local community both a person with the expertise to assist with the prescription and supervision of their program of exercise and aerobic training , and the location of a suitable community - based venue . physiotherapists and exercise science graduates are well placed to play a role in the development of poststroke community - based exercise programs that include aerobic exercise and provide additional skill training , as well as offering help and advice about ways of increasing physical activity throughout the day . the major forms of exercise to improve physical activity for individuals after stroke include , as well as aerobic fitness training [ 11 , 42 , 44 ] , functional task and balance training [ 1 , 41 , 5156 ] , and strength and endurance training [ 41 , 55 , 56 ] . a program can include , in addition to task - relevant exercises , a combination of aerobic , strengthening , and flexibility exercises ( see ) . active exercise can enhance physical activity and exercise tolerance if well prescribed , but exercises need to be sufficiently intensive and in weight - bearing positions . exercises that share similar biomechanical characteristics , for example , exercises that involve flexion and extension of hips , knees and ankles over the feet as a fixed base of support , are likely to enable a transfer of strength gains to improved stair walking , squatting , and standing up and sitting down . exercises in water may also have positive training effects , and when properly supervised can increase fitness . they may be particularly useful for people with painful arthritic joints who find it difficult to exercise . an interesting outcome of organized postdischarge exercise programs for chronic stroke patients has been the collegiality that develops among participants and the encouragement and support they provide to each other . similar observations regarding the social benefits of group community exercise programs have been reported by others [ 55 , 59 ] . increased self - esteem and a feeling of well - being and of being in control that is engendered by an exercise program can give individuals the confidence and drive to improve their performance in other activities . on completion of an exercise program on stationery bicycles , for example , participants reported increased self - esteem and several reported changes in their general behaviour one participant started gardening again and another could once again comb her hair . we know from findings of recent studies that it is possible , even several years after stroke , for people to make positive gains in strength , mobility , fitness , endurance , and skill in semisupervised community classes [ 5156 ] . several of these programs had an impact on activities such as getting on and off a bus and socializing with friends away from their homes , all activities considered meaningful to the individuals . interviews with participants and their families suggested that quality of life gains are critical factors in reintegrating stroke survivors into society . similarly , aerobic capacity improves with appropriate training even a long time after stroke [ 11 , 42 ] . methods used in different trials have included cycle ergometry , treadmill walking [ 63 , 64 ] , and a combination of aerobic and strengthening exercises [ 54 , 56 ] . although the effects are exercise - specific , generalization occurred in the improvements noted in general health and well - being that positively affected those activities the participants considered meaningful . a recent systematic review provides good evidence that aerobic exercise , at 5080% heart rate reserve , on 35 days a week for 2040 minutes should be an important component of stroke rehabilitation and postdischarge training . motivation to start exercising after discharge depends on several factors availability of community - based group exercise programs , previous exercise experience , knowledge about exercise and general health , and psychological factors [ 65 , 66 ] . availability of transport and/or dependency on another can affect participation as does absence of feedback . feedback in relation to improved balance , distance walked , or increased muscle strength may provide additional incentives for continued participation . exercise diaries or practice records can provide information to encourage individuals to continue to exercise after input from an instructor . social support is an important mediator of adherence and since effects of exercise are not always immediately apparent , social support is especially important at the beginning of a program . there is some evidence that most of those who participate in exercise programs for six months are likely to continue to do so . although it is evident that in general more intensive rehabilitation results in better recovery , evidence from studies over a number of years suggests that physical rehabilitation is not intensive or targeted enough to have a training effect or even to prevent secondary adaptive changes in muscle and metabolism that underlie the profound cardiovascular and muscular deconditioning . there is evidence that those who demonstrate improved walking at discharge are not necessarily functional walkers when faced with the energy requirements of real life nor are they confident enough to walk outside the house . it is becoming clear that the process of regaining independence and building up energy levels requires more active and intensive inpatient rehabilitation than commonly available , together with a comprehensive plan for a post - discharge continuation of physical activity and the development of exercise opportunities within the community . it should therefore be an expectation that clinical practitioners will follow specific guidelines based on this knowledge and on the evidence from clinical research , and that they will test functional outcomes of specific interventions at regular intervals using valid and reliable measures . for movement rehabilitation after stroke to achieve the goals expressed here , physiotherapists who have no background in exercise science or biomechanics need to upgrade their knowledge and skills in appropriate educational settings .

it is becoming increasingly clear that , if reorganization of brain function is to be optimal after stroke , there needs to be a reorganisation of the methods used in physical rehabilitation and the time spent in specific task practice , strength and endurance training , and aerobic exercise . frequency and intensity of rehabilitation need to be increased so that patients can gain the energy levels and vigour necessary for participation in physical activity both during rehabilitation and after discharge . it is evident that many patients are discharged from inpatient rehabilitation severely deconditioned , meaning that their energy levels are too low for active participation in daily life . physicians , therapists , and nursing staff responsible for rehabilitation practice should address this issue not only during inpatient rehabilitation but also after discharge by promoting and supporting community - based exercise opportunities . during inpatient rehabilitation , group sessions should be frequent and need to include specific aerobic training . physiotherapy must take advantage of the training aids available , including exercise equipment such as treadmills , and of new developments in computerised feedback systems , robotics , and electromechanical trainers . for illustrative purposes , this paper focuses on the role of physiotherapists , but the necessary changes in practice and in attitude will require cooperation from many others .

1. Introduction 2. Early Mobilization 3. Exercising during Rehabilitation 4. Continuing Exercise and Increasing Physical Activity after Discharge 5. Conclusion

PMC3996736

a ureteropelvic junction ( upj ) obstruction can be thought of as a restriction to flow of urine , from the renal pelvis to the ureter , which , if left uncorrected , leads to progressive renal deterioration . upj obstruction occurs in all paediatric age groups , but there tends to be a clustering in the neonatal period because of the detection of antenatal hydronephrosis and again later in life because of symptomatic occurrence . the surgical correction of upj obstruction has evolved on a number of fronts , with open surgical techniques yielding way to endoscopic , laparoscopic and robot - assisted approaches . whatever be the approach , dismembered pyeloplasty has been universally accepted because of its ( 1 ) broad applicability , including preservation of lower pole or crossing vessels , ( 2 ) excision of the pathologic upj and ( 3 ) successful reduction pyeloplasty . at times , dismembered pyeloplasty may be problematic whenever the renal pelvis is small and the ureteral length is inadequate . in such instances , a non - dismembered pyeloplasty can be performed to overcome this problem . in non - dismembered pyeloplasty , a spiral flap is created from the renal pelvis to repair the defect at the upj . such a flap is able to bridge the gap between the pelvis and healthy ureter over a distance of several centimetres . scardino and prince described a vertical flap that can be used in the situation of a dependent upj with a large , square - shaped extrarenal pelvis . the foley yv plasty was originally described for the correction of upj with a high ureteral insertion . the diagnosis of upj obstruction was firmly established based on history , physical examination , renal sonography , intravenous urography and scintigraphy . exclusion criteria included presence of active urinary tract infection and very poor renal function ( split renal function < 10% ) . the risks of the laparoscopic operation were fully explained to the parents of the children , which included post - operative infection , bleeding , failure of pyeloplasty , the need to convert to open surgery , damage to other viscera and adhesion formation . the first 5 mm endoscope port was inserted by open laparoscopy using a blunt hasson cannula through the umbilical crease . the abdomen was inspected in detail so as to plan to insert the remaining two 3/5 mm instrument ports . the peritoneum overlying the exposed kidney was incised just lateral to and above the colonic flexure . once the correct plane was identified , the renal capsule was traced into the renal sinus until the renal pelvis was identified . the adventitia around the proximal ureter and upj was cleared . whenever it was observed that dismembered pyeloplasty could not be easily performed foley 's yv plasty [ figure 1 ] was planned whenever it was observed that a tension free dismembered pyeloplasty was not possible in spite of all possible manoeuvres . the base of the v was positioned on the dependent , medial aspect of the renal pelvis and the apex at the upj . the incision from the apex of the flap , which represents the stem of the y , was then carried along the lateral aspect of the proximal ureter well into an area of normal calibre . the apex of the pelvic flap was brought to the inferior most aspect of the ureterotomy incision . the posterior walls were approximated utilising interrupted or running fine absorbable sutures with an 18 cm , 6/0 vicryl suture on a 3/8 round body needle . a 0.025 inch guide wire was then passed through the proximal ureter into the bladder . a 3 fr multi - length double pig - tail catheter was passed over the guide - wire into the bladder . the proximal end of the double pig - tail stent was then placed within the renal pelvis . the anastomosis was completed with approximation of the anterior walls of the pelvic flap and urethrotomy [ figure 2 ] . dilated pelvic calyceal system with small renal pelvis ( < 30cc ) operative steps of laparoscopic foly 's yv plasty . ( a ) rt upj showing dilated pelvis ( b ) apex of v being planned at upjunction ( c ) from the apex of v , the inferior limb of y being extended on the lateral margin of upper ureter ( d ) y - v plasty in progress ( e ) completion of y - v plasty post - operatively the drain was removed once the drainage was less than 5 cc/24 h. the catheter was removed the next day . children were followed up for urinary infection , and renogram was repeated after 3 months . during the study period january 2004 to december 2010 , 119 children prospectively underwent laparoscopic pyeloplasty for upj obstruction . seven of these children were antenatally diagnosed with hydronephrosis . of these , 108 children ( 63 male and 45 female ) with a mean age of 4.94 2.78 years underwent laparoscopic dismembered pyeloplasty and the remaining 11 children ( 5 male and 6 female ) [ table 1 ] with a mean age of 4.00 1.776 years underwent laparoscopic foley 's yv plasty . comparison of operative and post - operative parameters the surgical indication for foley 's yv plasty was a small sized renal pelvis ( mean renal pelvic volume 31.8 ml ) in five children , long narrow ureteric segment with a small renal pelvis ( mean renal pelvic volume 34.6 ml ) in three children and a high insertion of ureter with a small sized renal pelvis ( mean renal pelvic volume 30.4 ml ) in the remaining three children . in none of these 11 children crossing renal vessels were observed at the upj . in all these 11 children , the kidney was dissected from the surrounding fibro - fatty tissue to release the kidney and pull it downwards . the mean operating time for laparoscopic dismembered pyeloplasty was 87.22 7.69 min and the mean post - operative hospital stay was 106.29 22.76 h. in comparison , the mean operating time for laparoscopic foleyyv plasty was 77.27 5.18 min and mean post - operative hospital stay was 82.91 12.53 h. there were no major peri - operative complications noted and conversion to open was not necessary in any child . the drain was removed after 24 h in all the children and the per - urethral catheter removed after 48 h. the mean follow - up of these 11 children was 20.82 1.83 months . renogram done at 3 months post - operatively showed good drainage and improvement of renal function . open pyeloplasty ( opn ) has been the standard treatment for congenital or acquired upj obstruction in adults and children , with overall success rates of 90 - 100% . laparoscopic surgery has increasingly assumed a central role in the management of both benign and malignant urologic diseases . numerous studies have implicated that minimally invasive procedures can provide equivalent surgical outcomes with reduced morbidity when compared with their open counterparts . as a consequence , laparoscopy has become more integrated into mainstream practice and in some cases has evolved into the standard of care for certain procedures . a prime example of this latter point is the pyeloplasty procedure for the treatment of upj obstruction , which in present day is commonly performed via a minimally invasive ( laparoscopic or robotic ) approach . feasibility , safety and effectiveness of laparoscopic pyeloplasty in children was described with similar success rates as open procedure . ravish et al . , compared the results of laparoscopic and opn in children with primary upj obstruction . post - operative discomfort / pain was significantly less in the laparoscopic group and similarly the mean hospital stay was significantly shorter in the laparoscopic group . casale et al . , reported on a series of 26 children aged between 8 months and 15 years ( mean age 5 years ) who underwent trans - peritoneal laparoscopic pyeloplasty for an obstruction not caused by a crossing vessel . hynes dismembered pyeloplasty ( ah ) , while the remaining seven had a non - dismembered pyeloplasty in a heineke mikulicz fashion ( hm ) . the mean operative time was 3.1 and 2.5 h for ah and hm , respectively . four of the seven patients having non - dismembered procedures presented with acute flank pain within 3 days of stent removal . the ah pyeloplasty produced a 94% rate of resolution of upj obstruction , while the hm patients did poorly , with a success rate of only 43% ( p = 0.002 ; fisher 's exact test ) . singh et al . , evaluated the role of non - dismembered laparoscopic pyeloplasty and percutaneous endopyelotomy for upj obstruction with low volume renal pelvis . a total of 34 patients of laparoscopic non - dismembered pyeloplasty were compared with 26 patients of upj obstruction with pelvic volume less than 50 ml undergoing antegrade endopyelotomy . only operative time was significantly less in patients undergoing endopyelotomy ( p < 0.05 ) . they concluded that in patients with upj obstruction with low volume pelvis and good renal function , laparoscopic non - dismembered pyeloplasty was effective with significantly longer operative time compared with endopyelotomy . szydeko et al . , performed a retrospective study to compare the results of dismembered and non - dismembered y - v laparoscopic pyeloplasties and the complications observed after the two types of surgery . eighty - eight patients with upj obstruction underwent a laparoscopic operation . in two cases , anderson pyeloplasty ( lhap ) was performed on 50 patients , whereas a laparoscopic y - v pyeloplasty ( ly - vp ) was performed in 36 cases . the mean operative time for lhap was 219 min and for ly - vp 185 min . the mean hospital stay after lhap was 5.9 days and for ly - vp 5.3 days . , the mean operating time for laparoscopic foley 's yv plasty was 77.27 5.18 min , which was significantly less when compared with the laparoscopic dismembered pyeloplasty group . this was probably due to the need for extensive intra - corporeal suturing needed for dismembered pyeloplasty and also trimming of the hugely dilated renal pelvis . robotic - assisted surgery may be ideally suited for a pyeloplasty , allowing one to overcome the difficulties encountered with laparoscopic suturing . lee et al . , presented the first preliminary results of robotic - assisted laparoscopic trans - peritoneal pyeloplasty in children . they performed a retrospective case control study of 33 patients undergoing robot - assisted laparoscopic pyeloplasty ( ralp ) and 33 undergoing opn . mean operative time was significantly less for opn ( 181 vs 219 min for ralp , p = 0.031 ) . olsen and jorgensen reported their unique experience of retroperitoneal robotic - assisted pyeloplasty in 13 children with a median age of 6.7 years . this early experience has been corroborated by a number of centres , demonstrating that advanced laparoscopic re - constructive surgery is feasible even in children . a novel example of such advancement can be found in laparoendoscopic single - site ( less ) surgery . performing surgery through a single site of access holds the promise of decreased morbidity , lower blood loss , shorter convalescence and improved cosmesis . given the non - extirpative nature of the pyeloplasty procedure , this patient cohort has proven to be the ideal candidates for less surgery . there are now several studies in the published literature demonstrating the feasibility and efficacy of less pyeloplasty in both children and adults . although comparative studies have failed to demonstrate any objective advantage of the less pyeloplasty operation , it is important to realize that less surgery is still a fairly new technique . further technological developments and additional studies will ultimately define its role in the field of urologic surgery , and subsequently , its application for the pyeloplasty procedure . laparoscopic foley 's yv pyeloplasty is a safe and effective technique in appropriately selected cases of primary upj obstruction in children . the operative time and post - operative hospitalization was significantly less when compared with laparoscopic dismembered pyeloplasty . post - operative recovery of renal function was similar to those who underwent laparoscopic dismembered pyeloplasty .

introduction : laparoscopic dismembered pyeloplasty is an acceptable option for ureteropelvic junction ( upj ) obstruction in the paediatric population . we compared our results of laparoscopic dismembered and non - dismembered foley 's yv pyeloplasty.materials and methods : children presenting with hydronephrosis secondary to upj obstruction formed the study group . foley 's yv plasty was planned whenever it was observed that a tension free dismembered pyeloplasty was not possible in spite of all possible manoeuvres . children were followed up for urinary infection , and renogram was repeated after 3 months.results:during the study period , 108 children ( 63 male and 45 female ) with a mean age of 4.94 2.78 years underwent laparoscopic dismembered pyeloplasty and the remaining 11 children ( 5 male and 6 female ) with a mean age of 4.00 1.776 years underwent laparoscopic foley 's yv plasty . there were no major peri - operative complications noted and conversion to open was not necessary in any child . renogram done at 3 months post - operatively showed good drainage and improvement of renal function.conclusions:laparoscopic foley 's yv pyeloplasty is a safe and effective technique in appropriately selected cases of primary upj obstruction in children .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

PMC3722632

human immunodeficiency virus ( hiv)-1 infection has spread to all population groups in india and has reached epidemic proportions . the rate of progression of hiv-1 disease exhibits a remarkable variation among different individuals . many host genetic factors are now known to affect the disease progression rates , especially polymorphisms in genes encoding chemokine receptors . although , no studies on chemokine receptor polymorphisms have been reported in the endogamous population of state of maharashtra state so far , only one study has been carried out in healthy individuals of tribes and muslim ethnic groups of andhra pradesh , south india . certain members of the chemokine family of receptors serve as critical portals for the entry of hiv-1 into target cells . a mutant allele ( cc - chemokine receptor-5 [ ccr5]-32 ) of the -chemokine receptor gene ccr5 carrying a 32 base - pair deletion prevents cell invasion by the primary transmitting strain of hiv-1 . individuals who are homozygous for the ccr5-32/32 allele are highly resistant to hiv-1 infection ; the heterozygote state does not protect against hiv-1 infection , although , heterozygotes have been found to have significantly lower viral loads . early reports indicated that the ccr5-32 allele maybe absent in indigenous non - european populations . the ccr5_32 allele appears to have originated quite recently ( approximately 7,000 years ago ) in northeastern europe . although its frequency has now reached a relatively high level in europeans , e.g. , 16.3% in finns and 15.8% in moravians , it is not present among african populations , and is only so at low levels in the asian . hence , by application it is possible to evaluate the influence of the european population on the genetic constitution of others . in india population one study has been carried out in healthy individuals of the tribes and muslim ethnic groups of andhra pradesh , south india . majumder and dey , reported absence of ccr5-32 in various ethnic populations of india , both tribal and non - tribal , except for some populations of the northern and western regions where this allele may have been introduced by caucasian gene flow . although a few studies on chemokine , chemokine receptor , and dcsign exon 4 repeat number polymorphisms have been reported in north indian ( aryan descent ) hiv patients and healthy controls , there is a dearth of reports on the hiv patients with and without tuberculosis ( tb ) of south indian ( dravidian descent ) origin . ccr5-32 exhibit variable frequencies in distinct populations and possibly , their phenotypes depend on the ethnicity analyzed . the brazilian population presents a complex genetic background , characterized by a high degree of miscegenation . in major brazilian cities , such diversity is some time attributed to the positioning of the indian peninsula at the tri - junction of the three continents , viz . the contemporary indian population is stratified as tribal and non - tribal , i.e. , caste population . the origin of the caste in india is an enigma , though many are known to have a tribal origin . the maharashtra state of india forms a huge irregular triangle with its base on the west cost of india , overlooking the arabian sea . historically , the state is comprised of three sub - regions , western maharashtra , the vidarbha , and the marathwada . vidarbha lies on the eastern side and thus mainly contributes to the region broadly referred to as central india . apart from the tribal population , many other ethnic communities mainly hindus , muslim , buddhist and sikhs , inhabit the region . the vidarbha has a hoary past and has been under the domination of many hindus , muslims , and tribal - gond kingdoms . it is assumed that the relationship between these various populations may define the present genetic landscape of india . taking this assumption and geographical and ethnic diversity into account in the present study , we investigated the distribution of ccr5-32 alleles in tribes from the vidarbha region ( maharashtra ) central india . besides inhabiting the adjoining state , a substantial number of these people inhabit in few district of vidarbha . bhils are listed as adivasi residents of the states of gujarat , madhya pradesh , chhattisgarh , maharashtra , and rajasthan in western and central india as well as in tripura in far - eastern india on the border with bangladesh . bhils are divided into a number of endogamous territorial divisions , which in turn have a number of clans and lineages . most bhils now speak the language of the region they reside in , such as marathi and gujarati . the korkus are a typical tribal population from amravati district and found only in the satpuda mountain ranges spanning maharashtra and madhya pradesh . they are mainly concentrated in melghat a scheduled area of korku comprising 89% of the tribal population . the phasse are a sub tribe of the paradhi caste , which includes sub - castes like gav paradhi , berad - paradhi , gay - paradhi , chita paradhi . there are only three surnames among them , chauhan , pawar , and solanke . we sampled 15 samples from this group from akola district . a low cultivating caste of berar , who numbered 52,000 persons in 1911 , and belongs to the yeotmal , akola , and buldana districts . the andhs appear to be a non - aryan tribe of the andhra or tamil country , from which they derive their name . there were 8228 andh in andhra pradesh in 1991 . according to singh et al . , in their 2004 book people of india there are over 74,000 andhs in maharashtra . this tribe falls under the primitive tribes category and spread much over the central india . gond generally speaks gondi dialect , which belongs to the dravidian linguistic family , after indo - european , in india . brahmin is a class of priests and preachers of dharma and considered as the torch bearer of hinduism . majority of brahmin in maharashtra speak marathi , one of the major languages of indo - aryan linguistic group . we have collected 108 blood samples on whatman fta mini cards ( ge health - care , uk , ltd ) from 6 tribe 's populations and a caste population from the district of vidarbha region . every card was labeled with appropriate code as per tribes / caste and district with informed consent obtained from each volunteer . approximately , ( 200 l [ 2 - 5 drop ] ) of blood by veni - puncture was directly spotted on the fta mini card within a printed circle area and dried at room temperature . briefly , 100 ng of genomic dna was denatured at 94c for 10 min , following , which it was subjected to 30 cycles of denaturation , annealing , and extension . the reaction mixture of 50 l contained , 50 mmol kcl , 10 mmol tris - hcl , ph 8.3 , 800 mol dntps , 100 g / ml gelatin , 10 pmoles of each of the ccr5-specific primer forward : ccr5 f : ( 5-cctggctgtcgtccatgctg-3 ) and reverse : ccr5-r : ( 5-ctgatctagagccatgtgcac aactct-3 ) . , and 1.5 units of taq polymerase enzyme ( xcelris genomics , ahmedabad , india ) . the genotypes were visualized by running digested product on 2% agarose gel at 100 v for about 2 h and the results were recorded in gel documentation system . the ecori restriction enzymes digest the amplified polymerization chain reaction ( pcr ) product of 735 base pairs ( bps ) . the amplified product was digested with 10 u of ecori at 37c for 2 h. after digestion , the products were analyzed on a 2% agarose gel and bands were visualized on a ultraviolet ( uv)-transilluminator . pcr amplified a 735 bp region of genomic dna that spanned a 32-bp deletion differentiating the ccr5-_32 allele from its wild - type counterparts at the ccr5 locus . after restriction digestion with ecori , with wild gene yielded band at 332 bp and for mutated gene , the bands were at 332 and 403 figure 1 . cc - chemokine receptor-5 genotyping among the tribes lane 1 , 2 , 3 , 5 , 6 and 7 represent the pcr product from samples with homozygous wild type genotypes ( fragments of 332 bp wt / wt ) . lane 4 represents the -32 genotype ( with the presence of both fragment of 332 and 403 wt/32 ) statistical analysis of allele frequencies was performed using chi - square statistics . genotype distribution for polymorphism was first compared to predictable values from hardy weinberg equilibrium . in all cases , besides inhabiting the adjoining state , a substantial number of these people inhabit in few district of vidarbha . bhils are listed as adivasi residents of the states of gujarat , madhya pradesh , chhattisgarh , maharashtra , and rajasthan in western and central india as well as in tripura in far - eastern india on the border with bangladesh . bhils are divided into a number of endogamous territorial divisions , which in turn have a number of clans and lineages . most bhils now speak the language of the region they reside in , such as marathi and gujarati . the korkus are a typical tribal population from amravati district and found only in the satpuda mountain ranges spanning maharashtra and madhya pradesh . they are mainly concentrated in melghat a scheduled area of korku comprising 89% of the tribal population . the phasse are a sub tribe of the paradhi caste , which includes sub - castes like gav paradhi , berad - paradhi , gay - paradhi , chita paradhi . there are only three surnames among them , chauhan , pawar , and solanke . we sampled 15 samples from this group from akola district . a low cultivating caste of berar , who numbered 52,000 persons in 1911 , and belongs to the yeotmal , akola , and buldana districts . the andhs appear to be a non - aryan tribe of the andhra or tamil country , from which they derive their name . there were 8228 andh in andhra pradesh in 1991 . according to singh et al . , in their 2004 book people of india there are over 74,000 andhs in maharashtra . this tribe falls under the primitive tribes category and spread much over the central india . gond generally speaks gondi dialect , which belongs to the dravidian linguistic family , after indo - european , in india . brahmin is a class of priests and preachers of dharma and considered as the torch bearer of hinduism . majority of brahmin in maharashtra speak marathi , one of the major languages of indo - aryan linguistic group . we have collected 108 blood samples on whatman fta mini cards ( ge health - care , uk , ltd ) from 6 tribe 's populations and a caste population from the district of vidarbha region . every card was labeled with appropriate code as per tribes / caste and district with informed consent obtained from each volunteer . approximately , ( 200 l [ 2 - 5 drop ] ) of blood by veni - puncture was directly spotted on the fta mini card within a printed circle area and dried at room temperature . briefly , 100 ng of genomic dna was denatured at 94c for 10 min , following , which it was subjected to 30 cycles of denaturation , annealing , and extension . the reaction mixture of 50 l contained , 50 mmol kcl , 10 mmol tris - hcl , ph 8.3 , 800 mol dntps , 100 g / ml gelatin , 10 pmoles of each of the ccr5-specific primer forward : ccr5 f : ( 5-cctggctgtcgtccatgctg-3 ) and reverse : ccr5-r : ( 5-ctgatctagagccatgtgcac aactct-3 ) . , and 1.5 units of taq polymerase enzyme ( xcelris genomics , ahmedabad , india ) . the genotypes were visualized by running digested product on 2% agarose gel at 100 v for about 2 h and the results were recorded in gel documentation system . the ecori restriction enzymes digest the amplified polymerization chain reaction ( pcr ) product of 735 base pairs ( bps ) . the amplified product was digested with 10 u of ecori at 37c for 2 h. after digestion , the products were analyzed on a 2% agarose gel and bands were visualized on a ultraviolet ( uv)-transilluminator . pcr amplified a 735 bp region of genomic dna that spanned a 32-bp deletion differentiating the ccr5-_32 allele from its wild - type counterparts at the ccr5 locus . after restriction digestion with ecori , with wild gene yielded band at 332 bp and for mutated gene , the bands were at 332 and 403 figure 1 . cc - chemokine receptor-5 genotyping among the tribes lane 1 , 2 , 3 , 5 , 6 and 7 represent the pcr product from samples with homozygous wild type genotypes ( fragments of 332 bp wt / wt ) . lane 4 represents the -32 genotype ( with the presence of both fragment of 332 and 403 wt/32 ) statistical analysis of allele frequencies was performed using chi - square statistics . genotype distribution for polymorphism was first compared to predictable values from hardy weinberg equilibrium . in all cases , besides inhabiting the adjoining state , a substantial number of these people inhabit in few district of vidarbha . bhils are listed as adivasi residents of the states of gujarat , madhya pradesh , chhattisgarh , maharashtra , and rajasthan in western and central india as well as in tripura in far - eastern india on the border with bangladesh . bhils are divided into a number of endogamous territorial divisions , which in turn have a number of clans and lineages . most bhils now speak the language of the region they reside in , such as marathi and gujarati . the korkus are a typical tribal population from amravati district and found only in the satpuda mountain ranges spanning maharashtra and madhya pradesh . they are mainly concentrated in melghat a scheduled area of korku comprising 89% of the tribal population . the phasse are a sub tribe of the paradhi caste , which includes sub - castes like gav paradhi , berad - paradhi , gay - paradhi , chita paradhi . there are only three surnames among them , chauhan , pawar , and solanke . we sampled 15 samples from this group from akola district . a low cultivating caste of berar , who numbered 52,000 persons in 1911 , and belongs to the yeotmal , akola , and buldana districts . the andhs appear to be a non - aryan tribe of the andhra or tamil country , from which they derive their name . there were 8228 andh in andhra pradesh in 1991 . according to singh et al . , in their 2004 book people of india there are over 74,000 andhs in maharashtra . this tribe falls under the primitive tribes category and spread much over the central india . gond generally speaks gondi dialect , which belongs to the dravidian linguistic family , after indo - european , in india . brahmin is a class of priests and preachers of dharma and considered as the torch bearer of hinduism . majority of brahmin in maharashtra speak marathi , one of the major languages of indo - aryan linguistic group . we have collected 108 blood samples on whatman fta mini cards ( ge health - care , uk , ltd ) from 6 tribe 's populations and a caste population from the district of vidarbha region . every card was labeled with appropriate code as per tribes / caste and district with informed consent obtained from each volunteer . approximately , ( 200 l [ 2 - 5 drop ] ) of blood by veni - puncture was directly spotted on the fta mini card within a printed circle area and dried at room temperature . polymerase chain reaction was performed following previously prescribed protocol by . briefly , 100 ng of genomic dna was denatured at 94c for 10 min , following , which it was subjected to 30 cycles of denaturation , annealing , and extension . the reaction mixture of 50 l contained , 50 mmol kcl , 10 mmol tris - hcl , ph 8.3 , 800 mol dntps , 100 g / ml gelatin , 10 pmoles of each of the ccr5-specific primer forward : ccr5 f : ( 5-cctggctgtcgtccatgctg-3 ) and reverse : ccr5-r : ( 5-ctgatctagagccatgtgcac aactct-3 ) . , and 1.5 units of taq polymerase enzyme ( xcelris genomics , ahmedabad , india ) . the genotypes were visualized by running digested product on 2% agarose gel at 100 v for about 2 h and the results were recorded in gel documentation system . the ecori restriction enzymes digest the amplified polymerization chain reaction ( pcr ) product of 735 base pairs ( bps ) . the amplified product was digested with 10 u of ecori at 37c for 2 h. after digestion , the products were analyzed on a 2% agarose gel and bands were visualized on a ultraviolet ( uv)-transilluminator . pcr amplified a 735 bp region of genomic dna that spanned a 32-bp deletion differentiating the ccr5-_32 allele from its wild - type counterparts at the ccr5 locus . after restriction digestion with ecori , with wild gene yielded band at 332 bp and for mutated gene , the bands were at 332 and 403 figure 1 . cc - chemokine receptor-5 genotyping among the tribes lane 1 , 2 , 3 , 5 , 6 and 7 represent the pcr product from samples with homozygous wild type genotypes ( fragments of 332 bp wt / wt ) . lane 4 represents the -32 genotype ( with the presence of both fragment of 332 and 403 wt/32 ) genotype distribution for polymorphism was first compared to predictable values from hardy weinberg equilibrium . in all cases , our data on distribution of ccr5-32 mutation among the selected tribe and a caste is depleted table 1 . genotype and phenotype for the heterozygous mutation among the tribes sample suggested that it is either absent or present at low frequency 1.08% ( 1 in 93 tribe 's samples and 1 in 15 samples of a caste ) . none of the tribes and control caste was found to be homozygous for the ccr5-32 mutation , while the bhil tribe and control caste show the heterozygous for the ccr5-32 mutation in negligible frequency ( 0.034 ) . the analysis of suggested that the prevalence of the ccr5-32 is significantly low ( = 0.02 , p > 0.05 ) . the aggregate frequencies of the entire sample for the wild - type allele ccr5 and the ccr-5-32 variant were found to be 0.991 and 0.009 , respectively . among the tribes kolam , korku , paradhi , andh and while only the bhil tribe shows heterozygous genotype frequency ( 06.97% ) and allelic frequency ( 0.034 ) for ccr5/32 . only one bearer of this mutation was found in the blood sample of bhil tribe collected from yavatmal district of vidarbha region . genotypic distribution and gene frequencies of the ccr5 allele in different population samples of vidarbha region of maharashtra state no significant deviations from the hwe were observed ( p > 0.05 , chi - squared goodness of fit ) . table 1 shows the frequency of the ccr5-32 allele in the six tribes and one control caste brahmin population . this study describes the genotype and allele frequencies of the polymorphisms ccr5-32 in selected six tribal and one caste populations from vidarbha region . most importantly , however , this study is the first to be conducted in vidarbha that investigates the genetic polymorphisms ccr5-32 among different ethnic tribal population settlement in the districts of vidarbha region . ccr5 , a coreceptor for hiv - i virus , has been shown to be the most important for the hiv transmission . a 32-nucleotide deletion of ccr5 homozygous ( ccr5-32/32 ) display a high degree of the natural resistance to hiv transmission whereas ccr5-32 heterozygosity ( ccr5+/32 ) demonstrate a slower progression to acquired immunodeficiency syndrome ( aids ) than ccr5 wild type ( ccr5+/+ ) . however , this genetic mutation is found in caucasian rather than non - caucasian population including india . the ccr5-32 genotype frequency among our study tribes sample was absent or negligible , the average genotype frequency were common homozygous wt / wt ( 98.3% ) , heterozygous wt / mt ( 1.87% ) and rare homozygous mt / mt ( 0% ) , from the control group , revealing ccr5-32 allele frequency of 6.97% . the frequency of the ccr5-32 allele among our study population seems to be remarkably similar to previously reported frequencies in other asian populations . the ccr5-32 allele frequency among asians is very low in rajasthan indians ( 0.05% ) , andhra pradesh indians ( 0 - 0.03% ) , north indians ( 1.5% ) , south indians ( 1 - 3% ) , and ethnic population of kashmir ( 3 - 4% ) . a similar study conducted from the island of crete , greece showed allele frequency of 3.25% , with a 95% confidence interval ( ci ) for conformity with hardy - weinberg equilibrium of 0.74 - 5.7% . the ccr5-32 polymorphism is found all across europe at different allele frequencies , with a north to south decreasing gradient and lower distribution in the regions of southeast mediterranean . the frequency of the ccr5-32 allele in the studied tribal population is consistently similar with data reported from other populations with non - european ancestors . ccr5-32 allelic frequencies were not different when the self - reported racial characteristics of the individuals evaluated were considered . this allele has not been found among south american native indians , corroborating the hypothesis of a european origin of this allele and its introduction to the continent through migration . within the middle - eastern populations the frequency of the mutant ccr5-32 allele reached it 's the highest among iranians , 2.4% ; saudi , 2.1% ; and it 's the lowest among kuwaitis , 1% ; and the egyptians , 0.5% ; and is completely absent in individuals from the united arab emirates . our results suggest that the ccr5-32 allele is detected at very low frequency in studied tribal populations from vidarbha . the presence of low frequencies of ccr5-32 in an individual of bhil tribe ( 0.034 , value 0.017 ) in the present study implies that these communities may have a better resistance to hiv / aids than other studied tribe sample , as non show such mutation . the marginal presence of the allele seen in the studied tribal population could be due to gene flow from the people of european descent . however , ccr5-32 is completely absent in the populations from africa , oceania , and the americas . however , lack of the homozygous ccr5-32 mutation and the low prevalence of heterozygous ccr5-32 mutations suggest that the indians are highly susceptible to hiv / aids , and this correlates with the highest number of hiv / aids infected individuals in india .

background : genetic relationships among the ethnic groups are not uniform across the geographical region . considering this assumption , we analyzed the frequency of the cc - chemokine receptor-5 ( ccr5)-32 allele of the ccr5 chemokine receptor , which is considered a caucasian marker , in bhil tribal and brahmin caste sample sets from the population.materials and methods:108 blood samples were collected from 6 tribe 's populations and a caste population from the district of vidarbha region.results and discussion : the presence of low frequencies of ccr5-32 in an individual of bhil tribe ( 0.034 , 2 value 0.017 ) in the present study implies that these communities may have a better resistance toward human immunodeficiency virus ( hiv)/acquired immunodeficiency syndrome ( aids ) than the other studied tribe sample , as non - show such mutation.conclusion:the marginal presence of the allele seen in the studied tribal population could be due to gene flow from the people of european descent . however , lack of the homozygous ccr5-32 mutation and the low prevalence of heterozygous ccr5-32 mutations suggest that the indians are highly susceptible to hiv / aids , and this correlates with the highest number of hiv / aids infected individuals in india .

Introduction Material and Methods None Population Blood sample collection and DNA extraction Amplification and restriction digestion of DNA Genotyping for CCR5 -32 polymorphism Statistical analysis Observation and Results Discussion and Conclusion

PMC3743068

heart failure ( hf ) causes a significant economic burden , morbidity , and mortality . the primary cause of hf hospitalization ( hfh ) is volume overload which is treated using diuretic therapy . further , ace - inhibitors and -blockers are known to reduce mortality in hf patients . implantable medical devices , such as pacemakers , implantable cardioverter defibrillator ( icd ) , and cardiac resynchronization therapy defibrillator ( crt - d ) , can provide daily measurements of several diagnostics such as intra - thoracic impedance ( imp ) , atrial fibrillation ( af ) burden and rate control information , and night heart rate , heart rate variability , and patient activity can identify when patients are risk for hf events and could potentially be used unilaterally or in a combined fashion for informed patient management . in the past decade multiple studies have reported combining implantable device diagnostics to identify patients at risk of hf events and death . the objective of this study was to develop and validate a single - hf risk score derived by combining information from multiple device diagnostic parameters in a bayesian belief network ( bbn ) framework to improve the ability to identify when patients are at risk for hfh . multiple physiological processes interact in a complex manner during hf with a high degree of uncertainty in the severity of the manifestation of the disease that may or may not require hospitalization . the bbn approach allows for uncertain reasoning to estimate the probability of an hfh under a set of given diagnostic evidence . the development set included data available from the ofisser ( n = 269 ) , italian clinicalservice project ( n = 174 ) , and connect ( n = 478 ) studies . the validation set included data available from the partners - hf ( n = 650 ) , fast ( n = 134 ) , precede - hf ( n = 52 ) , and sense - hf ( n = 474 ) studies . patient data were included in the data analysis cohorts if the patient had > 90 days of device diagnostic data that includes intra - thoracic impedance monitoring . details for each study and additional data inclusion criteria for this analysis are detailed in the appendix . the studies were divided into development and validation data sets based on the chronological order in which data from the studies were made accessible for this investigation . the method for computing diagnostic information is the same in all the devices included for the data analysis . each cardiovascular hospitalization was carefully adjudicated for signs and symptoms of hf which included the administration of i.v . or oral diuretic during the hospitalization . since a dynamic risk score for hfh was the focus of this study , death was not used as an endpoint in the data analysis . implanted medical devices monitor several clinical diagnostic parameters that may include imp , af burden , ventricular rate during atrial fibrillation ( vraf ) , ventricular tachycardia ( vt ) episodes , patient activity ( act ) , day and night heart rate ( nhr ) , and heart rate variability ( hrv ) ( figure 1 ) . these parameters are monitored continuously and the device stores sample data points for each parameter daily . imp is a surrogate measure for blood volume or pulmonary capillary wedge pressure , with an increase in fluid volume leading to a reduction in imp . hrv is the standard deviation of 5 min median of atrial intervals during a 24 h period , with reducing hrv implying increases in sympathetic tone . nhr is the average heart rate between midnight and 4 am and is a measure for resting heart rate . act is the number of minutes in a 24 h period the patient is active and is a surrogate of functional capacity . af burden is measured as total duration of fast atrial rate during a 24 h period , with atrio - ventricular conduction ratio 2:1 . the device also records the % of crt pacing delivered in a day , number of vt episodes and whether the patient received a defibrillation shock . figure 1the schematic for computation of the combined risk score using the different hf - related diagnostic variables in the medtronic crt - d system . the schematic for computation of the combined risk score using the different hf - related diagnostic variables in the medtronic crt - d system . features were extracted from the diagnostics parameters to ascertain an evidence level for each diagnostic parameter on a daily basis ( appendix ) . a higher value of optivol fluid index implied a higher level of evidence for hf . low or decreasing trend in act or hrv and high or increasing trend in nhr were considered as evidence for hf . if any two of the five arrhythmia / therapy related criteria were met it identified a higher evidence level for worsening hf . the thresholds for the trend indexes which look for sustained increases or decreases in the measurements of nhr , act , and hrv were determined in the development set data . a bbn framework was used to combine the evidence from each diagnostic parameter ( figure 1 ) . on any day a certain set of diagnostic criteria is met which is categorized to different evidence levels as shown in appendix . the evidence level for each diagnostic parameter is then used to generate the hf risk score for the day using a lookup table defined by the bbn model using data from the development set . monthly evaluations were simulated every 30 days , similar to the evaluation used in the partners - hf study , beginning on the 60th day from start of available diagnostic data . each monthly evaluation included : ( i ) a retrospective look at maximum value of the diagnostic risk score in the last 30 days to ascertain the patient status into the diagnostic evaluation groups , and ( ii ) a prospective assessment for the first hfh in the next 30 days . a monthly evaluation was included only if there was > 30 days of device data and clinical follow - up following the diagnostic evaluation , thus excluding deaths from the analysis . the risk score was categorized into three diagnostic evaluation groups : high , medium , and low . the first natural break after the top 10% of the risk score in the development set was chosen as the threshold for the high group . the rest of the risk scores were divided into two similar sized groups at a natural breakpoint with the hfh event rate < 0.5% in the low group in the development set . the high and medium monthly diagnostic evaluation groups were compared with the low group for time to first hfh in the next 30 days using the anderson gill model , an extension of the cox proportional hazards model that accounts for multiple evaluations in patients . the model was adjusted for baseline variables ( age , gender , nyha , history of coronary artery disease , mi , af , diabetes , and hypertension ) and baseline medications ( ace - i / arb , diuretics , -blockers , and anti - arrhythmic drugs ) in the validation data set . a sensitivity and specificity analysis sensitivity ( and specificity ) is defined as the number of evaluations with score ( or < ) threshold and hfh ( or no hfh ) event in next 30 days divided by the total number of evaluations with hfh ( without hfh ) in next 30 days . the sensitivity and specificity computations are adjusted for multiple evaluations in patients using generalized estimating equation ( gee ) with an exchangeable correlation structure . the development set included data available from the ofisser ( n = 269 ) , italian clinicalservice project ( n = 174 ) , and connect ( n = 478 ) studies . the validation set included data available from the partners - hf ( n = 650 ) , fast ( n = 134 ) , precede - hf ( n = 52 ) , and sense - hf ( n = 474 ) studies . patient data were included in the data analysis cohorts if the patient had > 90 days of device diagnostic data that includes intra - thoracic impedance monitoring . details for each study and additional data inclusion criteria for this analysis are detailed in the appendix . the studies were divided into development and validation data sets based on the chronological order in which data from the studies were made accessible for this investigation . the method for computing diagnostic information is the same in all the devices included for the data analysis . each cardiovascular hospitalization was carefully adjudicated for signs and symptoms of hf which included the administration of i.v . or oral diuretic during the hospitalization . since a dynamic risk score for hfh was the focus of this study , death was not used as an endpoint in the data analysis . implanted medical devices monitor several clinical diagnostic parameters that may include imp , af burden , ventricular rate during atrial fibrillation ( vraf ) , ventricular tachycardia ( vt ) episodes , patient activity ( act ) , day and night heart rate ( nhr ) , and heart rate variability ( hrv ) ( figure 1 ) . these parameters are monitored continuously and the device stores sample data points for each parameter daily . imp is a surrogate measure for blood volume or pulmonary capillary wedge pressure , with an increase in fluid volume leading to a reduction in imp . hrv is the standard deviation of 5 min median of atrial intervals during a 24 h period , with reducing hrv implying increases in sympathetic tone . nhr is the average heart rate between midnight and 4 am and is a measure for resting heart rate . act is the number of minutes in a 24 h period the patient is active and is a surrogate of functional capacity . af burden is measured as total duration of fast atrial rate during a 24 h period , with atrio - ventricular conduction ratio 2:1 . the device also records the % of crt pacing delivered in a day , number of vt episodes and whether the patient received a defibrillation shock . figure 1the schematic for computation of the combined risk score using the different hf - related diagnostic variables in the medtronic crt - d system . the schematic for computation of the combined risk score using the different hf - related diagnostic variables in the medtronic crt - d system . features were extracted from the diagnostics parameters to ascertain an evidence level for each diagnostic parameter on a daily basis ( appendix ) . a higher value of optivol fluid index implied a higher level of evidence for hf . low or decreasing trend in act or hrv and high or increasing trend in nhr were considered as evidence for hf . if any two of the five arrhythmia / therapy related criteria were met it identified a higher evidence level for worsening hf . the thresholds for the trend indexes which look for sustained increases or decreases in the measurements of nhr , act , and hrv were determined in the development set data . a bbn framework was used to combine the evidence from each diagnostic parameter ( figure 1 ) . on any day a certain set of diagnostic criteria is met which is categorized to different evidence levels as shown in appendix . the evidence level for each diagnostic parameter is then used to generate the hf risk score for the day using a lookup table defined by the bbn model using data from the development set . monthly evaluations were simulated every 30 days , similar to the evaluation used in the partners - hf study , beginning on the 60th day from start of available diagnostic data . each monthly evaluation included : ( i ) a retrospective look at maximum value of the diagnostic risk score in the last 30 days to ascertain the patient status into the diagnostic evaluation groups , and ( ii ) a prospective assessment for the first hfh in the next 30 days . a monthly evaluation was included only if there was > 30 days of device data and clinical follow - up following the diagnostic evaluation , thus excluding deaths from the analysis . the risk score was categorized into three diagnostic evaluation groups : high , medium , and low . the first natural break after the top 10% of the risk score in the development set was chosen as the threshold for the high group . the rest of the risk scores were divided into two similar sized groups at a natural breakpoint with the hfh event rate < 0.5% in the low group in the development set . the high and medium monthly diagnostic evaluation groups were compared with the low group for time to first hfh in the next 30 days using the anderson gill model , an extension of the cox proportional hazards model that accounts for multiple evaluations in patients . the model was adjusted for baseline variables ( age , gender , nyha , history of coronary artery disease , mi , af , diabetes , and hypertension ) and baseline medications ( ace - i / arb , diuretics , -blockers , and anti - arrhythmic drugs ) in the validation data set . a sensitivity and specificity analysis sensitivity ( and specificity ) is defined as the number of evaluations with score ( or < ) threshold and hfh ( or no hfh ) event in next 30 days divided by the total number of evaluations with hfh ( without hfh ) in next 30 days . the sensitivity and specificity computations are adjusted for multiple evaluations in patients using generalized estimating equation ( gee ) with an exchangeable correlation structure . all statistical analyses were performed using sas version 9.2 ( sas institute , inc . , cary , nc , usa ) . the development data set consisted of 921 patients with an average follow - up duration of 10.6 5.8 months with 28 deaths and 68 patients ( 7.4% ) with hfhs at a rate of 0.14 per patient year . a total of 9790 patient - months of data was analysed of which there were 91 months with hfhs providing an event rate of 0.9% . the validation data set consisted of 1310 patients with an average follow - up duration of 8.1 5.0 months with 33 deaths and 110 patients ( 8.4% ) with hfhs at a rate of 0.22 per patient year . a total of 10 655 patient - months of data was analysed of which there were 163 months with hfhs providing an event rate of 1.5% . the baseline characteristics of the patients in the study are shown in table 1 . table 1baseline demographics of patients in the development and validation setsdevelopment set ( n = 921)validation set ( n = 1310)mean age ( sd)68 ( 11)67 ( 11)male gender ( % ) 6974nyha ( % ) i24 ii1922 iii7670 iv34ischaemic ( % ) 6361myocardial infarction ( % ) 4348hypertension ( % ) 7062diabetes ( % ) 3738history of af ( % ) 2132lvef < 35% ( % ) 9692device type ( % ) icd04 crt - d10096baseline medications ( % ) ace / arb7084beta - blockers8788diuretics7787digoxin2933aldosterone antagonist2622aad1822anti - platelet or anticoagulant8661 warfarin3325 baseline demographics of patients in the development and validation sets the event rates , expressed as a percentage of monthly evaluations that were followed by an hfh in the next 30 days , for the low , medium , and high evaluation groups in the development and the validation data sets are presented in table 2 . the hazard ratios for the comparison of the event rates in the medium and high groups with respect to the low group are also shown in table 2 . figure 2 shows the kaplan meier plot for time to first hfh in the 30 days following monthly diagnostic evaluation in the validation data set . in the validation data set , a total of 163 monthly evaluations ( 1.5% ) were followed by an hfh in the next 30 days . of the 1100 monthly evaluations when the risk score was in the high group , 75 ( 6.8% ) were followed by an hfh in the next 30 days . the risk score was in the high group in at least one monthly evaluation in 446 patients ( 34% ) . monthly diagnostic evaluations with a risk score in the high group were 10 times ( hr : 10.0 ; 95% ci : 6.415.7 , p < 0.001 ) more likely to have an hfh in the next 30 days compared with monthly evaluations with a risk score in the low group . results are similar if the model is adjusted for the presence of hfh in the last 30 days ( hr : 8.2 ; 95% ci : 5.113.1 , p < 0.001 ) . table 2comparison of event rates between different evaluation groups within the development and validation setsdata setevaluation groupsevaluations ( % ) patientshf hospitalizations ( % of evaluations)hazard ratio ( 95% ci)p - valuedevelopment ( n = 921)low4525 ( 46)80215 ( 0.3)referencemedium4018 ( 41)83347 ( 1.2)3.7 ( 2.0 , 6.7)<0.001high1247 ( 13)40529 ( 2.3)6.2 ( 3.1 , 12.3)<0.001validation ( n = 1310)low4838 ( 45)108528 ( 0.6)referencemedium4717 ( 44)114260 ( 1.3)2.1 ( 1.3 , 3.4)0.001high1100 ( 10)44675 ( 6.8)10.0 ( 6.4 , 15.7)<0.001the high group consisted of risk scores > 20% and the low group consisted of risk scores 5% . figure 2kaplan meier curves for time to first hf hospitalization after monthly diagnostic evaluation for the different risk score groups for the validation set . comparison of event rates between different evaluation groups within the development and validation sets the high group consisted of risk scores > 20% and the low group consisted of risk scores 5% . meier curves for time to first hf hospitalization after monthly diagnostic evaluation for the different risk score groups for the validation set . figure 3 shows the event rates for individual diagnostic evidence levels described in appendix and the combined risk score for the validation set . while each of the diagnostic element has the capability of stratifying patients at risk for hfhs , the combined risk score improves the ability to identify when patients are at a higher than normal risk and when patients are at lower than normal risk for hfhs . figure 3event rates for different levels of evidence for each diagnostic parameter and the combined risk score . event rates for different levels of evidence for each diagnostic parameter and the combined risk score . the number of times each of the individual diagnostic criteria was triggered as per cent of monthly evaluations with a risk score in the different risk score groups in the validation set is shown in figure 4a . each diagnostic parameter exceeded threshold significant proportion of times when the risk score is in the high group with reduced patient activity ( evidence level 2 in appendix ) and high optivol fluid index ( evidence level 3 and 4 in appendix ) exceeding threshold most often . figure 4b shows the number of diagnostic parameter that were triggered at the same time when the risk score was in the low , the evidence level criteria for the trigger of the diagnostic parameters for figure 4b were same as that used in figure 4a . when the risk score was in the low group , very often none of the diagnostic parameters triggered a high - evidence level , whereas three different diagnostic parameters triggered a high - evidence level most often when the risk score was in the high group . figure 4(a ) per cent of monthly evaluations high - evidence level criteria was met for each diagnostic parameter in different risk score groups . ( b ) the distribution of number of diagnostic parameter that were triggered , based on evidence level criteria in ( a ) , when the risk score was in the low , medium , and ( a ) per cent of monthly evaluations high - evidence level criteria was met for each diagnostic parameter in different risk score groups . ( b ) the distribution of number of diagnostic parameter that were triggered , based on evidence level criteria in ( a ) , when the risk score was in the the 25-percentile , median , and 75-percentile of the risk score distribution in the validation set were 3.8 , 5.5 , and 11.5% , respectively . the receiver operating characteristic ( roc ) curve , plotting the gee estimates of sensitivity and specificity , for the validation set is shown in figure 5 . evaluations done every 30 days in the monthly evaluation scheme contribute one data point for the sensitivity and specificity calculations . in the validation set , the threshold between the low and medium risk groups ( score of 5% ) had a sensitivity and specificity of 82.8 and 45.8% , respectively , and the threshold between the medium- and high-risk groups ( score of 20% ) had a sensitivity of 46.0% , i.e. 46% of the months with hfhs were preceded by a high-risk score , and specificity of 90.2% , i.e. 10% of the months with no hfhs were also preceded by a figure 5receiver operating characteristics curve plotting the sensitivity vs. specificity in a 30-day evaluation framework for the validation set . receiver operating characteristics curve plotting the sensitivity vs. specificity in a 30-day evaluation framework for the validation set . when monthly evaluations were in low-risk group , < 0.6% had hfhs in the next 30 days , i.e. a negative predictive value of 99.4% for the low risk can be used to triage for patients who are at lower than average risk for hf requiring hospitalization . a very high - risk group , i.e. a group with high positive predictive value , can be formed for risk scores > 40% that occurs in 2% of all monthly evaluations with 14.2% having hfhs in next 30 days . similarly , another very high - risk group would be evaluations with a risk score in high group for > 14 of 30 days , which happens in 3% of the evaluations with 11.9% having hfhs in the next 30 days . high in the 30 days prior to months with hfh was 7 11 days . the study presented the development and validation of a novel dynamic hf risk score derived from combining diagnostic parameters monitored in implantable devices . patients who achieve a high - risk state on any day in the last 30 days are 10 times more likely to be hospitalized for hf in the next 30 days compared with patients who had a low risk on each of the last 30 days . most of these risk scores identified a static risk at baseline or in an in - hospital setting , i.e. identify which patient is at risk for the development of hf or mortality . the dynamic hf risk score developed in this study , can identify when a high - risk patient is at higher risk of an hfh in an ambulatory setting , thus providing incremental information beyond what is provided by a static risk score . the dynamic hf risk score is time - varying and the same patient may be at high and low risk at different periods of time depending on the status of continuously monitored diagnostic parameters in the implanted device . management based on a dynamic risk score is similar in approach to recently reported intra - cardiac pressure or intra - thoracic impedance measurements . the key difference is incorporation of the multiple diagnostic parameters to form a combined diagnostic with the intention of improving the overall accuracy of the diagnostic . long - term ambulatory monitoring using implantable devices featuring remote access and wireless alerting capabilities enables the dynamic assessment of the hf status , thus providing the opportunity to optimize treatment strategies for hf in a timely fashion . like any diagnostic ( weight , temperature , ecg , etc . ) , diagnostic information must be coupled with appropriate clinical actions in order to improve outcomes in hf patients . whether therapeutic interventions based on the dynamic hf risk score is safe and effective in improving outcomes in hf patients need prospective evaluation . several randomized controlled studies for management of hf patients based on diagnostic information have yielded inconsistent results . each of the diagnostic parameters in implantable devices correspond to one or more of the basic hf assessment metrics such as fluid status , functional capacity , resting tachycardia , autonomic balance , arrhythmia , and non - adherence . combining multiple parameters into a single - risk score makes it a simple to use triaging scheme indicating when a patient needs more attention in an ambulatory setting . a transition to high - risk state can proactively initiate collection of more clinical and symptomatic information over the telephone or in person to facilitate a diagnostic decision . further , having information regarding which device parameters caused transition to high - risk state may lead to more clarity on treatment options . for example , a high - risk score caused by decrease in intra - thoracic impedance as well as new onset af with poor rate control at the same time may indicate a very specific treatment plan : acute control of the fluid status , if necessitated by additional evidence , followed by improved chronic management of heart rate , once euvolaemia is achieved , to prevent future occurrences . a clinical action may not always need a medication adjustment ; it can also be counselling for non - adherence or deciding to monitor the patient more often in the clinic . the chosen diagnostic criteria were optimized for simplicity of a quick visual review of each diagnostic variable . the bbn approach also combines multiple diagnostic parameters , but it does so in a more rigorous decision - making framework that mimics clinical decision - making with elements of uncertain reasoning , causal relationships , and differential reasoning in the same framework . the bbn approach generates a single - risk score that can be used for initial triage without having to go through each diagnostic variable , thus improving the simplicity . the risk score is a continuous number allowing for the choice of flexible thresholds for obtaining optimal performance . further , feature set definition for individual diagnostic variables can be improved without compromising the simplicity , e.g. relative change in activity , nhr , and hrv can be incorporated in addition to absolute thresholds . finally , more diagnostic variables with orthogonal information , e.g. biomarkers , intra - cardiac pressures , can be easily added into the framework without needing data from all the variables in the same study to create a revised framework . the absolute risk of an hfh in a 30-day period following a monthly evaluation with a high - risk state was only 6.8% . thus , a high - risk state should not be used unilaterally to make treatment decisions as it may lead to over - reaction as it happened in the dot - hf study . the absolute risk is low primarily because of the low rate of hfhs in this ambulatory monthly evaluation framework ( overall event rate of 1.5% ) , the denominator being all monthly evaluations in all patients . when the event rate is higher , for example , in evaluating readmissions for hf , the absolute risk is also higher . the absolute risk for events with milder symptoms of hf not requiring hospitalization will be higher . the hf risk score categorizes the baseline risk of 1.5% into three groups , one with a higher risk ( 6.8% ) and one with a lower risk ( 0.6% ) , with the middle group being similar to the overall risk of 1.5% . although the absolute risk is low , the relative risk between the high- and low - risk groups is high . thus , the risk score can be used as a tool to triage patients who may need more attention ( e.g. more frequent follow - up ) . randomized control studies of intensive follow - up - based hf management have yielded varying results ; however , a risk score - based follow - up may improve the efficiency of disease management programmes by spending more resources on patients in a high - risk state and fewer resources on patients in a low - risk state . the retrospective analysis was done by pooling data from multiple studies in order to increase the sample size for the development and validation sets . with the exception of the fast study , most of the data included in the study was from within the first year of the device life , thus the results may not reflect the performance of the risk score during the later years of the device life . serial assessment of clinical diagnostic data related to hf , such as weight , blood - pressure , and bnp , was not performed in a consistent manner in all the studies . thus , comparison of the dynamic risk score to previously described clinical risk scores could not be performed , and the adjustment for other clinical variables in the statistical analysis was limited to baseline history collected in the studies . the incremental value of an hf risk score over clinical diagnostic measurements can not be established in this data set . it is hypothesized that the dynamic assessment of an hf risk score provides an ambulatory triage mechanism to indicate when to gather additional clinical information to evaluate the patient status in a timely manner to improve the efficacy of disease management programmes . the retrospective analysis was done by pooling data from multiple studies in order to increase the sample size for the development and validation sets . with the exception of the fast study , most of the data included in the study was from within the first year of the device life , thus the results may not reflect the performance of the risk score during the later years of the device life . serial assessment of clinical diagnostic data related to hf , such as weight , blood - pressure , and bnp , was not performed in a consistent manner in all the studies . thus , comparison of the dynamic risk score to previously described clinical risk scores could not be performed , and the adjustment for other clinical variables in the statistical analysis was limited to baseline history collected in the studies . the incremental value of an hf risk score over clinical diagnostic measurements can not be established in this data set . it is hypothesized that the dynamic assessment of an hf risk score provides an ambulatory triage mechanism to indicate when to gather additional clinical information to evaluate the patient status in a timely manner to improve the efficacy of disease management programmes . we developed and validated a method for combining multiple device - derived diagnostic parameters into a single - dynamic hf risk score which may be evaluated in an ambulatory setting to triage patients at a higher risk for hf events in the next 30 days . future studies are needed to prospectively evaluate whether timely clinical actions initiated by the stratification of hf patients using the hf risk score on a regular basis can reduce hfhs . m.c . salary is supported by the national institute for health research biomedical research unit at the royal brompton hospital , london . funding to pay the open access publication charges for this article was provided by medtronic inc . conflict of interest : dr cowie : research grants from medtronic , and honoraria from medtronic , st jude medical and boston scientific . dr crossley : consultant for medtronic , boston scientific , and cardiac control systems ; lecturing income from medtronic , boston scientific and sanofi ; research support from medtronic , boston scientific and st jude medical . dr abraham : consulting fees from medtronic , st jude medical , biotronik , cardiomems , and novartis . : consultant for st jude medical and employed for clevland clinic . w.h.w.t . : grants from national institutes of health and abbott laboratories . j.k . : patents ( planned , pending , or issued ) from medtronic . g.c . : granted from medtronic and received payment for lectures including service on speakers bureaus from medtronic . m.c . : consultant for medtronic and received payments for lectures including service on speakers bureaus from medtronic .

backgroundwe developed and validated a heart failure ( hf ) risk score combining daily measurements of multiple device - derived parameters.methodsheart failure patients from clinical studies with implantable devices were used to form two separate data sets . daily hf scores were estimated by combining changes in intra - thoracic impedance , atrial fibrillation ( af ) burden , rapid rate during af , % crt pacing , ventricular tachycardia , night heart rate , heart rate variability , and activity using a bayesian model . simulated monthly follow - ups consisted of looking back at the maximum daily hf risk score in the preceding 30 days , categorizing the evaluation as high , medium , or low risk , and evaluating the occurrence of hf hospitalizations in the next 30 days . we used an anderson gill model to compare survival free from hf events in the next 30 days based on risk groups.resultsthe development data set consisted of 921 patients with 9790 patient - months of data and 91 months with hf hospitalizations . the validation data set consisted of 1310 patients with 10 655 patient - months of data and 163 months with hf hospitalizations . in the validation data set , 10% of monthly evaluations in 34% of the patients were in the high - risk group . monthly diagnostic evaluations in the high - risk group were 10 times ( adjusted hr : 10.0 ; 95% ci : 6.415.7 , p < 0.001 ) more likely to have an hf hospitalization ( event rate of 6.8% ) in the next 30 days compared with monthly evaluations in the low - risk group ( event rate of 0.6%).conclusionan hf score based on implantable device diagnostics can identify increased risk for hf hospitalization in the next 30 days .

Background Methods Data set and event definitions Diagnostic parameters Combined diagnostics Statistical analysis Results Discussion Limitations Conclusions Funding

PMC3947701

patients ' time and costs during illness and health care treatment are relevant aspects to include in a complete analysis of the social costs of health interventions . time and costs related to patients ' travels from home to their health care providers are also relevant when the social implications of treatment options are assessed in , for example , cost - effectiveness analyses . travel time and costs vary for the individual patient depending on the type of treatment provided , the frequency of contacts with health care providers , the traveled distance , and the mode of travel . while the need to include travel time and costs is widely accepted , details about how travel time and costs should be included have not been resolved [ 24 ] . a number of studies have investigated the travel time and costs for patients who are invited to attend hospital clinics as part of screening programs for , for example , cervical cancer , colorectal cancer , breast cancer , aortic aneurysm , and diabetic retinopathy , and for services that require frequent hospital attendance , for example , anticoagulation management [ 1012 ] . rheumatic arthritis ( ra ) is a chronic disease that requires life - long treatment and frequent contacts with health care providers to monitor and adjust medical treatment . treatment options that require frequent hospital visits may impose nontrivial travel time and costs on patients . however , no data on travel time and cost for ra patients have been reported in the scientific literature , although patients ' time and travel costs have been considered in a few economic evaluations of ra interventions , for example . an often - used strategy is to include patients ' travel time and costs as a simple average without any attempt to obtain detailed information of the variation among individual patients ' travel time and costs . the objective of this study was to explore the frequency of contacts with different types of health care providers for danish outpatients in care for ra , and assess average travel time and costs during a three - month period . as part of this study , we developed a strategy to obtain information from patients on their travel time and costs . patients with rheumatoid arthritis as defined by the acr 1987 criteria were identified among consecutive patients attending 11 danish rheumatology outpatient clinics between july 2006 and july 2007 . relevant patients were invited to participate in a patient - reported questionnaire study aimed at describing health - related quality of life , resource use , and time and costs spent on seeking health care treatment . the participating departments consisted of a mix of university and local hospitals distributed across the country . clinical staff at the participating clinics identified consecutive patients and administered a questionnaire booklet that patients were encouraged to complete and return during the visit at the outpatient clinic . patients who agreed to complete the booklet were given a prepaid envelope to return it by mail or were allowed to return the questionnaire to the clinical staff . , we were able to ensure that only the data from the first returned questionnaire were included in the analysis . in addition to information about travel time and costs , the questionnaire booklet asked for data on various social and life style factors including self - assessment of current health status using a global health score on a visual analogue scale ( vas ) , the health assessment questionnaire ( haq ) , and the eq-5d . in addition , clinical staff recorded information about the patient 's clinical status including disease duration , disease activity ( das-28 score and serum c - reactive protein ( crp ) level ) , and use of ra medication . this part of the questionnaire consisted of one page with two sections of questions ( see figure 1 ) . the danish phrasing of questions was inspired by recommendations by a uk working party on patient - reported costs ( figure 1 ) . the first section asked the question : how many times during the last 3 months have you visited the following health care providers , and what did it cost you on average per visit ? please provide the cost in dkk from your home to the place of treatment and return . the following six types of health care providers were specified ( one row for each):rheumatology outpatient clinic ( for blood tests , collection of medication , training and contacts with nurses , etc.),other outpatient clinics , general practitioner , privately practicing medical specialist , hospital admission , emergency department . rheumatology outpatient clinic ( for blood tests , collection of medication , training and contacts with nurses , etc . ) , other outpatient clinics , general practitioner , privately practicing medical specialist , emergency department . for each provider , there were two columns with the following headings : number of visits during the last three months ; average transport cost per visit ( dkk ) . how do you normally travel to the following health care providers and how long time do you usually spend on travel per visit ? please state the time used in minutes from your home to the treatment site and return . the same six types of health care providers were specified and columns were provided to respond specifically to each of six different modes of travel ( walking / biking ; bus , train , metro ( i.e. , public transport ) ; hospital - provided transport ; taxi ; private car ; ambulance ) . the questionnaire was pilot - tested in a small , convenient sample and slightly edited for clarity before the main study . briefly , age was categorized in three groups as 50 years or younger , 5175 years , or 76 years or older . cohabiting was categorized as living alone ( single ) or cohabiting ; residential area was categorized as rural villages / smaller cities or larger cities ; education was categorized according to duration as none / short ( 10 years or less ) , medium ( 1113 years ) , or long ( 13 + years ) ; body mass index ( bmi ) as underweight ( < 18.5 ) , normal ( 18.525 ) , overweight ( 2535 ) , or obese ( > 35 ) . labor market attachment was answered according to 10 categories and then recoded as in or out of the labor market . disease duration was categorized as 02 years , 310 years , or 10 + years according to the date of first diagnosis and date of questionnaire completion ; crp levels were categorized as normal or elevated ; biological treatment was classified as yes or no . the scores of patient global vas and the eq-5d vas ranged from 0 to 100 . a low score on global vas and a high eq-5d vas score indicated good health . all cost data were uplifted from 2006 - 2007 price level to 2013 price level by multiplying the reported cost data by the ratio of the national consumer price index published by statistics denmark ( http://www.statistikbanken.dk/pris6 ) january 2007 ( 112.4 ) and june 2013 ( 130.0 ) . the national currency was converted to euro assuming a currency rate of 100 dkk ~ 13.33 ( 1 ~ 7.50 dkk ) . some patients presumably misunderstood whether they should include the latest visit at the outpatient clinic in the three - month number of visits at a rheumatology outpatient clinic and provided a zero or missing number ; thus , 35 respondents replied 0 and for 44 respondents a value was missing , but the patient had provided valid information about average travel costs . a total of 392 respondents did not provide valid travel expenses but provided valid information about mode of travel to the rheumatology outpatient clinic . further 192 respondents did not provide any information about the number of visits , travel expenses , or mode of travel to the rheumatology outpatient clinic but provided other data in relation to travel . since a visit to the rheumatology outpatient clinic was a condition for participating in the study , we replaced all missing numbers of rheumatology outpatient visits with a conservative one ( 1 ) visit . missing information about visits to other outpatient clinics , general practice , privately practicing specialists , and hospital admissions and visits at an emergency department was interpreted as zero visits . some patients who provided a valid number of visits did not report their average travel expenses . in the reporting of the raw data we present only the provided information ( i.e. , only responses with nonmissing items ) . in the analysis of travel costs , missing information about travel costs was replaced by the mean costs calculated from those who had reported a valid number of visits and a valid cost . all numbers of visits were within the expected range ( max 90 visits , that is , once a day over 3 months ) . we considered replacing or deleting the single response indicating 90 visits and the three responses indicating 60 visits but decided against this and retained the actual responses . travel expenses over 100 ( n = 23 ) were inspected more closely . some of these related to travel in private cars over long distances . however , the seven patients who provided the highest travel expenses ( up till 500 ) provided no information about travel time . these data appeared unrealistic and most likely due to the patient misunderstanding the question . as these few extreme observations had undesirable influences on the estimated mean figures , we replaced the seven observations with travel expenses over 200 with the population mean . the average number of visits at different health care providers and the average travel time and costs per return visit are reported as number of valid responses , mean , standard deviation , and 25 , 50 , and 75 percentiles . modes of travel and time are reported as the number and proportion of individuals who use each of the different travel modes and the average travel time for those who use the particular travel mode . we aggregated the reported travel times for each individual and report the total travel time for those who have reported any travel data to the destination . we examined differences in travel time and costs by gender , age , cohabiting status , residential area , education , labor market attachment , disease duration , crp level , bmi , and biological treatment by bivariate comparisons using parametric tests . as neither the travel time nor the costs were normally distributed due to many individuals with low costs / time and few with very large costs / time , we reanalyzed the data using nonparametric tests , but these produced similar results . given the high number of observations and the preference for reporting cost data as averages , we report here only the applied parametric tests ( t - test / anova ) . of the 3704 patients invited to complete the questionnaire booklet , 549 patients ( 15% ) did not return the booklet and 308 ( 8% ) patients did not complete the travel questions . the analysis was thus based on replies from 2847 patients ( 77% of those invited and 90% of those who returned the questionnaire ) . chi - squared tests indicated that the study sample was younger than both groups of nonresponders ( p < 0.01 ) , more were married / cohabiting ( p < 0.01 ) , living within a city area ( p = 0.02 ) , had longer education ( p < 0.01 ) , and were in the labor market ( p < 0.01 ) . more individuals in the study sample had normal crp levels ( p = 0.04 ) and received treatment with biological medication ( p = 0.01 ) . t - tests indicated that the study population reported worse health than those who did not respond to the travel questions but responded to the haq , eq-5d vas , and eq-5d tto ( all p < 0.01 ) . no difference could be observed between respondents and noncompleters in terms of gender ( p = 0.29 ) , duration of ra ( p = 0.44 ) , or bmi ( p = 0.26 ) . during the three - month period , the 2847 patients reported on average 2.8 ( sd 4.0 ; median 2 ) visits to a rheumatology outpatient clinic for consultations with doctors or nurses , blood tests , and collection of medication ( table 2 ) . the median visits frequency was two while 25% of the sample had three or more visits during the three - month period . average travel cost per visit to the rheumatology clinic was = 13 ( sd 20 ) . 1144 patients ( 40% ) had at least one visit to their general practitioner and this group had on average 2.3 visits with the general practitioner during the three - month period . as expected , mean travel costs were lower for visits to general practitioners ( 4 ) and private medical specialists ( 10 ) than to outpatient clinics ( 1113 ) and hospital admissions ( 16 ) . many ( 63% ) patients travelled to the health provider by private car , while 11% used bus , train , or metro , and 7% walked or biked ( table 3 ) . the average travel time to the rheumatology outpatient clinic was 73 minutes per visit . table 4 provides estimates of the aggregated average three - month travel costs and time for all health care providers . the average patient had 4.4 ( sd 5.7 ) contacts with health care providers during the three - month period and spent 4.6 hours and 56 when travelling to and from the health care providers . the mean contact frequency , mean travel time , and mean costs were similar for various subgroups , although the average number of contacts was higher for women , those living in larger cities , those out of the labor market , and those on biological treatment . average travel times were higher for patients aged 5075 years , out of the labor market , with elevated crp level , and on biological treatment . mean travel costs were higher for patients living in larger cities , with elevated crp level , and on biological treatment . patients from a single hospital ( with a large rural catchment area ) reported statistically significant higher travel time and expenses ( data not shown ) . in regression analyses , none of the patient characteristics correlated strongly with the number of contacts , travel time or costs , and only a small proportion of the variation could be explained by such models ( r < 0.03 ) ( data not shown ) . a larger proportion of the variation was explained by the regression models when the number of contacts with health care providers was included as an explanatory variable , although none of the other estimated parameters reached statistical significance . these results were unchanged when variables for hospitals were introduced and when the analysis was conducted separately for each hospital ( data not shown ) . in this study , we obtained data on patients ' travel time and costs using a self - complete questionnaire developed for the purpose . patients with ra attending at least one outpatient clinic for treatment within a 3-month period had on average 4.4 contacts with health care providers and spent 4.6 hours and 56 on transport to and from these providers . the majority of the contacts were to the rheumatology outpatient clinic ( on average 2.8 times during the three - month period , 3.4 hours , and 41 ) . the most frequent mode of travel was private car , thereafter public bus , train , or metro . during the same period of time , nearly 40% of the patients also visited their general practitioners and those that did had on average 3.2 contacts during three - month period and spent about one hour and 6 dkk on travel to their gp . overall , the costs and amount of time that these patients spent on travel in connection with health care treatment are not surprising . frequent contacts with health care providers use more of patients ' time and increase travel expenses . we were unable to identify strong associations between travel time and costs and patient characteristics , and we found significant variations at only one of the participating hospitals . social and demographic factors had relatively little influence on travel time and costs for rheumatic patients . ra patients living in larger cities , with elevated crp level , and on biological treatment had more contacts and spent more time and expense on travels than other patients , but the association was not particularly strong and these variables were unable to explain much of the observed variation in travel time and costs . one hypothesis could be that patients living in larger cities are more likely to respond to symptoms than patients from rural areas . we expected greater variation in travel time and expense between different population groups and disease characteristics . it may be that none of the variables tested have any influence on patients ' travel time and costs . thus , although travel time and costs are influenced by the distance between the patient 's residence and the health provider , the geographical distribution of providers may be broadly similar ( at least in denmark ) , reflecting fairly equal access to care . another explanation might be that variations in travel time and costs are large , and this study sample had insufficient statistical power to identify relevant associations . the sample size was considerable ( n = 2847 ) ; however , we do not consider this a primary explanation . there were no problems reported in understanding what information was requested , but a considerable proportion ( 8% ) did not answer the transport questions . lack of knowledge about the exact travel time and costs is the most likely explanation , but the questions may have been unclear for some respondents particularly for those using private car as the mode of travel . here they would have needed to take into account the distance driven , fuel costs , and other running costs of the car , and not all patients may have known these . public transport such as bus , train , and metro is easier to cost as a certain amount is paid for the ticket . judging from the distribution of the cost data , however , there appeared to be a reasonable relation between travel time and travel cost . an alternative to the focus on travel costs could have been to ask patients to provide information only on travel distance , as done in other studies [ 1013 ] . patients are likely to have a fairly good idea of the distance to the health care provider , but this approach would give less precise estimates of costs based on distance traveled . while transport by private car and taxis could be estimated by applying a predetermined cost per km , the cost related to bus , trains , and metro would be more complicated and would need a detailed knowledge of the fee structure of these transport modes . the frequency of visits to the six types of health care providers appeared to be straightforward to answer . the 3-month interval was chosen to be able to make a reasonable estimate of the annual cost and was short enough that patients would remember a visit and the transport mode . we interpreted missing replies to indicate no use of health providers within the previous three months . in future studies , it might be considered to ask patients to indicate zero as an indication of no contacts . however , the detailed data on travel time fits well with expectations based on the relatively dense distribution of health care facilities within a relatively small country like denmark . also , our sample came from 11 of the 28 outpatient rheumatology hospital departments in denmark . unlike several other studies on patients travel costs [ 1012 ] , we decided not to assign a monetary value on travel time , as the data collection did not include a measure of value of time or income of the patients . we did have information on education and labor market attachment and could have imposed a stratified measure of hourly income obtained from the national statistics bureau . however , we should ideally use a measure of opportunity cost and that is less simple to obtain , as it would require information about the patient 's time use if they had not been visiting a health care provider . if we followed the recommendations of the human capital approach and assumed that all contacts with health care providers should be valued according to the opportunity cost of leisure time ( personal income net of tax ) , we would have derived at an average cost of transport corresponding to 95 per patient per three - month period . in this analysis , some studies have also considered the travel costs of accompanying persons . from an economic perspective , the relevance of including the cost of accompanying persons can be discussed and it will be highly dependent on the individual patient 's need to be accompanied . in the case of most ra patients in outpatient care , we assume that an accompanying person is not required . in this study more than 70% of the eligible patients responded to the travel time and cost questions . however , a substantial proportion of the patients ( 8.3% ) did not provide valid response . . recall bias may be important as we asked patients to report their use of health care services during a three - month period . there may be some element of under- or overreporting although it is difficult to assess the direction and consequences . we applied a fairly simplistic approach to missing items although more advanced methods could have been employed ( e.g. , multiple imputation ) . such a more advanced approach would ensure larger variability in the data in comparison with the approach we used . however , a crucial prerequisite for assessment is some explanation as to the pattern of missing data . here we assumed that those patients who did not provide valid responses may be represented by those who provided valid response . a brief inspection using logistic regression to identify different social characteristics in those with and without valid responses did suggest a systematic pattern in patients with missing responses . another potential bias is the fact that out of the 3700 patients identified eligible for the study , 15 percent did not respond to the questionnaire at all and further 8 percent who responded to the questionnaire did not provide answers on the travel time and cost questions . we were able to identify difference between responders and nonresponders , and it appeared that responders were younger , more likely to be married / cohabiting , living in a city area , had longer education , and were in the labor market . it is difficult to assess the consequences of such systematic pattern in the nonresponders , but it may provide some explanation as to the reasons for non - response , namely , that information about travel time and cost may not be relevant or important and therefore patients have avoided providing answers on these questions . the results of this study suggest that patients with rheumatoid arthritis in active outpatient care have on average 4.4 contacts with health care providers within a 3-month period and spend 4.6 hours and 56 on travelling to and from these providers . these findings are important when analyzing the social impact of treatment for patients with rheumatoid arthritis .

objectives . to investigate travel time , and travel cost related to contacts with health care providers for patients with rheumatoid arthritis ( ra ) during a three - month period . methods . patient - reported travel time and travel cost were obtained from 2847 patients with ra . eleven outpatient clinics across denmark recruited patients to the study . data collected included frequency , travel time and travel costs for contacts at rheumatology outpatient clinics , other outpatient clinics , general practitioners , privately practicing medical specialists , inpatient hospitals and accident and emergency departments . results . over a 3-month period , patients with ra had on average 4.4 ( sd 5.7 ) contacts with health care providers , of which 2.8 ( sd 4.0 ) contacts were with rheumatology outpatient clinics . private car and public travel were the most frequent modes of travel . the average patient spent 63 minutes and 13 on travelling per contact , corresponding to a total of 4.6 hours and 56 during the 3-month period . there was great variation in patient travel time and costs , but no statistically significant associations were found with clinical and sociodemographic characteristics . conclusion . the results show that patients with ra spend private time and costs on travelling when they seek treatment . these findings are particularly important when analyzing social costs associated with ra .

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

PMC3805316

conjugated microporous polymers ( cmps ) and related materials , such as covalent triazine - based frameworks ( ctfs ) and porous aromatic frameworks ( pafs ) , are a fascinating class of materials that can , in some cases , combine microporosity with useful physical properties that arise from their electronic conjugation . cmps typically comprise aromatic organic units covalently bonded to three or four neighboring units . insoluble cmps and related polymers , such as pafs , hence have commonly been envisaged as extended 3- or 4-connected networks that contain rings but only a small number of end - groups . if this picture is true , then cmps , ctfs , pafs , and their like , would be the closest organic polymer analogues of well - known inorganic materials such as boron nitride , zinc oxide , and ( alumino)silicates , materials whose structures are also based on 3- or 4-connected networks . alternatively , however , these materials could also resemble highly branched polymers or dendrimers , which are tree - like molecules with a relatively large number of end - groups but , typically , no closed rings . structural hypotheses and models aside , it is essentially unknown if cmps , ctfs , and pafs can be best described as highly branched polymers or as extended networks , or even as small but insoluble oligomers , which might nonetheless exhibit permanent microporosity . this uncertainty over structure mainly stems from the amorphous or poorly crystalline nature of the experimental samples , coupled with their total lack of solubility . however , the need to understand structure property relationships is highlighted by the recent discovery of new porous materials , such as soluble , hyperbranched cmps and porous dendrimers , organic molecules of intrinsic microporosity , and other discrete organic molecules that can show high levels of microporosity in the amorphous , solid state . hence , extended networks are not a prerequisite for microporosity , and network structures can not simply be assumed because a given material is microporous , particularly since typical cmp structures , which lack any deliberate solubilizing functionality , would be expected to become insoluble at quite modest molecular weights . it is important , therefore , that methods are developed to elucidate the molecular structures of cmps , rather than simply inferring them based upon observations of permanent microporosity . the experimental determination of the number of end - groups , for example by solid - state nmr , should in principle allow us to differentiate between , e.g. , dendrimers and highly extended condensed networks . in practice , however , this is difficult because side - reactions might cleave such end - groups , making them invisible to nmr and artificially lowering the end - group - to - molecular unit ratio . for example , in nickel - coupled yamamoto polymerizations of tetrahedral aryl halides , it is possible that halogen end groups might be removed by metal - catalyzed dehalogenation . additionally , entrained gases , physisorbed water vapor , or residual catalyst can all lead to difficulties in the precise determination of end - group concentration by elemental analysis . in this study , we take an alternative approach and focus on optical absorption and fluorescence spectra for cmps as a handle on their molecular structure . we believe that this is a useful strategy , because the electronic properties of cmps are in any case central to many of their most interesting applications . we demonstrate that the optical properties of cmps give a unique insight into the structural elements present in the cmp . furthermore , we show that this structural knowledge can also be exploited in terms of engineering the optical properties of cmps and related compounds . we concentrate here on cmps based on the polymerization of pyrene monomers , but the general approach should be broadly applicable to other cmps and compounds such as ctfs and pafs . thin layer chromatography ( tlc ) was performed using precoated aluminum sheets with silica gel 60 f254 ( merck ) and visualized by uv light ( = 254 or 280 nm ) . solution h nmr spectra were collected on a bruker uxnmr / xwin - nmr 400 mhz spectrometer while the solid - state c nmr spectrum for the ecmp was collected a 9.4 t bruker dsx nmr 400 mhz spectrometer equipped with a 4 mm hxy triple - resonance mas probe ( in double - resonance mode ) . ftir spectra for the ecmp and its precursors were collected on a bruker tensor 27 spectrometer equipped with a specac attenuated total reflectance module . gel permeation chromatography ( gpc ) utilized a lc 1120 hplc pump , a pl - els 1000 evaporative light scattering detector , a pl gel 5 mm mixed - c gpc column and midas autosampler ( polymer laboratories ltd . thf was used as the eluent with flow rate of 1.00 ml / min at 40 c and polystyrene as the standard . full synthetic details and nonoptical characterization results are given in section esi-1 of the supporting information . solution measurements for the soluble materials were obtained at 0.04 and 0.004 mg / ml ( 1,3-linear pyrene polymer and scmp ) and 1.5 mg / ml ( py(5 ) dendrimer ) respectively in dcm . for the same soluble materials thin film samples were prepared by dissolving 20 mg ( 1,3-linear pyrene polymer and scmp ) or 6 mg ( py(5 ) dendrimer ) of the compounds in 1 ml of dcm , after which the resulting solution was added to a quartz cuvette held at an angle and left overnight to evaporate . the resulting film on the inside of the cuvette provided a range of film thicknesses for analysis ( thickest at the bottom of the cuvette ) . measurements for solid - state powders were obtained by grinding the sample with kbr . vis spectra were obtained using a shimadzu uv-2550 uv vis spectrophotometer running the uvprobe software , version 2.34 . all spectra were obtained as absorbance measurements from 200800 nm , with scan speed set to fast and using a slit width of 5 nm . film and solution samples were measured in a quartz cuvette as a transmission measurement . solid powdered samples were analyzed using the isr-2200 integrating sphere attachment with a quartz solid sample holder as diffuse reflection measurement . emission and excitation spectra were obtained on a shimadzu rf-5301pc spectrofluorophotometer running rfpc software , version 2.04 . spectra were obtained using a fast scan speed and with sensitivity set to high . slit widths were adjusted so as to maximize the signal - to - noise for each sample . solution samples were analyzed in a quartz cuvette with the standard cell holder attachment . film samples were analyzed adhered to the wall of a quartz cuvette placed in the solid ( powder ) holder attachment . powder samples were analyzed in a quartz solid sample holder held in the solid ( powder ) sample holder attachment . data was exported to excel for further processing , and second order diffraction peaks of the excitation wavelength were manually removed from the spectra and the long wavelength baseline set to zero . the excited state properties of pyrene oligomer model clusters were calculated using a six - step approach . first , for each cluster model we performed a conformer search using the opls - aa forcefield and the low - mode sampling algorithm as implemented in macromodel 9.3 ( for more details about the conformer search see section esi-2 of the supporting information ) second , the ground state singlet ( s0 ) geometries of the low - energy conformers found in the conformer search were optimized using ground state density functional theory ( dft ) calculations . third , where possible , the harmonic frequencies at these optimized s0 geometries were calculated using the same dft setup to verify that the optimized structures correspond to proper minima on the s0 energy surface . fourth , the excitations at the optimized s0 geometry were calculated using time - dependent density functional theory ( td - dft ) . fifth , the lowest singlet excited state of each oligomer was relaxed using td - dft , to obtain its minimum energy geometry . finally , frequency calculations on the excited state minima were performed for selected oligomers to verify that they correspond to proper minima on the td - dft excited state energy surface . for the dft / td - dft calculations mainly the range - separated hybrid xc - functional cam - b3lyp was used and in some selected cases also the plain hybrid xc - functional b3lyp ( mainly to study the ground state energetics ) , where for all excitation calculations the tamm all the dft / td - dft calculations employing the cam - b3lyp xc - functional used a combination of nwchem 6.0 ( vertical excitation energies ) and gamess - us version 1 october 2010 r1 ( vertical excitation energies , excited state optimizations ) and the 6 - 31 g * * split - valence basis - set . all the dft calculations employing the b3lyp xc - functional were performed using the turbomole 6.3.1 code , and employed the double- dzp basis - set . the pyrene - based cmp network ( scheme 1b ) obtained by homopolymerization of 1,3,6,8-tetrabromopyrene ( see scheme 1a for atom labeling of pyrene ) has a reported brunauer emmett teller ( bet ) surface area of 1508 m / g and is highly fluorescent . note , solubilizing tert - butyl groups on the 7-position of structures c and e have been removed for clarity . the pyrene - based cmp network is a member of a broad family of related pyrene - based polymers and oligomers reported previously in the literature . this family of structures includes a 1,3-linear polymer based on the yamamoto coupling of 1,3-dibromo-7-tert - butylpyrene ( scheme 1c ) , first - and second - generation dendrimers ( py(5 ) and py(17 ) , respectively ) based on pyrene coupled via the 1,3,6 and 8 positions ( scheme 1e ) , and a branched statistical copolymer of 1,3,6,8-tetrabromopyrene and 1,3-dibromo-7-tert - butylpyrene . the latter copolymer combines porosity with solubility in selected solvents , and we hence refer to this as a soluble cmp or scmp . finally , we note that the synthesis of a linear polymer , obtained through coupling of pyrene via its 2 and 7 positions , has also been reported . however , this material is less closely related to our pyrene cmp than other pyrene polymers and oligomers because the pyrene monomer is linked through positions other than 1 , 3 , 6 , and/or 8 , and it has very bulky aryl substituents on the 4,5,9 and 10 positions . as such hence , we have considered four possible basic architectures : a linear soluble pyrene polymer , perfectly branched , soluble pyrene dendrimers , a branched soluble pyrene polymer ( the scmp ) , and a branched pyrene cmp network . as a first step in understanding the structure of the pyrene - based cmp network , we prepared samples of the insoluble cmp network , and the soluble 1,3-linear polymer , py(5 ) first - generation dendrimer , and scmp . we also synthesized a novel copolymer ( ecmp , for expanded - cmp ) via the cross - coupling of 1,3,6,8-tetrabromopyrene and 1,3-diboronic ester-7-tert - butylpyrene ( scheme 2 ) . unlike the scmp , which is a statistical copolymer based on similar monomers and where , for example , two 1,3-substituted pyrene monomers ( bifunctional units ) might link directly to each other , the ecmp will have an alternating copolymer structure , where 1,3,6,8- substituted pyrene monomers ( tetrafunctional units ) are only linked to 1,3-substituted pyrene monomers and vice versa . a further immediate difference between ecmp and scmp is the lack of solubility for ecmp : by contrast , the analogous scmp statistical copolymer is soluble in a range of common solvents . full synthetic details and characterization data are available in the supporting information for all these materials ( section esi-1 ) . we next measured the absorption and fluorescence spectra for these samples in solution , where possible , as a thin film cast from solution ( again , where possible ) , and as a powder mixed with kbr ( the only option for the insoluble cmp and ecmp ) . these measurements were compared with those reported previously in the literature for the 1,3-linear polymer material ( solutions and thin films ) , for the py(5 ) material ( solution ) , for the scmp material ( in solution ) , and for the insoluble cmp ( powder ) . this is the first systematic comparison of the solid - state and solution phase spectra of these materials . we concentrate here mostly on the fluorescence spectra , partly because the powder absorption spectra appeared significantly broadened relative to thin films , possibly due to scattering effects related to the polymer particle size distribution ( there is also a small red shift in the fluorescence peak position between the thin film and powder spectra , e.g. , 10 nm in the case of 1,3-linear polymer material , possibly also due to scattering , but with no real effect on the spectrum shape , for more details see section esi-3 of the supporting information ) . moreover , in the case of a structurally heterogeneous material , such as the insoluble pyrene cmp / ecmp and scmp are likely to be , the absorption spectrum will be a convolution of the absorption spectra of a range of different structural elements ( chromophores ) , all of which displaying vibrational broadening . as a result , figure 1 shows the normalized experimental fluorescence spectra for the various powder samples . the insoluble cmp network fluorescence spectrum is significantly red - shifted compared to the other materials , with the wavelength of the fluorescence maximum increasing as follows : 1,3-linear polymer ( 479 nm , 2.6 ev ) < py(5 ) dendrimer ( 501 nm , 2.5 ev ) < scmp ( 526 nm , 2.4 ev ) < ecmp ( 530 nm , 2.3 ev ) < insoluble cmp ( 618 nm , 2.0 ev ) . it is apparent that the chromophores responsible for fluorescence in the cmp network and ecmp are quite distinct from those from the fluorescent chromophores in the 1,3-linear polymer and the py(5 ) dendrimer . it is also clear that the insoluble cmp fluorescence chromophore is absent in the analogous soluble polymeric materials most obviously in the 1,3-linear polymer , which does not fluoresce at all in this wavelength range . these fluorescence spectra demonstrate that at least part of the insoluble pyrene cmp network and the ecmp structures are fundamentally different in character from those of the 1,3-linear pyrene polymer and the py(5 ) dendrimer . experimental fluorescence spectra for the 1,3-linear pyrene polymer ( purple ) , py(5 ) pyrene dendrimer ( blue ) , scmp ( cast film green , precipitated film orange ) , ecmp ( light red ) and cmp ( dark red ) , clearly showing the red shift in the spectra . in all cases we suggest that it is unlikely that these profound differences in the fluorescence spectra can be ascribed simply to the size or effective molecular weight of the conjugated pyrene system . for example , from the solution data of figueira - duarte et al . , we know that the difference in fluorescence energy of the two dendrimers py(5 ) and py(17 ) is less than 0.1 ev while the system more than triples in size . similarly , we know , based on solution data for the 1,3-linear pyrene dimer and trimer oligomers and the 1,3-linear pyrene polymer , that the fluorescence energy is effectively converged with the pyrene dimer . it is thus unlikely that simply increasing the number of conjugated units can explain the large observed red shift in fluorescence for the insoluble pyrene cmp , or indeed even the much smaller red shift observed for ecmp . we also believe that these differences do not arise from agglomeration related effects . we studied two different scmp samples ; one sample obtained through precipitation with an antisolvent ( orange line in figure 1 ) and one obtained through slow evaporation of the solvent ( green line in figure 1 ) . both samples differ strongly in density and porosity to gases ( precipitated materials have much higher porosity than cast materials , where the precipitated material absorbs nitrogen in micropores while the cast material is impervious to nitrogen ) and thus probably in degree of agglomeration . the difference in the position of the peak maximum for cast and precipitated scmp ( 8 nm , 0.03 ev ) is , however , small , especially in comparison with the red shift between the 1,3-linear polymer and the pyrene cmp ( see section esi-3 , supporting information ) . a comparison of the experimental solid - state ( powder ) absorption spectra ( see figure s3 , section esi-3 of the supporting information ) , while complicated somewhat by the peak - broadening problems discussed above , also suggests that the bulk of the cmp and ecmp structure are different from that of the 1,3-linear pyrene polymer and the pyrene py(5 ) dendrimer . not only are the first absorption maxima of the cmp ( 417 nm , 3.0 ev ) and the ecmp ( 430 nm , 2.9 ev ) red - shifted compared to both the 1,3-linear polymer ( 370 nm , 3.4 ev ) and py(5 ) dendrimer ( 397 nm , 3.1 ev ) , but the cmp and ecmp peak are also much broader . the dendrimer absorption spectrum reaches 25% of the maximum intensity of the first peak at 477 nm ( 2.6 ev , i.e. 0.5 ev below the peak maximum ) while the absorption spectrum of the cmp reaches the same 25% already at 605 nm ( 2.0 ev , i.e. 1.0 ev below the peak maximum ) . part of this 0.5 ev of extra broadening of the cmp absorption spectrum might be due to a difference in the inherent vibrational broadening of the cmp and the dendrimer and/or a difference in the scattering - related broadening highlighted above . however , the extent of the broadening also suggest , like for the fluorescence data , the presence of chromophores in the insoluble pyrene cmp and ecmp that absorb at wavelengths longer than the peak maximum ( i.e. , abs > 417 nm and > 430 nm respectively)that is , a much greater red shift than suggested by the peak maximum alone . again , solution data appear to rule out an explanation for this red shift based on an increased conjugation length . in summary , both solid - state absorption and fluorescence spectra suggest that a significant component of the insoluble pyrene cmp and ecmp are structurally different to both the 1,3-linear polymer and the py(5)/py(17 ) dendrimer . we therefore decided to use computational chemistry calculations on cluster models of the polymer as a means to rationalize these qualitative observations and to elucidate the type of chromophore that might explain the large observed spectral red shift in the pyrene cmp and ecmp networks . the various cluster models that we investigated included fragments of linear polymers , fragments of dendrimers , and closed pyrene rings comprising between three and six pyrene units . we predicted the absorption and fluorescence spectra for the various cluster models and , where relevant , considered a range of conformers . in keeping with kasha s rule , we assumed in the fluorescence calculations that transitions from higher singlet excited states ( e.g. , s2 ) to s1 are so fast that fluorescence occurs in appreciable yield only from the latter s1 state . we first focused on the materials for which we have good knowledge of the molecular topology : that is , the 1,3-linear pyrene polymer and the py(5 ) dendrimer . in previous work , we demonstrated that td - cam - b3lyp gives a good match to experimental absorption and fluorescence spectra of linear 1,3-oligomers and 1,3-linear polymer in solution after applying a rigid downward shift to account for the effect of environment ( i.e. , in this case , solvent ) and an inherent bias of the cam - b3lyp density functional that results in the general overestimation of excitation energies . moreover , we showed that the spectral features converge rapidly with oligomer length , and that chains of six pyrene units ( in the case of absorption ) and just two pyrene units ( in the case of fluorescence ) give peak maxima predictions that are close to those of an infinite polymer . also , it was found that the tert - butyl groups could be omitted without significantly changing the results . since we are interested here in solid - state versions of the various polymers , we repeated this earlier analysis for both the 1,3-linear pyrene polymer and the py(5 ) dendrimer ( scheme 1c and 1e , respectively ) , and compared td - cam - b3lyp predictions with experimental data ( figures s4 and s7 in section esi-3 of the supporting information ) for thin films . we focus on films for these solution - processable materials , rather than powders , because the thin film data have , as discussed above , a much better resolution for the absorption spectra . we found that td - cam - b3lyp correctly predicts the relative ordering of the first absorption peak maximum of the 1,3-linear polymer versus the first absorption peak maximum and shoulder of the py(5 ) dendrimer , and also the relative positions of the fluorescence peak maxima for both materials . for thin film data , application of a rigid downward shift of 0.5 ev to the td - cam - b3lyp predictions resulted in a good absolute match between the predicted and observed positions of spectral features ( see figure 2 ) , and hence , all computational spectroscopic results hereafter include the same absolute shift . as the absorption and fluorescence maxima of the powder samples are further red - shifted by 0.1 ev relative to those of thin - film samples ( see discussion above and section esi-3 of the supporting information ) , this may mean that our calculated corrected spectra need to be further red - shifted by a similar amount when comparing to powder spectra . comparison of the experimental ( solid lines ) and td - cam - b3lyp predicted absorption ( dashed lines ) and fluorescence ( insert ) spectra of 1,3-linear pyrene polymer ( purple ) and the py(5 ) pyrene dendrimer ( blue ) . for systems such as the 1,3-linear pyrene polymer and the py(5 ) dendrimer there are only a limited number of possible conformers that all lie relatively close in energy . for example , the lowest excitation energy of the py(5 ) dendrimer varies by less than 0.05 ev between the five different conformers ( see also figure s9 in section esi-4 of the supporting information for an example of the effect of oligomers in the case of 1,3-linear pyrene oligomers ) . next , we considered an alternative chromophore structure that is strongly related to the 1,3-linear pyrene polymer but which has not , to our knowledge , been prepared experimentally : the 1,8-linear polymer ( see scheme 1d ) . nonetheless , this substitution pattern is ( theoretically ) possible , and would result from the linear yamamoto coupling of 1,3,6,8-tetrabromopyrene , followed by debromination from the remaining two brominated positions per pyrene unit . 1,3,6,8-tetrabromopyrene does not have a c4 axis , but only a c2 axis perpendicular to the plain of the ring ; that is , it is hence , the relative orientation of the pyrene units in the chain results in the 1,3- and 1,8-linear pyrene polymers being structurally different and having different optical properties . the 1,8-oligomers , and by extrapolation the 1,8-linear polymer , is predicted to have spectral features that are slightly red - shifted compared to the 1,3-linear polymer , but rather similar to the dendrimer ; fluorescence at 500 nm ( 2.5 ev ) and two strong absorption features at 400 nm ( 3.1 ev ) and 375 nm ( 3.3 ev ) , respectively ( see also table 1 and figure s9 in section esi-4 , supporting information ) . 1,8-linear polymers and oligomers thus do not appear to make good candidates for the red - shifted spectral features in the cmp and ecmp . having considered linear polymers and dendrimers , we next considered possible cluster fragments of extended networks containing rings . , has been invoked previously in a structural model for paf-1 based on isomorphous replacement in an amorphous silica model . here it is known from topological considerations that every 4-connected network will have at least a certain fraction of rings in this size - range . for the reasons outlined above , the orientation of the pyrene units in the ring lead to structurally different rings . the long sides ( i.e. , 18 or 36 ) and short sides ( i.e. , 13 or 68 ) of the pyrene units give rise to l(ong ) and s(hort ) edges , respectively . these l and s edges can combine into rings made of either just one type of edge ( a ) or a combination of both types of edges ( b ) . henceforth , we label such rings by the number of pyrene units in the ring , followed by the orientation of the edges . for example , the two possible a - type rings formed from four pyrene units are labeled 4llll and 4ssss , respectively , while an example of a possible b - type ring is 4lsls ( see also figure 3 ) . because every pyrene unit has two l and two s sides , a mixture of different ring configurations is always expected to occur in the extended material . dft - optimized lowest energy configurations of 4llll , 4ssss and 4lsls rings ( atoms represented as blue spheres indicate where the rings would connect to the rest of the amorphous pyrene network in the pyrene cmp / ecmp ) . we also considered a number of distinct conformers for the 4- and 5-rings ( typically 2 or 3 , distinguished from each other by a superscript number at the end of the ring label : that is , 4llll for the lowest energy , or first , conformer of the 4llll ring , 4llll for the second most stable , etc . this was done to sample the possible conformations rings could have when they form part of an extended framework . competing bonding requirements make it unlikely that in an extended material all rings would be in their lowest energy conformation . figure 4 shows the 4ssss and 4ssss conformers ( 4ssss is shown in figure 3 ) and illustrates that the difference between different conformers lies in the relative orientation of the different pyrene units ( e.g relative to the plane of the ring up down up down for 4ssss , flat - down - flat - up for 4ssss and flat - up down - optimized lowest energy configurations of the 4ssss and 4ssss conformers ( 4ssss shown in figure 3 ) . all values are in ev and , as discussed in the text , rigidly shifted downwards by 0.5 ev . for all 4- and 5-ring chromophores , the observed spread in excitation energies for the different conformers is presented with the value for the lowest energy conformer in bold . for the linear polymers and dendrimers , the position of table 1 summarizes our predictions for the location of the fluorescence and the first absorption peaks for the different rings ( see table s2 in section esi-5 ( supporting information ) for the unshifted energies ) . focusing first on the lowest energy conformers of each ring type , we observe that the lowest absorption and fluorescence peak of the 3sss and 3lll rings lie at significantly lower energy ( and hence longer wavelength ) than those predicted for the different linear polymer and dendrimer cluster models . in the case of the other ring types , the position of the lowest absorption and fluorescence peak is , however , generally very similar to those predicted for the structurally related polymers ( i.e. , the 1,3-linear polymer for the pure s rings and the 1,8-linear polymer for the pure l rings ) . that said , the energy of the lowest absorption peak is always slightly lower than that of the corresponding linear oligomer of the same length ( i.e. , 4ssss vs the 1,3-coupled tetramer , see figure s9 in section esi-4 , supporting information ) . the only exception is the lowest absorption peak ( but not fluorescence peak ) of the 5sssss ring , which is red - shifted by 0.4 ev compared with both the 1,3-coupled linear pentamer and the 1,3-linear polymer . surprisingly , the lowest energy conformers of most rings built from more than three pyrene units thus show generally only minor evidence of ring - strain in their spectra . the higher energy conformers of the rings ( lying 2060 kj/(mol pyrene ) higher in energy ) , however , show a different picture . for these conformers , the position of the first absorption and fluorescence peaks more specifically , for each ring - size , we found that the more strained the conformer that is , the higher its energy with respect to the lowest energy conformer the lower in energy and higher in wavelength the fluorescence peak and first absorption peak are predicted to lie . clearly , then , the presence of such strained rings could give rise to a red shift in both the fluorescence and absorption spectra of cmp and ecmp networks . for the cmp network specifically , the measured fluorescence spectrum fits well with the fluorescence energies predicted for the more strained 4- and 5-rings , and suggests that such rings might be the chromophores responsible for the large red shift in the cmp fluorescence . regarding the absorption spectrum , this again will be a convolution of the absorption spectra of a range of different chromophores . in principle , the red shift and broadening of the cmp absorption spectrum could be explained by a combination of different ring - sizes , configurations , and conformations as present in an amorphous network . we propose that the bulk of the rings would be moderately strained 4- , 5- , and 6-rings , and that these chromphores are responsible for the shift of the first absorption maximum to 3.0 ev ( 310 nm ) . a smaller fraction of more strained rings could account for the chromophores responsible for broadening the cmp absorption spectrum to even longer wavelengths , the most strained of which would be the strained 4- and 5-ring chromophores responsible for the fluorescence . in line with this evidence of rings in cmps , energetic estimates ( discussed in the supporting information , section esi-6 ) suggest that the formation of rings , even small or strained ones , is energetically feasible during the kinetically driven cmp synthesis which involves irreversible bond - formation , especially when one takes into account that some of the polycondensation might take place after phase separation within the porous , precipitated network phase . overall , we thus believe that a structural model of the cmp should contain the above - described rings ( and indeed our previous preliminary model of the pyrene cmp contained rings ) . the likely microscopic link between strain and the spectroscopic red shift are the pyrene smaller torsion angles that is , flatter structures are expected to result in increased overlap between the -systems on adjacent pyrene units and hence enhanced conjugation , which would explain , for example , the observed red shift in the absorption energies . indeed we find that the strained rings that display a large red shift have considerably smaller average torsion angles than those of their chain counterparts ; e.g. , 42 for 3lll and 46 for 4ssss compared with 70 for long 1,3- and 1.8-linear chains . likewise , those rings that have a similar absorption on - set as the linear - polymers also have average torsion angle comparable to that of linear chains ; e.g. , 67 for 4ssss . see supporting information , section esi-5 , for more information about the torsion angle distributions of the rings . small torsion angles , in the absence of rings , could also result in a significant red shift ( at least in the case of the absorption spectrum , see for example data for linear chains in figure s10 in section esi-7 of the supporting information ) . however , it is difficult to envisage where the strain required for the formation of these small torsion angles should originate from , if not from the presence of rings . a comparison of the data in table 1 and the experimental fluorescence spectrum of the pyrene cmp in figure 1 suggests that fluorescence occurs from only the most strained subset of chromophores and not from the less strained chromophores ( though there might be a shoulder at 540 nm , 2.8 ev ) , despite the fact the less strained chromophores do appear to contribute to the absorption spectrum . our calculations , discussed in more detail in the supporting information ( section esi-8 ) , suggest that this might be because an excited ring - based system can lower its energy significantly by moving the excited state from larger rings to smaller rings ( e.g. , 5lllll * + 4llll 5lllll + 4llll * , where the asterisk denotes the ring upon which the excited state is localized ) and , for a given ring - size , from a less strained conformer to a more strained conformer ( e.g. , 4llll * + 4llll 4llll + 4llll * ) . assuming that excited state transport is generally fast relative to excited state relaxation , then excited state relaxation and fluorescence would indeed only occur at the most strained chromophores in the material , even though all chromophores to some extent contribute to absorption . the possible shoulder at 540 nm ( in the range where fluorescence is predicted to occur from unstrained 4ssss- and 6ssssss / llllll - rings ) might then be indicative of those cases where excited state transport is too slow and/or strained rings too far away from the absorption chromophore for the excited state to reach the most strained ring before fluorescence . in this scenario , the appearance of multiple fluorescence peaks or shoulders could potentially be a marker for material heterogeneity ( e.g. , the spatial distribution of small rings or linear fragments , see below , over the bulk of the material ) . for the ecmp , the measured fluorescence spectrum matches the fluorescence energies predicted for moderately strained 4ssss and 4lsls rings ( configurations by definition including short edges , see figure 3 , in - line with the fact that the bifunctional 1,3-pyrene units , by definition , will be incorporated short in any ring that is formed . thus , perhaps counterintuitively , expanding the cmp by inserting bifunctional units between the tetrafunctional units does not seems to lead to an apparent change in the size of the smallest ring in the network , but only the degree of strain that these small rings are under . the lower strain could be either ( i ) the direct result of having bifunctional units in the ring that are relatively free to move , since they do not form part of any other rings , or ( ii ) an environmental effect due to other rings surrounding the small ring being larger than in the related cmp . the fact that no shift in the size of the smallest ring is observed fits with the idea that the pyrene ecmp and cmp are kinetic and not thermodynamic products . a similar shift in the fluorescence spectrum , as for the ecmp , interpretation of spectra for scmp is considerably more difficult because scmp is a statistical copolymer of tetrafunctional and bifunctional units , and hence the material can contain fragments of tetrafunctional units linked to tetrafunctional units as well as bifunctional units linked to bifunctional units . the presence of the latter chains could be an explantion to why the scmp , in contrast , to ecmp is soluble in common organic solvents . the spectral similarities between both materials suggest that moderately strained 4- and 5-rings also exist in scmp , and that they are the chromophore responsible for the scmp fluorescence . the excess in fluorescence of scmp relative to ecmp in the 450480 nm range , coincident with the measured and predicted fluorescence maxima of the 1,3-linear pyrene polymer , might be the fingerprint of linear - chain rich , unbranched domains , that are absent , as discussed above , in the strictly alternating ecmp . the occurrence of this fluorescence excess also suggests that the chain - rich domains are large enough that at least some of the excitations originally generated there do not diffuse to more strained chromophores before radiative de - excitation to the ground state . finally , a slight fluorescence excess at long wavelengths , at least for the precipitated scmp sample ( flu > 550 nm ) might also suggests the possible presence of scmp-1 domains rich in tetrafunctional units ( i.e. , cmp - like domains ) . on the basis of the above analysis , the absorption and fluorescence spectra of cmps and related polymers should in principle be able to be engineered by the incorporation of different sizes of rings or rings with different degrees of strain . the ecmp and scmp are examples of how expanding rings / reducing strain by incorporating bifunctional monomers allows one to blue shift the absorption and fluorescence spectra . this analysis is also in - line with the blue - shift observed between the pyrene cmp network and the ordered alternating 1:2 copolymer of tetrafunctional 1,3,6,8-tetrabromopyrene and bifunctional 1,4-benzene diboronic acid . copolymers with other ratios than 1:2 might yield intermediate blue - shifted spectra as long as the bifunctional monomer is homogenously incorporated in the cmp . we have presented examples here for pyrene - based materials , but we believe that these general principles should hold for a much broader class of polymer network materials . finally , a combination of the approach developed here with the polymatic tool for building structural models of ( amorphous ) polymers of colina and co - workers could in the future lead to improved structural models of cmps and related materials . here the optical spectra of the material would be another constraint for the structural model to fulfill , just as the material s surface area , pore - volume etc . a combination of theoretical and experimental spectroscopy provides an unrivalled insight into the atomic structure of porous conjugated polymers , where hitherto structures have been largely a matter of speculation and hypothesis . specifically , we show that , in the case of an insoluble pyrene cmp network , the significant red shift in its absorption and fluorescence spectra relative to related pyrene - based materials can be rationalized by the presence of strained rings in a network structure . the incorporation of strained rings can , in principle , be exploited to tune the absorption and fluorescence spectra of a polymer , and hence optimize this for potential applications such as photocatalysis and photovoltaics . certainly , these structural effects will be central in strategies which seek to control photophysical properties in porous organic polymers by band gap engineering .

the photophysical properties of insoluble porous pyrene networks , which are central to their function , differ strongly from those of analogous soluble linear and branched polymers and dendrimers . this can be rationalized by the presence of strained closed rings in the networks . a combined experimental and computational approach was used to obtain atomic scale insight into the structure of amorphous conjugated microporous polymers . the optical absorption and fluorescence spectra of a series of pyrene - based materials were compared with theoretical time - dependent density functional theory predictions for model clusters . comparison of computation and experiment sheds light on the probable structural chromophores in the various materials .

Introduction Experimental Section Results and Discussion Conclusions

PMC3981354

bowel obstruction is a common surgical admission around the world usually resulting from post surgical adhesions or abdominal hernias . conversely , small intestinal tumors , such as midgut carcinoids , are uncommon neoplasms and an infrequent cause of intestinal obstruction . although the small bowel is the most common site for these tumors , the overall incidence is still less than two per 100,000 of all medical or surgical admissions , however the actual reported incidence is closer to eight per 100,000 based on autopsy studies . a preexisting small bowel neoplasm can prohibit the passage of an accidentally ingested foreign object and produce an obstruction that neither the neoplasm nor the foreign body could have produced alone . we hereby report an interesting case in which a small piece of foreign material accidentally ingested by a patient lead to the detection of an asymptomatic midgut carcinoid in the proximal ileum that would have otherwise eluded detection for several years . a previously healthy , 39-year - old male presented to the emergency department ( ed ) with a less than 24 hour complaint of severe cramping abdominal pain and two episodes of emesis within the past five hours . his past medical history is significant only for hypertension for which he was noncompliant and not taking any medication . social history was relevant for cigarette smoking of one pack per day and for beer drinking several times per week but he denied ever being intoxicated . his last drinking event was a couple of cans of beer two days prior to his presentation . review of systems was negative except for the gastrointestinal complaint resulting in his emergency room ( er ) visit . the patient was employed as a mechanic and had previously been active until the onset of his current symptoms . at presentation , his pulse was 98 beats / min , his blood pressure was 186/88 and his temperature was 99.0 degrees fahrenheit . his chest was clear to auscultation , heart sounds were regular and without murmur , and he had no peripheral edema or cyanosis . his bowel was diffusely tender to deep palpation , without focal tenderness ; and no ventral or inguinal hernias were found . the patient displayed mild voluntary guarding however ; there were no obvious rebound or other peritoneal signs . his laboratory analysis was remarkable only for a white blood count of 19.810 with left shift ( 28% bands ) . plain films of the abdomen showed dilated small bowel loops with multiple air fluid levels , highly characteristic of a distal small bowel obstruction . a computed tomography ( ct ) scan of the abdomen with iv and po contrast revealed dilated , contrast - filled , small bowel loops , a short segment of small bowel with thickened bowel wall , and a small metallic ring within the lumen ( figure 1 ) . the colon was collapsed and mesenteric adenopathy was noted ( figure 2 ) . figure 1metallic piece in the lumen of the thickened small bowel . metallic piece in the lumen of the thickened small bowel . the patient was resuscitated with 2 liters of intravenous lactated ringer 's and then taken to the operating room ( or ) for an exploratory laparotomy . upon entering the abdomen , dilated loops of small bowel were immediately encountered that had a tapioca - like studding pattern , particularly around the proximal jejunum . an obvious stricture was noted in the distal jejunum / proximal ileum which displayed the characteristics of a transition point in which the distal bowel segment was decompressed , and the proximal segment was dilated and edematous . palpationrevealed a patchy , white , firm , napkin - ring - like deformity in the bowel wall with thickening and contracture of the surrounding mesentery ( figure 3 ) . a hard , subcentimeter nodule with surrounding desmoplastic reaction was visualized , this was consistent with a metastatic mesenteric lymph node . several other lymph nodes were firm and easily palpable or visible within the remainder of the small bowel mesentery . running the entire length of the bowel revealed two more palpable subserosal masses within the jejunum / ileum just distal to this site , each measuring less than one centimeter . a segmental en bloc small bowel resection to include all three lesions and the mesentery was performed . the adjacent remaining mesentery was then carefully explored and dissected for any additional metastatic lymph nodes , three were identified and removed . a cholecystectomy was also performed without difficulty . the specimen was immediately dissected in the pathology lab ( figure 4 ) . upon opening the bowel wall a dense , intraluminal mass , with an associated tight luminal obstruction additionally , a tab from a beer can was seen immediately proximal to the intraluminal mass ( figure 5 ) . figure 4specimen opened to reveal the primary carcinoid tumor and the adjacent beer can lid induced mucosal damage . specimen opened to reveal the primary carcinoid tumor and the adjacent beer can lid induced mucosal damage . figure 5beer can lid retrieved from the small bowel lumen adjacent to the carcinoid tumor . the final pathology revealedx a multifocal , low - grade neuroendocrine tumor ( carcinoid ) with multiple drop metastases throughout the segment of intestine and metastatic mesenteric lymph nodes . both the intraluminal mass and lymph nodes stained positive for chromogranin and synaptophysin , but were negative for ki-67 . the patient 's recovery was uneventful with the return of normal bowel function on the fourth postoperative day . he was discharged home on the fifth postoperative day . upon questioning at a postoperative visit , the patient recalled that two days prior to his admission he had dropped a beer tab into the beer can that he was drinking and he must have swallowed it without knowing . his 24 hour urine was collected and submitted for analysis of 5-hiaa levels , this was within normal limits . carcinoids are rare neoplasms , first reported by langhans in 1867 and first described by lubarsch in 1888 . the term carcinoid ( karzinoide , meaning cancer - like in german ) was originally given by oberndorfer to this tumor for its ' histological malignant appearance , but benign and indolent clinical course when compared to that of other cancers . patients with midgut carcinoid commonly present with vague and non - specific symptoms , which often results in clinical detection being delayed for years . as time progresses , the most common presenting symptom is intense , episodic , abdominal pain , which is typically indicative of advanced disease . this pain may be due to intermittent intussusceptions of an affected segment , mesenteric ischemia due to buckling of the fibrosed mesentery or arterial elastosis , or frank obstruction . unlike other cancers , the size of the primary tumor does not necessary correlate with presence of metastatic disease ( with the exception of appendiceal carcinoids ) . often times , the diagnosis is made only after the development of carcinoid syndrome ( flushing , wheezing , and diarrhea ) due to the serotonin produced escaping hepatic clearance when cancer cells metastasize to the liver or retro - peritoneum strictures . admission for intestinal obstruction can be due to a variety of causes and is a frequent event in hospitals around the world . in one series intestinal obstructions accounted for 3% of all surgical admissions and up to 20% of all acute abdominal conditions requiring admission by other observers . post surgical adhesions are the most common etiology of acute mechanical obstruction of the small bowel ( sbo ) and account for 6080% of all cases of intestinal obstruction in the developed world . hernias are recognized as the second most common etiology of mechanical small bowel obstruction in developed countries . malignant small bowel obstruction is the third , and a much less frequent , cause of hospital admission in developed countries . in one series , it accounted for less than 5% of small intestinal obstructive cases , but can make up to 7.4% of all admissions for small bowel obstructions . although most small bowel tumors found at autopsy are benign ( 75% ) , those that present with symptoms or are found during surgery are usually malignant . the most common tumor of the small bowel is an adenoma , which is generally asymptomatic . several theories attempt to explain the low incidence of tumorigenesis in the small bowel including rapid turnover of mucosal cells , rapid transit of luminal contents , low bacterial counts , and the alkalinity of chime . population clusters do exist , but potential risk factors for the development of malignant disease of the small intestine are poorly defined . masses causing intraluminal obstruction or extrinsic compression are rarely obstructive in the small intestine due to the liquid nature of the small bowel enteric contents . even a very small luminal channel is sufficient for the passage of all residuals of digestion , with the exception of an indigestible non - yielding foreign object . in this patient , a preexisting and previously undetected , asymptomatic small intestinal neoplasm caused the retention of a foreign body resulting in the complete obstruction of the small bowel . such an unusual combination of factors resulting in a bowel obstruction has not widely been reported in literature due to the rarity of such a condition . a foreign object that is small enough to pass through the esophagus and pylorus is unlikely to cause obstruction anywhere else along the lower gastrointestinal tract . therefore , when a foreign object is found to be obstructing at the mid portion of the small bowel , suspicions of a preexisting small bowel neoplasm should be raised and a timely exploratory surgery should be conducted .

bowel obstruction is a common surgical admission around the world . on the other hand , small intestinal tumors , such as midgut carcinoid , are uncommon neoplasms and an infrequent cause of intestinal obstruction leading to hospitalization . a foreign body is an extremely rare cause of intestinal obstruction and when ingested , foreign bodies most often lodge in the narrowest portion of the gastrointestinal tract . narrowing of the small bowel due to a neoplasm can prohibit the passage of an accidentally ingested foreign object and produce an obstruction that neither the neoplasm nor the foreign body could have produced alone . we hereby report a case in which an accidentally ingested piece of foreign material leads to the finding of a small , early stage , asymptomatic , midgut carcinoid cancer in the proximal ileum that would have otherwise eluded detection for several years .

Introduction Case Report Discussion

PMC4664710

this transcriptome shotgun assembly project has been deposited at ddbj / embl / genbank under the accession gdkm00000000 . high yielding rice cultivars need high quantities of nitrogen which is usually supplied in the form of urea fertilizers . rice cultivars show diversity in their nitrogen use efficiency which influences the yield of particular cultivars . nitrogen use efficiency is influenced by nitrogen uptake , transport , vacuolar storage , utilization and remobilization . so , it is necessary to understand the molecular basis of nitrogen use efficiency of high yielding rice cultivars for understanding the basis of their high yield . the high throughput rna - seq analysis enables understanding of the functioning of all genes at a particular time and location . in this work rna - seq analysis is employed for understanding the genomic basis behind nitrogen use efficiency of the high yielding indica rice cultivar rpbio-226 . rp bio-226 seeds were surface sterilized with 0.1% hgcl2 for 30 min , rinsed thoroughly with distilled water and germinated on murashige and skoog medium that lacked a nitrogen source . ten day old seedlings were transferred to 0.1 mm urea containing murashige and skoog medium . total rna was isolated from the leaves and rna concentration and purity were estimated with a nanodrop spectrophotometer . mrna was purified and fragmented at elevated temperature ( 94 c ) in the presence of divalent cations . first strand cdna synthesis was carried out by fragmented mrna and reverse transcribed with superscript iii reverse transcriptase by using random hexamers . illumina adapters were ligated to the cdna molecules after end repair and addition of a base . the library was amplified using 8 cycles of pcr for enrichment of adapter ligated fragments . the prepared library was quantified using qubit and validated for quality by running an aliquot on high sensitivity bioanalyzer chip . the effective sequencing insert size is 130580 ; the inserts are flanked by adapters whose combined size is 120 bp . transcriptome sequencing was carried out with the illumina_nextseq500 system ( illumina , san diego , ca ) . the preprocessing of the reads was performed with fastqc and the adapters were removed with fastx toolkit , . samtools ( ver 0.1.18 ) and snpeff ( ver 4.1 ) were used for creation of variation report with a mapping quality of > 30 and read depth of > 20 as cutoffs , . de novo transcriptome assembly was performed with velvet and oases softwares , ( table 1 ) . we have planned to analyze the transcriptome of rp bio 226 in comparison with other rice cultivars for understanding the variation in nitrogen use efficiency of rice .

rice yield is greatly influenced by the nitrogen and rice varieties show variation in yield . for understanding the role of urea nutrition in the yield of elite indica rice cultivar rpbio-226 , the urea responsive transcriptome was sequenced and analyzed . the raw reads and the transcriptome shotgun assembly project has been deposited at ddbj / embl / genbank under the accession gdkm00000000 . the version described in this paper is the first version , gdkm01000000 .

Direct link to deposited data Experimental design, materials and methods

PMC4672979

a 43-year - old man with a history of hypertrophic obstructive cardiomyopathy ( hocm ) was admitted to our hospital due to recently aggravated exertional dyspnea and palpitations . transthoracic echocardiography revealed asymmetrical inter - ventricular septal hypertrophy with systolic anterior motion ( sam ) of the anterior mitral valve leaflet ( fig . the septal wall thickness was 25 mm , the left ventricular outflow tract flow velocity was 4.6 m / sec , and the pressure gradient was > 90 mmhg . the end - diastolic and end - systolic volumes of the left ventricle were 71 ml and 25 ml , respectively . an elongated anterior leaflet of the mitral valve showed sam , resulting in grade iii eccentric mitral regurgitation toward the left atrial posterior wall . 12-lead electrocardiography , a sinus rhythm with left ventricular hypertrophy was confirmed , indicating a repolarization abnormality . right thoracotomy was performed with a 4.5-cm incision in the fourth intercostal space , and four additional ports were inserted at the third ( left arm , chitwood clamp ) , fourth ( left atrial retractor ) , and sixth ( right arm ) intercostal spaces . a 3-cm radial incision was made in the amvl from the right fibrous trigone to the left fibrous trigone , taking care to prevent aortic valve injury . through the incised amvl asymmetric septal hypertrophy was present , but neither papillary muscle anomaly nor fusion was noted . septal myectomy was then performed diffusely at the nadir of the right aortic sinus and cautiously continued leftward to prevent a conduction injury ( fig . the total weight of the excised muscle was 11 g. the amvl incision was repaired using a continuous running suture . an ink test using methylene blue showed an adequate coaptation surface , and a water test revealed no residual mitral regurgitation . in order to prevent possible micro - air embolism in the left chamber , the left atrium was filled with blood before closure of the transatrial incision . after weaning from cardiopulmonary bypass , intraoperative transesophageal echocardiography performed immediately after weaning from cardiopulmonary bypass revealed mild mitral regurgitation ( grade i ) with a well - relieved left ventricular outflow tract obstruction ( pressure gradient , 15 mmhg ) without sam , as confirmed by the cardiologist on our team . the patient remained in the intensive care unit for 18 hours , and no postoperative complications occurred . postoperative transthoracic echocardiogram findings before discharge showed no left ventricular outflow tract obstruction ( vmax , 1.9 m / sec ) and only mild mitral regurgitation . hocm is a heterogeneous disease , in which the aortic outflow tract is obstructed by an abnormally thickened ventricular septum . since morrow introduced transaortic myectomy for the treatment of hocm , which has a complicated pathophysiology , resection - plication - release repair has become the standard treatment . due to the challenge of septal visualization using the transmitral approach , septal myectomy for left ventricular outflow tract obstruction relief in addition , improved resolution of sam via the transaortic valve approach has decreased the occurrence of mitral regurgitation due to loss of coaptation with the posterior leaflet . however , the transmitral approach decreases the risk of aortotomy and aortic valve damage and speeds up postoperative recovery . moreover , in cases of extensive mitral valve pathology , the transmitral approach allows more complex mitral valve repair and mitral valve replacement . furthermore , as in our present case , mitral regurgitation with redundant amvl can be sufficiently treated with amvl incision and repair . as experience with minimally invasive mitral valve repair has been gained in several centers , minimally invasive transmitral septal myectomy through an amvl incision has become possible via a robotic approach , as first described by khalpey et al . . in our center , we have successfully treated four cases to date , including the present case , by septal myectomy through a minimally invasive right mini - thoracotomy approach using the automated endoscope system for optimal positioning ( aesop ; computer motion inc . , santa barbara , ca , usa ) in two cases and the da vinci system in two cases . the aesop technology has advantages for the transmitral approach but requires a long instrument with a limited view , which makes the performance of this delicate procedure more difficult . in addition to the well - known cosmetic advantages of minimally invasive approaches , the flexible joints and three - dimensional scope of the da vinci system allow for insertion inside the left ventricle below the plane of the mitral valve , which enables precise inspection of the submitral apparatus and a better view for determining the amount of septum that should be removed than is obtained using the transaortic approach . in summary , minimally invasive transmitral septal myectomy is a good surgical option for mitral repair in selected cases as an alternative to the conventional transaortic approach , because it can provide not only cosmetic advantages but also better visibility of the subvalvular apparatus and easier extension with good results .

a 43-year - old man with symptomatic hypertrophic obstructive cardiomyopathy ( hocm ) was admitted to our hospital with aggravated exertional dyspnea and successfully treated with robotic transmitral septal myectomy . minimally invasive transmitral septal myectomy may be a feasible surgical option for the treatment of hocm in selected cases as an alternative to transaortic myectomy .

CASE REPORT DISCUSSION

PMC4513759

dentifrices have been a source of concern and subject of study for many professionals , since it is one of the main resources used in daily oral care by the population . dentifrices have different components , such as detergents , fluoride , therapeutic ingredients , flavors , and abrasives . among the abrasives , the most common are calcium carbonate and silica.1 these abrasives have an important role in cleaning teeth , removing bacteria and stains from the tooth surface . however , the dentifrice should promote optimal tooth surface cleaning with minimal abrasion,2 since dentifrices with high amounts of abrasives can damage hard tissue , soft tissue and restorations , causing gingival recession , cervical abrasion , and dentin hypersensitivity.3,4 currently , patients increasingly value esthetic and seek simplified and safe results for their oral problems.5 thus , the composite resins have been widely used in restorative treatments . however , it should be considered that the success and longevity of these restorations are related to the material , the dentist , and the patient.6 the patients are responsible to care for their dietary habits , preventive measures , availability of fluoride , and oral hygiene since carious lesions have been the most common cause of direct composite resin restorations replacements.7 studies have been conducted to evaluate the surface roughness of composite resins caused by the abrasivity of some dentifrices . amaral et al.8 evaluated the action of abrasive dentifrices on esthetic restorative materials after simulated toothbrushing cycles . the researchers found a significant difference between the abrasivity of dentifrices , but not among the composite resins . the dentifrices that used silica and carbonate were less abrasive compared to the ones containing bicarbonate . liljeborg et al.9 assessed the acrylic surface roughness caused by different values of relative dentin abrasivity ( rda ) , which is the abrasivity characteristic of dentifrices provided by the manufacturer . in this study , an association was analyzed between the value of rda , provided by the manufacturer , with the surface roughness ( ra ) found for each dentifrice after simulated toothbrushing . dentifrice with low rda ( 40 ) showed higher ra ( 5.73 m ) , while the other with high rda ( 130 ) obtained ra of 1.84 m ; a third dentifrice with the lowest value of rda ( 30 ) showed a ra value of 1.13 m . a large supply of new dentifrices has been available on the market nowadays , which makes the choice more difficult for patients . furthermore , the speed of production has surpassed independent studies without ties to manufacturers.10 concerned about the integrity of composite resin restorations from the abrasivity of dentifrices available , this study aims to answer the following questions : ( a ) does toothbrushing with dentifrice change the surface roughness of composite resins ? ( b ) is the surface roughness the same , regardless of the type of dentifrice or composite resin evaluated ? therefore , the aim of the study was to evaluate , in vitro , the surface roughness of two composite resins submitted to simulated toothbrushing with three different dentifrices in periods of 6 months , 1 year , and 2 years . the null hypothesis is that toothbrushing with dentifrices does not influence the surface roughness of composite resins . the composite resins empress direct ( ivoclar vivadent , schaan , liechtenstein ) and z350 xt ( 3m / espe , st . the composite resin specimens were made using a silicone matrix with orifices of 5 mm in diameter and height . a polyester strip was then placed on the composite resin followed by a glass plate in order to obtain a flat surface . the composite resin was then light - cured with the light - emitting diode light unit radii - cal ( sdi , australia ) for 20 s at a distance of 1 mm from the surface of the specimen . the composite resin surface in contact with the polyester strip received the toothbrushing with dentifrices . for each composite resin , 36 specimens where fabricated and stored in distilled water at 37c for 24 h to complete the polymerization and simulate conditions of the oral cavity environment . the 36 specimens of each composite resin were randomly divided into three groups ( n = 12 ) . each group was subject to simulated toothbrushing with three different dentifrices : oral - b pro - health whitening ( obw ) , colgate sensitive ( cs ) pro - relief , colgate total clean mint 12 ( ct12 ) . the initial surface roughness of each specimen was measured with a roughness tester sl-201 ( mitutoyo surftest analyzer , tokyo , japan ) . three consecutive measurements of the specimen were taken in different regions ( one central , one right , and one left ) , with cut - off of 0.25 , obtaining the mean average from the three measurements . the simulated toothbrushing machine developed by idea institute of pucrs was used for this study . each specimen was fixed in the center ( orifice ) of an acrylic plate ( 55 mm 25 mm 4 mm ) , respectively for the diameter and height , enabling the test surface to remain 1 mm beyond the edge of the orifice , which housed the specimen . each plate was placed in an acrylic tank , which was attached to the brushing machine by metal pins . the acrylic tank was filled with a mixture composed of 1 g of dentifrice paste per 1 ml of distilled water . soft bristle classic colgate ( colgate - palmolive , so bernardo do campo , sp , brazil ) toothbrushes were used , with a load of 200 g. the speed of brushing was 250 cycles per minute , carried out in 5,000 cycles , 10,000 cycles , and 20,000 cycles of simulated toothbrushing , cumulatively , that corresponds to approximately 6 months , 12 months , and 24 months of brushing,11 respectively . after every brushing cycle , the specimens were washed in running water and ultrasonically cleaned in distilled water for 10 min , followed by drying with compressed air . the surface roughness reading was perpendicular to the brushing direction of the toothbrush bristles . for the correct positioning of the specimen in the brushing machine and to always ensure readability in the same direction ( perpendicular to the brushing ) , a mark with a diamond bur and high - speed handpiece was made on the border of each specimen . from each of the composite resins , the 12 specimens were randomly divided into three groups ( n = 4 ) , each group being subjected to the simulated toothbrushing with dentifrice as described above . of the four samples , one was analyzed by sem before simulated toothbrushing , the second after 5,000 cycles , the third after 10,000 cycles , and the fourth after 20,000 cycles of simulated toothbrushing . the specimens were dried in dehumidifier with silica gel for 72 h , metallized with gold ( balzers , liechtenstein ) and observed under a scanning electron microscope ( jsm 6060 , eindhoven , netherlands ) at 4,000 magnification for qualitative analysis of the surface . three - way analysis of variance ( anova ) was used to test the effect of composite resin , dentifrice , and brushing time on the surface roughness , followed by tukey 's test at a significance level of 5% . the composite resins empress direct ( ivoclar vivadent , schaan , liechtenstein ) and z350 xt ( 3m / espe , st . the composite resin specimens were made using a silicone matrix with orifices of 5 mm in diameter and height . a polyester strip was then placed on the composite resin followed by a glass plate in order to obtain a flat surface . the composite resin was then light - cured with the light - emitting diode light unit radii - cal ( sdi , australia ) for 20 s at a distance of 1 mm from the surface of the specimen . the composite resin surface in contact with the polyester strip received the toothbrushing with dentifrices . for each composite resin , 36 specimens where fabricated and stored in distilled water at 37c for 24 h to complete the polymerization and simulate conditions of the oral cavity environment . the 36 specimens of each composite resin were randomly divided into three groups ( n = 12 ) . each group was subject to simulated toothbrushing with three different dentifrices : oral - b pro - health whitening ( obw ) , colgate sensitive ( cs ) pro - relief , colgate total clean mint 12 ( ct12 ) . the initial surface roughness of each specimen was measured with a roughness tester sl-201 ( mitutoyo surftest analyzer , tokyo , japan ) . three consecutive measurements of the specimen were taken in different regions ( one central , one right , and one left ) , with cut - off of 0.25 , obtaining the mean average from the three measurements . the simulated toothbrushing machine developed by idea institute of pucrs was used for this study . each specimen was fixed in the center ( orifice ) of an acrylic plate ( 55 mm 25 mm 4 mm ) , respectively for the diameter and height , enabling the test surface to remain 1 mm beyond the edge of the orifice , which housed the specimen . each plate was placed in an acrylic tank , which was attached to the brushing machine by metal pins . the acrylic tank was filled with a mixture composed of 1 g of dentifrice paste per 1 ml of distilled water . soft bristle classic colgate ( colgate - palmolive , so bernardo do campo , sp , brazil ) toothbrushes were used , with a load of 200 g. the speed of brushing was 250 cycles per minute , carried out in 5,000 cycles , 10,000 cycles , and 20,000 cycles of simulated toothbrushing , cumulatively , that corresponds to approximately 6 months , 12 months , and 24 months of brushing,11 respectively . after every brushing cycle , the specimens were washed in running water and ultrasonically cleaned in distilled water for 10 min , followed by drying with compressed air . the surface roughness reading was perpendicular to the brushing direction of the toothbrush bristles . for the correct positioning of the specimen in the brushing machine and to always ensure readability in the same direction ( perpendicular to the brushing ) , a mark with a diamond bur and high - speed handpiece from each of the composite resins , 12 additional specimens were obtained , as previously described . the 12 specimens were randomly divided into three groups ( n = 4 ) , each group being subjected to the simulated toothbrushing with dentifrice as described above . of the four samples , one was analyzed by sem before simulated toothbrushing , the second after 5,000 cycles , the third after 10,000 cycles , and the fourth after 20,000 cycles of simulated toothbrushing . the specimens were dried in dehumidifier with silica gel for 72 h , metallized with gold ( balzers , liechtenstein ) and observed under a scanning electron microscope ( jsm 6060 , eindhoven , netherlands ) at 4,000 magnification for qualitative analysis of the surface . the values of surface roughness were submitted to the kolmogorov - smirnov normality test . three - way analysis of variance ( anova ) was used to test the effect of composite resin , dentifrice , and brushing time on the surface roughness , followed by tukey 's test at a significance level of 5% . three - way anova analysis showed that the dentifrice ( p = 0.044 ) and the brushing time ( p = 0.000 ) had a significant effect on the surface roughness . the composite resin was not significant ( p = 0.381 ) and the interaction among the factors was not significant ( p > 0.05 ) . ct12 had the lowest surface roughness mean ( 0.269 m ) , which was not significantly different from cs ( 0.300 m ) . the highest surface roughness mean was obtained with obw ( 0.390 m ) , which was not significantly different from the cs ( table 3 ) . the highest surface roughness mean was obtained after 20,000 brushing cycles ( 0.584 m ) , differing statistically from the other cycles . the 5,000 cycles ( 0.297 m ) and 10,000 cycles ( 0.354 m ) had no statistical difference . the lowest surface roughness mean was obtained for the measurement performed at baseline ( 0.046 m ) , differing statistically from the other cycles ( table 4 ) . the sem images of figure 1a - d correspond to the z350 xt composite resin . there was substantial surface smoothness of the composite resin ( figure 1a ) before toothbrushing . after 5,000 cycles of brushing , dentifrice obw ( figure 1d ) caused significant surface roughness of the composite resin compared to dentifrice ct12 ( figure 1b ) and cs ( figure 1c ) . ( a - d ) surface morphology of composite resin z350 xt after 5,000 cycles of simulated toothbrushing ( 4,000 ) ( a ) 0 cycles ; ( b ) abrasion with colgate total 12 clean mint ; ( c ) abrasion with colgate sensitive pro - relief ; ( d ) abrasion with oral b pro - health whitening . the increasing number of brushing cycles caused a higher removal of the resin matrix and thus greater exposure of the filler particles . surface morphology of composite resin empress direct after simulated toothbrushing abrasion with colgate total 12 clean mint ( 4,000 ) ( a ) 0 cycle ; ( b ) 5,000 cycles ; ( c ) 10,000 cycles . the sem images of figure 1a - d correspond to the z350 xt composite resin . there was substantial surface smoothness of the composite resin ( figure 1a ) before toothbrushing . after 5,000 cycles of brushing , dentifrice obw ( figure 1d ) caused significant surface roughness of the composite resin compared to dentifrice ct12 ( figure 1b ) and cs ( figure 1c ) . ( a - d ) surface morphology of composite resin z350 xt after 5,000 cycles of simulated toothbrushing ( 4,000 ) ( a ) 0 cycles ; ( b ) abrasion with colgate total 12 clean mint ; ( c ) abrasion with colgate sensitive pro - relief ; ( d ) abrasion with oral b pro - health whitening . the increasing number of brushing cycles caused a higher removal of the resin matrix and thus greater exposure of the filler particles . surface morphology of composite resin empress direct after simulated toothbrushing abrasion with colgate total 12 clean mint ( 4,000 ) ( a ) 0 cycle ; ( b ) 5,000 cycles ; ( c ) 10,000 cycles . the clinical wear of a restoration may result from numerous factors , such as functional and centric contacts , attrition of the food bolus , and areas of interproximal contact . the toothbrush abrasion , however , has been an important phenomenon in regards to wear of composite resins , which is most commonly seen on the buccal surface.12 hence , the marketing of various dentifrices , the abrasivity of three different dentifrices was evaluated in this study : ct12 , containing silica ; cs pro - relief , composed of calcium carbonate and bicarbonate ; obw , based on silica and titanium dioxide . the dentifrice factor was significant and directly influenced the surface roughness results , rejecting the null hypothesis . this finding concurs with other studies that found that the action of toothbrushing associated with the use of abrasive dentifrices has been responsible for an increase on surface roughness of composite resins.8,13 - 18 the lowest surface roughness mean was obtained with the dentifrice ct12 ( 0.269 m ) , confirming barbieri et al.18 study that the same dentifrice was responsible for a low abrasion of composite resins analyzed . the ct12 contains silica as an abrasive agent that exhibits low abrasivity.1 amaral et al.8 study concluded that dentifrices based on silica or calcium carbonate are less abrasive than those containing sodium bicarbonate . this possibly justifies the fact that the dentifrice cs , which has bicarbonate in the composition , caused a higher surface roughness compared to ct12 , although no statistical difference was found . the dentifrice obw showed the highest ra ( 0.390 m ) mean , different from ct12 . this result corroborates the findings of amaral et al.8 and barbieri et al.18 , in which the whitening dentifrices also results in higher surface roughness both containing silica abrasives in combination with other agents . the study of menezes et al.4 found a significant difference in ra caused by whitening dentifrices on bovine root dentin , stating that the change of ra depends on the dentifrice used . according to cury et al.1 there are dentifrices with low , medium , and high abrasivity . among the lower abrasivity are the dentifrices in the form of gel , containing silica as the abrasive agent . however , when this mineral is combined with other abrasives such as calcium carbonate , sodium pyrophosphate , titanium oxide , and sodium phosphate , it is considered a high abrasive dentifrice . this finding is consistent with the results of this study , in which the dentifrice obw , responsible for a statistically significant abrasivity , has silica as an abrasive agent in combination with titanium dioxide , classifying it as a highly abrasive dentifrice.1 according to gusmo et al.,19 the main cause factor of abrasivity is not limited to the type nor quantity of abrasive present in the dentifrice , but the physical characteristics of the mineral as the size and the shape of the particles . silica , for example , when used in fine particles with regular shapes , preserves its mild abrasivity mineral characteristic , but when coarse and irregular particles are incorporated , it is highly abrasive . thus , only the gel or cream formula , or the type of the abrasive agent present in the dentifrice has not been sufficient to characterize its abrasivity to the tooth structures and composite resins . the simulated toothbrushing abrasion is considered an established model in the literature , because it is an important in - vitro wear factor , able to simulate a clinical condition . according sexson and phillips,20 for each session of brushing thus , maintaining an oral hygiene based on two daily sessions of brushing , 10,000 cycles are performed by the end of 1 year . in the present study , the highest surface roughness mean was obtained after 20,000 cycles , the equivalent of 24 months of brushing ( 0.584 m ) , differing statistically from 5,000 cycles , equivalent to 6 months of brushing ( 0.297 m ) and 10,000 cycles , equivalent to 12 months of brushing ( 0.354 m ) . this finding concurs with other studies.13,15,16,18 in the present study , a nanohybrid composite resin ( empress direct ) and a nanocomposite resin ( z350 xt ) were used , and there was no statistical difference in surface roughness among the composite resins . senawongse and pongprueksa,14 after evaluating the surface roughness of micro - hybrid , nanohybrid , and nanocomposite resins , also found no significant difference among the materials , although the nanocomposite resin demonstrated fewer irregularities . on the other hand , suzuki et al.,15 after evaluating the surface roughness of nanohybrid and nanocomposite resins in 50,000 cycles of simulated toothbrushing , concluded that there was significant difference in rates of roughness of composite resins . the nanohybrid composite resins had higher ra , and nanocomposite resins showed better results , especially filtek supreme xt , which was considered a clinically successful composite resin . this finding is not equivalent with the present study , in which both composites ( empress direct and z350 xt ) performed similarly to a performance evaluation of 24 months ( 20,000 cycles ) . this difference in results among studies is probably related to the amount of toothbrushing cycles performed on each study . according to the manufacturer , the composite resin z350 xt consists of a combination of zirconia and silica nanoparticles in nanoclusters reducing the interstitial space between the particles and increasing the amount of load . the resin matrix with nanoparticles results in better physical properties and greater wear resistance , which means that during the abrasion these nanoclusters wear at a speed similar to the surrounding resin matrix . the result is a smooth , glossy , and durable polished surface . through the analysis of the sem images , it was found that both composite resins had higher surface irregularities , higher resin matrix wear , and exposure of the filler particles when brushed with the dentifrice obw . these wear patterns are commonly found in evaluations by sem after abrasive test.13 - 16,18 regarding the clinical significance of this study , it is evident that the surface roughness of composite resins changes according to the type of dentifrice used and the time of brushing , and the whitening dentifrice obw had greater capacity to change the surface of composite resins . within the limitations of the methodology used , it was possible to conclude that : the composite resins z350 xt and empress direct showed similar surface roughness after all cycles of simulated toothbrushingthe higher the brushing time , the higher the surface roughness of composite resinsthe dentifrice obw caused a higher surface roughness in both composite resins . the composite resins z350 xt and empress direct showed similar surface roughness after all cycles of simulated toothbrushing the higher the brushing time , the higher the surface roughness of composite resins the dentifrice obw caused a higher surface roughness in both composite resins .

background : the aim of the study was to evaluate , in vitro , the surface roughness of two composite resins submitted to simulated toothbrushing with three different dentifrices.materials and methods : totally , 36 samples of z350xt and 36 samples of empress direct were built and randomly divided into three groups ( n = 12 ) according to the dentifrice used ( oral - b pro - health whitening [ obw ] , colgate sensitive pro - relief [ cs ] , colgate total clean mint 12 [ ct12 ] ) . the samples were submitted to 5,000 , 10,000 or 20,000 cycles of simulated toothbrushing . after each simulated period , the surface roughness of the samples was measured using a roughness tester.results:according to three - way analysis of variance , dentifrice ( p = 0.044 ) and brushing time ( p = 0.000 ) were significant . the composite resin was not significant ( p = 0.381 ) and the interaction among the factors was not significant ( p > 0.05 ) . the mean values of the surface roughness ( m ) followed by the same letter represent no statistical difference by tukey 's post - hoc test ( p < 0.05 ) : dentifrice : ct12 = 0.269a ; cs pro- relief = 0.300ab ; obw = 0.390b . brushing time : baseline = 0,046 ; 5,000 cycles = 0.297b ; 10,000 cycles = 0.354b ; 20,000 cycles = 0.584c.conclusion:z350 xt and empress direct presented similar surface roughness after all cycles of simulated toothbrushing . the higher the brushing time , the higher the surface roughness of composite resins . the dentifrice obw caused a higher surface roughness in both composite resins .

Introduction Materials and Methods Selection of composite resin specimens Experimental groups Analysis of surface roughness Simulated toothbrushing Analysis by scanning electron microscopy (SEM) Statistical analysis Results Analysis by SEM Discussion Conclusions

PMC3963413

proprioceptors consist of position ( static ) and movement ( kinesthetic ) receptors . in insects and crustaceans , chordotonal organs are the structures that provide that information to the cns . not all chordotonal organs span a joint but they can still monitor joint movements due to their attachment on the apodemes ( tendon like structures ) which span the joint and move in association with the skeletal muscle and joint articulation . typically a chordotonal organ has 60 - 100 or more sensory neurons embedded within an elastic strand , neurons that signal static joint position , direction and speed of movement . the input from chordotonal organs at each joint and leg is then centrally processed allowing coordinated movements by the animal . the forces that leg muscles produce during isometric and isotonic contractions are detected by tension receptors associated with muscle fibers and their attachments to apodemes7 - 9 . in the crustacean walking leg protocols that follow we present methodology for recordings from primary sensory neurons that monitor proprioception and the neurons that respond to forces generated by muscle fibers . a technique for activating leg movements and quantifying force generation is also presented , as well as anatomical techniques that can be used to characterize the arrangement of these peripheral nervous system structures . the procedures demonstrated below enable structural and functional analysis of the neurons that innervate both types of receptors relative to their location on a chordotonal elastic strand and apodeme . to illustrate , we use the propodite - dactylopodite ( pd ) chordotonal organ , the organ that spans the distal most segment of the crab leg . though detailed electrophysiological studies began in the 1930s and are still being carried out today , some aspects have become known about the segmental connections of proprioceptors in the various joints and their roles in coordinated control of muscles10 - 16 . establishing the structure - function relationship between the proprioceptive organs , muscles and the nervous system will further help define these roles . for instance , labeling the somata and distal endings of tension neurons inserted into the apodeme will reveal their location relative to muscle fibers . we present three staining techniques for crustacean legs that can be used in research or academic laboratories . methylene blue staining provides suitable contrast for muscles and nerves and is recommended as a simple technique for students to learn anatomy . labs that have fluorescence microscopy setups can accomplish more selective neuronal staining by briefly exposing the nerves to the vital dye 4-di-2-asp . the third alternative is cocl2 backfill , which stains and fixes the neurons , and does not require fluorescence imaging . though it is labor and time intensive , this staining process gives high contrast and specificity for the nerves that are filled . together these techniques can be used for comparing various chordotonal organs , not only within a limb or between limbs , but also among other crustacean and insect species . blue crabs ( c. sapidus ) used in physiological recordings and for anatomical staining are readily available all around the southern and southeastern border of the united states . this species serves as a representative of the chordotonal and tension nerve arrangements found in most crabs . laboratories on the west coast will prefer to use the much larger dungeness crab ( cancer magister ) for these experiments . hold the crab across the carapace from behind , and avoiding the claws , pinch the proximal part of the meropodite with forceps . use caution when handling blue crabs as they are rather aggressive and very fast . the anatomical location of chordotonal organs ( hatched regions ) are superimposed on this schematic . the double arrow head indicates where to transect the leg for the pd nerve experiments . cut a large window in the cuticle on the pigmented ( lateral ) side of the propodite with a scalpel with a # 11 blade ( figure 2 and 3 ) . remove the cuticle layer by sliding the scalpel blade beneath and parallel to the cuticle . this severs the muscle fibers attached to the cuticle . using the same technique cut a smaller window on the pigmentless ( medial ) side of the propodite , but leave the condyle ( the socket joint or hinge between segments ) attachment intact . remove muscle fibers that obscure your view of the organ from both sides of the tendon . once this has been accomplished , firmly reattach the preparation to the dish with the pigment side ( lateral ) facing up ( figure 4 ) . follow the pd organ nerve in the propodite as far proximally as possible in order to free - up a long length of nerve ( 1.5 cm ) for recording purposes . this is best done while the pd nerve is still attached to the main leg nerve . after separating the pd nerve from the main leg nerve with the aid of glass needles , take note about where the extreme flexion and extension positions are and a half - way point for later use . once the prep dish is placed on the stage , you will need to adjust the position of the high intensity illuminator beam to best visualize the preparation . position the micromanipulator so the attached suction electrode assembly will have easy access to the saline bath and preparation . the suction electrode is constructed as shown in an online video . to detect neural activity , draw the cut end of the pd nerve into the suction electrode . move the dactyl throughout extended and flexed positions for several cycles with the aid of a glass probe or wooden dowel . next observe activity when the dactyl is pinned in the extended , flexed , and mid positions . the medial side has a soft texture that can be felt by pinching gently in the meropodite region with a fingernail . the excitor motor nerve that innervates the opener muscle also innervates the stretcher muscle in the carpopodite . in order to stimulate the opener muscle the stretcher motor nerve in the carpodite region is isolated and stimulated with a suction electrode . remove a section of the cuticle in the carpus region on the inner side ( medial side , figure 5a ) . cut the apodeme of the bender muscle and remove the muscle carefully as not to pull the main leg nerve out of the leg cavity ( figure 5b , c note the arrows where to bender apodeme is separated ) . the main leg nerve and a branch to the stretcher muscle find the nerve branching from the main nerve bundle to the stretcher muscle ( figure 5e , at arrow ) . this can be cut close to the muscle and pulled into a suction electrode to stimulate ( figures 5f and g , arrow depicts the branch ) . tease out a section of the nerve that projects towards the opener without pinching the nerve . pull the motor nerve into the suction electrode and then stimulate it . to expose the opener muscle and tension nerve remove the closer muscle and the ventral region of the propodite . cut off the closer muscle with a scalpel with a # 11 blade in the propodite ( figure 6 ) . note : do not cut deeply in the proximal region because it might damage the opener motor nerve . figure 6 . cut the cuticle on the ventral half of the propodite to expose the closer muscle so it can be removed . exchange the saline with fresh cooled saline throughout the dissection process to keep the neurons alive . for further dissection , place the preparation dish under a dissecting microscope and use fiber - optic illumination . carefully cut the closer tendon from its attachment to the dactyl using sharp - pointed medium - size scissors . be very careful not to disturb the branches to the opener muscle from the main leg nerve which should be clearly visible . remove and discard the closer muscle and tendon . this hole will be used later to hook the metal pin on the tension ( force ) transducer , but it is necessary to make it at this stage of the procedure . locate the nerve branch that projects to the opener muscle and apodeme in the distal region of the opener muscle . observe the tension nerve that arises from the distal end of the apodeme and proceeds to the motor nerve bundle . to detect neural activity , fill a suction electrode with crab saline and examine for the neural correlate of passive tension on the opener apodeme . while recording electrical activity from the tension nerve , rotate the dactyl rapidly into an extended joint ( i.e. stretching the opener muscle ) . firmly attach a metal hook so that the tip of the hook goes through a small hole in the dactyl . note : make sure the transducer is at a 90 angle each time so that the pin is perpendicular to the transducer for maximal detection of the force generated . pull the hook and dactyl so that it is at about a 45 angle of the opener joint . now stimulate the motor nerve at 100 hz for 250 msec and measure the force as well as the firing frequency of the tension nerve . place the joint into a fully extended position so that the opener muscle fibers are flaccid . stimulate the motor nerve at 100 hz for 250 msec and measure the force as well as the firing frequency of the tension nerve . there may be some force measured by the transducer due to this passive stretch of the muscle . stimulate the motor nerve at 100 hz for 250 msec and measure the force as well as the firing frequency of the tension nerve . after measuring a force , proceed in a series of various frequencies : 20 , 40 , 60 , 80 , and 100 hz for 8 - 10 sec in each joint position . arrange the preparation so that the hook on the force transducer can easily be pushed out of the hole in the dactyl . now stimulate the motor nerve at 100 hz with a continuous stimulation of 5 sec . after the first second or less , as tension has built up on the opener muscle , push the pin out of the hole in the dactyl . examine the tension nerve recording before and after release of the pin holding the dactyl . modulatory effects of neuroactive substances such as octopamine , serotonin , and proctolin , which alter the output of the tension neurons , can be determined by adding these molecules to the bathing media over the exposed opener muscle and repeating the experiments . use a pipette and drip 1 - 2 ml over the preparation . methylene blue technique dilute one part methylene blue chloride stock solution ( 0.25% ) with two parts of distilled water.add this mixture to five parts of buffered saline . thoroughly irrigate the area of interest.dissect a leg according to the protocol in section 1 . incubate the preparation in the methylene blue solution at 12 - 13 c.examine the preparation every 10 - 15 min with the dissecting microscope using low intensity illumination to follow the progress of the staining . in some cases staining will be completed in an hour , in others it may take overnight . dilute one part methylene blue chloride stock solution ( 0.25% ) with two parts of distilled water . examine the preparation every 10 - 15 min with the dissecting microscope using low intensity illumination to follow the progress of the staining . in some cases staining will be completed in an hour , in others it may take overnight . 4-di-2-asp technique fluorescent dyes can be used to back - fill the pd neuron or directly stain the preparation from the bath . however a suitable microscope with abilities to view the fluorescent stain is required.dissect a leg according to a protocol in section 1.incubate the preparation in a 10 m concentration of 4-di-2-asp solution and leave the preparation in the refrigerator for 15 min . use just enough solution to cover the preparation.photograph the preparations quickly and avoid over exposure to the mercury light . fluorescent dyes can be used to back - fill the pd neuron or directly stain the preparation from the bath . dissect a leg according to a protocol in section 1 . incubate the preparation in a 10 m concentration of 4-di-2-asp solution and use just enough solution to cover the preparation . photograph the preparations quickly and avoid over exposure to the mercury light . cobalt chloride technique expose the pd nerve according to the protocol in section 1 and keep it immersed in cold saline.make a petroleum jelly well to hold the cocl2 . if any cocl2 spills into the saline bath the entire preparation will stain black , and the preparation should be discarded.slip a small cut of a polystyrene sheet , to make a plastic platform , and pin it in such a manner that it will not float away or become immersed in the saline bath ( figure 7 ) . figure 7 . click here to view larger image.eject petroleum jelly from a fine hypodermic needle fastened to a disposable syringe , then make a barrier ( a circle might work well ) , which should be about 1 - 1.5 mm high except for a shallow " v " at midpoint where the nerve will be draped across , on top of the slip of polystyrene.make a small puddle of saline on either side of the barrier near the " v " . taking care not to stretch or pinch the nerve , lift the nerve carefully from the dish and place it in the saline puddle in the petroleum jelly well.work quickly so that the section of nerve does not dry out , carefully eject petroleum jelly to cover the exposed nerve . now test the barrier with saline and make sure it is not leaking.blot away saline on the inside of the barrier and see if it fills up when the bath saline is high around the wall of petroleum jelly to be sure that the two sides are isolated from one another ( figure 8) . click here to view larger image.wick away the saline in the well using a piece of tissue paper . avoid letting the paper wrap up the nerve.make a new puddle using a few small drops of distilled water , and then cut the nerve end . be very careful not to pull the nerve through the barrier when making the cut . the osmotic shock of the distilled water will " balloon " the axons.within 30 sec add a small drop of cocl2 to the water , soak up this solution , and then add enough cocl2 to form a puddle over this shortened section of nerve ( figure 9 ) . preparations are best kept refrigerated at 13 c for 12 - 24 hr . the cut nerve swells up in the presence of water that facilitates and accelerates the movement of dye molecules through the axons into the neuron cell bodies . blot away the cobalt solution using a tissue and wash away the remnants of cobalt with several changes of saline.transfer the isolated preparation to a small glass petri dish containing about 10 ml of crab saline . good metal tools are not to be used to handle the preparation after this step ( you should use specific tools that are not to be used at a later time for physiology).add 1 - 2 drops of ammonium sulfide ( nh4)2s to the saline . cap the ( nh4)2s bottle tightly and place back into the hood . observe the reaction in the preparation under a dissecting microscopewithin 1 - 2 min , cobalt - filled neurons and their processes should begin to appear because they will stain black.after 5 - 10 min , replace the development solution with fresh saline.make certain that the development solution you have poured into the sink drain is followed by running tap water for a few minutes or in a waste bottle with a lid.pour out the saline and fix the nerve preparation for about 15 min with two changes of bouin s solution fixative . for larger tissues like the opener muscle ( to stain tension neurons ) , increase the duration of fixation to 30 min.dehydrate in a series of ascending order of ethanol concentration beginning at 70% ( i.e. 70% , 80% , 90% , 100% ) . about 10 min at each concentration is sufficient for small tissues.after about 10 - 15 min in two changes of 100% ethanol , clear the tissue by replacing ethanol with 100% methyl salicylate . the preparation will stay in this solution permanently for repeated viewing . with time the filled cells will become more apparent because the surrounding tissue will become clearer . intensification methods can be used to help prevent fading over time.use the same process to fill the tension nerve with cocl2 . however , change ethanol solution multiple times in each ethanol dehydration step and incubate the preparation inside alcohol for about 20 min for each step to thoroughly dehydrate the muscles and allow them to clear well . expose the pd nerve according to the protocol in section 1 and keep it immersed in cold saline . make a petroleum jelly well to hold the cocl2 . if any cocl2 spills into the saline bath the entire preparation will stain black , and the preparation should be discarded . slip a small cut of a polystyrene sheet , to make a plastic platform , and pin it in such a manner that it will not float away or become immersed in the saline bath ( figure 7 ) . eject petroleum jelly from a fine hypodermic needle fastened to a disposable syringe , then make a barrier ( a circle might work well ) , which should be about 1 - 1.5 mm high except for a shallow " v " at midpoint where the nerve will be draped across , on top of the slip of polystyrene . make a small puddle of saline on either side of the barrier near the " v " . taking care not to stretch or pinch the nerve , lift the nerve carefully from the dish and place it in the saline puddle in the petroleum jelly well . work quickly so that the section of nerve does not dry out , carefully eject petroleum jelly to cover the exposed nerve . now test the barrier with saline and make sure it is not leaking . blot away saline on the inside of the barrier and see if it fills up when the bath saline is high around the wall of petroleum jelly to be sure that the two sides are isolated from one another ( figure 8) . make a new puddle using a few small drops of distilled water , and then cut the nerve end . . the osmotic shock of the distilled water will " balloon " the axons . within 30 sec add a small drop of cocl2 to the water , soak up this solution , and then add enough cocl2 to form a puddle over this shortened section of nerve ( figure 9 ) . preparations are best kept refrigerated at 13 c for 12 - 24 hr . the cut nerve swells up in the presence of water that facilitates and accelerates the movement of dye molecules through the axons into the neuron cell bodies . blot away the cobalt solution using a tissue and wash away the remnants of cobalt with several changes of saline . transfer the isolated preparation to a small glass petri dish containing about 10 ml of crab saline . good metal tools are not to be used to handle the preparation after this step ( you should use specific tools that are not to be used at a later time for physiology ) . add 1 - 2 drops of ammonium sulfide ( nh4)2s to the saline . observe the reaction in the preparation under a dissecting microscope within 1 - 2 min , cobalt - filled neurons and their processes should begin to appear because they will stain black . after 5 - 10 min , replace the development solution with fresh saline . make certain that the development solution you have poured into the sink drain is followed by running tap water for a few minutes or in a waste bottle with a lid . pour out the saline and fix the nerve preparation for about 15 min with two changes of bouin s solution fixative . for larger tissues like the opener muscle ( to stain tension neurons ) , dehydrate in a series of ascending order of ethanol concentration beginning at 70% ( i.e. 70% , 80% , 90% , 100% ) . after about 10 - 15 min in two changes of 100% ethanol , clear the tissue by replacing ethanol with 100% methyl salicylate . the preparation will stay in this solution permanently for repeated viewing . with time intensification methods can be used to help prevent fading over time . use the same process to fill the tension nerve with cocl2 . however , change ethanol solution multiple times in each ethanol dehydration step and incubate the preparation inside alcohol for about 20 min for each step to thoroughly dehydrate the muscles and allow them to clear well . hold the crab across the carapace from behind , and avoiding the claws , pinch the proximal part of the meropodite with forceps . use caution when handling blue crabs as they are rather aggressive and very fast . the anatomical location of chordotonal organs ( hatched regions ) are superimposed on this schematic . the double arrow head indicates where to transect the leg for the pd nerve experiments . cut a large window in the cuticle on the pigmented ( lateral ) side of the propodite with a scalpel with a # 11 blade ( figure 2 and 3 ) . remove the cuticle layer by sliding the scalpel blade beneath and parallel to the cuticle . this severs the muscle fibers attached to the cuticle . using the same technique cut a smaller window on the pigmentless ( medial ) side of the propodite , but leave the condyle ( the socket joint or hinge between segments ) attachment intact . remove muscle fibers that obscure your view of the organ from both sides of the tendon . once this has been accomplished , firmly reattach the preparation to the dish with the pigment side ( lateral ) facing up ( figure 4 ) . follow the pd organ nerve in the propodite as far proximally as possible in order to free - up a long length of nerve ( 1.5 cm ) for recording purposes . this is best done while the pd nerve is still attached to the main leg nerve . after separating the pd nerve from the main leg nerve with the aid of glass needles , take note about where the extreme flexion and extension positions are and a half - way point for later use . once the prep dish is placed on the stage , you will need to adjust the position of the high intensity illuminator beam to best visualize the preparation . position the micromanipulator so the attached suction electrode assembly will have easy access to the saline bath and preparation . the suction electrode is constructed as shown in an online video . to detect neural activity , draw the cut end of the pd nerve into the suction electrode . move the dactyl throughout extended and flexed positions for several cycles with the aid of a glass probe or wooden dowel . next observe activity when the dactyl is pinned in the extended , flexed , and mid positions . the medial side has a soft texture that can be felt by pinching gently in the meropodite region with a fingernail . the excitor motor nerve that innervates the opener muscle also innervates the stretcher muscle in the carpopodite . in order to stimulate the opener muscle the stretcher motor nerve in the carpodite region is isolated and stimulated with a suction electrode . remove a section of the cuticle in the carpus region on the inner side ( medial side , figure 5a ) . cut the apodeme of the bender muscle and remove the muscle carefully as not to pull the main leg nerve out of the leg cavity ( figure 5b , c note the arrows where to bender apodeme is separated ) . the main leg nerve and a branch to the stretcher muscle find the nerve branching from the main nerve bundle to the stretcher muscle ( figure 5e , at arrow ) . this can be cut close to the muscle and pulled into a suction electrode to stimulate ( figures 5f and g , arrow depicts the branch ) . tease out a section of the nerve that projects towards the opener without pinching the nerve . pull the motor nerve into the suction electrode and then stimulate it . to expose the opener muscle and tension nerve remove the closer muscle and the ventral region of the propodite . cut off the closer muscle with a scalpel with a # 11 blade in the propodite ( figure 6 ) . note : do not cut deeply in the proximal region because it might damage the opener motor nerve . figure 6 . cut the cuticle on the ventral half of the propodite to expose the closer muscle so it can be removed . exchange the saline with fresh cooled saline throughout the dissection process to keep the neurons alive . for further dissection , carefully cut the closer tendon from its attachment to the dactyl using sharp - pointed medium - size scissors . be very careful not to disturb the branches to the opener muscle from the main leg nerve which should be clearly visible . remove and discard the closer muscle and tendon . this hole will be used later to hook the metal pin on the tension ( force ) transducer , but it is necessary to make it at this stage of the procedure . locate the nerve branch that projects to the opener muscle and apodeme in the distal region of the opener muscle . observe the tension nerve that arises from the distal end of the apodeme and proceeds to the motor nerve bundle . to detect neural activity , fill a suction electrode with crab saline and draw the cut end of the opener tension nerve into the electrode . examine for the neural correlate of passive tension on the opener apodeme . while recording electrical activity from the tension nerve , rotate the dactyl rapidly into an extended joint ( i.e. stretching the opener muscle ) . firmly attach a metal hook so that the tip of the hook goes through a small hole in the dactyl . note : make sure the transducer is at a 90 angle each time so that the pin is perpendicular to the transducer for maximal detection of the force generated . pull the hook and dactyl so that it is at about a 45 angle of the opener joint . now stimulate the motor nerve at 100 hz for 250 msec and measure the force as well as the firing frequency of the tension nerve . place the joint into a fully extended position so that the opener muscle fibers are flaccid . stimulate the motor nerve at 100 hz for 250 msec and measure the force as well as the firing frequency of the tension nerve . bend / flex the joint so that it is fully flexed ( ~90 ) . in this position . there may be some force measured by the transducer due to this passive stretch of the muscle . stimulate the motor nerve at 100 hz for 250 msec and measure the force as well as the firing frequency of the tension nerve . after measuring a force , proceed in a series of various frequencies : 20 , 40 , 60 , 80 , and 100 hz for 8 - 10 sec in each joint position . arrange the preparation so that the hook on the force transducer can easily be pushed out of the hole in the dactyl . now stimulate the motor nerve at 100 hz with a continuous stimulation of 5 sec . after the first second or less , as tension has built up on the opener muscle , push the pin out of the hole in the dactyl . examine the tension nerve recording before and after release of the pin holding the dactyl . modulatory effects of neuroactive substances such as octopamine , serotonin , and proctolin , which alter the output of the tension neurons , can be determined by adding these molecules to the bathing media over the exposed opener muscle and repeating the experiments . methylene blue technique dilute one part methylene blue chloride stock solution ( 0.25% ) with two parts of distilled water.add this mixture to five parts of buffered saline . thoroughly irrigate the area of interest.dissect a leg according to the protocol in section 1 . incubate the preparation in the methylene blue solution at 12 - 13 c.examine the preparation every 10 - 15 min with the dissecting microscope using low intensity illumination to follow the progress of the staining . in some cases staining will be completed in an hour , in others it may take overnight . dilute one part methylene blue chloride stock solution ( 0.25% ) with two parts of distilled water . thoroughly irrigate the area of interest . dissect a leg according to the protocol in section 1 . examine the preparation every 10 - 15 min with the dissecting microscope using low intensity illumination to follow the progress of the staining . in some cases staining will be completed in an hour , in others it may take overnight . 4-di-2-asp technique fluorescent dyes can be used to back - fill the pd neuron or directly stain the preparation from the bath . however a suitable microscope with abilities to view the fluorescent stain is required.dissect a leg according to a protocol in section 1.incubate the preparation in a 10 m concentration of 4-di-2-asp solution and leave the preparation in the refrigerator for 15 min . use just enough solution to cover the preparation.photograph the preparations quickly and avoid over exposure to the mercury light . fluorescent dyes can be used to back - fill the pd neuron or directly stain the preparation from the bath . incubate the preparation in a 10 m concentration of 4-di-2-asp solution and leave the preparation in the refrigerator for 15 min . cobalt chloride technique expose the pd nerve according to the protocol in section 1 and keep it immersed in cold saline.make a petroleum jelly well to hold the cocl2 . if any cocl2 spills into the saline bath the entire preparation will stain black , and the preparation should be discarded.slip a small cut of a polystyrene sheet , to make a plastic platform , and pin it in such a manner that it will not float away or become immersed in the saline bath ( figure 7 ) . click here to view larger image.eject petroleum jelly from a fine hypodermic needle fastened to a disposable syringe , then make a barrier ( a circle might work well ) , which should be about 1 - 1.5 mm high except for a shallow " v " at midpoint where the nerve will be draped across , on top of the slip of polystyrene.make a small puddle of saline on either side of the barrier near the " v " . taking care not to stretch or pinch the nerve , lift the nerve carefully from the dish and place it in the saline puddle in the petroleum jelly well.work quickly so that the section of nerve does not dry out , carefully eject petroleum jelly to cover the exposed nerve . now test the barrier with saline and make sure it is not leaking.blot away saline on the inside of the barrier and see if it fills up when the bath saline is high around the wall of petroleum jelly to be sure that the two sides are isolated from one another ( figure 8) . click here to view larger image.wick away the saline in the well using a piece of tissue paper . avoid letting the paper wrap up the nerve.make a new puddle using a few small drops of distilled water , and then cut the nerve end . be very careful not to pull the nerve through the barrier when making the cut . the osmotic shock of the distilled water will " balloon " the axons.within 30 sec add a small drop of cocl2 to the water , soak up this solution , and then add enough cocl2 to form a puddle over this shortened section of nerve ( figure 9 ) . preparations are best kept refrigerated at 13 c for 12 - 24 hr . the cut nerve swells up in the presence of water that facilitates and accelerates the movement of dye molecules through the axons into the neuron cell bodies . blot away the cobalt solution using a tissue and wash away the remnants of cobalt with several changes of saline.transfer the isolated preparation to a small glass petri dish containing about 10 ml of crab saline . good metal tools are not to be used to handle the preparation after this step ( you should use specific tools that are not to be used at a later time for physiology).add 1 - 2 drops of ammonium sulfide ( nh4)2s to the saline . cap the ( nh4)2s bottle tightly and place back into the hood . observe the reaction in the preparation under a dissecting microscopewithin 1 - 2 min , cobalt - filled neurons and their processes should begin to appear because they will stain black.after 5 - 10 min , replace the development solution with fresh saline.make certain that the development solution you have poured into the sink drain is followed by running tap water for a few minutes or in a waste bottle with a lid.pour out the saline and fix the nerve preparation for about 15 min with two changes of bouin s solution fixative . for larger tissues like the opener muscle ( to stain tension neurons ) , increase the duration of fixation to 30 min.dehydrate in a series of ascending order of ethanol concentration beginning at 70% ( i.e. 70% , 80% , 90% , 100% ) . about 10 min at each concentration is sufficient for small tissues.after about 10 - 15 min in two changes of 100% ethanol , clear the tissue by replacing ethanol with 100% methyl salicylate . the preparation will stay in this solution permanently for repeated viewing . with time the filled cells will become more apparent because the surrounding tissue will become clearer . intensification methods can be used to help prevent fading over time.use the same process to fill the tension nerve with cocl2 . however , change ethanol solution multiple times in each ethanol dehydration step and incubate the preparation inside alcohol for about 20 min for each step to thoroughly dehydrate the muscles and allow them to clear well . expose the pd nerve according to the protocol in section 1 and keep it immersed in cold saline . make a petroleum jelly well to hold the cocl2 . if any cocl2 spills into the saline bath the entire preparation will stain black , and the preparation should be discarded . slip a small cut of a polystyrene sheet , to make a plastic platform , and pin it in such a manner that it will not float away or become immersed in the saline bath ( figure 7 ) . eject petroleum jelly from a fine hypodermic needle fastened to a disposable syringe , then make a barrier ( a circle might work well ) , which should be about 1 - 1.5 mm high except for a shallow " v " at midpoint where the nerve will be draped across , on top of the slip of polystyrene . make a small puddle of saline on either side of the barrier near the " v " . taking care not to stretch or pinch the nerve , lift the nerve carefully from the dish and place it in the saline puddle in the petroleum jelly well . work quickly so that the section of nerve does not dry out , carefully eject petroleum jelly to cover the exposed nerve . blot away saline on the inside of the barrier and see if it fills up when the bath saline is high around the wall of petroleum jelly to be sure that the two sides are isolated from one another ( figure 8) . make a new puddle using a few small drops of distilled water , and then cut the nerve end . be very careful not to pull the nerve through the barrier when making the cut . the osmotic shock of the distilled water will " balloon " the axons . within 30 sec add a small drop of cocl2 to the water , soak up this solution , and then add enough cocl2 to form a puddle over this shortened section of nerve ( figure 9 ) . preparations are best kept refrigerated at 13 c for 12 - 24 hr . the cut nerve swells up in the presence of water that facilitates and accelerates the movement of dye molecules through the axons into the neuron cell bodies . blot away the cobalt solution using a tissue and wash away the remnants of cobalt with several changes of saline . transfer the isolated preparation to a small glass petri dish containing about 10 ml of crab saline . good metal tools are not to be used to handle the preparation after this step ( you should use specific tools that are not to be used at a later time for physiology ) . add 1 - 2 drops of ammonium sulfide ( nh4)2s to the saline . observe the reaction in the preparation under a dissecting microscope within 1 - 2 min , cobalt - filled neurons and their processes should begin to appear because they will stain black . after 5 - 10 min , make certain that the development solution you have poured into the sink drain is followed by running tap water for a few minutes or in a waste bottle with a lid . pour out the saline and fix the nerve preparation for about 15 min with two changes of bouin s solution fixative . for larger tissues like the opener muscle ( to stain tension neurons ) , dehydrate in a series of ascending order of ethanol concentration beginning at 70% ( i.e. 70% , 80% , 90% , 100% ) . after about 10 - 15 min in two changes of 100% ethanol , clear the tissue by replacing ethanol with 100% methyl salicylate . the preparation will stay in this solution permanently for repeated viewing . with time intensification methods can be used to help prevent fading over time . use the same process to fill the tension nerve with cocl2 . however , change ethanol solution multiple times in each ethanol dehydration step and incubate the preparation inside alcohol for about 20 min for each step to thoroughly dehydrate the muscles and allow them to clear well . when the pd organ is stretched by fully extending the joint , activity in the pd nerve is robust during the movement as shown for the first second in figure 10 . this activity is from the static position sensitive neurons ( second half of the recording shown in figure 10 ) . movement evokes a response during displacement , and the firing is mostly present during the stretching of the chordotonal strand ( figure 11 ) . this is an approach to demonstrate different populations of sensory neurons being recruited for positions or types of movements . typical amplitudes range from 0.25 - 1.5 mv , but these values are dependent on the resistance ( i.e. tightness ) of the suction electrode seal . frequency of spikes in the various size ranges can also be graphically represented for analysis . forces generated by the opener muscle with respect to the stimulation frequency can be compared by superimposing the respective voltage - time traces on top of each other ( figures 13a and b ) . activity of the tension nerve can then be correlated with the amount of relative force generated at each stimulation frequency , and for each joint position . as in the pd nerve , a variety of spike amplitudes are seen in response to contraction of the opener muscle ( figure 13c ) . anatomical arrangement of neurons in the walking leg is clearly observed with methylene blue staining ( figure 14 ) . the entire course of the tension nerve and stretcher motor nerve are shown in figure 15 . individual neurons of the pd nerve are shown with higher contrast using 4-di-2-asp ( figure 16 ) and cocl2 ( figure 17 ) backfill techniques . at high magnification the sensory endings can be seen inside of the supportive scolopales ( figures 16b ) . figure 10.move and hold at 0. the dynamic neurons are robust in firing during the movement and spikes from static sensitive neurons are present while the joint is held open . figure 11.rapid open and closing from fully flexed to extended ( 90 - 0 ) position . the joint is fully extended and then quickly moved to a flexed position and then held still . the relative forces that are developed with the joint fully flexed and stimulated at the various frequencies . ( a ) voltage - time traces from the force transducer are shown with each stimulation frequency . ( b ) the traces in panel a are superimposed in different colors for ease in comparison . ( c ) voltage - time traces of electrical activity recorded from the tension nerve when the motor nerve was stimulated at 80 hz . note the regular pattern of the stimulus artifacts as compared to the neural activity . also , note the various amplitudes of the neural responses . figure 15.the tension nerve arising from distal end ( red arrows ) and joining the motor nerve ( green arrow).click here to view larger image . ( a ) a back - fill of the pd nerve in cancer magister with 4-di-2-asp . neurons that were filled with cocl2 and processed ( a ) . traced outline of the stained preparation shown ( b ) . the purpose of this set of experiments is 1 ) to teach and exhibit the fundamental principles of extracellular recordings from an identifiable proprioceptive organ and tension nerve and 2 ) to stress the importance of anatomical mapping in relation to physiological function of particular sensory systems . this experimental approach and the animal models utilized are inexpensive and relatively easy to conduct in neurophysiology teaching laboratories . the neurons of chordotonal organs are of two specific functional types , those that respond to movement and those responding to static positions . single cell recordings from a variety of chordotonal organs , no matter which joint is examined , have shown this to be the case . indeed , chordotonal organs associated with the antennal joints of lobsters reveal the same two sensory types and basic anatomy . in addition to there being two neuron types ( movement and position ) , the neurons share the same anatomical arrangement on their respective elastic strands . the large somata located proximally on the strand tend to belong to the dynamic movement sensitive neurons . the pd joint only contains a single chordotonal organ while there are two chordotonal organs in the carpus - propodus ( cp ) and merus - carpus ( mc ) joints . the dissection to expose proprioceptive structures in blue crabs ( c. sapidus ) for electrophysiological recording requires a strategy that allows joint movements to take place in the natural positions while recording from sensory neurons . the tension nerve for the opener muscle in the walking leg is a very fine nerve made up of several neurons . unless care is taken , the tension nerve as well as the motor nerve innervating the muscle to be stimulated , can be damaged during this dissection . for optimal recordings recordings are readily accessible in a student laboratory using a 30 - 40x magnification dissecting microscope and low - end micromanipulators . future experiments that would be interesting to pursue with the joint chordotonal organs would be to examine the structural and physiological profiles during leg regeneration in various species at different stages in the life cycle as a follow up to an initial study that used cancer magister . questions remaining to be addressed are 1 ) does the distribution and organization of regenerated neurons depend on the age of the animal when regenerating a limb , 2 ) are the axonal projections to the cns ( ventral nerve cord ) in a regenerating limb functional or does it take time and joint use to establish functional connections , and 3 ) what happens to the severed axons proximal to the autotomy plane when the limb is autotomized ? crustaceans conform to environmental conditions and their surrounding temperature , but it is unclear how they maintain coordination within a neural circuit as neurons alter their activity in response to temperature changes . a slow rate of change might allow the animal some time for acclimatization whereas a rapid change may not29,30 . physiological changes in ph or osmolarity due to metabolism , behavior , or environmental impact may present similar challenges to neural circuits involved in proprioception . these crustacean preparations are ideal for addressing these types of problems because their function is well characterized at a single cell level . in this protocol we have demonstrated the physiological importance of tension neurons in monitoring force generated by the opener muscle . these tension receptors can be traced to their location within the apodeme by using staining procedures . these neurons , as in mammals , detect force at various levels and recruit additional neurons as the force increases . the frequency in activity is related to the stimulation frequency of the motor neuron until saturation in reception is reached . using a quick release protocol with the flexed dactylus joint , tension activity quickly disappears but then returns upon regaining tension in a fully extended joint . various neuromodulators can be applied to the preparation to see how it effects the development of force and neuronal response . one of the important aspects is how the neural responses are processed and integrated in the central nervous system and their impact on activity of motor neurons . the techniques we have shown allow one to start to address more information about the tension ( sensory ) nerve - motor neuron circuit function , i.e. signal in an intact leg to the ganglion and back to the muscle . the staining procedures demonstrated are key to understanding the physiology of sensory neurons that innervate proprioceptive organs . anatomical arrangement of the neurons based on function and the size of the soma are similar in the various chordotonal organs within the crab legs . it is not known if similar neuronal arrangements are also found in other crustacean species or insects . combining physiological recordings from single cells and mapping the location allows direct structure function relationships . the long term preservation of the anatomical arrangement with cocl2 staining and fixation allows one to repetitively make measures and assess the structural arrangement . proprioception and tension reception of skeletal muscles are sensory modalities that enable coordinated behaviors and responses to external and internal environment for articulated animals in a variety of skeletal muscle configurations . the muscle receptor organ in the abdomen of the crayfish is another well - documented preparation ( see the crawdad project ; http://www.crawdad.cornell.edu/ ) for teaching purposes of proprioception with only two neurons per abdominal hemi - segment . being able to record from single neurons to sensory nerve bundles provides further details that aid in understanding the basic principles of sensory reception . these relatively simple crustacean preparations allow one to address fundamental aspects of proprioception and tension monitoring , with the potential to determine the neural circuits that enable central integration of proprioceptive and other sensory inputs .

the primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement , and muscle tension . electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation , and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve . in addition , we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation . staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans . comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function . three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory .

Introduction Protocol 1. Dissection and Recording Electrical Activity from the Propodite-dactylopodite (PD) Nerve 2. Recording Electrical Activity from the Tension Nerve while Monitoring Force Generation 3. Staining Peripheral Nervous System Structures in Crustacean Walking Legs Representative Results Discussion Disclosures

PMC4370114

at present , global environmental changes have been linked to the geographical distribution and dynamics of malaria . as with the simulations , the depictions of anthropogenic global and regional climate changes on health impact of malaria can alter malaria epidemiology landscape in receptive areas of southeast asia , south america , and east africa [ 15 ] . such climate changes driven by greenhouse gas and land use change have been projected to significantly affect the spread of malaria in tropical africa before 2050 . the causal links between environmental change and human health are complex because the cause - effect relationships are often indirect and dynamic over space and time . malaria is caused by five human malarial parasites including plasmodium falciparum , p. vivax , p. malariae , p. ovale , and p. knowlesi . the malaria ecology is the integrated science of studying the interactions of malaria parasites circulating in their reservoir hosts , anopheles vectors and humans , and the constraints of which the adaptation of malaria parasites is regulated by the activities of humans and anopheles vectors , as well as by the biological and physical environments of anopheles vectors in a niche , a habitat , or an ecosystem . actually , the dynamics of the malaria ecology is constrained geographically and seasonally by ecological relationships in nature . what happened to breakdown the dynamics of the malaria ecology stems from two phenomena , which include anthropogenic climate and land use changes [ 614 ] . apart from that are driven by the natural processes , the land use changes driven by human activities have the potential effects on climate , whether globally , regionally , or locally , and can modify or manipulate the ecology of malaria as well as other vector - borne diseases [ 8 , 1517 ] . the main drivers contributing greatly to malaria epidemiology landscape changes include deforestation , dam construction , irrigation , stream diversion , agricultural land use change , and unplanned urbanization [ 814 ] . having the potential effects on the health impact of malaria , these human - induced changes can induce direct changes in the geographical distribution of malaria incidence and anopheles vectors [ 6 , 7 , 9 , 10 , 13 , 1821 ] , as well as the vectorial capacity of anopheles vectors [ 22 , 23 ] . also , such indirect changes in environmental conditions including shaded environments affect the availability of breeding sites and feeding activities [ 24 , 25 ] . taken together , if expected to accompany climate change or other human - induced changes , such anthropogenic land use change contributes substantially to the potential risks for malaria transmission in receptive areas due to the combination of malaria transmission dynamics , the susceptibility of human populations , and the geographical distribution of malaria vectors . in present review , the authors focused radically on the expansion of rubber plantations as the agricultural land use changes by delineating a unique landscape structure ( i.e. , the pattern and extent ) of the ecotope of malaria - associated rubber plantations ( mrp ) . this agricultural intensification is considered the main driver that has the potential effects on malaria transmission dynamics occurring in forest - related and forest fringe - related malaria in thailand [ 26 , 27 ] . the mrp ecotope , on the other hand , can be depicted as the hotspot of modeling malaria transmission dynamics upon the mrp factors underlying human settlements and movement activities ( e.g. , revisiting rubber plantation polygon(s ) and routine rubber plantation practices ) , health behaviors , land use / land cover change , malaria vector population dynamics , and agrienvironmental and climatic conditions . of note , this review synthesized the novel concepts and perspectives on applied landscape ecology and epidemiology of malaria , as well as approaches to determine the degree to which the mrp ecotope as fundamental landscape scale can establish malaria infection pocket(s ) . the challenge is that the advancement of malaria ecotoping in any hotspots pertaining to malaria epidemiology landscape change is to integrate systemic and uniform approaches and tools for modeling malaria transmission by making use of remotely sensed satellite imagery or landscape aerial photography using unmanned aerial vehicle ( uav ) , global positioning systems ( gps ) , and geographical information systems ( gis ) . thailand is located in the southeast asia and bordered by cambodia , lao people 's democratic republic , myanmar , and malaysia . the country is also constituted of the greater mekong subregion ( gms ) that includes cambodia , lao pdr , myanmar , people 's republic of china ( yunnan , prc ) , thailand , and vietnam . it has a land area of 51,311,501.92 hectares ( approximately 513,115 km ) ( see table s1 in supplementary material available online at http://dx.doi.org/10.1155/2015/909106 ) and a population of 64,785,909 people as of 2013 . from the 1980s to the 1990s , thailand had lost forest cover of between 13.0 million hectares and 14.8 million hectares . however , in the early 2010s , the forest land has been increased up to 17.6 million hectares as a result of the continuation of the policy implementation on reforestation , rehabilitation , and restoration across five regions of the country ( see table s1 ) . the propagation of upland perennial agriculture attributed to human activities has the potential effects on reduction or loss of biodiversity and habitats , surface water hydrology , soil erosion , and carbon sink and flux [ 1114 ] . for instance , the expansion of perennial agriculture ( e.g. , rubber and other mixed oil palms or orchards ) is a trade - off if expected to accompany land reform and human settlement / resettlement through the policy - driven economy and social / human development . this phenomenon is explained by the disturbance and fragmentation of the forest land affected by increasing agricultural land . that is , the affected forest land is influenced by which the perennial agriculture , whether or not it is irrigated , is cultivated around or close to the forests and to which human settlements and activities are related . however , this agricultural land use change is in turn a driving force influencing the functioning ecosystem and services of the forests and the connectivity of forest patches . here , the propagation of rubber plantations is used as an example of land use / land cover change that has the potential effects on the risks for malaria transmission in thailand or elsewhere in the gms countries including myanmar and malaysia [ 26 , 27 , 29 , 30 ] . the land area covered with as many as rubber plantation polygons is intensively exploited by land management strategy to propagate rubber plantations by private - owned smallholdings or estates . this topographically shaped landscape scale can be specifically defined by a unique landscape structure . in thailand , this is a topic of interest because the people not only exploit suitable and sustained productions of the natural rubber and wood but also pose the risks for malaria , especially in transmission control areas ( tcas ) of the south and east of thailand as shown in figures 1(a ) and 1(b ) . this phenomenon can lead not only to the changes of malaria landscape ecology and epidemiology , but also to the consequences of the implementation of currently available malaria control strategies , as well as surveillance systems and tools , at both national and subnational levels within the endemic gms countries implementing the national malaria control programs ( nmcps ) [ 26 , 27 , 30 , 31 ] . the disease is caused by two main human malaria parasites , p. falciparum and p. vivax , and to lesser extent by p. malariae and p. ovale . malaria epidemiology normally relates the infections to the causes of malaria in any infected individuals and to the risk factors attributed to malaria among the vulnerable populations involved in agriculture . this malaria epidemiology landscape can be envisaged as the endemic localities in which local people render the occupational and behavioral exposures susceptible to the infections as those who reach remotely inhabited areas of upland agriculture although infested with malaria vectors . if their settlement is located far from the forest land , the adulthood infection rather than the childhood infection occasionally occurs due to revisiting or performing workmen 's forest activities whether seasonally or periodically at the forests infested with anopheles vectors [ 26 , 27 , 29 , 32 , 33 ] . most malaria - contracted adults acquire the infection through outdoor biting of anopheles vectors during the night time in the absence or lack of preventive measures . if their settlement is located around or close to the forest land , both adulthood and childhood infections occasionally occur through outdoor or indoor biting of anopheles vectors during the night time in the absence or lack of preventive measures [ 26 , 27 , 29 , 33 ] . this is a reason why the epidemiologic landscape of forest - related and forest fringe - related malaria relates the schoolchildren and working groups to the occupation involved in agriculture . the questions are raised about why the risk of mrp occurs only in some valleys or hills and how we could comprehend a topologically detailed and accurate graphic presentation of the expansion of rubber plantation polygons on the valleys and hills , regulating that risk of mrp . to comprehend the pattern and extent of mrp ecotopes that relate malaria transmission risks to the expansion of rubber plantations , we synthesized novel concepts and perspectives on applied landscape ecology and epidemiology of malaria . understanding a geographically defined malaria ecotope previously described by sorosjinda - nunthawarasilp and bhumiratana is essential to demarcate the scope of any mrp ecotope . the mrp ecotope can be defined as a land area covered with the patches of forests and rubber plantations as it is geographically associated with the infestation or reinfestation of anopheles vectors . the mrp ecotope has a unique landscape structure topologically shaped not only by the diverse land use types ( or level i to iii categories of land use ) including rubber trees , whether monocultured or mixed with other perennial trees , waterway , water body , and forests , but also by the natural evaluation and inclination . in panels ( a ) to ( c ) , the topographic land use maps show the current status of the expansion of rubber plantations in three different malaria - endemic provinces including phang - nga ( south ) , trat ( east ) , and kanchanaburi ( central ) . phang - nga ( panel ( a1 ) ) and trat ( panel ( b1 ) ) have many land areas of traditional rubber plantation practices ( rpps ) , whereas kanchanaburi ( panel ( c1 ) ) exhibits growing trend of increased land areas of nontraditional rpps . details of mrp ecotopes with traditional and nontraditional rpps are described later . among these malaria - endemic provinces , phang - nga is a good example of the expansion of rubber plantations into the forest lands such as disturbed forests . a contour map of malaria ecotope or mrp ecotope illustrated with contour lines shows valleys and hills in which malaria transmission is likely to be dynamic as a result of the expansion of rubber plantations on the different elevations within the thung kha ngok tca of phang - nga ( panel ( a2 ) ) . since then large - scale development of rubber - planted land area has the effects on shifted land use and land cover , the expansion of rubber plantations has changed , instantly rather than gradually , with the change of the infestation of anopheles vectors . however , the persistence of rubber plantations with dense canopy can gradually recreate shaded environments under suitable agrienvironmental climatic conditions . if connected to the forest land infested with anopheles vectors , the extent of rubber plantation polygons can bring about malaria landscape change . that is , the extended forest cover manages to promote the adaptation and survival of local potent anopheles vectors and human - vector interactions , but not always to facilitate the dispersal of the populations of anopheles vectors although they interact with each other in the habitats with breeding and foraging . however , not all the patches covered with rubber plantation polygons are infested with anopheles vectors . even if a patch of rubber plantation polygon(s ) is infested or reinfested with some anopheles vectors , a probable human - vector contact site is not definitely understood unless malaria or mrp ecotope is well defined . therefore , we need to understand what are the linkages to which the vulnerable persons including rubber farmers , rubber plantation workers , and accompanied persons acquire the infections over space and time despite the fact that the coverage of malaria control strategies used in the nmcp is household - level implemented . figure 2 shows malaria risks are associated with routine rpps during the night time as often this phenomenon is seen in the mrp ecotope with traditional rpps in the south and east of thailand . during seasonal harvestation of the natural rubber , rubber farmers or rubber plantation workers , whether or not they reside in an mrp ecotope , do not always sleep under nets as they have person - time sleeping hours less than that of general people who normally reside in the densely populated areas of the village . however , both low and high malaria risks for the adulthood infection stem from the strong likelihood that probable human - vector contact occurs at multiple locations and time periods during which anopheles vectors seek any blood meals . for example , high malaria risk occurs when any susceptible persons prefer the night time routine rpps , for example , rubber tapping , rubber coagulation , or rubber sheet processing , at their rubber plantation farm infested with anopheles vectors . during the practices , they do not perceive or beware of the malaria risk by the adoption of personal protection behaviors or boarder preventive measures . that is , if they practice longer time at night , there is the strong likelihood of malaria risk . rubber farmers or rubber plantation workers involved in most traditional rpps normally schedule routine rpps ( e.g. , rubber tapping and rubber sheet processing ) , starting from early wet season and extending to cool season ( figure 2(a ) ) . in a given rubber plantation farm ( or a rubber plantation polygon ) that produces the natural rubber sheets , skillful rubber plantation workers ( 2 persons ) can tap up to 400 to 500 rubber trees per night and , continually , harvest the rubber milk to process the rubber sheets . tapping a rubber tree varies from one to two minutes before getting started a new one . if they start to tap the rubber trees around 21:00 h , it is possible that they will acquire frequent exposure to bites of anopheles vectors around 03:00 h or to be exposed continuously to multiple bites from 21:00 to 06:00 h. this is why the probable human - vector contact site is not always close to the house during which seasonal harvestation is operated . the occurrence of probable human - vector contact is likely to be dynamic if there is the availability of anopheles breeding sites and feeding activities on different altitudes and inclinations . the malaria risk is more likely to be dynamic if there is difference in human movement patterns during nighttime rubber plantation practices ( figure 2(b ) ) , which include nighttime rubber tapping ( hours ) and rubber tapping patterns ( days ) such as alternate - day tapping and 2 - 3 days after a day of recovery . if expected to accompany risk behaviors , these human movement activities can pose the risk for malaria infections , depending on how any vulnerable persons acquire the multiple bites of anopheles vectors at multiple locations . in general , the seasonal malaria transmission relating to seasonal harvestation occurs in most mrp ecotopes of the southern and eastern thailand . the seasonal harvestation normally takes about 10 months of harvestation ( starting from may to february of the next year ) , and then a 2-month of recovery persists between march and april . in the east of thailand , there is a tendency of schedule change due to local climate change . if the rubber trees shade their leaves as continuously exposed to cool weather , a recovery phase of harvestation will vary from as early as mid january to mid march , and then seasonal harvestation will start as early as in late march . eventually , the dynamics of malaria transmission occurring in the diverse mrp ecotopes of the eastern thailand maybe relate local climate change to the temporal and spatial distributions of vector populations and the seasonal harvestation of the natural rubber . taken together , the mrp ecotope encompasses the epidemiological complex setting in which the malaria transmission risk is relatively associated with geospatial distributions and behaviors of the anopheles vectors , human settlement , and movement activities such as revisiting rubber plantation polygons infested with anopheles vectors and routine rpps . that is why we need to comprehend mrp ecotope to better understand the stratification of malaria risks in certain endemic localities or transmission areas for surveillance , prevention , and control . as illustrated earlier by figures 1 and 2 , land areas covered with traditional rubber plantations in the south and east of thailand are situated on different altitudes up to 200 meters above sea level ( masl ) and slopes up to 35 degrees . the rubber trees manage normally to grow under the climatologically and geologically suitable conditions and to persistently yield the natural rubber after 7 to 20 years of the plantation . on the other hand , the mrp ecotope with traditional rpps becomes epidemiological complex setting in which the malaria infection pocket ( mip ) is likely to be established in space and time through the interconnections that can pose probable human - vector contact . understanding the topography of diverse mrp ecotopes with traditional rpps is needed not only to analyze what characteristics of mrp ecotope with traditional rpps are connected to establish an mip but also to determine the degree to which local anopheles vectors are adapted to local environment of the mrp ecotope . in this regard , we can classify two subecotopes of mrp ecotope with traditional rpps ( figures 3(a ) and 3(b ) ) ; both subecotopes a1 and a2 depend radically on the infestation or reinfestation of anopheles vectors . , we need to comprehend the biology and ecology of local anopheles vectors adapted to local environments . the subecotope a1 ( figure 3(a ) ) is the mrp ecotope with traditional rpps that has an extremely high potential for the establishment of mip whereby the persistence of breeding sites and feeding activities of anopheles vectors exit in rubber plantation polygons . rubber plantations whether mixed with oil palms , fruit orchards , or other perennial trees the subecotope a1in which land use types are heterogeneous is located at less than 200 masl or between 100 and 200 masl , around or near the forests . similar to that of the subecotope a1 , the subecotope a2 has a high potential for the establishment of mip and is located at less than 100 masl . the presentation of the subecotope a2 is shown for phang - nga ( figure 3(a ) ) and trat ( figure 3(b ) ) . also , the existence of the ecotope b , namely , waterway or water body , plays the significant role for the infestation or reinfestation of anopheles vectors ( figure 4 ) . waterway is a narrow pathway of water that is constantly moving , as the current is regulated by season variation or by man - made dam or irrigated water body . water body is a source of usually fresh water that is issuing , whether naturally or man - made , from the ground , and is an inland body of standing water whether small or large , or shallow or deep . as essential for the suitable microclimate including humidity and temperature , both waterways and water bodies are required for the adaptation and survival of anopheles vectors . most mrp ecotopes , which are confined to the tcas of malaria - endemic provinces of the south and east of thailand , cover both subecotopes a1/a2 and ecotope b. these ecotopes serve as the places that are connected to establish the mip(s ) . nonetheless , if these ecotopes are more diverse , the probable human - vector contact will more likely be dynamic and , eventually , the mip will not truly be specific to a rubber plantation polygon . more specifically , we need to comprehend mrp multifactors such as agrienvironmental climatic conditions and vector population dynamic that are associated with malaria transmission dynamics in the mrp ecotope with traditional rpps . in most land areas of traditional rpps in the south ( figure 3(a ) ) and east ( figure 3(b ) ) , the normal growth of clonal rubber trees requires agrienvironmental climatic conditions such as heavy rain ( average annual precipitation : > 1,400 mm ) , optimal temperature ( average annual temperature : approximately 2628c ) , and optimal humidity ( average annual humidity : > 65% ) . meanwhile , if there exist the reservoir and irrigation surrounding or within the mrp ecotope ( figure 4(a ) : panel ii ) , the agrienvironmental climatic conditions will promote the infestation or reinfestation of anopheline mosquitoes including malaria vectors . as for malaria vector surveillance in the mrp ecotopes with traditional rpps , we also leverage needed data / information of what are relevant to the infestation of primary malaria vectors and what are epidemiologically linked with malaria transmission risks . still , diverse groups of anopheles vectors can be adapted to local environments favorable to breeding and foraging in both subecotopes a1 and a2 . for example , the ground surveys in 2014 demonstrated that most mrp ecotopes with traditional rpps are infested with the anopheles vectors . the suspected rather than primary / secondary vectors are predominantly found in both subecotopes a1 and a2 as shown in figures 3(a ) and 3(b ) . the abundance and distribution of three primary vectors , including an . dirus , seemed to be regulated by seasonal and geospatial variations as similar to that observed by several reports [ 3437 ] . as shown in figures 1to 3 , most mrp ecotopes with traditional rpps are close to forests or confined to the valleys or sloping hills that exit waterways at the altitude less than 100 masl or between contour lines , 100 to 200 masl . aconitus , all of which are commonly found through both indoor and outdoor collections using human landing catch in a wide range of densities between dry and wet - cool seasons . if confined to the valleys or sloping hills at the altitude less than 100 masl , there exist as many as adult numbers and densities of an . stephensi can breed in widespread breeding places both in the urban and rural settings in south and west asia [ 38 , 39 ] . in the rural settings , the larvae can be found in stream pools and margins , ponds , puddles , irrigation channels , seepage canals , catch basins , and springs . in the urban climates , they can be found in a variety of artificial containers such as cisterns , wells , tubs , ornamental ponds , fountains , and sewage . by contrast , a variety of anopheles vectors which are endogenous to mrp ecotopes with traditional rpps in the south and east of thailand prefer breeding in natural breeding habitats in the wild rather than breeding in artificial containers in the wild or rural and urban settings . dirus , are likely to breed their progeny in the waterways such as brook and/or stream . the abundance and distribution of anopheles larva populations found in natural breeding habitats are dynamic due to seasonal and geospatial variations . among these vectors , maculatus is a common vector that distributes widely across the south of thailand and peninsular malaysia . maculatus can breed in the wild in various breeding habitats such as water pockets formed on the bank of rivers and waterfalls , shallow pools , and slow flowing streams generally located at 100400 m from the nearest human settlement . based on anopheles larval surveys , if adapted to local environments that similarly happened in the wild , an . maculatus can breed in the man - made reservoir ( figure 4(b ) : panel i ) and artificial container like plastic bowl ( figure 4(b ) : panel ii ) . which is endogenous to the subecotope a2 of bang ma mrp ecotope ( figure 3(a))is adapted well to breed its progeny in a water - containing plastic bowl , as well as to cobreed with aedes albopictus . it is very interesting to note the colonization of two taxa in this niche , showing that an . maculatus progeny which has one 3rd larva and six 4th instar larvae accompanies with more than 50 larvae of ae . albopictus in plastic bowl but also its changes in breeding characteristics remain to be established . the mrp ecotopes with nontraditional rpps have been resulted from the agricultural intensification of rubber plantations instead of crop plantations and other perennial agriculture in the northeast , north , and central thailand , as shown in figures 1(c ) and 3(c ) . many rice fields , mixed orchards , or crop plantations in upland areas of the northeast , north , and central thailand have been converted to the newly planted areas of rubber plantations . still , nontraditional rpps require suitable agrienvironmental climatic conditions . for instance , it requires average annual precipitation , approximately 1,2001,400 mm , but annual average for total rainfalls , approximately 120150 days . unlike in traditional rpps , the seasonal harvestation of the natural rubber in the nontraditional rpps normally starts from march to october ( or 8 months of harvestation ) while recovering from november to february of the next year ( or 4 months of recovery ) . as with land and water management strategies , the mrp ecotopes with nontraditional rpps are likely to interconnect human settlements and activities and vector biology and ecology . the nontraditional rpps are likely to plant the rubber trees especially confined to riverine or irrigated areas such that the suitable microclimate will eventually create the rubber plantations with dense canopy or shaded environment . subsequently , the mrp ecotope with nontraditional rpps nurtured with waterway and/or water body is linked with the adaptation and diversification of anopheline taxa including malaria vectors that maybe develop gradually the species richness ( the number ) and evenness ( the abundance ) . if microenvironments are managed to resuscitate this mrp ecotope with nontraditional rpps , a plethora of anopheles mosquitoes will be proxy measure of the infestation or reinfestation of anopheles vectors . eventually , the reemergence of introduced malaria that possibly occurs in the mrp ecotopes with nontraditional rpps is explained by the interconnections underlying the dynamics of malaria transmission , the geographical distribution of anopheles vectors , and the susceptibility of human populations . the baseline entomological data are needed for malaria vector surveillance and control to determine the extent to which the infestation and reinfestation of anopheles vectors occur in newly planted areas before and during the seasonal harvestation of the natural rubber . continuations of larval survey along with environmental observations will help explain temporal and spatial distribution of local anopheles mosquitoes that can infest in waterways or water bodies confined to newly planted areas as in figure 4(a ) ( panels i and ii ) . the waterway rather than water body serves as breeding site for anopheles vectors is commonly found to be slow - running brook or stream with the vegetation . nonetheless , it is very difficult to estimate the retention time of egg hatchability and development of first to fourth instar larvae due likely to the species diversity and season variation . are likely to distribute along the brook or stream margins with the vegetation during wet - cool season , as well as to aggregate in small pools or water pockets close to the margins of the brook or stream during dry season . particularly in dry season during which the stream normally runs very slowly , anopheles larvae can be found in shallow sand or mud beds of the brook or stream whether or not the aquatic plants or plant debris are present . during wet - cool season , the infestation of anopheles spp . can exhibit diverse groups of anopheles spp . including both nonpotent and potent malaria vectors . in general , seasonal regulation of local anopheles mosquitoes adapted to local environment is an ecological driver that influences the abundance , distribution , and survival of anopheles larvae . regarding this , we can therefore classify two subecotopes of the mrp ecotope with nontraditional rpps ( figure 3(c ) ) ; both subecotopes c1 and c2 depend radically on the infestation or reinfestation of anopheles spp . that are adapted to local environments . the subecotope c1 is the mrp ecotope with nontraditional rpps that has a moderate - to - high potential for the establishment of mip whereby the availability of breeding sites and feeding activities of anopheles vectors exists in rubber plantation polygons . rubber plantations whether or not mixed with fruit orchards , forested plantations , and other field crops can manipulate anopheles infestation or reinfestation . as seen in figure 3(c ) , most rubber plantation polygons are located at less than 200 masl , around or near the forests although nurtured with the waterways . in most tcas in the northeast , north , and central thailand , many mrp ecotopes with nontraditional rpps are around or close to the forests to which anopheles vectors are sessile . also , the malaria incidence is epidemiologically linked with currently developed malaria cases or history of malaria infections in the past . based on our experiences , malaria vector surveillance in some sampled sites of the subecotope c1 of kanchanaburi ( figure 3(c ) ) showed that both nonpotent and potent anopheles spp . there is the likelihood that the subecotope c1 corresponds to the abundance and distribution of diverse anopheles taxa including primary vectors , an . larva numbers of anopheles spp . can be found both in dry and wet - cool seasons in waterways and/or water bodies as in figure 4(a ) . among the adapted taxa , larva numbers of an . minimus are more likely to be found in slow - running streams in dry season than in wet - cool season . perhaps water flows and diets influence the abundance and distribution of larvae of these two species . dirus whether or not the indoors or outdoors collections by human landing catch are performed during wet - cool season . the subecotope c2 is the mrp ecotope with nontraditional rpps that has a low potential of the establishment of mip whereby the availability of breeding sites and feeding activities of anopheles vectors exists irregularly in rubber plantation polygons . rubber plantations whether or not mixed with fruit orchards , rice fields , and other field crops can manipulate the anopheles infestation or reinfestation . this ecotope of which any autochthonous malaria cases are absent is located at less than 100 masl on upland crop plantation areas with the irrigation as in figure 4(a ) or confined to the riverine or irrigated areas with agricultural practices . the subecotope c2 can reflect the land use change in the newly planted areas confined to the northeast , north , and central thailand which are unknown or nonendemic for malaria . for instance , many mrp ecotopes with nontraditional rpps in the northeast are close to thai - cambodia border or thai - lao border or mekong river . based on our malaria vector surveillance data , both anopheles larvae and adults hyrcanus can be commonly found during the night time than during the early sunset or during the early morning . both outdoor and indoor collections of anopheles adult mosquitoes can be performed on this nocturnal appearance during which continuations of adult survey along with environmental observations are conducted . the abundance and distribution of anopheles adult mosquitoes obtained by adult vector survey are likely to correspond to that entomological data obtained by larval survey in this subecotope . as to bridge malaria landscape ecology and epidemiology [ 41 , 42 ] , the mrp ecotope can be defined as a fundamental land unit , or it is considered as small as landscape scale of analysis used in landscape ecology [ 4345 ] . the mrp ecotope ( spatial ) is suited to determine the degree to which the agricultural land use changes attributed to the expansion of rubber plantations ( nonspatial ) have the potential effects on malaria transmission risks . the mrp ecotoping , on the other hand , can also serve the holistic assessment framework whether such negatively ecosystemic outcomes contribute substantially to malaria transmission dynamics in thailand and , internationally , within the other gms countries . as mentioned earlier , the relative land use types of the mrp ecotope including rubber plantation polygons , waterways , and water bodies serve as the spatial data set which are essential for the identification and characterization of mrp ecotope confined to the tca , based on both land use and contour maps . the identification is to define a land area geographically associated with the infestation or reinfestation of anopheles vectors . the characterization is to define a land area that has the potential risk for malaria transmission . for instance , the topographic land use maps employ levels i to iii land use information originally obtained from the satellite imagery . when analyzed for any tca at the subdistrict or village level , the landscape features of the mrp ecotope exhibit the degree to which anopheles vectors can infest or reinfest in respective valleys or sloping hills covered with rubber plantations and nurtured with waterways and/or water bodies . basically , the malaria control stratification system used in the registry of the tca at the subdistrict or village level should be currently or up - to - date reviewed by the nmcp 's implementers or the infection control personnel as seen in figure 1 . based on the landscape parameters or spatial and nonspatial data available for the delineation of the mrp ecotope , a contour map of the tca illustrated with contour lines will better help us to permit the coverage land area infested with anopheles vectors as seen in figure 3 , in the presence or absence of any currently developed malaria cases . as seen in figures 2(b ) , 3 , and 5 , the landscape structure of anopheles - infested land area is relatively related to the specific ridge of hills with low - to - moderate steepness of the slope that generate waterways , especially brooks and streams , or perhaps fork . more essentially , hillside slope and valley(s ) confine the sloping waterways that move slowly down or flowing downward on a gap or pass . if covered with rubber plantations mixed or not mixed with other perennial trees and orchards , the responsible valleys or hills can confine the mip of corresponding rubber plantation polygon . however , the ground truths especially for which the attribute data of probable human - vector contact are relevant to rubber plantations polygons are needed to demarcate the georeferences using the gps and to triangulate the spatial data obtained from the standard land cover classification systems of remotely sensed satellite imagery [ 17 , 46 ] or landscape aerial photography . the landscape aerial photography ( lap ) is the advanced technology of aerial imagery of the perspective landscape scale such that the uav can provide high resolution of land use map as compared to that created by the remotely sensed satellite imagery ( table 1 ) . the lap which is applied to a coverage area map of this georeferenced mrp ecotope that established the malaria events between 2013 and 2014 ( table 2)is demonstrated by using uav and followed by the programmed trajectory [ 4749 ] . the uav using the multirotors employs the projective camera that virtually provides a coverage area map pertaining to a series of aerial photos with high resolution . more significantly , the spatial data can be deduced from any locations at which the ground survey can not reach . as seen in figures 5(a ) and 5(b ) , the multirotors programmed with the trajectory can perform aerial imagery survey in which the on - board camera can record a series of 210 snapshots over a coverage area of 1.5 km at the altitude above 300 meters . the klong khak mrp ecotope encompasses the unique landscape features of the expansion of the rubber plantations on the valleys and hills infested with anopheles vectors ( figure 5(a ) ) and has the high potential for malaria transmission ( figure 5(b ) ) that exhibits the mips within a 2 km land area . mosaic techniques are essential for uav image processing and construction of a mosaic image to obtain a broad landscape , based radically on the georeference and anchor image of the partially aerial snapshots ( figure 5 ) . because there are pairs of partially uncalibrated snapshots that overlap weakly perspective views , image mosaics of corresponding sidelap and overlap views are analyzed to present pairs of strongly perspective views relevant to the georeference . a feature - based technique [ 5052 ] is commonly referred to as a linear feature - based noniterative method for the joint estimation of all images . the technique is the most widely used to perform image registration , that is , pairwise registration of the anchor images and between pairs of the nonanchor images , and hence construction of registered image pairs used in the joint algorithm . as seen in figures 5(a ) and 5(b ) , constructing image mosaics from 210 snapshots is an essential step for leveraging spatial data whether or not the mrp ecotope has the unique landscape features pertaining to the establishment of mip . based on the construction of image mosaics mentioned above , a high - resolution perspective view represents a present landscape structure of the mrp ecotope or mip confined within the mrp ecotope . the ground surveys are also needed to validate the spatial data obtained from lap using uav . the uav - based maps are georeferenced using the gps ; that is , both validated spatial and nonspatial data can be manipulated precisely and timely as described below . more evidently , table 2 and figure 5(b ) show a profile of the klong khak mrp ecotope that exhibits the 2013 - 2014 events of the malaria infections in relation to rubber plantation practices . the infographic presentations of the mip confined to this klong khak mrp ecotope can be performed on the different platforms of the gis - based uav and google earth applications . as in figure 5(b ) , the validated spatial data initially obtained by the uav - based lap and mosaic technique are displayed on the arcgis software applications or the google earth applications and then synchronized with the attributes of malaria events . regarded as the relative land use type of the klong khak mrp ecotope , the rubber plantation polygons correspond to probable human - vector contact sites because routine rpps during the night time by rubber farmers or rubber plantation workers rendered them susceptible to multiple bites of anopheles vectors including infective bite(s ) at multiple locations . obviously , different rubber plantation polygons contribute to epidemiologic patterns of the infection over a time period . malaria transmission occurs as the event of plasmodium infection , whether single or mixed , in a point manner or an intermittent manner rather than the sequential event of a continuous infection . malaria infection is considered as an individual newly infected with either of four plasmodium spp . this mrp ecotope exhibits the strong evidence that the availability of breeding sites and feeding activities of primary anopheles vectors relates to the single malaria infections , that is , any individuals infected with the only one type of the plasmodium species over a time period . that is , this mrp ecotope relates the occupational and behavioral exposures to render the adults susceptible to outdoor bites of anopheles vectors including infective bite(s ) . the childhood infection may be acquired through outdoor biting if the children or younger persons are accompanied persons involved in rpps during the night time . like the forest - related malaria in thailand , the mrp ecotope exhibits seasonal malaria transmission ; that is , the incidence is relatively higher during wet season than during dry season and hence it increases with increasing age . once seasonal transmission of autochthonous malaria occurs in the mrp ecotopes that are confined to the tca at the subdistrict level , it does not mean that all the malaria infections are epidemiologically linked with the same source of the infection . thus , in a given mrp ecotope , malaria transmission occurs as the event of the infection in one or more mips . moreover , the profiling of the mrp ecotope and mip could benefit the advantages of uav imagery rather than the remotely sensed satellite imagery ( table 1 ) . still , the systemic and uniform approaches to mrp ecotoping require both the epidemiological data obtained from routine malaria surveillance systems and household surveys and the entomological data obtained by anopheles larva and/or adult surveys . the rubber forestry is a land management strategy by which the people benefit from the exploitations of propagating rubber plantations and harvesting the natural rubber and wood . thailand is a case study such that there are coincided phenomena that land use / land cover changes influenced by increasing land areas for the rubber forestry are related to increased risks for malaria although the vertical implementation of the nmcp has been gradually achieving the overall reduction of malaria mortality and morbidity , and the program is moving toward the preelimination phase of malaria control . the nmcp employs the malaria control strategies suited to stratify the tca at the subdistrict or village level . this stratification is based radically on both the epidemiological data obtained by the malaria surveillance systems that employ the notification of laboratory - confirmed malaria infections or case numbers reported by active and passive surveillance systems and the entomological data obtained by the malaria vector surveillance that monitors the infestation / reinfestation of anopheles vectors . however , malaria transmission dynamics occurs continually in the tcas where the mrps coexist with the changes of malaria landscape ecology and epidemiology . more importantly , the effective and sustained primary prevention strategies are still desired because the people involved in the rubber forestry sector do not know what renders them susceptible to the malaria infections . regarding this , if large - scale malaria control is required , the landscape of forest - related or forest fringe - related malaria needs to be logically analyzed in completeness , correctness , and timeliness . as for malaria vector surveillance that achieves the targets or desired outcomes , the ecotope - based entomological surveillance ( ees ) can provide the proof that the mip is the hotspot suited to determine the extent to which the responsible anopheles vectors and their counterparts can infest or reinfest within the mrp ecotope as described elsewhere . from global and regional perspectives , the gms countries are being shifted to the world largest natural rubber producers that exhibit growing trend of increase in land areas of rubber plantations , as the coordinators of the nmcps and other public health personnel involved in policy formulation and strategic deployment might have adopted the interventions and services suited to the target populations in the rubber forestry sector . as mentioned earlier , such ees approaches can provide the strong evidence that the informative malaria ecotope can be modeled for malaria transmission dynamics to link between human - vector - parasite interactions and human - environment - vector interactions in receptive areas of land use and land cover change . nonetheless , the ees requires the needed data / information of the malaria infections or the incidence in order to analyze the vulnerability in how the diverse groups of anopheles vectors potentially transmit the malaria parasites vertically to humans in the mrp ecotope , based on the time lag and probable human - vector contact . by which once the establishment of mip is monitored within the tca or across tcas at the subdistrict level . in addition to the approaches and methods described elsewhere [ 41 , 42 , 53 ] , this approach can be optionally applied to or used as a promising tool for the identification and characterization of mip . if properly validated , this approach will ease decision - making process of prioritizing most risk areas ; that is , this leads to the right direction of the selection of management activities and strategies to be implemented to the target areas and populations at risk by the national - level and subnational - level coordinators of the nmcp . also , it will help develop the downstream protocols , procedures , and tools for decision - making implementation , evaluation , and monitoring of the effectiveness of those selected strategies and services that can lead to the reduction of the operational costs for surveillance , prevention , and control . the delineation of mrp ecotope can be optionally suited to determine the scope of very large infographic data relating to human , agent , vector , and environment . in practice for example , a 1 1 km grid of the mrp ecotope that corresponds to the latitude and longitude can be used as small as landscape scale or the unit of assessment of the malaria infections in humans and anopheles vectors as well as of insecticide resistance in anopheles vectors . as shown in figure 3 , the contour maps show the scalable mrp ecotopes cover geographically defined transmission area of as small as 500 m , in which at least one mip exists . the configuration of mrp ecotope map illustrated with contour lines allows us to infer the parameters specific for anopheles vector - infested rubber plantation polygons that lie between successive contour lines . more interestingly , such specific locations to which rubber plantation polygons on the valleys or sloping hills and the steepness of slopes are confined are likely to regulate the abundance and distribution of potent anopheles vectors between adjacent contour lines . the operational and research endeavors for malaria prevention and control should be paid attention to harmonizing the global platform of the georeferenced mrp ecotope , whether or not it is mapped on different platforms of applicable gis . in particular , the nmcps can adopt the georeferenced mrp ecotope through the development of malaria ecotope database and management when the tca at the subdistrict or village level is logically analyzed and targeted for the preelimination and elimination phases .

the agricultural land use changes that are human - induced changes in agroforestry ecosystems and in physical environmental conditions contribute substantially to the potential risks for malaria transmission in receptive areas . due to the pattern and extent of land use change , the risks or negatively ecosystemic outcomes are the results of the dynamics of malaria transmission , the susceptibility of human populations , and the geographical distribution of malaria vectors . this review focused basically on what are the potential effects of agricultural land use change as a result of the expansion of rubber plantations in thailand and how significant the ecotopes of malaria - associated rubber plantations ( mrp ) are . more profoundly , this review synthesized the novel concepts and perspectives on applied landscape ecology and epidemiology of malaria , as well as approaches to determine the degree to which an mrp ecotope as fundamental landscape scale can establish malaria infection pocket(s ) . malaria ecotoping encompasses the integrated approaches and tools applied to or used in modeling malaria transmission . the scalability of mrp ecotope depends upon its unique landscape structure as it is geographically associated with the infestation or reinfestation of anopheles vectors , along with the attributes that are epidemiologically linked with the infections . the mrp ecotope can be depicted as the hotspot such that malaria transmission is modeled upon the mrp factors underlying human settlements and movement activities , health behaviors , land use / land cover change , malaria vector population dynamics , and agrienvironmental and climatic conditions . the systemic and uniform approaches to malaria ecotoping underpin the stratification of the potential risks for malaria transmission by making use of remotely sensed satellite imagery or landscape aerial photography using unmanned aerial vehicle ( uav ) , global positioning systems ( gps ) , and geographical information systems ( gis ) .

1. Overview of Malaria Epidemiology Landscape Changes and Risks 2. Current Status of Forest-Related and Forest Fringe-Related Malaria Landscape in Thailand 3. MRP Landscape Epidemiology 4. MRP Landscape Ecology 5. Profiling of MRP Ecotope and Malaria Infection Pocket 6. Perspectives

PMC4668187

ankle joints have a complicated anatomical and mechanical structure , and can have develop , which are among the most frequent injuries during normal physical activities1,2,3 . ankle trauma can be categorized as acute or chronic ; conservative treatment with rehabilitation training is crucial because chronic lateral instability of the ankle hinders mobility and reduces ambulatory status and balance capabilities4,5,6 . frequent sprains of the ankle joint are caused by loss of muscular stability or inappropriate treatments , and collateral pain is reported as the most common cause7 . patients with chronic ankle instability ( cai ) have reduced balance capabilities , making it difficult to abruptly turn around or halt . this functional instability is due to damage of the mechanoreceptors in the joint region after ankle injury6 . mechanical injuries can result in a limited range of motion , anthropometric changes , ligament laxity , and degenerative changes4 , 5 , 8 . therefore , appropriate rehabilitation to enhance mobility is beneficial for the recovery of myofunction in both the general public and athletes2 , 9 . in particular , patients with a severe reduction in proprioception due to an ankle sprain require improvement in muscular strength and postural control10 , 11 . in this respect , resistance training is safe and effective in the prevention and rehabilitation of ankle sprains with cai , and is a useful method for functional improvement12 . complex training of major joints should be performed efficiently14 , and should result in pain relief , restoration of range of motion , recovery of muscle strength and endurance , and improvement in postural control15 . the present study was conducted to provide baseline data for an efficient rehabilitation exercise program . isokinetic muscular strength was analyzed after 8 weeks of a strategic strength resistance exercise program in male subjects in their twenties who had pain or functional damage in the ankle joint . this study recruited 22 male subjects who were orthopedically diagnosed with a functional injury of the ankle joint , and performed a strategic strength resistance exercise program for 8 weeks . prior to enrollment , the purpose of this study and experimental methods were disclosed to all subjects , and participants voluntarily gave their signed informed consent . the study was approved by the hufs university and the institution in which it was performed , and complied with the ethical standards of the declaration of helsinki . the average physical characteristics of the participants were : age 26.414.16 years old , height 179.107.31 cm , and weight 75.848.07 kg . prior to the test , the degree of instability of each ankle joint was measured using the ankle score scale ( ass ) , which is comprised of 8 subjective questions regarding pain , swelling , instability , stiffness , stair - climbing , running , work activities , and support ability . the muscular mobility of plantar flexion and dorsiflexion in the ankle were measured using isokinetic testing equipment ( humac norm testing and rehabilitation system , csmi solutions , usa ) . to avoid hyperflexion or hyperextension injury during the test , each subject set their own range of motion by individual calibration , and the measurements were digitized and quantitated using the software provided by the manufacturer . to analyze comprehensive isokinetic myofunction in the ankle , values of peak torque at 30/sec and peak torque per body weight quadriceps ratio of the ipsilateral thigh , peak torque of extension and flexion was measured , and the flexion to extension value was converted to a percentile ( % ) . the 8-week strategic strength resistance exercise program focused on the improvement of muscular strength , proprioceptive ability , and functional ability to improve complex instabilities of the ankle joint . the training consisted of three sets per week , with a total of 24 sets , at 60 minutes per set , and an increase in intensity every 2 weeks , following the progressive overload principle . in addition , the muscular strength program consisted of weight training , plyometric exercises without a load , and thera - band exercises to strengthen muscles in the lower limbs , such as the quadriceps , hamstring , and gastrocnemius . exercises to enhance proprioceptive ability were performed to improve the range of motion of the ankle joints , and props were used to improve static and dynamic postural controllability . , usa ) for windows . for all measurements , the average and standard deviation were calculated , and a paired t - test was adopted to test the differences in isokinetic myofunction ; a medical questionnaire was complete before and after the 8-week strategic strength resistance exercise program . results from the medical examination by interview before and after the 8-week program are shown in table 1table 1.results of the medical examination by interview regarding the ankle jointfactorsbefore programafter programpain15.00.017.82.6***swelling6.62.410.00.0***instability17.22.621.62.4***stiffness4.31.34.61.0stair climbing8.12.59.12.0running5.92.08.82.2***work activities10.31.310.61.7support ability4.31.35.00.0*total71.68.687.46.2***unit : point , values are the meansd , * p<0.05 , * * * p<0.001 . the overall scores for the 8 questions regarding the ankle joint before and after participation in the program showed a statistically significant improvement ( p<0.001 ) from 71.638.62 to 87.446.21 , respectively . unit : point , values are the meansd , * p<0.05 , * * * p<0.001 peak torque measurements in plantar flexion and dorsiflexion of the ankle before and after the 8-week program are shown in table 2table 2.measurements of the isokinetic peak torque of the anklesbefore programafter programpfright87.325.6118.528.5*left93.934.0112.729.3dfright25.95.834.16.9**left27.35.534.88.0*unit : nm , values are the meansd , pf : plantar flexion , df : dorsi flexion , * p < 0.05 , * * p < 0.01 . unit : nm , values are the meansd , pf : plantar flexion , df : dorsi flexion , * p < 0.05 , * * p < 0.01 measurements of the peak torque per body weight in plantar flexion and dorsiflexion are shown in table 3table 3.measurements of the peak torque / body weight of the anklesbefore programafter programpfright124.838.3149.633.7left133.846.9142.739.7dfright37.19.143.47.8left39.68.744.59.2unit : % , values are the meansd , pf : plantarflexion , df : dorsiflexion . the overall trend showed that the measurements increased , but this was not statistically significant . unit : % , values are the meansd , pf : plantarflexion , df : dorsiflexion analysis of the isokinetic hamstring : quadriceps ratio in plantar flexion and dorsiflexion is shown in table 4table 4.analysis of the isokinetic hamstring : quadriceps ratio of the anklesbefore programafter programright29.34.932.212.9left31.98.133.216.0unit : % , values are the meansd . despite the lack of statistical significance , both the left and right ratios increased in the post - program measurements . weakness or unstable mobility in extension and flexion can cause instabilities in the major joints of the lower limb17 . patients with cai have difficulty with fundamental physical activities such as postural control and gait performance , and have limited functional activities , including postural sway ; there is a risk of muscle injury because range of motion is decreased due to weakening and imbalance of lower limb strength6 . the prevention of instability using self - evaluation with a medical questionnaire is desirable because untreated instability can induce abnormal external forces on the ankles , repeatedly resulting in an increase in instability factors18 . a medical examination by interview and the functional ankle instability questionnaire this study showed that an 8-week strategic strength resistance exercise program significantly improved pain , swelling , instability , and support ability of the ankle joint , as well as other factors that stabilized following the program . isokinetic strength testing mostly measures strength exhibition and balance ratios in the lower limb , and can be utilized for predicting isokinetic strength , relative diagnosis , and the possibility of occurrence20 . studies conducted on strength , balance , and functionality have revealed that weakening of the ankle joint can worsen functional instability and the possibility of reinjury21 , and that a combined exercise program is beneficial in the improvement of complex instability of the lower limb22 . in addition , studies have shown that strength of plantar flexion is significantly different between jazz dance and ballet at an angular speed of 120/sec , while strength of dorsiflexion is significantly different between jazz dance and ballet at an angular speed of 30/sec23 . another study reported that dorsiflexion strength is higher in a concentric exercise group compared with an eccentric exercise group , when 12 male athletes with chronic ankle sprain were treated with 8-week isokinetic exercise programs24 . despite the differences in experimental methods , the majority of the results of previous studies that investigated abnormalities of ankles are consistent with the results of the present study . in the present study , a medical examination by interview was performed and the change in isokinetic strength of the ankle was measured before and after a strategic strength resistance exercise program . peak torque , relative strength , and isokinetic hamstring : quadriceps ratios for right plantar bilateral dorsiflexion increased . notably , the differences in the answers to the medical questionnaire and the peak torque were statistically significant . thus , a strategic strength resistance exercise program is necessary for the functional stability of the ankle joint , and active treatments are recommended to prevent or treat chronic impairments . strategic and efficient exercise intervention programs are needed to maintain strength in ankle joint reinjury .

[ purpose ] the purpose of this study was to investigate the effects of a strategic strength resistance exercise program on the isokinetic muscular function of the ankle joint . [ subjects ] this study included 22 males in their twenties who were diagnosed with functional injury of the ankle joint . [ methods ] to strengthen plantar flexion and dorsiflexion of the ankle joint , 8 weeks of weight , resistance band , and plyometric training , and training using props were performed . [ results ] a medical examination by interview indicated that pain , swelling , instability , running , and support capacity of the ankle joint significantly improved with the strategic strength resistance exercise program . for the isokinetic peak torque of the ankles , significant differences were observed in right plantar flexion and bilateral dorsiflexion . [ conclusion ] the strategic strength resistance exercise program is highly recommended for the functional stability of the ankle joint . efficient exercise therapy is useful for muscle damage prevention , muscle strengthening , and functional interventions .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION