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polyunsaturated fatty acids (pufas), such as linoleic acid (la, cis, cis-9,12-octadecadienoic acid), are considered as being the precursors for the synthesis of flavors, mainly lipid - derived aldehydes and alcohols. although pure commercial la has been extensively used as a model substrate in research for the synthesis of lipid - derived flavors, its high cost limited its use at the industrial level [1, 2 ]. hence, edible oils could be alternative and economical sources for la, the pufa of interest. among the most common commercial edible oils, safflower oil (so) is characterized by its high content (70 to 80%) of la, its availability, and its low cost. the literature [1, 4, 5 ] suggested the necessity of hydrolyzing the edible oils, prior to their use as sources of la for the production of flavors. although the use of hydrolyzed edible oils, including sunflower and safflower, for the synthesis of hexanal has already been reported in literature, little information was available on the nature of the hydrolysis process. the harsh conditions of high pressure and temperature of the conventional process of hydrolysis of edible oils could lead to the polymerization of fats, resulting in extremely dark free fatty acids (ffas) that would limit its industrial use [6, 7 ]. the use of enzymes for the hydrolysis of oils, with a high content of pufas, could be hence of great interest since enzyme - catalyzed hydrolytic reactions offer simple operational procedure, high fatty acid selectivity, and low cost as compared to their chemical counterparts [2, 7 ]. lipases (triacylglycerol (tag) hydrolases, ec 3.1.1.3) are by far the most commonly used enzymes for the hydrolysis of oils, resulting into ffas, glycerols, and acylglycerols. because of its ability to efficiently hydrolyze edible oils, the lipase from candida rugosa (crl) has been extensively reported in literature [2, 7, 8 ]. moreover, freitas. showed that the hydrolyzed soybean oil, obtained by lipase from porcine pancreas (ppl), has the highest la content among the investigated enzymes. although commercial ppl and crl have been commonly reported in literature [2, 8 ] for their use in the hydrolysis of oils, the lipase from thermomyces lanuginosus (lipozyme tl i m) is rather widely used for the synthesis of structured lipids ; however, its immobilization on granular silica carrier makes it an attractive lipase, since it facilitates its dispersion, recovery, and reusability as well as providing better stability as compared to its free counterpart. the overall objective of this study was to determine the capacity of selected commercial lipases to hydrolyze so for the liberation of free la for the application as a flavor precursor. the specific objectives were to investigate the effects of selected parameters on the degree of hydrolysis of so and to determine the composition of the hydrolyzed so in free fatty acids as well as mono-, di-, and triacylglycerols. edible safflower oil (so) was purchased from a local market ; its composition in fatty acids (%) was determined by gas chromatography (gc) to be 7.5, 3.8, 16.5, and 72.2 of c16:0, c18:0, c18:1, and c18:2, respectively. two non - immobilized lipases from candida rugosa type vii (crl) and from porcine pancreas type ii (ppl) as well as sodium methoxide (ch3ona) were purchased from sigma chemical co. (st. louis, mo). the hydrolytic activity for crl was 1,449 unit / mg solid enzyme, whereas that for ppl was 59.45 unit / mg solid enzyme when olive oil is used at ph 7.7. in addition, an immobilized lipase from thermomyces lanuginosus (lipozyme tl i m), with 20 unit / mg solid enzyme, was obtained from novo nordisk (copenhagen, denmark). both ppl and lipozyme tl i m are 1,3-regiospecific lipases, whereas crl is a nonspecific lipase. polyoxyethylene sorbitan monolaurate (tween-20), sodium hydroxide (naoh), sigma 7 - 9, whatman 1ps filter, and organic solvents of high - performance liquid chromatography (hplc) grade were purchased from fisher scientific (fair lawn, nj). 12 n hydrochloric acid (hcl), 12 n sulfuric acid (h2so4), and 1 n standard hcl solution were also purchased from fisher scientific. fatty acid methyl ester (fame), thin - layer chromatography (tlc), and hplc standards were purchased from nu - chek prep (elysian, mn). the hydrolytic reaction was carried out in 125 ml erlenmeyer flasks, incubated at 45c, with continuous shaking at 250 rpm, using an orbital shaker - incubator (new brunswick scientific co., inc., edison, nj). the final so concentration (6 mm) in the enzymatic reaction medium was calculated on the basis of its content in trilinolein (tla), as determined by gc. the reaction mixture consisted also of 0.5% (v / v) tween-20 and sufficient tris - hcl buffer solution (0.1 m) to adjust the final volume of the mixture to 30 ml. the ph for crl, ppl, and lipozyme tl i m was 7.2, 7.7, and 8.0, respectively. the enzymatic reaction was initiated by the addition of 1 ml of either crl or ppl suspension (100 mg solid enzyme / ml), prepared in 0.1 m tris - hcl buffer solution at ph 7.2 and ph 7.7, respectively, or by a direct addition of 100 mg of granular lipozyme tl i m to the reaction mixture. in the case of crl, the enzyme suspension was homogenized for 10 sec, prior to its addition to the reaction mixture, using the sonicator ultrasonic processor (model xl2020, heat systems, inc., blank samples, containing all components except the enzyme preparation, were carried out in tandem with the enzymatic trials. at specific intervals, flasks were removed from the orbital shaker - incubator and the degree of hydrolysis was determined by titration, using mettler - toledo dl58 automated titrator (mettler - toledo, schwerzenbach, switzerland) equipped with a 10 ml burette and a sample changer st20a. the equivalent point was determined by measuring the ph change, with the use of a dg101-sc ph electrode, which was calibrated prior to its use. standard hcl solutions of 0.1 and 0.2 n were used to standardize 0.1 and 0.2 n naoh solutions, respectively. a standardized solution of 0.1 n naoh was used for the titration, when the ph of the reaction mixture was greater than 6.7 ; however, a 0.2 n naoh standardized solution was used at ph lower than or equal to 6.7. in order to meet the defined application, preliminary tests were conducted to adjust the equivalent point method 90005 (labx software). the degree of hydrolysis was defined as the percentage weight of ffas in the sample divided by the initial weight of so sample and calculated according to the following equation : (1)oil hydrolysis%=vtevtommwwso100,where vto and vte were the volumes of naoh solution used, respectively, for the sample at time 0 and at the equivalent point ; [m ] is the molar concentration of naoh solution used (0.1 n or 0.2 n) ; mw is the molecular weight of linoleic acid (la) (280.45 g / mol) ; wso is the initial weight of the so sample. the effect of reaction time, 0 to 8 h, on the hydrolysis of so was investigated, using crl, lipozyme tl i m, and ppl as biocatalysts. the effect of enzyme concentration, 0.0 to 4.0 mg solid enzyme / ml reaction mixture, on the hydrolysis of so was studied, at constant reaction time of 3 h for crl and ppl and 6 h for lipozyme tl i m. using the selected lipases, the degree of hydrolysis of so was investigated over a wide range of substrate concentrations, 0 to 8 mm. the enzymatic reaction was carried out for 3 h for crl and ppl and 6 h for lipozyme tl i m, using the optimal biocatalyst amount of 1.3, 2.0, and 3.3 mg solid enzyme / ml reaction mixture for crl, ppl, and lipozyme tl i m, respectively. in order to favor the hydrolysis of so, the effect of temperature, 30 to 55c, on the hydrolytic reaction, catalyzed by the investigated biocatalysts, was investigated. the optimized conditions used for crl and ppl were 1.3 and 2.0 mg solid enzyme / ml reaction mixture, with 6 and 1 mm of so, respectively, and 3 h of reaction time. for lipozyme tl i m, 3.3 mg of granular enzyme / ml reaction mixture, 7 mm of so, and 6 h of reaction time were used. under the optimized reaction conditions, described previously, the effect of ph on the hydrolysis of so by the selected lipases was investigated, using different buffer solutions (0.1 m) of potassium phosphate (ph 6.5 to 6.7) and tris - hcl (ph 7.0 to 8.7). after hydrolysis, the flasks were withdrawn from the orbital shaker - incubator and the enzymatic reaction was halted by the addition of drops of 1 n hcl solution (ph 3.4). the hydrolyzed so (hso) was extracted 3 times with hexane (1 : 1, v / v), with rigorous agitation for 5 min ; the reaction mixture was then left for decantation for 10 min. all traces of protein, water, and tween-20 were eliminated by suction filtration, using a whatman 1ps filter paper. the hexane was evaporated, using a speedvac system (model aes2010, thermo savant, holbrook, ny). any residual traces of hexane were removed by a gentle stream of nitrogen and the recovered hso was stored at 80c for further analyses. the recovered hso was subjected to high - performance liquid chromatography (hplc) analysis, according to the procedure developed in our laboratory. the hplc system was a beckman system (beckman instruments inc., san ramon, ca), equipped with a computerized integration and data handling system (model 126), using a 32 karat software version 8.0 and a uv - vis diode - array detector (dad - model 168). the recovered hso was separated on a normal - phase- (np-) zorbax rx - sil column (250 4.6 mm, i.d. the absorbance was measured simultaneously at 205 and 234 nm to monitor the presence of any linoleic acid hydroperoxides (hpods) in the recovered hso, resulting from the la autooxidation. the isocratic eluent system was a mixture of hexane/2-propanol / acetic acid (990 : 10 : 1, v / v / v) at a flow rate of 1.0 ml / min, for a total elution time of 26 min. aliquots of so and hso, obtained by crl, were analyzed by thin - layer chromatography (tlc) on silica gel 60 plates, with fluorescent indicators (whatman, fisher scientific). the tlc was carried out with a saturated solvent mixture of hexane / diethyl ether / acetic acid (140 : 60 : 1, v / v / v). the tlc plates were visualized under visible light, after their spraying with 20% (v / v) sulfuric acid. acylglycerols standard (ags - std), composed of 25% equal amount of methyl linoleate, mono-, di-, and trilinolein, as well as a mixture of free fatty acid standards (la / oa - std), containing 67% linoleic acid and 33% oleic acid, was used as references. retention factor (rf), defined as the migration distance of a component over that of the solvent, was used to characterize the different components of so and hso. the high - performance liquid chromatography (hplc) analysis of hso, obtained by crl, was carried out according to the method developed in our laboratory. the separation of different components was performed on an agilent zorbax sb - c18 reversed - phase column (250 4.6 mm, i.d. a volume of 20 l sample was diluted to 320 l with isopropanol. the diluted sample was filtered and 20 l of the filtrate was subjected to hplc analysis. the elution of the injected sample was carried out by a gradient solvent system, using methanol as solvent (a) and isopropanol as solvent (b). the elution was initiated by an isocratic flow of 100% of solvent a for 10 min, followed by a 10 min linear gradient to 40 and 60% of solvents a and b, respectively, and then to 100% of solvent b for 10 min period. the elution was maintained for an additional period of 5 min before reverting it to the initial conditions (100% solvent a), followed with an equilibration period of 10 min for the next sample. the flow was 1 ml / min and the detection was performed at 215 nm for monitoring the lipid components. so was also analyzed by hplc to take into account the presence of any trace amounts of ffas, mono-, and diacylglycerols. fatty acid methyl esters (fames) of so and hso were prepared according to badings and de jong to determine their composition in fa and ffa, respectively. for the preparation of fames of so, fifty mg of so was diluted in 0.6 ml of hexane, followed by the addition of 60 l of 2 m sodium methoxide in 20% methanol. the mixture was incubated in reciprocal shaking water - bath (model 25, precision scientific, chicago, il) at 65c. after 20 min of incubation, 1 ml of 10% sulfuric acid solution prepared in absolute methanol was added, followed by its incubation at 85c for 30 min. for the preparation of the free fames of hso, fifty mg of the recovered hso was diluted in 0.6 ml of hexane, followed by the addition of 1 ml of 20% hcl solution prepared in absolute methanol. the mixture was incubated in reciprocal shaking water - bath at 85c for 15 min. the fames of so and hso were then extracted 3 times with 4 ml hexane. the mixture was centrifuged (1,600 rpm, 5 min) and the upper layer was recovered, where the organic solvent was evaporated with the use of a speedvac system (model aes1010, savant, holbrook, ny). the residual traces of the organic solvent were removed by a gentle stream of nitrogen. the recovered fames were diluted, with hexane, prior to gas chromatography (gc) analysis. the fatty acid composition of so as well as the free ones of the hso, obtained by crl, was analyzed, by gas - liquid chromatography (gc), using agilent 6890 series (gc) system (agilent technologies, wilmington, nc), equipped with computerized integration and data handling (gc chemstation g2075aa, version a.09.03, agilent) software and a flame ionization detector (fid). the separation of the different fames was performed on a hp - innowax polyethylene glycol fused capillary column (30 m 0.25 mm, i.d. 0.25 m film thickness, agilent), with a flow rate of carrier gas (he) of 1 ml / min. the oven temperature was programmed as follows : 150c during the first 1 min and then it increased to 200c, at 10c / min ; the temperature was then increased to 220c, at 1c / min, and it was held for 5 min before a final increase to 225c, at 1c / min, where it was held for 5 min. the flow rates for the hydrogen and air were set at 40 and 400 ml / min, respectively. was calculated as the standard deviation (sd) of triplicate trials divided by their mean multiplied by 100. one - way analysis of variance (anova) test was used to determine the differences among several groups, followed by the holm - sidak test for pairwise comparisons. for one - way anova, using the selected lipases, the time course for the enzymatic hydrolysis of safflower oil (so) is shown in figure 1. over a wide range of reaction time (0 to 6 h), the use of ppl as biocatalyst showed that the level of hydrolysis of so after 3 and 6 h was 36.9 and 41.8%, respectively. with the use of crl as biocatalyst, the degree of hydrolysis of so significantly (p 0.05). on the other hand, the immobilized lipozyme tl im - catalyzed reaction showed a significant (p 0.05) decrease in the degree of hydrolysis after an additional 2 h of reaction time reaching 86.7%. pairwise comparisons of the degrees of hydrolysis of so, obtained at 3 h for ppl and crl and 6 h for lipozyme tl i m, were found to be statistically significant (p 0.05) change in the degree of hydrolysis when the amount of enzyme was increased to 3.3 mg solid enzyme / ml reaction mixture. the results also indicated that the degree of hydrolysis of so by ppl, with an enzyme concentration lower than 0.7 mg solid enzyme / ml reaction mixture, was deviated from the hyperbolic michaelis - menten plot ; however, the degree of hydrolysis was significantly (p 0.05) increase to 36.9%, with 3.3 mg solid enzyme / ml reaction mixture. using lipozyme tl i m as biocatalyst, a limited but significant (p 0.05) change in the degree of hydrolysis. although the use of lipozyme tl i m resulted in the highest degree of hydrolysis of so, it required 1.7- and 2.5-time amount of enzyme as compared to that of ppl and crl, respectively. moreover, while there was a statistical significant (p 0.05) difference in the degree of hydrolysis of so between crl and tl i m at 1.3 and 3.3 mg solid enzyme / ml reaction mixture, respectively. the literature [7, 17, 18 ] reported similar findings for the hydrolysis of various vegetable oils by crl. the limited increase in the degree of hydrolysis at higher enzyme concentrations may be due to the enzyme - saturation at the interface between the oil and the aqueous phase in which any further increase in the amount of enzyme would show negligible effect [7, 18, 19 ]. o'connor and bailey indicated that the hydrolysis of tributyrin by ppl exhibited a sigmoidal curve, which was attributed to cooperative interactions between the lipase and its coenzyme, present in its preparation ; these authors suggested that ppl could be allosterically regulated by effectors. on the basis of these experimental findings, the optimum amount of crl, ppl, and lipozyme tl i m for the hydrolysis of so was determined to be 1.3, 2.0, and 3.3 mg solid enzyme / ml reaction mixture, respectively, and was used consequently throughout this study. using the investigated lipases, the degree of hydrolysis of so was determined over a wide range of substrate concentrations, 0 to 8 mm. the experimental findings (figure 3) suggest that the lipolytic activity of the investigated lipases deviated from the hyperbolic michaelis - menten plot. using ppl as biocatalyst, the degree of hydrolysis significantly (p 0.05) decrease in the degree of hydrolysis of so from 90.6 to 84.1% with the increase in substrate concentration, from 1 to 6 mm, before a significant (p 0.05) difference in the degree of hydrolysis of so with the increase in substrate concentration from 1 to 8 mm. in addition, there was an insignificant (p > 0.05) difference in the degree of hydrolysis between that for crl, with 6 mm of so, and that for ppl and tl i m, using 1 and 7 mm of so, respectively. serri. reported that the degree of hydrolysis of palm oil by crl decreased steadily with the increase in substrate concentration ; it was suggested that such behavior may be due to the saturation of the active site of lipase by the oil phase, limiting hence the availability of the enzyme for the substrate. kwon and rhee reported similar findings for the free and immobilized crls, with tributyrin concentration greater than 3 and 4% (v / v), respectively ; this was attributed to an increase in the hydrophobicity of the system, reducing hence the activity of the lipase. o'connor and bailey proposed that the enzyme inhibition at higher substrate concentrations may be due to an interaction between the insoluble substrate, such as tributyrin, and the lipase. in contrast, noor. reported that the degree of hydrolysis of palm oil by lipase sp398, from h. lanuginosa, increased linearly as a function of oil concentration. chew. indicated similar findings, upon the hydrolysis of palm olein by lipozyme tl i m. based on the experimental findings, the optimum substrate concentration for crl, ppl, and lipozyme tl i m was determined to be 6, 1, and 7 mm, respectively, and was used for further optimization of so hydrolysis. the effect of temperature on the enzymatic hydrolysis of so was investigated. using crl as biocatalyst, the results (figure 4) show a statistical significant (p 0.05) increase in the degree of hydrolysis of so from 30 to 45c, followed by a significant (p 0.05) decrease to 80.4% at 55c. the experimental findings could be explained by the fact that the immobilization of the enzyme may have restricted its movement by its contact with the support, enhancing hence its rigidity ; as a result, the stability of the enzyme is enhanced with respect to extreme temperatures. while there was a statistical significant (p 0.05) difference in the degree of hydrolysis between crl and tl i m at 45 and 50c, respectively., who reported that the optimum temperature for the hydrolysis of palm oil was 45c ; these authors suggested that the significant decrease in hydrolysis at temperatures higher than 45c could be due to the disruption of the enzyme tertiary structure, which could result in its denaturation. similar findings were also reported by fadilolu and sylemez, when olive oil was hydrolyzed by celite - immobilized crl. in contrast, maidina. indicated that the hydrolysis yield of so by crl at 45c was less than half of that obtained at 30c ; these authors concluded that the temperature was a crucial parameter for crl, affecting not only the enzyme activity and its stability but also the state of the reaction medium and/or interface. the differences in the optimal temperature for crl, obtained by maidina. and those reported in figure 4, could be due to the choice of the surfactant used for the stabilization of the interface. in addition, kwon and rhee investigated the temperature effect on the hydrolysis of tributyrin and triolein, using free and immobilized crl, where the optimum temperature for the free and immobilized enzyme was 50 and 60c, respectively. goswami. reported that the optimum temperature for the hydrolysis of castor oil by crl was 35c. these authors suggested that the temperature change affected the rate of the lipase - catalyzed hydrolysis of vegetable oils and the thermal inactivation of the enzyme itself, indicating that, at low temperatures, the rate of thermal inactivation of lipase was negligible, whereas that of the hydrolysis of oil increased as a function of temperature increase ; however, at high temperatures the rate of thermal deactivation of lipase became more prominent and hence there was a decrease in the rate of the hydrolysis of oil. based on the experimental findings, further studies were performed for crl, ppl, and lipozyme tl i m, using 45, 35, and 50c as their optimum temperature, respectively. a change in ph is known to have an effect on the ionization of both free substrate and enzyme. in order to maximize the hydrolysis of so by the investigated lipases, the enzymatic trials were carried out in a wide range of ph, varying from 6.5 to 8.7. the results (figure 5) show that crl exhibited its highest degree of hydrolysis (84.1%) at ph 7.2, whereas ppl reached its maximum (91.6%) at ph of 8.2 ; however, further increase or decrease in the ph resulted in an overall significant (p 0.05) increase to 90.2% at ph 8.0 ; nevertheless, at ph greater than 8.0, the degree of hydrolysis significantly (p 0.05) difference in the degree of hydrolysis among the selected lipases. on the basis of the experimental findings, crl was selected as the most appropriate biocatalyst for the hydrolysis of so. the so and its hydrolyzed product (hso) were first analyzed by tlc (figure 6), using acylglycerols standard (ags - std), composed of 25% equal amount of methyl linoleate (ch3-la), monolinolein (mla), dilinolein (dla), and trilinolein (tla), as well as a mixture of free fatty acid standards (la / oa - std), composed of 67% linoleic acid (la) and 33% oleic acid (oa) ; the retention factor (rf) was used to characterize and to identify the different components of so and hso. figure 6 shows that only two bands at close positions were obtained for so and hso, with the first one having rf value of 0.27 and 0.37 for so and hso, respectively, characteristic of ch3-la, and the mixture of la / oa, whereas the second band has rf value of 0.80 and 0.84 for so and hso, respectively, characteristic of the triacylglycerols (tags). there was a lack in mono- (mags) and diacylglycerols (dags) presence in the hso, which could suggest that the hydrolysis of so resulted mainly in ffas and trace amount of tags. figures 7(a) and 7(b) demonstrate the elution profile of the hplc analysis of ags - std and la / oa - std, respectively, whereas figures 7(c) and 7(d) show that of so and hso, respectively. peaks # 4, 7, 8, and 12 (figure 7(a)) correspond to mla, ch3-la, dla, and tla, respectively, whereas, peaks # 1, 5, and 6 (figure 7(b)) correspond to linoleic and oleic acids. the tags of so were identified as peaks # 9 to 16 (figure 7(c)), with peak # 12 characteristic of tla. after hydrolysis of so by crl, the experimental results (figures 7(c) and 7(d)) show a significant decrease in tags, with the appearance of two new major peaks, # 5 and 6, which were characterized along with peaks # 1, 2, and 3 as ffas. using a calibration curve and a linear equation of y = 2000000x + 729216, with r of 0.9813, the trilinolein concentration in the hso was determined to be 8.19 mm (data not shown). the absence of mags and dags in the hso could be due to the nonspecificity of crl as opposed to the other specific lipases, such as ppl and lipozyme tl i m [2, 23 ]. the relative content of ffas in so and hso (table 1), obtained by hplc, was found to be 1.4 and 91.1%, respectively, with a net value of 89.7% for the ffas in hso which is close to the optimum degree of hydrolysis of so (84.1%), determined by titration. gas chromatography (gc) analysis of hso was carried out to determine its relative composition in ffas (data not shown). the composition of ffas of hso was determined to be c16:0, c18:0, c18:1, and c18:2, with a relative content of 8.3, 3.6, 15.3, and 72.8%, respectively. the relative fa composition of hso was found to be close to that of so ; these experimental findings could be attributed to the nonspecificity of crl. in contrast, freitas. reported that while the soybean oil has a low percentage of oleic and palmitic acids in its composition, high concentrations of these fas were found in the crl - hydrolyzed soybean oil. the results gathered in this study showed that, among the investigated catalysts, the lipase from candida rugosa was found to be the most appropriate one for the hydrolysis of safflower oil (so) ; its use resulted in high concentrations of free linoleic acid (la) that can be used as a substrate for the production of the flavor precursors, linoleic acid hydroperoxides. the overall experimental findings could contribute to the development of a bioprocess for the synthesis of natural flavor precursors by a biotechnological approach that is economically viable.	commercial lipases, from porcine pancreas (ppl), candida rugosa (crl), and thermomyces lanuginosus (lipozyme tl i m), were investigated in terms of their efficiency for the hydrolysis of safflower oil (so) for the liberation of free linoleic acid (la), used as a flavor precursor. although ppl, under the optimized conditions, showed a high degree of hydrolysis (91.6%), its low tolerance towards higher substrate concentrations could limit its use for so hydrolysis. in comparison to the other investigated lipases, lipozyme tl i m required higher amount of enzyme and an additional 3 h of reaction time to achieve its maximum degree of so hydrolysis (90.2%). on the basis of the experimental findings, crl was selected as the most appropriate biocatalyst, with 84.1% degree of hydrolysis. the chromatographic analyses showed that the crl - hydrolyzed so is composed mainly of free la.
cardiac anesthesia is associated with ischemia - reperfusion injury. trying to limit the extent and mitigate the consequences of ischemia, volatile agents have been shown to improve outcome in cardiac surgery. despite these data, volatile anesthesia is not the most widely used technique for cardiac anesthesia. in the previous issue of critical care, steurer and these data have to be placed in the context of already - accumulated evidence and potential clinical applications. anesthetic gases are fascinating drugs : first - generation anesthetic gases, especially halothane, had significant cardiac side effects, increasing the risk of malignant arrhythmia and sensitizing the heart to catecholamines. further generations, especially sevoflurane, have been shown to have interesting neuro- and cardioprotective properties. in the last 10 to 15 years, a wealth of data has shown that peri - operative administration of halogenated agents is associated with myocardial protection and better outcome. darwin proposed that species adapt to environment, the better - adapted breed having the better chances of survival. interestingly, at the tissue, cell, mitochondrion, and possibly the gene level, there is a dynamic adaptation to the environment. this led to the concept of preconditioning, a mechanism in which brief sublethal periods of ischemia will provide protection from a subsequent lethal ischemia and mitigate the effect of ischemia - reperfusion. as stated by friedrich nietzsche, ' what does not destroy me, makes me stronger '. the organism or the tissue will acquire some ' injury - resistant ' phenotype for a certain period of time. ischemic pre - conditioning is an interesting and powerful concept, but its clinical applicability is limited by the already - jeopardized myocardium. all hypnotics decrease myocardial consumption and may favor the myocardial oxygen supply / demand balance. on top of these macrohemodynamic effects, halogenated agents have intrinsic cardio- and neuroprotective effects that are similar to those of ischemic pre - conditioning. at a basic science level, the myocardial protection effects of sevoflurane involve apoptotic mrna inhibition, neuromodulation, cytokine / inflammation modulation, redox - sensitive path ways, endothelial preservation, ion channels, notch signaling pathways, and probably other mechanisms to be discovered. this translates clinically as a decrease in post - cardiac surgery troponin levels, lower inotropes requirements, and better cardiac output. preconditioning is well established, and volatile agents are now recommended as the agent of choice by the american heart association for high - risk patients. however, pre - conditioning is difficult to apply once the injury has already been established. interestingly, brief periods of ischemia at the onset of the reperfusion period are associated with cardioprotection, leading to a myocardial infarction size decrease similar to or slightly inferior to that of ischemic pre - conditioning. isoflurane has been shown to improve remodeling after coronary artery occlusion in rats. in a similarly designed animal model of circulatory arrest published last year in this journal, sevoflurane administered immediately after the return of spontaneous circulation decreased myocardial damage. the mechanisms of volatile agent post - conditioning are being explored by several laboratories around the world. lemoine and colleagues showed on in vitro right atrial appendages that the human heart is sensitive to desflurane - associated post - conditioning. the data from steurer and colleagues offer the ' first in man ' evidence of post - conditioning and potential myocardial protection in a clinical setting : sevoflurane administered in the intensive care unit (icu) arrival for 4 hours. interestingly, there was still an effect even with a 2- to 3-hour gap after the onset of ischemia (aortic clamping) and a 1- to 2-hour gap between the start of reperfusion (aortic cross - clamp release) and the administration of sevoflurane (upon arrival to the icu). further questions remain to be answered to optimize and assess the clinical magnitude of myocardial protection provided by post - conditioning (table 1). some research questions regarding sevoflurane post - conditioning sevoflurane and isoflurane are very flexible generic anesthetic drugs administered daily by numerous anesthetists around the world. the demonstration of cardio - or neuroprotective effects (or both) when applied after the ischemia will make them interesting in myocardial ischemia, cardiogenic shock, or even traumatic brain injuries. it has been shown to be associated with fast awakening / weaning times after the drug is stopped. the anesthesia - conserving device has made the delivery simple and adaptable on ' regular ' icu - type ventilators. however, technical aspects such as gas analyzers, concentration measurement, and exhausted gas scavenging (most icu - type ventilators release the exhaust gases into the environment) need to be taken into account. educational aspects must include nurse training, dosage, availability of dantrolene, and training for the unlikely event of a malignant hyperthermia reaction. in addition, data show plasma fluoride concentration (39 25 mol / l) close to safety limits (50 mol / l) at 24 and 48 hours after 9 hours of sevoflurane. sevoflurane administration beyond 12 to 24 hours needs to be assessed in terms of fluoride plasma concentration and nephrotoxicity. most cardiac surgery patients require a maximum of a few hours of mechanical ventilation (if any). in these circumstances, sevoflurane sedation in the cardiac icu may be a very flexible option with an additional myocardial protection benefit. sevoflurane and possibly other volatile anesthetic agents show promising post - conditioning properties after cardiac surgery. this opens a new field of investigations and potential therapies aiming at mitigating secondary myocardial injury after the primary injury is done. more data are necessary to assess the magnitude conferred by this protection, its clinical relevance, the window of opportunity, and the collateral protection on other organs.	volatile anesthetic agents have been used for decades in the peri - operative setting. data from the past 15 years have shown that pre - injury administration of volatile anesthetic can decrease the impact of ischemia - reperfusion injury on the heart, brain, and kidney. recent data demonstrated that volatile agents administered shortly after injury can decrease the ischemia - reperfusion injury. several questions need to be answered to optimize this therapeutic target, but this is a promising era of secondary injury mitigation.
the emission of ultrasonic vocalizations (usvs) is a major means of communication used by rats [1 - 4 ]. three types of rat usvs have been identified so far, which are categorized according to their average peak or dominant frequency, namely the 50-khz usvs. while 40-khz usvs are emitted by rat pups, 22-khz and 50-khz usvs detailed descriptions of the origin, neurobiological, and pharmacological mechanisms of usv emission by young and adult rats, along with their acoustic features, are provided elsewhere in this issue. several lines of evidence have demonstrated that rats emit usvs in response to a wide range of stimuli that may produce either euphoric (positive) or dysphoric (negative) states. rat pups emit 40-khz usvs, termed also distress calls, when separated from their mother and litter, since they perceive this situation as stressful and threatening. in the young (post - weaning rats) and adult rats, stressful and threatening situations initiate emission of 22-khz usvs, whereas appetitive and pleasurable situations promote emission of 50-khz usvs. taken together, these findings envision usvs as a powerful tool in preclinical studies of affect, motivation, and social behavior. emission of usvs by rats can be also influenced by certain classes of drugs [10 - 15 ]. this observation is of paramount interest, as it indicates that measurement of rat usvs can have relevance not only to neurobiological mechanisms, but also to neuropharmacology and psychopharmacology. in line with this, rat usvs are currently being used at the preclinical level to study the effects elicited by different classes of drugs, and to study the neurobiological mechanisms of these effects. usvs appear particularly suitable for neuropharmacological studies, as they have a marked ethological component and are emitted by rats in natural situations. therefore, the use of usvs can provide straightforward experimental models that usually do not necessitate extensive training on the part of researchers and/or use of other complex procedures (e.g. food or water deprivation), which may affect the intended results. this review summarizes the potential use of rat usvs in neuropharmacology and drug evaluation, and discusses the strengths and limitations of usvs - based experimental paradigms. attention will be paid to the different classes of drugs that can be studied by recording and analysis of rat usvs, and to the different experimental models that can be paired with and complemented by evaluation of rat usvs. when separated from their mother and littermates, rat pups emit usvs which comprise broad range of frequencies between 30 and 65 khz, and are generally referred to as distress calls or 40-khz usvs, based on their average peak frequency. rat pups develop the ability to emit these usvs shortly after birth and maintain it until weaning, at about 18 days of age. in order to understand the behavioral significance of 40-khz usvs, it has to be considered that newborn rat pups are completely dependent on their mother for surviving, and that they emit 40-khz usvs specifically in response to isolation from the nest and mother. these observations have suggested that the emission of 40-khz usvs in response to isolation may be regarded as a direct correlate of distress and/or anxiety in the rat pup. this view is also corroborated by the results of other independent studies that have shown how anxiolytic drugs reduce the number of 40-khz usvs emitted by isolated rat pups. interestingly, different classes of anxiolytic drugs are able to elicit this effect, including those most commonly used in the clinic such as benzodiazepines and agonists of the serotonin 5-ht1a receptors [17 - 19 ] (table 1). moreover, the emission of 40-khz usvs by isolated rat pups can be attenuated by drugs that possess combined anxiolytic and antidepressant properties, such as selective inhibitors of the serotonin transporter (ssris) (table 1). further support to the hypothesis that emission of 40-khz usvs may be a juvenile homologue of anxiety in rat pups comes from the evidence that this behavioral response is exacerbated when pups are either administered pentylenetetrazole (ptz), a drug that possess anxiogenic properties, or exposed to low temperatures that act as a stressful stimulus. taken together, these findings indicate that evaluating the magnitude of 40-khz usvs emission in isolated rat pups may represent a useful tool for screening the anxiolytic properties of drugs. notwithstanding these findings, it has to be pointed out that while measuring 40-khz usvs in rat pups may provide a reliable experimental model for evaluating benzodiazepine - like drugs and agonists of the serotonin 5-ht1a receptors, the same paradigm may not be suited for screening the anxiolytic properties of other classes of drugs. in this regard, previous studies have shown that certain drugs that have clinically relevant anxiolytic potential (e.g. clonidine, an agonist of norepinephrine 2 receptors) amplify, instead of attenuating, the emission of 40-khz usvs by isolated rat pups (table 1). therefore, a possible occurrence of false negative results should be carefully considered when utilizing this behavioral paradigm for evaluating the anxiolytic properties of new drugs. moreover, it is interesting to mention that, although drug screening studies usually focus on the number of vocalizations emitted, drugs can also influence the duration of 40-khz usvs in isolated rat pups, (table 1). a recent study that compared the effects of various drugs with anxiolytic properties on emission of 40-khz usvs has indicated that the duration of these usvs may be more sensitive to the anxiolytic effects of drugs than the number of vocalizations emitted. in addition, the same study has suggested that the measurement of duration of 40-khz usvs could enable easier discrimination of anxiolytic effects from potential sedative effects of drugs than the number of vocalizations. therefore, a combined analysis of both the numbers and the duration of 40-khz usvs emitted by isolated rat pups could help to show anxiolytic effects of drugs that are not adequately revealed by recording the number of vocalizations only. besides the data demonstrating that 40-khz usvs emitted by isolated rat pups are sensitive to the anxiolytic properties of drugs, further experimental findings have been collected that suggest a link between these usvs and anxiety - like states. previous studies have consistently demonstrated that different neurotransmitters known to participate in the pathophysiology of anxiety, such as -aminobutirric acid (gaba), endogenous cannabinoids, and glutamate, can modulate the emission of 40-khz usvs by isolated rat pups. moreover, experiments performed in the tsukuba genetic model of low (tle) and high (the) emotional rat pups have shown that the emission of 40-khz usvs stimulated by isolation is significantly more marked in the pups, which also show a more pronounced anxiety - like phenotype at adulthood, compared with tle pups. furthermore, a reduced binding of the antagonist flumazenil to the benzodiazepine receptor has been described in the brain of rat pups subjected to isolation, and a similar finding has been observed in patients suffering panic disorders by means of single - photon emission computed tomography (spect) studies with the antagonist iomazenil. this latter finding has led to the hypothesis that the emission of 40-khz usvs elicited by maternal separation in rat pups could have face validity towards the separation - induced anxiety that can occur in children, although this has to be conclusively demonstrated. taken together, these findings indicate that 40-khz usvs emitted by isolated rat pups could serve as a model suited not only for the screening of anxiolytic drugs, but also for studying the neurobiological mechanisms of anxiety, the individual traits, and the neuro - developmental factors that may promote the manifestation and influence the severity of this pathology. in addition to being suited for the study of anxiety - like states and their pharmacological treatment, 40-khz usvs emitted by isolated rat pups may be of interest for the investigation of all those conditions, either pharmacological or pathological, that may harm the fetus and the newborn during the prenatal and perinatal phases of development. this experimental use of 40-khz usvs is supported by evidence showing that the ability of rat pups to vocalize in a species - typical manner when isolated from the nest is dependent on the integrity of the central nervous system (cns) (see for a discussion). therefore, modifications of this behavioral response can be assumed as indicative of neurological impairments in the affected pups. furthermore, the use of 40-khz usvs triggered by isolation allows to study not only those insults that may directly target the pup after birth (e.g., perinatal asphyxia and hypoxia), but also those factors (e.g., infections) and maternal behaviors (e.g., substance abuse) that can harm the fetus during gestation. modifications in the number of 40-khz usvs emitted and their acoustic features have been reported in isolated rat pups previously exposed to diverse neurological insults, such as febrile convulsions, global asphyxia, perinatal hypoxic - ischemic encephalopathy, and gestational exposure to lipopolysaccharide [30 - 33 ]. interesting findings have also been obtained in rat pups delivered by dams allowed to drink ethanol during gestation. the pups exposed to ethanol have been reported to emit lower numbers of 40-khz usvs following isolation, compared to non - exposed pups. also, changes in vocalization in ethanol - exposed pups were found to be associated with other behavioral impairments, such as loss of righting reflex, reminiscent of some of the symptoms observed in infants suffering from fetal alcoholic syndrome (fas). similar deficits in the emission of 40-khz usvs following isolation have been reported in rat pups delivered by dams treated with cocaine during pregnancy. moreover, it is worth mentioning the results of other studies which have demonstrated that drugs of abuse that may be toxic for the brain impair the emission of 40-khz usvs by isolated rat pups when administered in the post - natal period. such results have been reported for ethanol, 3,4-methylenedioxymethamphetamine (mdma, or ecstasy), and morphine [35 - 37 ]. besides the relevance to pharmacological and toxicological insults, it is noteworthy that the analysis of 40-khz usvs emitted by isolated rat pups in different situations may also be used for investigating the pups ability to communicate with their mother, which is a critical process in neurobehavioral development. in this regard, it is worth mentioning that an increased emission of 40-khz usvs has been observed in isolated rat pups subjected to prenatal stress. moreover, a decreased emission of these usvs has been reported in rats that underwent early maternal separation. interestingly both these experimental models reproduce factors that can critically influence the proper development of social and communicative functions. the findings discussed so far suggest that parameters of 40-khz usvs emitted by rat pups in response to isolation may be regarded as a behavioral correlate of neurological and neuropsychiatric abnormalities. however, other lines of evidence suggest that the emission of 40-khz usvs by isolated rats pups may be critically influenced by the functioning of the cardiovascular system, and that a decrease in venous return may trigger these usvs. as mentioned earlier, the emission of 40-khz usvs by isolated rat pups can be powerfully stimulated by exposure to cold, which is associated with an increase in blood viscosity and a reduction in heart rate. moreover, the administration of antihypertensive drugs, such as the above mentioned clonidine, chlorisondamine (a ganglionic blocker that elicits vasodilation and bradycardia), and sodium nitroprusside, (a directly acting dilator of arteries and veins) has been demonstrated to stimulate the emission of 40-khz usvs by isolated rat pups (table 1). the possible influence of cardiovascular function on the emission of 40-khz usvs deserves consideration. on the one hand, this might contribute to explanation of the false negative results that may be observed in this paradigm after the administration of anxiolytic drugs (see above). in line with this, it can be hypothesized that the effects elicited by clonidine on 40-khz usvs in isolated rat pups could stem from the antihypertensive, rather than anxiolytic, properties of the drug. on the other hand, the emission of 40-khz usvs by isolated rat pups could be envisioned as a relevant behavioral parameter to be used in the investigation of cardiovascular development and toxicity. however, this has not specifically been addressed yet, and exhaustive experimental studies need to be performed to corroborate this proposed use of 40-khz usvs. taken together, the available experimental evidence indicates that the emission of 40-khz usvs by isolated rat pups may represent a valid experimental tool for both the screening of new drugs, in particular anxiolytic drugs, and the investigation of the factors that can harm the newborn and influence its development. although the concept that rat pup 40-khz usvs may be a straightforward correlate of anxiety has been questioned, and some limitations, such as the occurrence of false negative results, may complicate the use of rat 40-khz usvs for drug evaluation, they still represent a significant measure of developing anxiety in rat pups. young and adult rats emit the so - called 22-khz usvs in response to a wide series of stimuli they perceive as threatening ; therefore 22-khz usvs are often referred to as these vocalizations are characterized by a relatively constant frequency and a long duration (up to seconds). it needs to be mentioned that the duration of 22-khz usvs is significantly longer than that of both 40-khz and 50-khz usvs, that are generally of 20 - 150 ms in duration. rats can also emit 22-khz usvs with a shorter duration. with regard to these latter usvs, it is interesting to mention that a recent study has demonstrated that rats that self - administer binges of cocaine emit short 22-khz usvs. furthermore, earlier investigations have observed the emission of short 22-khz usvs in nave rats subjected to handling and in rats exposed to foot shocks. while these latter findings seem to suggest that short 22-khz usvs may be produced in response to aversive stimuli, the precise behavioral significance of these usvs is still unclear, and studies that evaluate the emission of 22-khz usvs usually rely on those vocalizations that have a long duration (see for a discussion on the possible communicative function of the short 22-khz usvs). the use of 22-khz usvs in neuropharmacology and drug screening chiefly involves the study of anxiolytic and mixed anxiolytic / antidepressant drugs. moreover, the emission of 22-khz usvs can be evaluated, together with rats performance in other behavioral paradigms, in investigations aimed at elucidating the mechanisms and neuronal circuits that are involved in neuropsychiatric disorders (e.g. anxiety, depression). different types of experimentally - delivered stressful stimuli, such as air puffs, electric foot - shocks, and loud noises, can stimulate the emission of 22-khz usvs, and these stress - induced vocalizations have been proposed to model an anxiety - like state in the rat. in general, the use of 22-khz usvs for the screening of anxiolytic drugs relies on conditioning paradigms, in which rats are expected to vocalize in anticipation of a stressful stimulus, usually an electric foot - shock, to which they have previously been exposed [47 - 50 ]. although different experimental protocols exist, which may differ with regard to the apparatus used and the duration of conditioning, the procedure to attain the conditioned emission of 22-khz usvs by rats is generally performed as follows. rats are firstly individually placed in a soundproof chamber and randomly exposed to a train of unavoidable foot - shocks [47 - 49 ]. to facilitate conditioning, in some variants of this procedure, foot - shocks are paired with other contingent stimuli, either visual or acoustic [47 - 49 ]. conditioning to foot - shocks is then repeated until the test day, when rats are exposed to the same chamber but no foot - shocks are delivered. in the course of this experimental paradigm, rats will progressively develop environmental conditioning, and will emit anticipatory 22-khz usvs when re - exposed to the environment where unavoidable foot - shocks were previously delivered. this behavioral response is based on the fact that rats will perceive this situation as threatening since it has been previously associated with the presentation of unavoidable stressful stimuli [47 - 49 ]. when this procedure is applied to drug screening, the endpoint is to evaluate whether drugs attenuate or suppress the emission of 22-khz usvs by rats conditioned to this situation. other studies have demonstrated that drugs with clinically relevant anxiolytic properties, such as the benzodiazepine diazepam and the 5ht1a receptor agonists buspirone and gepirone, effectively attenuate the emission of 22-khz usvs by rats conditioned to stress (table 2). further support to a possible link between stress - induced 22-khz usvs and anxiety comes from studies showing that the emission of these vocalizations is usually associated with behaviors that are thought to indicate an anxiety - like state in rats (e.g. freezing, decreased rearing), and is exacerbated by the administration of the anxiogenic drug ptz. in line with this finding, it is also noteworthy that some studies have demonstrated that drugs that attenuate stress - induced 22-khz usvs are often also able to counteract anxiety - like behaviors in well - standardized behavioral paradigms, such as the elevated - plus maze (epm) or the vogel conflict test [53 - 55 ]. taken together, these observations indicate that the emission of stress - induced 22-khz usvs can be considered an appropriate experimental model to be used in the evaluation of the anxiolytic properties of drugs. nevertheless, this paradigm has some limitations which have to be carefully considered in order to properly interpret the results obtained. studies with benzodiazepines have demonstrated that while diazepam significantly attenuates the emission of stress - induced 22-khz usvs by rats, other drugs in this class (e.g. chlordiazepoxide, zolpidem) are significantly less effective. moreover, diazepam has often been reported to act on stress - induced 22-khz usvs when administered at doses close to those at which the drug elicits sedative effects. taken together, these findings indicate that the effects of benzodiazepines on the emission of stress - induced 22-khz usvs by rats may vary with the specific drug evaluated. in addition, they suggest that complementary tests to evaluate the presence of sedation should be performed when benzodiazepine - like drugs are screened by measuring their effects on stress - induced 22-khz usvs. additional pharmacological studies have suggested that the emission of 22-khz usvs by rats conditioned to stress could also mimic panic - like anxiety, and that this paradigm may represent a particularly useful tool for testing drugs designed for panic - like disorders. a significant attenuation of the emission of stress - induced 22-khz usvs has been reported in rats that were administered either with ssris, such as citalopram, fluoxetine, paroxetine, or with the benzodiazepine alprazolam, all of which have specific indication for the treatment of panic - like anxiety. in line with what discussed above, the efficacy of alprazolam has been found to be higher than that of other benzodiazepines, which would further corroborate the proposed link between stress - induced 22-khz usvs and panic - like anxiety. the vast majority of drug screening studies that have investigated the emission of 22-khz usvs by rats conditioned to stress have employed acute administration protocols. few studies have been performed so far that have evaluated the effects of chronic drug administration on stress - induced 22-khz usvs, and these studies have yielded mixed results. on the one hand, rats chronically administered with benzodiazepine - like drugs have been reported not to develop tolerance to the drug - induced decrease in the emission of stress - induced 22-khz usvs. on the other, the emergence of tolerance to this effect has been described in rats conditioned to stress and chronically treated with novel anxiolytic agents, such as agonists of the neuro - tensin nts1 receptor. therefore, while the attenuation of stress - induced 22-khz usvs stands as a valid and widely used paradigm for the evaluation of the anxiolytic properties of drugs, all the above described limitations should be carefully considered. in particular, the thorough elucidation of the chronic effects of drugs on these vocalizations appears particularly relevant to the development of new anxiolytics, as the clinical treatment of anxiety and its related disorders may often require drugs that are able to maintain their effects after a long - term treatment. in addition to the use in the screening of anxiolytic drugs, stress - induced 22-khz usvs may be of interest for the investigation of the neurobiological bases of emotionality, as well as for the characterization of the pharmacological and pathological factors that may influence the emotional state of rats. evaluating the emission of stress - induced 22-khz usvs has been a useful tool in neuroanatomical and neuropharmacological studies that have contributed to characterization of brain circuits engaged in the manifestation of anxiety - like behaviors in rats. this approach has demonstrated that various neurotransmitter systems (e.g., gaba, norepinephrine, serotonin, neurokinin-1, neurotensin) and brain regions (e.g., forebrain cholinergic nuclei, periaqueductal gray, medial prefrontal cortex) participate in these behaviors. a sustained emission of stress - induced 22-khz usvs has also been reported in rats subjected to withdrawal from different drugs, including diazepam, and substances of abuse such as cocaine, ethanol, or opiates [65 - 68 ]. these findings indicate that stress - induced 22-khz usvs may be a valid tool for investigating the anxiety - like states associated with drug withdrawal, and for elucidating the role these states may have in drug relapse and establishment of drug dependence. finally, the emission of stress - induced 22-khz usvs has been used in longitudinal studies aimed at investigating the influence of prenatal and perinatal insults on emotionality in later life, with particular attention to the development of anxyous- and depressive - like phenotypes. these studies have yielded interesting results which show how rats exposed to stressful events in early life, such as maternal separation, maternal immune activation reminiscent of perinatal infections, or isolation rearing, display significant changes in the emission of stress - stimulated 22-khz usvs in the adulthood, revealed by either an increase or a decrease in the number of vocalizations compared with non - exposed rats [69 - 72 ]. it is important to note that in the case of maternally - separated rats, changes in the emission of stress - induced 22-khz usvs have been found to positively correlate with the presence of a depressive - like phenotype, measured as an increase in immobility time in the forced swimming test. besides the study of neuropsychiatric diseases and their treatment, 22-khz usvs can also be used as a complementary behavioral parameter in experimental models of pain. in general, in these models, the emission of 22-khz usvs can either be spontaneous or elicited by the application of mechanical stimuli. the magnitude of the vocalization, measured either as the number of 22-khz usvs emitted per time unit, or the total number of seconds of vocalization, has been regarded as a direct correlate of the nociceptive response. independent studies have demonstrated a sustained emission of 22-khz usvs by rats subjected to different experimental models of pain, such as electric stimulation of the tail, formalin - induced paw inflammation, freund s adjuvant - induced arthritis, intramuscular injection of cephalosporins, and paw incision model of postoperative pain [73 - 76 ]. moreover, it has been demonstrated that the emission of 22-khz usvs by rats subjected to experimental chronic pain can be attenuated by the administration of drugs that possess clinically relevant analgesic properties, such as morphine and non - steroidal anti - inflammatory drugs (nsaids). additional studies have shown that novel drugs and natural substances with proposed analgesic activity also are able to modulate the emission of 22-khz usvs associated with experimental chronic pain in the rat. taken together, these findings envision the evaluation of 22-khz usvs as a potentially useful tool for the development of novel analgesic therapies. however, it has to be acknowledged that the existence of a link between the emission of 22-khz usvs and pain states in the rat is still disputed, and some of the results obtained by previous investigations on this issue require further consideration. some of the studies that have demonstrated a sustained emission of usvs in rat models of pain have failed to unambiguously identify the 22-khz usvs of long duration as the vocalization subtype specifically emitted under the experimental circumstances evaluated. in fact, the emission of either 22-khz usvs of short duration or usvs with a frequency contained between 60 and 80-khz has also been recorded in experimental models of pain. while this finding could be an artifact of either the experimental procedure or the usvs recording equipment used, if clearly demonstrated, it would challenge the idea that 22-khz usvs of long duration are the usvs subfamily whose emission is specifically associated with pain states. in addition, studies that have evaluated the emission of 22-khz usvs by rats subjected to experimental pain have yielded mixed results. experiments performed in rat models of arthritis have shown that procedures such as the extension or the compression of the knee can stimulate the emission of 22-khz usvs [78, 79 ]. conversely, other studies failed to observe any significant emission of 22-khz usvs by rats subject to freund s adjuvant - induced arthritis. furthermore, a detailed study that has compared the emission of 22-khz usvs across experimental rat models of inflammatory pain (formalin test), neuropathic pain (partial sciatic nerve ligation), and visceral pain (referred hyperalgesia), has found no positive correlation between the emission of 22-khz usvs and other behaviors that are indicative of pain in these models, such as licking and hind paw withdrawal. however, as mentioned earlier in this review, others have reported a sustained emission of 22-khz usvs by rats subjected to these and others models of chronic pain [73 - 79 ]. in considering these results together, it is important to remark that the studies on 22-khz usvs and pain described here often differed with regard to the experimental procedures used. experiments performed in rat models of arthritis have evaluated either the spontaneous emission of 22-khz usvs, or the vocalization in response to the application of mechanical stimuli [13, 77 - 80 ]. in a similar fashion, studies with the formalin test have evaluated behavioral responses and 22-khz usvs emission by using different concentrations of the irritant [74, 81 ]. while experimental discrepancies could have certainly contributed to the mixed results reported in the literature, it is still controversial whether the emission of 22-khz usvs could be considered a parameter that directly relates to the intensity of nociception in rats. nevertheless, 22-khz usvs are currently widely used as an additional behavioral measure in the study of experimental chronic pain and, in this regard, it is noteworthy that recent findings have suggested that these vocalizations could model not only nociception, but also the affective component of pain. pain involves both a sensory component, which depends on the transmission of nociceptive stimuli, and an affective component, which consists of the desire to attenuate the intensity of pain, and eventually terminate it. in consideration of the above discussed relationship between 22-khz usvs and anxiety - like states, it could be possible that these vocalizations may represent an index of distress or anxiety associated with pain. this interpretation could contribute to explaining the inconsistent results obtained in studies that have evaluated the emission of 22-khz usvs in rats subjected to experimental pain. it is conceivable that the intensity of nociception would not necessarily match the level of distress associated with pain. furthermore, the relevance of 22-khz usvs to the affective component of pain is also indirectly substantiated by the finding that morphine can attenuate the emission of 22-khz usvs in rat models of pain, because morphine exerts its analgesic effects not only by acting at the level of the nocicpeptive pathways, but also by improving the affective perception of pain. in addition to their use in the screening of anxiolytics and analgesics, rat 22-khz usvs could also be a useful tool in the development of other classes of drugs. stress - induced 22-khz usvs can be used, in combination with other behavioral measures, for evaluating the pharmacological profile of antipsychotic drugs, based on the different ability of so called classical (e.g., clozapine) antipsychotics to attenuate conditioned responses. the evaluation of the effects of antipsychotics on stress - induced 22-khz usvs can also disclose the presence of additional anxiolytic properties of these drugs. previous experiments have demonstrated that the emission of 22-khz usvs that occurs during conditioned fear is associated with changes in peripheral physiological parameters such as increase in blood pressure and heart rate. moreover modifications in the acoustic parameters, but not numbers, of 22-khz usvs have been shown to positively correlate with blood pressure and heart rate. (see for emotional expression containing concurrent emission of vocalization and manifestation of emotional autonomic symptoms). taken together, these findings would suggest that the evaluation of the acoustic features of stress - induced 22-khz usvs could represent a complementary, non - invasive, method for the performance of cardiopharmacology experiments in rats. nevertheless, it has still to be determined whether changes in 22-khz usvs may be indicative of predictable cardiovascular changes, and may be a suited tool in the screening of new drugs. the effects of various drugs on the emission of 22-khz usvs in different experimental models are summarized in table 2. fifty - khz usvs are characterized by a high peak frequency, usually contained within 3580 khz, and a short duration, usually within 10150 ms. young and adult rats emit these usvs in response to, or anticipation of, pleasurable stimuli such as mating, heterospecific play, namely the procedure known as tickling by a human hand that follows rough - and - tumble play of juveniles, and non - aggressive social encounters with conspecifics. these lines of evidence have suggested that the emission of 50-khz usvs may represent a behavioral parameter that indicates positive affect in the rat. in line with this, 50-khz usvs are increasingly being used in experiments for investigating those situations and factors that may promote the manifestation of positive affective states [2 - 4,9 ]. interestingly, previous pharmacological studies have demonstrated that the emission of 50-khz usvs can be modulated by certain drugs that possess rewarding and addictive properties. a sustained emission of 50-khz similar findings have been reported in rats subjected to environmental conditioning to amphetamine or morphine. these results have been later replicated, and confirmed by showing that other substances of abuse, such as cocaine, mdma, and nicotine, as well as the psychostimulant caffeine, may influence the emission of 50-khz usvs in rats [11,14,91 - 94 ]. taken together, these findings would suggest that 50-khz usvs may represent a behavioral response that can be measured in studies concerned with the motivational effects of drugs of abuse, either alone or in combination with other paradigms such as conditioned place preference (cpp), and drug self - administration. the evaluation of 50-khz usvs during self - administration appears a promising strategy that may disclose the emotional components inherent to drug abuse, and the factors that may promote relapse and reinstatement of drug taking. a detailed discussion of the usefulness and relevance of 50-khz usvs to drug self - administration is provided elsewhere in this issue. studying the motivational properties of drugs by means of 50-khz usvs may nevertheless have some limitations that need to be carefully considered. in this regard, it is noteworthy that evidence has been accumulated which demonstrates that the effects of drugs of abuse on the emission of 50-khz usvs by rats may significantly differ. on the one hand, many independent studies have consistently demonstrated that the dopaminergic psychostimulants amphetamine and cocaine elicit the emission of a high number of 50-khz usvs immediately after their administration to rats. on the other hand, mdma, morphine, and nicotine, which also possess rewarding and addictive properties previous results obtained in rats which engaged in playful social contacts have suggested that the number of vocalizations emitted may code the affective significance of 50-khz usvs. based on this, the results summarized above would suggest that the number of 50-khz usvs emitted immediately after drug administration should not be considered a straightforward correlate of the motivational properties of drugs, at least in the case of non - dopaminergic drugs. it is still unclear why certain drugs that have rewarding properties fail to stimulate the emission of 50-khz usvs immediately after their administration. one possible explanation of this could rely on the mechanism(s) by which rewarding drugs elicit their central effects, and on the crucial influence of dopamine transmission in the nucleus accumbens (nacc) shell on the emission of 50-khz usvs. while a thorough discussion of how different rewarding drugs act at the molecular level goes beyond the aims of the present review, it has to be mentioned that dopaminergic psychostimulants promote a massive release of dopamine in the nacc shell, but other rewarding drugs elicit this effect to a lower extent. besides, experimental evidence also exists showing that norepinephrine and glutamate may modulate the emission of 50-khz usvs. based on these considerations, it is probable that the effects of drugs with rewarding properties that elicit emission of 50-khz usvs recorded immediately after their administration may differ in their action from the general effects these substances have on both dopamine and non - dopaminergic neurotransmitters. interestingly, drugs that fail to stimulate a sustained emission of 50-khz usvs by rats may nevertheless influence other features of these vocalizations. previous experiments have shown that mdma, morphine, and nicotine can modify the acoustic features (peak frequency and bandwidth) of the 50-khz usvs that are recorded immediately after their administration (fig. it has been suggested that the maximum peak frequency, together with the number of vocalizations emitted, could code the affective significance of 50-khz usvs. moreover, different subtypes of 50-khz usvs have been isolated, and it has been proposed that each of them could have a different behavioral significance in terms of rats affective state. interestingly, mdma, morphine, and nicotine, while failing to elevate the total number of 50-khz usvs emitted by rats, have been shown to modify the number of certain subtypes of these vocalizations (table 3). notwithstanding these considerations, no convincing information is currently available that links the acoustic features of drug - stimulated 50-khz usvs to the motivational properties of drugs. similarly, no sufficient evidence has been collected so far to conclude that drug - induced changes in the relative numeric proportion of the various 50-khz usvs subtypes may reflect different aspects of the motivational properties of drugs. in this regard, it is worth mentioning that previous studies have suggested that the trill subtype of 50-khz usvs, may be a selective indicator of drug - induced positive affect, since a sustained emission of this usvs subtype has been reported following amphetamine administration. however, more recent investigations that have examined the effects of amphetamine, methamphetamine, and morphine on the different subtypes of 50-khz usvs have led to mixed results, and showed either a decrease or no changes in the number of trill vocalizations after administration of these drugs. on these bases, additional studies are needed to clarify whether modifications in the acoustic features and subtypes of drug - induced 50-khz usvs may somehow relate to the motivational properties of rewarding drugs. although the precise significance of the 50-khz usvs emitted by rats immediately after drug administration may need further discussion, clearer data have been obtained in experiments that have examined the long - term effects of drugs on 50-khz usvs. as mentioned earlier, previous studies have demonstrated that rats treated with either amphetamine, mdma, morphine or nicotine in a novel environment exhibit a sustained emission of 50-khz usvs when later re - exposed to the environment where the drug was administered. similar findings have also been observed in rats previously trained to self - administer cocaine. taken together, and considering that 50-khz usvs are emitted in anticipation of pleasurable stimuli, these effects are likely to reflect environmental drug conditioning and, in turn, to indicate the desire for drug by previously drug - experienced rats. moreover, a comparative study of the long - lasting changes in 50-khz usvs emitted by rats subjected to drug withdrawal and re - exposed to a previously drug - paired environment has demonstrated that this response was significantly more marked and persistent in morphine - treated rats than in rats treated with amphetamine, mdma, or nicotine. these results would be in line with the ability of opiates to induce intense craving, which can persist long after drug discontinuation. therefore, these findings would suggest a possible use of conditioned 50-khz usvs as a tool for investigating long - term effects of drugs of abuse and craving associated with drug withdrawal. taken together, the results obtained with conditioned 50-khz usvs would suggest that these vocalizations could be a more reliable indicator of the motivational properties of drugs of abuse than the 50-khz usvs recorded immediately after drug administration. besides being used for evaluation of drug effects in relation to dependence, the emission of 50-khz usvs could be a relevant behavioral parameter to be included in longitudinal studies that investigate the effects of early drug exposure on later changes in emotionality. in this regard, it is interesting to mention that a wealth of studies performed in different species of experimental animals have demonstrated a positive association between the emergence of delayed cognitive development, emotional abnormalities and the exposure in early life to drugs of abuse, such as cocaine, ethanol, and -tetrahydrocannabinol (thc) [112 - 114 ]. these considerations also apply to the human setting, as similar findings have been obtained in clinical and epidemiological studies [115, 116 ]. in this regard, it is also worth mentioning that the exposure to drugs of abuse often begins as early as during gestation, in the case of children born from dependent mothers, or at adolescence [117, 118 ], both of which are critical phases in brain development. as of today, the only studies available concerned with the long - term effects of early drug exposure on the emission of 50-khz usvs at later life stages have investigated how prenatal ethanol exposure may affect the ability of rats to vocalize in response to behavioral challenges at adolescence or adulthood. however, these studies have obtained mixed results. on the one hand, prenatal exposure to ethanol has been reported not to affect the emission of 50-khz usvs by adult rats during copulatory behavior. on the other hand, an increased emission of both 50-khz and 22-khz usv in response to a mild stress has been reported in adolescent rats exposed to ethanol during the prenatal period, compared with non - exposed rats. it is possible that methodological issues such as the protocol of ethanol administration, or the specific stimulus used to elicit usvs could underlie these discrepancies. nevertheless, in light of the behavioral significance of 50-khz usvs discussed elsewhere in this review, the evaluation of 50-khz usvs appears a potentially relevant behavioral measure to be included in longitudinal studies that assess the long - term neurobehavioral effects caused by the exposure to drugs of abuse in early life. while the study of drugs of abuse is currently one the fields where rat 50-khz usvs are most extensively used, experimental evidence suggests that these vocalizations may be relevant also to other aspects of neuropharmacology. in this regard, interesting results have been obtained by studies that have examined the emission of 50-khz usvs stimulated by tickling. briefly, tickling consists of manipulation of rats according to a specific protocol in order to reproduce the features of the rough - and - tumble play that occurs among juvenile rats [8, 122, 123 ]. previous studies have observed that rats differ in regard to number of 50-khz usvs emitted in response to tickling and that, accordingly, they can be classified in two populations, one displaying a high and the other a low emission of 50-khz usvs. rats that display the low emission of 50-khz usvs in response to tickling present both a phenotype and a genotype that are reminiscent of those featuring autism in humans, when compared with rats that respond to tickling by emitting the high number of 50-khz usvs [125, 126 ]. in fact, the former rats have been shown to engage in less social contacts with their conspecifics, and to show a differential expression of the gene encoding for the glutamate n - methyl - d - aspartate (nmda) receptor, the altered function of which has been proposed to be critically involved in the pathophysiology of autism. pharmacological experiments performed in this putative model of autism have demonstrated that glyx-13, a drug acting as a partial agonist of the glycine site of the ndma receptor, effectively counteracts the deficits in 50-khz usvs emission observed following tickling. glyx-13 is currently under clinical investigation [128, 129 ], which lends support to the idea that 50-khz usvs may represent a useful behavioral measure in the screening and preclinical development of new drugs to be used in the treatment of neuropsychiatric diseases. interesting results on tickling - induced 50-khz usvs have also been obtained in a recent study that has suggested that deficits in the emission of these vocalizations could be a behavioral parameter indicative of negative symptoms in pharmacological models of schizophrenia. in line with previous findings, this study found that the administration of phencyclidine and mk-801 (also known as dizocilpine), two nmda receptor antagonists that are widely used to induce schizophrenia - like phenotypes in rats, attenuates the emission of 50-khz usvs in rats subjected to tickling. the same study observed that buspirone, which has a clinically relevant potential in counteracting the negative symptoms of schizophrenia, effectively reversed the deficit in tickling - induced 50-khz usvs. this finding is of great interest, as it would substantiate the relevance of 50-khz usvs for the experimental modeling of the negative symptoms of schizophrenia. however, it has to be mentioned that aripiprazole, which also has clinical indication for the treatment of negative symptoms of schizophrenia, had no beneficial effects in the same rat model. therefore, further studies should be performed to ascertain whether deficits in 50-khz usvs may be considered a good behavioral correlate of experimental psychosis, and which of the negative symptoms of schizophrenia can be specifically modeled by changes in tickling - induced 50-khz usvs. another potential use of tickling - induced 50-khz usvs could be as a tool for evaluating the neurobiological mechanisms of anxiety and depression and, in turn, the anxiolytic and antidepressant properties of drugs. this is suggested by previous experiments which have demonstrated that the number of 50-khz usvs emitted in response to tickling may predict the behavior of rats in tests that evaluate anxiety- and depression - like behaviors, such as epm, fear conditioning, forced swimming test, and sucrose preference. however, up to now no studies have been performed that have specifically addressed a possible link between the emission of tickling - induced 50-khz usvs and the effects of anxiolytic or antidepressant drugs. nevertheless, additional experimental evidence exists which supports the relevance of 50-khz usvs to the evaluation of the anxiolytic properties of drugs. as a previous study has demonstrated, rats subjected to social isolation exhibited a sustained emission of 50-khz usvs when exposed to a conspecific, and the magnitude of this vocalization was higher than that recorded from non - isolated rats. also, diazepam has been reported to further amplify the emission of 50-khz usvs in this experimental model. this result suggests that the emission of 50-khz usvs by socially - isolated rats may be sensitive to the effects of anxiolytic drugs, at least in the case of benzodiazepines, and could potentially be of relevance to the screening of these drugs. another recent study has suggested that the emission of 50-khz usvs could be an index of positive affect in experimental mania, by showing that 50-khz usvs stimulated by amphetamine, whose administration can induce mania - like states in rodents, could be reversed by lithium and tamoxifen, both of which possess a clinically relevant antimanic potential [137, 138 ]. the proposed relationship between 50-khz usvs and mania these results appear very interesting, and suggest that evaluating the emission of 50-khz usvs could provide a simple paradigm to study mania at the preclinical level, which could in turn enable significant progress beyond the current state of knowledge, since the available experimental models of mania are largely unsatisfactory. finally, it is noteworthy that, as mentioned above, rats emit 50-khz usvs during copulatory behavior [140 - 142 ]. this has allowed, in the first place, to use the emission of 50-khz usvs in studies that have investigated neurobiological mechanisms of sexual behavior and its affective components. moreover, the emission of 50-khz usvs during copulatory behavior has recently been exploited as a tool to investigate the occurrence of changes in phonation that may accompany neurodegeneration in experimental models of parkinson disease. to summarize, the experimental evidence discussed above suggests that 50-khz usvs could be of potential relevance to the study of both the neurobiology of different neuropsychiatric disorders and the screening of new drugs to be used in the treatment of these disorders. moreover, while the emission of 50-khz usvs is chiefly dependent on brain emotive mechanisms rather than peripheral mechanisms, further studies should be performed to find out whether measurements of 50-khz usvs could as well have some experimental use for studying processes outside of the central nervous system. several lines of experimental evidence have accumulated to suggest that recording and analysis of usvs emitted by rat pups or by young / adult rats could represent a useful tool in neuropharmacology, which may also have potential implications for other areas of pharmacology. the evaluation of rat usvs can be used either as a self - standing technique, or be combined with other validated models in behavioral pharmacology that are employed to reproduce the features of neurological and psychiatric disorders. importantly, the emission of usvs is sensitive to the effects of different classes of drugs, hence providing a potential tool to be used in drug screening studies. nevertheless, the use of rat usvs as an experimental model in neuropharmacology has some intrinsic limitations, which have to be carefully considered. some of the aspects related to the behavioral significance of rat usvs and their translation to human communication and expression of affect are still to be elucidated. moreover, the effect of drugs on the emission of usvs may differ from those the same drugs elicit in other behavioral paradigms. finally, certain drugs that possess clinically relevant effects may have a negligible or unexpected influence on the emission of usvs. notwithstanding these limitations, rat usvs stand as a new promising tool in neuropharmacology, particularly in light of their marked ethological component, and they create a possibility of combining them with other well - standardized behavioral paradigms, without the need for extensive training and complex animal manipulations. dr. nicola simola gratefully acknowledges the sardinian regional government for financial support (p.o.r. operational programme of the autonomous region of sardinia, european social fund 20072013 axis iv human resources, objective l.3, line of activity l.3.1 avviso di chiamata per il finanziamento di assegni di ricerca). part of the studies discussed in this review were supported by fondazione banco di sardegna (grants 2013.1321 and 2014.0395 awarded to nicola simola). funding sources had no further role in study design ; in the collection, analysis and interpretation of data ; in the writing of the manuscript ; and in the decision to submit the paper for publication.	several lines of evidence indicate that rats emit ultrasonic vocalizations (usvs) in response to a wide range of stimuli that are capable of producing either euphoric (positive) or dysphoric (negative) emotional states. on these bases, recordings of usvs are extensively used in preclinical studies of affect, motivation, and social behavior. rat usvs are sensitive to the effects of certain classes of psychoactive drugs, suggesting that emission of rat usvs can have relevance not only to neurobiology, but also to neuropharmacology and psychopharmacology. this review summarizes three types of rat usvs, namely 40-khz usvs emitted by pups, 22-khz usvs and 50-khz usvs emitted by young and adult animals, and relevance of these vocalizations to neuropharmacological studies. attention will be focused on the issues of how rat usvs can be used to evaluate the pharmacological properties of different classes of drugs, and how rat usvs can be combined with other behavioral models used in neuropharmacology. the strengths and limitations of experimental paradigms based on the evaluation of rat usvs will also be discussed.
red phosphorus is nonvolatile, insoluble, and unabsorbable, and therefore nontoxic when ingested. yellow phosphorus (also referred to as white phosphorus), on the other hand, is a severe local and systemic toxin causing damage to gastrointestinal, hepatic, cardiovascular, and renal systems. we describe here a case of acute yellow phosphorus poisoning that led to fulminant hepatic failure and eventually, the patient recovered with conservative management. a 3-year - old girl was brought to the hospital with an alleged history of accidental consumption of an unknown quantity of rodenticide paste (ratol, containing 3% yellow phosphorus). she was made to rinse her mouth with salt water and remained asymptomatic until 8 hours when she started vomiting. she was then brought to the hospital 14 hours after poisoning due to persistent vomiting. there was no history of jaundice, abdominal pain, breathlessness, or oliguria. on admission, the child was conscious and afebrile with a heart rate of 122/minute and respiratory rate of 32/minute. the liver was palpable 2 cm below the costal margin, soft, and with rounded margins. results of the investigations done on the day of admission and subsequently are shown in table 1. stages of hepatic encephalopathy and investigation reports on the second hospital day, the child developed fever and was noticed to be sleeping excessively and not taking feeds. a diagnosis of stage 1 hepatic encephalopathy was made and the patient was given intravenous (iv) fluids with 10% dextrose, iv cefotaxime vitamin k, injection ranitidine, and oral ampicillin. intake and output was strictly monitored and blood glucose was measured six - hourly. despite initiation of antihepatic failure therapy, the child progressed to stage 3 encephalopathy on day 3 and had malena for which she was administered fresh frozen plasma. the sensorium started improving on the 8 hospital day, with a simultaneous improvement in her laboratory parameters [table 1 ]. yellow phosphorus is an inorganic substance used in military ammunition, fire crackers, fertilizers, and as rodenticide. after absorption, it is distributed to all tissues, particularly the liver, and the peak level is reached after 2 to 3 hours of toxic oral ingestion. bile salts are important for absorption of phosphorus. because of water content and low oxygen tension, phosphorus remains stable in gut for longer period. phosphorus is a general protoplasmic poison causing cardiac, hepatic, renal, and multiorgan failure. the first stage occurs during the first 24 hours in which patient is either asymptomatic or has signs and symptoms of local gastrointestinal irritation. there may be mild elevation of liver enzymes and bilirubin in this stage. the third stage (advanced) patients may present with acute hepatic failure, coagulopathy, and deranged liver function, as was witnessed in our patient. central nervous system effects include changes in mental status like confusion, psychosis, hallucinations, and coma. gastric lavage with potassium permanganate is recommended to convert the phosphorus to relatively harmless oxides. fernandez and canizares in a series of 15 patients have reported a mortality of 27%, confirming that yellow phosphorus is extremely lethal when ingested. the indiscriminate use of yellow phosphorus in the manufacture of fireworks should be eliminated. since rodents are developing resistance to rodenticides containing warfarin, rat poisons containing yellow phosphorus are making a big come - back. the yellow phosphorus rodenticides pose a special problem in that the product directions suggest that the paste be applied to bread to enable ingestion by rodents, thus making it appealing to children as well.	we report a case of three year old girl, who was brought to hospital for accidental consumption of rat - poison (3% phosphorus). the patient was asymptomatic for first 48 hours. later on she developed the symptoms of hepatic failure. she was managed conservatively and was discharged after 14 days.
the term fibromatosis encompasses a broad variety of lesions, divided into (1) superficial (fascial), (2) deep, which are musculo - aponeurotic abdominal or extra - abdominal, intra - abdominal and others involving various organs of the body and (3) fibromatosis of infancy and childhood. they present as nodular soft tissue masses and thus are amenable to fine needle aspiration cytology (fnac). in cytologic smears, a spectrum of cytologic features may be seen based on the site aspirated and the phase or age of the lesion. therefore, initial diagnosis of fibromatosis may be challenging, but recurrent lesions can aptly be spotted on fnac. we present a case of 37-year - old male who presented with a superficial infiltrative type of fibromatosis. a 37-year - old male presented with the presence of a right supraclavicular mass since 20 days. it was gradually progressive, but not associated with pain or numbness. on examination, it was 3 3 cm in size, firm to feel and fixed to the underlying deep muscle. adjacent vessels carotid, subclavian and brachial plexus were preserved. magnetic resonance imaging (mri) was performed, which showed a t1 t2 hypo - intense lesion in the inferior aspect of right supraclavicular region with focal areas of calcification. the lesion was diffusely infiltrating the adjacent soft tissue [figure 1 ]. magnetic resonance imaging (mri) showing a t1 t2 hypointense lesion in the right supraclavicular region which is diffusely infiltrating the adjacent soft tissue fnac from the mass was performed which revealed moderately cellular smears and showed scattered spindle to fusiform cell clusters, stromal fragments [figure 2a ] and collagen matrix material. these spindle cells were embedded in or were in proximity to the densely eosinophilic collagen matrix material [figure 2b and 2c ]. the cells were oval to spindle shaped with tapering ends, bipolar nuclei, bland nuclear chromatin and inconspicuous nucleoli [figure 2d ]. based on the morphology a cytologic diagnosis of spindle cell lesion possibly benign was offered. cytology smears showing fibromatosis ; (a) bland spindle cells embedded in hypocellular dense stromal fragment (h and e, 200) ; (b) spindle cells seen within stromal fragments (h and e, 200) ; (c) oval to spindle cells embedded in metachromatically staining matrix material (mgg, 400) ; (d) oval to spindle cells with bland nuclear chromatin and inconspicuous nucleoli (h and e, 400) it can be localized or infiltrative and multicentric and can involve internal tissues and organs as the mesentery and the retroperitoneum (often associated with familial intestinal polyposis), the breast, and almost every organ or region, including bones, meninges and central nervous system. a number of classification schemes have been proposed for fibromatoses based on location, histologic appearance and age of the patient. based upon the location, these are divided as superficial and fascial, which include palmer, planter and penile fibromatoses and those which are deep and musculo - aponeurotic include abdominal and intra - abdominal fibromatoses. based on the age, they are classified into congenital and acquired types. based on histologic appearances, there are few cases series as well as case reports of fnac in fibromatoses, which includes cases of fibromatosis colli, infantile myofibromatosis and fibromatosis of breast.[57 ] fnac from fibromatoses may yield a spectrum of cytologic findings. depending upon the age and location of the lesion, different cytologic features may be present ranging from the cellular, mildly atypical aspirate mimicking low grade sarcoma to acellular benign appearing aspirate that can be seen in superficial fibromatosis. in such cases, the aspirate should be more vigorous as the dense connective tissues are not easy to sample by fine needle. two to three passes should be taken in different directions to avoid the same track and to obtain adequate cellularity. the differential diagnosis of fibromatoses ranges from a reactive fibrous lesions secondary to inflammation to a low grade fibrosarcoma. this includes scar or keloid, mesenchymal repair, nodular fasciitis, schwannoma, leiomyoma, solitary fibrous tumor (sft), gastrointestinal stromal tumor (gist), fibrosarcoma, synovial sarcoma and malignant melanoma. malignant tumors such as fibrosarcoma display increased cellularity, cytologic atypia and mitosis, which may overlap with features of proliferative phase of fibromatosis. the nuclei in a schwannoma are wavy and serpentine in contrast to plump nuclei of fibromatosis. aspirates of leiomyoma are usually paucicellular and have cells with blunt or cigar - shaped nuclei. the number of stromal cells is less than those observed in fibromatosis, the cells display more stellate configurations and a greater degree of inflammation may be noted. deep fibromatosis shows a strong nuclear positivity for -catenin immunostaining, which can be of help in differentiating it from other myofibroblastic proliferations. smooth muscle neoplasms are positive for desmin and majority of gist show positivity for c - kit. therefore, cell block may be obtained from the aspirated material and the recommended panel includes at least -catenin, c - kit, cd34 and desmin to differentiate fibromatosis from other entities with morphologic overlaps. fnac is useful in providing a tentative pathologic diagnosis of these tumor - like lesions, which under the appropriate clinical setting can be used for management, if surgical intervention is contraindicated or not warranted. based on clinical history, physical findings, radiological data and cytologic observations, a correct diagnosis can be rendered in most cases.	fibromatosis form a spectrum of clinicopathologic entities characterized by the infiltrative proliferation of fibroblasts that lack malignant cytologic features. the fibromatosis can be localized or infiltrative and multicentric and can involve internal tissues and organs as the mesentery, retroperitoneum, breast, and almost every organ and region of the body, including the bones, the meninges and the central nervous system. we report a case of 37-year - old male who presented with a right supraclavicular mass with superficial infiltrative type of fibromatosis and fine needle aspiration cytology (fnac) was performed. we report this case because of limited literature of fnac in fibromatosis and quick role of fnac in the diagnosis of fibromatosis.
such a management is challenging because of difficulties in accessing the lesion, risks for damages of neighboring organs, and risks for massive blood loss due to an extensive vascularity. preoperative tumor embolization has been proposed, exposed however the patients for ischemic neuropathy that can result in motor and sensory deficits in the pelvis and lower extremities. we present a new surgical concept for management of ilio - sacral tumors based on a combined anterior laparoscopic approach and posterior open resection of the tumor. a 33-year - old woman, 6 weeks after vaginal delivery, complained of lumbosacral dysesthesia, which led her gynecologist to have a lumbo - pelvic ct performed. this revealed an intensive vascularized mass of the sacrum reaching from the foramina l5 to s3, crossing the sacro - iliac joint and extending into the iliac bone. mri and angiography were suggestive of a chondromatous process (figs. 1 and 2). ct guided biopsies confirmed a cartilaginous process, which in synopsis with the images was graded as gi chondrosarcoma. neuropelveological assessment diagnosed a s2 - 4 radiculopathy right with vulvodynia, coccygodynia, low back pain and bladder hypersensitivity, and a l5-s2 irritative sciatica without signs for neurogenic damages. orthopedic examination was unremarkable with normal gait. as there is no effective adjuvant treatment for low grade chondromatous lesions, the patient after extensive repeated discussion opted for resection of the tumor and reconstruction realizing that mutilation was unavoidable due to the involvement of sacral nerve roots. massive pelvic congestion due to postpartum situation was considered as an additional risk factor for hemorrhage. the patient signed informed consent forms for the procedure and authorization for communication as a case report (figs. for the procedure, the patient was placed in unstable lateral decubitus to allow free access anteriorly for laparoscopy as well as the back by rotating the patient without need for new sterile preparation. for the laparoscopy, one 10 mm trocar was placed in the navel to introduce a 10 mm/0 hdtv optic and three further 5 mm - trocars were placed in the lower abdomen. for identification of the different sacral nerves roots (snr), intraoperative electrical stimulation using a laparoscopic probe with a current fixed by 250 s/35 hz/4 v was applied to the nerves. the procedure was started with the full exposure of the os sacrum and the right pelvic sidewall by dissection of the right pararectal space. after transection of the sacral hypogastric fascia, the medial and caudal limits of the tumor and as well as the snr were identified. while the snr l#5, s#1 and s#2 were attached on the tumor, the snr s#3 and s#4 did not show any contact with the tumor and were exposed in order to avoid their damages during the rest of the procedure. the next step consisted in the full exposure of the pelvic ureter followed by the coagulation / transection of the internal iliac and the lateral sacral vessels. all cardinal vessels below the tumor were also transected including the pudendal and inferior gluteal vessels. the sciatic nerves were identified just before its entry through the great sciatic foramen. to reduce risk for bleeding during the gluteal dissection, the superior gluteal vessels were dissected at the level of the suprapiriform level of the great sciatic foramen, separated from the superior gluteal nerve, and coagulated selectively. after dissection of the right femoral nerve, the major psoas muscle was transected twice proximally and distally to the tumor to permit en bloc resection. also the nerves roots l5, s1 and s2 were kept in contact to the tumor. after complete anterior exposure of the tumor, two gigli saws were passed from anterior to posterior by passing with laparoscopic forceps through the foraminas l5s1 and s1s3 for transection of the sacrum medial to the tumor. the entire laparoscopic part of the procedure was based on the principle of non - touching en - bloc resection of the tumor. for posterior resection of the ilium, the incision for the open posterior approach included the exit points of the 2 inserted gigli saw as extended cranially for the later insertion of the pedicle screws and caudally below the sciatic notch for the iliac osteotomy in analogy to a juder approach. the defect was replaced with a structural fresh frozen femoral allograft and stabilization performed by lumbo - ischial screw / rod fixation. laparoscopic control excluded any residual bleeding and permit closure of the peritoneum after introduction of a robinson drainage. to avoid risk for postoperative bladder overdilatation blood loss was estimated to < 1000 cm ; the patient received a total of 2 units erythrocytes. histologic examination of the specimen showed an osteochondrosarcoma with tumor - free margins. at 17 months because of the denervation of the nerve root l5 and below, she mostly uses two canes, but she has a functioning quadriceps. operative treatment of tumors in the sacroiliac area is among the most challenging musculoskeletal tumor surgeries. because almost all the deaths from chondromas result from local recurrence, greater effort to obtain adequate surgical excision has been made over the last decades. most common surgical approach used are the posterior, posterolateral, anterior and posterior, anterior and lateral combined approach. in tumor of the sacrum, exposition is mostly obtained by a posterior midline incision ; lateral osteotomies are usually performed through the sacral foramina using a threadwire saw and kerrison rongeurs. although various reports analyzed en - bloc excision of sacral tumors, there are still technical problems to improve protection of nerve roots and pelvic organs and reduce intraoperative bleeding. in posterior approach, because first elevation of the sacrum allows dissection of presacral structures, risk for damages intrapelvic structures is high. wound infections, neurologic deficits, pelvic instability, and cerebrospinal fluid leakage are the main complications of sacrectomy. extensive hemorrhage is the most serious complication since it may threaten the life of the patient and jeopardize the outcome of surgery. angelini reported about sacral resection with a mean blood loss by 2961 ml (10008000 ml). in a further retrospective study on 173 patients, 39.88% of the patients had blood loss greater than 3000 ml. in one study of nine patients with total sacrectomies, the blood loss ranged between 4.5 and 17 l (mean, 6.3 l). in another report three sacral tumor resections were performed with blood losses of 9250, 7500, and 9600 ml. in one larger - scale study of 29 patients who underwent partial or total sacrectomies the median blood loss was 3.9 l and the maximum blood loss was 37 l. it is obvious that primary control on blood supply of the sacrum and parameters may reduce intraoperative blood loss. the results of several studies suggest preoperative arterial embolization and aortic balloon occlusion, but indications remain uncertain. because of numerous pelvic anastomoses, single closure of the internal iliac artery does not protect from hemorrhage. we opted for a primary closure of all parametric vessels including the internal iliac artery, the gluteal and the sacral vessels by laparoscopic before starting with bone resection, that vascular control do not present major difficulties for pelvic surgeons trained in laparoscopic retroperitoneal surgery. in our patient, this first step decrease considerably blood loss despite the massive postgravid pelvic varicosis. laparoscopic dissection of the pelvic organs not only had protect the patient from visceral lesions, but had also permitted in same time to exclude abdominal metastases, and to resect the bones according to the limits of the tumor with in turn macroscopic tumor - free margins. the introduction of the gigly saw through the different sacral foramens had also contributed to a tumor - adapted and precise transaction of the bone under laparoscopic control. high amputation of the sacrum inevitably results in damages of the sacral nerve roots, the pudendal nerve and the coccygeal plexus. however, it is well - known that simultaneous abdominosacral resection circumvents many of problems for such advanced procedures since it provides good exposure of the intra - abdominal structures, and avoids damage to the sacral nerve roots. similarly, laparoscopic approach had permitted an optimal exploration of the entire lumbosacral plexus before resection : the nerve roots l5, s1 and s2 were resected en bloc with the tumor, while the roots s3 and s4 (bladder functions) below the tumor, as well as the femoral nerve (extension and stabilization of the knee during locomotion) above the tumor, were respected for a maximum reduction of postoperative functional morbidity. the neuropelveology is a new field of medicine that deals with pathologies of the pelvic nerves. neuropelveologists are trained in laparoscopic neurosurgery to the pelvic nerves. in the presented patient, because of particular high risk for intraoperative hemorrhage few weeks after delivery, primary laparoscopic approach appeared to be a good way for a optimal preparation of orthopedics sacroiliac resection with macroscopic tumor - free margin, by a maximal reduction of risks for intraoperative hemorrhage and pelvic organs damages. marc possover did the project development, data collection and management, data analysis, manuscript writing / editing and surgery.key learning pointreduction of morbidity during surgery for iliosacral tumors.	introductionsacral tumor often involves en bloc surgical resection with tumor - free margins and functional reconstruction challenges. such a management is challenging because of difficulties in accessing the lesion, risks for damages of neighboring organs, and risks for massive blood loss. in posterior approach, because first elevation of the sacrum allows dissection of presacral structures, such risks for damages intrapelvic structures and hemorrhage are especially high.presentation of casewe report here about a laparoscopic assisted posterior resection of a ilio - sacral chondrosarcoma in a women, 6 weeks after vaginal delivery. primary laparoscopic approach consisted in dissection of the ureter and of the colon with control to the pelvic vessels and nerves and determination of limits of the resection. the iliac osteotomy was performed from posterior approach with saw and osteotomes at the predetermined extralesional level. the defect was replaced with a structural fresh frozen femoral allograft and stabilization performed by lumbo - ischial screw / rod fixation.discussionsurgical time was about 360 min. no intra - postoperative complications occurred. blood loss was estimated to about 1000 cm3. histologic examination of the specimen showed tumor - free margins. at 8 months follow - up, the patient appears to be without recurrence. because of the denervation of the nerve root l5 and below, she mostly uses two canes, but she has a functioning quadriceps. continence and voiding functions for urine and stool have fully recovered.conclusionprimary laparoscopic approach appeared to be a good way for preparation orthopedics sacroiliac resection to reduce postoperative morbidity, intraoperative blood loss and better assure macroscopic tumor - free margins.
synthetic biology is the code name for engineering using the machinery of the cell, from tinkering with existing organisms all the way to the design of life from scratch (: 11). in the wake of the transition of biology from a taxonomy - based to an information - based discipline, [synthetic ] biologists dream of controlling the machinery of life like engineers control device layouts on a computer chip (ibid. :, the well - known researcher j. craig venter appears unrivalled in his capacity to attract venture capital. he and his team are working on a long - term project involving the transplantation of minimal genomes (i.e. genomes stripped of all dispensable genes) made from synthesized dna into host cells whose genomes have been removed. we re moving from reading the genetic code to writing it, venter himself explains. on 31 may 2007 he caused a stir when the us patent and trademark office published the patent application his institute had filed on the creation and various useful applications of a new life form called mycoplasma laboratoriums (j.). the claims are very broadly formulated ; specific mention is made of the creation of new synthetic organisms for the production of biofuels like hydrogen and ethanol. at present, such applications may sound futuristic, but venter, it seems, wants to send a message to the general public that his enterprises (not only the non - profit j. craig venter institute but also the company synthetic genomics, inc. ; all patent rights will be assigned to the latter) intend to play a key role in solving the urgent problems of energy supply and climate change. though the spotlight of publicity seems to be trained on venter, he is not the only person who is active in the emerging field of synthetic biology. a growing clique of young enthusiasts is clustering around such luminaries as tom knight, drew endy, jay keasling and george church, who specialize in designing and constructing artificial biological systems, ideally from scratch, and who thereby take a more bottom - up approach to synthetic biology [21, 44 ]. in their view, the implementation of this task calls for standardization. leading synthetic biology researchers from mit, harvard and california are busily engaged in building a library (or catalogue) of interchangeable standard parts called biobricks, or pieces of dna with known functions, from which practitioners can draw at will to construct new life forms. by putting together carefully selected building blocks it is possible to design genetic circuits capable of regulating successive reaction steps in metabolic pathways for the production of valuable substances within the cell. the insertion of such circuits in a microbial host organism can turn the latter into a tiny biochemical factory. the rise of synthetic biology can be seen as a continuation, and interim culmination, of some longer - term trends that are characteristic of major strands in the development of western science and technology. historians of science have pointed out that in embracing the engineering ideal of controlling life contemporary synthetic biologists may be regarded as heirs to the visionary american biologist jacques loeb, who was pursuing a similar ambition as early as 1900 [5, 12, 40, 45 ]. technology of living substance, which would enable man to act as a creator of new life forms. for some, the ambition to control is inextricably linked with the reconceptualization of biological life [... ] as informational, in parallel with a similar conviction in nanotechnology that the physical world can be manipulated as if it were information (: 104). synthetic biology can indeed be seen as the culmination of a long - standing and still ongoing trend of progressive the informational view of life that has dominated molecular biology since watson and crick unravelled the structure of dna in 1953 is increasingly being materialized in tangible technological applications, even to the point of blurring the distinction between living matter and information. the historical roots of synthetic biology can be traced even farther back. several practitioners justify their new field by quoting the famous statement by physicist richard feynman : 61).1 feynman s dictum echoes similar statements by eighteenth - century luminaries such as giambattisto vico (verum et factum convertuntur or the true and the made are convertible) and immanuel kant (reason has insight only into what it itself produces according to its own plan ; see : 19). the idea that there is a very close connection between knowing and making, between understanding an object and the ability to create or (re-)assemble it, is not at all foreign to the tradition of western science and philosophy. an interesting illustration of what one could dub the vico - kant - feynman principle is provided by nineteenth - century synthetic organic chemistry (: 168). in several respects, no wonder then that some contemporary practitioners attempt to draw heuristic lessons from this example [6, 63 ]. synthetic biology may also have inherited some of the cultural ambivalences and reservations pertaining to the natural - versus - artificial dichotomy from synthetic chemistry. synthetic biology adopts the epistemological approach of synthetic chemistry, but pursues it with the resources of modern information technology. in informational terms, feynman s dictum boils down to von neumann s motto if you ca nt compute it you do nt understand it!.2 new life forms can be designed by writing informatization of the biological world may have a disenchanting effect on our view of life. cells and organisms are seen as computers that can be easily (re)programmed according to our wishes. rather than evolving naturally, many are worried that life will lose its special meaning when such reductionist views prevail. a case in point the european directive on the legal protection of biotechnological inventions (directive 98/44/ec) gives the following definition of the key object of protection :... biological material means any material containing genetic information and capable of reproducing itself or being reproduced in a biological system (art. notice that not just genes and cells but complete organisms can be brought under this definition. during political debates on the implementation of the directive in the netherlands in 20032004, several members of parliament expressed concern that the new legislation would reduce living beings to the status of biological material. in response to this concern, cabinet ministers pointed out that all living beings consist of biological material, but of course are more than just biological material this facile defence is rather disingenuous, however, as organisms clearly meet the definition of biological material given in the directive and on that count must be held to be such material (besides, the directive declares entire organisms to be patentable). there is no escape from the conclusion that in modern patent law plants and animals are being reduced to the status of raw material or carrier of genetic information. synthetic biology puts heavy pressure on many of the culturally entrenched distinctions and demarcations that are constitutive of our symbolic order. it shifts or blurs the boundaries between matter and information, life and non - life, nature and artefact, organic and inorganic, creator and creature, the evolved and the designed. in science and technology studies, entities that challenge the settled boundaries of nature and society are often designated as monsters. like the creations of synthetic biology, victor frankenstein s creature was a prime example of a monster in this particular sense.3 whenever culturally sanctioned boundaries are breached by such monsters, researchers are quickly accused of playing god or of treading in frankenstein s footsteps. indeed, in recent times these types of accusations have triggered societal debates on the moral and symbolic implications of synthetic biology in which the question about the meaning of life has also found a prominent place. by following the tropes of playing god and emulating frankenstein in their discursive settings, i therefore also hope to gain a useful contextual angle from which to deal with the latter question. the rise of synthetic biology can be seen as a continuation, and interim culmination, of some longer - term trends that are characteristic of major strands in the development of western science and technology. historians of science have pointed out that in embracing the engineering ideal of controlling life contemporary synthetic biologists may be regarded as heirs to the visionary american biologist jacques loeb, who was pursuing a similar ambition as early as 1900 [5, 12, 40, 45 ]. technology of living substance, which would enable man to act as a creator of new life forms. for some, the ambition to control is inextricably linked with the reconceptualization of biological life [... ] as informational, in parallel with a similar conviction in nanotechnology that the physical world can be manipulated as if it were information (: 104). synthetic biology can indeed be seen as the culmination of a long - standing and still ongoing trend of progressive the informational view of life that has dominated molecular biology since watson and crick unravelled the structure of dna in 1953 is increasingly being materialized in tangible technological applications, even to the point of blurring the distinction between living matter and information. several practitioners justify their new field by quoting the famous statement by physicist richard feynman : 61).1 feynman s dictum echoes similar statements by eighteenth - century luminaries such as giambattisto vico (verum et factum convertuntur or the true and the made are convertible) and immanuel kant (reason has insight only into what it itself produces according to its own plan ; see : 19). the idea that there is a very close connection between knowing and making, between understanding an object and the ability to create or (re-)assemble it, is not at all foreign to the tradition of western science and philosophy. an interesting illustration of what one could dub the vico - kant - feynman principle is provided by nineteenth - century synthetic organic chemistry (: 168). in several respects, no wonder then that some contemporary practitioners attempt to draw heuristic lessons from this example [6, 63 ]. synthetic biology may also have inherited some of the cultural ambivalences and reservations pertaining to the natural - versus - artificial dichotomy from synthetic chemistry. synthetic biology adopts the epistemological approach of synthetic chemistry, but pursues it with the resources of modern information technology. in informational terms, feynman s dictum boils down to von neumann s motto if you ca nt compute it you do nt understand it!.2 new life forms can be designed by writing the informatization of the biological world may have a disenchanting effect on our view of life. cells and organisms are seen as computers that can be easily (re)programmed according to our wishes. rather than evolving naturally, many are worried that life will lose its special meaning when such reductionist views prevail. a case in point the european directive on the legal protection of biotechnological inventions (directive 98/44/ec) gives the following definition of the key object of protection :... biological material means any material containing genetic information and capable of reproducing itself or being reproduced in a biological system (art. notice that not just genes and cells but complete organisms can be brought under this definition. during political debates on the implementation of the directive in the netherlands in 20032004, several members of parliament expressed concern that the new legislation would reduce living beings to the status of biological material. in response to this concern, cabinet ministers pointed out that all living beings consist of biological material, but of course are more than just biological material (, 23 ff). this facile defence is rather disingenuous, however, as organisms clearly meet the definition of biological material given in the directive and on that count must be held to be such material (besides, the directive declares entire organisms to be patentable). there is no escape from the conclusion that in modern patent law plants and animals are being reduced to the status of raw material or carrier of genetic information. synthetic biology puts heavy pressure on many of the culturally entrenched distinctions and demarcations that are constitutive of our symbolic order. it shifts or blurs the boundaries between matter and information, life and non - life, nature and artefact, organic and inorganic, creator and creature, the evolved and the designed. in science and technology studies, entities that challenge the settled boundaries of nature and society are often designated as like the creations of synthetic biology, victor frankenstein s creature was a prime example of a monsters, researchers are quickly accused of playing god or of treading in frankenstein s footsteps. indeed, in recent times these types of accusations have triggered societal debates on the moral and symbolic implications of synthetic biology in which the question about the meaning of life has also found a prominent place. by following the tropes of playing god and emulating frankenstein in their discursive settings, i therefore also hope to gain a useful contextual angle from which to deal with the latter question. responding to the news that craig venter and his team had filed for a patent on mycoplasma laboratorium, the leader of the etc group, pat mooney, declared in june 2007:for the first time, god has competition. venter and his colleagues have breached a societal boundary, and the public hasnt even had a chance to debate the far - reaching social, ethical and environmental implications of synthetic life.. for the first time, god has competition. venter and his colleagues have breached a societal boundary, and the public hasnt even had a chance to debate the far - reaching social, ethical and environmental implications of synthetic life.. however, it is definitely not the first time that an assertion of this or a similar kind has been made. the recombinant - dna debate in the 1970s already gave rise even then to a book entitled playing god. in recent decades this accusation has been levelled innumerable times at classical biotechnologists (e.g.). similarly, genetic engineering in its classical form has been charged with breaching critical boundaries. for members of the etc group, apparently, such considerations fail to constitute a persuasive reason to abstain from hammering the same message home time and again, as is shown by the frequency with which the expression playing god continues to appear in their publications and press releases (see for instance). journalists also tend to resort to this metaphor when reporting new developments in the life sciences. when, for example, back in 1999 business week discussed venter s newly initiated research on the minimal genome, it chose the title playing god in the lab. and in may 2007 newsweek printed a lead story on synthetic biology, with a portrait of a thoughtful, upward - looking craig venter adorning the cover alongside the boldly printed words playing god. this expression has meanwhile become, as publicist and science journalist philip ball remarks, a lazy journalistic clich and an alarmist slogan. however, it is a clich that may still enable writers or ngos to attract the attention of a large audience. in discussions on biotechnology and synthetic biology, alongside and in combination with allusions to the presumed arrogance of playing god, a name is very often invoked that many scientists consider a tainted f - word : frankenstein. in fact, the frankenstein theme is closely entwined with the motif of playing god. as mary shelley herself wrote in 1831 in the introduction to her gothic novel frankenstein ; or, the modern prometheus : frightful must it be ; for supremely frightful would be the effect of any human endeavour to mock the stupendous mechanism of the creator of the world. (: 9). the main character of the novel, victor frankenstein, ultimately brought disaster upon himself and his loved ones by indulging in the unhallowed arts of bestowing animation upon lifeless matter (ibid. : 53). he aspired to become greater than his nature [would ] allow (ibid. : in other words, frankenstein wanted to play god and was as severely punished for his transgression as prometheus, who had stolen fire from the gods. as a dutch literary critic succinctly explains : the moral seems clear, and is more relevant than ever in the 21st century, which is dominated by the advancing genetic and bio - technologies : do not play god and beware of the dangers of technology. in his book frankenstein s footsteps, jon turney calls the story of frankenstein the governing myth of modern biology (: 3). mary shelley herself took pains to point out that the theme of her gothic novel was not entirely the product of her own imagination. in fact, the subject matter resonated with scientific ideas that had circulated around 1800, such as erasmus darwin s views on the spontaneous generation of life4 and luigi galvani s ideas of using electricity to animate lifeless matter:perhaps a corpse would be re - animated ; galvanism had given token of such things : perhaps the component parts of a creature might be manufactured, brought together, and endued with vital warmth. (: 8).5 perhaps a corpse would be re - animated ; galvanism had given token of such things : perhaps the component parts of a creature might be manufactured, brought together, and endued with vital warmth. (: 8).5 this brief summary can not possibly do justice to the many subtleties and layers of meaning that lie hidden in mary shelley s novel. in the popular imagination, based more on retellings and especially on hollywood movies than on the original novel, the frankenstein story has been reduced to the straightforward, one - dimensional tale of a mad scientist who created a hideous and rampaging monster. the original story has been turned into the frankenstein myth and much of the ambiguity of the novel has been lost. one of the effects of this process is that victor frankenstein s noble intentions, including his wish to banish disease from the human frame and render man invulnerable to any but a violent death (: 42) have been eclipsed. langdon winner and stephen jay gould argue that frankenstein s greatest moral failure was that he refused to take proper responsibility and care for the creature he himself had put into this world (: 306314 ;). ellen ter gast, finally, identifies frankenstein s secretiveness and his refusal to communicate with his scientific colleagues and the wider public as a further shortcoming (: 116, 132). bioethicist bernard rollin, for example, referred to the frankenstein story when discussing the ethical and social aspects of animal biotechnology. at the height of the british food scare around genetically modified foods in 1998, the prince of wales single - handedly poured oil on troubled waters by launching the term incidentally, in 1999, when discussing his plans to construct an artificial bacterium with a minimal genome, venter himself declared with some bravado : shelley would have loved this! (: 110). however, forewarned by the furore caused by dolly the cloned sheep, he was smart enough to first solicit ethical advice from a panel of leading bioethicists and theologians which he himself installed, the so - called ethics of genomics group led by arthur caplan and mildred cho. in the event, the panel saw no fundamental objections against venter s plans to construct a minimal genome and eventually delivered the desired positive advice [15, 64 ]. ironically, journalist chris mooney commented that victor frankenstein could have saved himself a lot of trouble, had he conducted his affairs in the same smart way as venter:if only victor frankenstein had had some media savvy, he might have been j. craig venter. rather than living in dread of his appalling creature, he could have assembled a panel of bioethicists and theologians to bless it, applied for a swiss government grant to research it, and hired an investment bank to explore an initial public offering if only victor frankenstein had had some media savvy, he might have been j. craig venter. rather than living in dread of his appalling creature, he could have assembled a panel of bioethicists and theologians to bless it, applied for a swiss government grant to research it, and hired an investment bank to explore an initial public offering venter is indeed a pre - eminent example of the modern bio - entrepreneur, who deftly combines scientific and commercial objectives. to victor frankenstein, however, although the name of mary shelley s tragic hero is invoked in connection with classical biotechnology as well as with synthetic biology, philip ball takes the view that the comparison with frankenstein s unhallowed arts seems much more appropriate for the latter field:compared with conventional biotechnology and genetic engineering, the risks involved in synthetic biology are far scarier. whether you approve of them or not, gmos are more like patients with an organ transplant than frankenstein s monster. there is no sense in which genetic engineers are making lifebut that is what synthetic biologists propose to do, if indeed they have not already done so. compared with conventional biotechnology and genetic engineering, the risks involved in synthetic biology are far scarier. whether you approve of them or not, gmos are more like patients with an organ transplant than frankenstein s monster. there is no sense in which genetic engineers are making lifebut that is what synthetic biologists propose to do, if indeed they have not already done so. ball later went back on this statement, as we will see below. to avoid unwelcome associations with dr frankenstein s pursuits and to escape the charge of playing god, many current practitioners of synthetic biology plead humility and deny that their activities resemble anything that might come close to creating or thus mit professor george church elaborates on the purportedly humble comparison of synthetic biologists with simple engineers, even if he does not seem entirely willing to give up the coveted epithet of intelligent designers:were acting as engineers, possibly as intelligent designers. the religiously - inclined would not put humans in the same league with the intelligent designer, or god. as creative as we become, and as industrious and as good as we are at designing and manufacturing living things, which we ve been doing since the stone age no matter how good we get at that, it s like calling a candle a supernova. a candle is not a supernova ; it s not even in the same league. and we, as intelligent designers, are not in the same league as the intelligent design forces that started the whole shebang. we re not designing sub - atomic particles from scratch ; we re not designing galaxies. we re really not even designing the basic idea of life ; we re just manipulating it. (; my italics). the religiously - inclined would not put humans in the same league with the intelligent designer, or god. as creative as we become, and as industrious and as good as we are at designing and manufacturing living things, which we ve been doing since the stone age no matter how good we get at that, it s like calling a candle a supernova. a candle is not a supernova ; it s not even in the same league. and we, as intelligent designers, are not in the same league as the intelligent design forces that started the whole shebang. we re not designing sub - atomic particles from scratch ; we re not designing galaxies. we re really not even designing the basic idea of life ; we re just manipulating it. (; my italics). to which one might reply that in this sense victor frankenstein was not designing life (the idea of life) from scratch either : the dissecting room and the slaughterhouse furnished many of my materials (...) (: 55). if the construction of artificial life forms only deserves to be called creation of life when it is created literally out of nothing (creatio ex nihilo), then we can be pretty sure that this elusive aim will never be achieved. belatedly, venter also takes a more modest stance. while he used to compare himself with frankenstein, he now insists that he is not in the business of creating life, but is merely engaged in modifying life to come up with new life forms. perhaps with the cover page of newsweek in mind, he now dismisses any suggestion that he is trying to play god as his collaborator hamilton smith once gave a less timid and less evasive reply to the charge that he was playing god : we do nt play. equally defiant is the response of james watson, the doyen of molecular biology, to the same allegation : if scientists do nt play god, who else is going to?. for the etc group, such declarations simply expose the incurable hubris from which many molecular and synthetic biologists suffer. it seems that synthetic biologists can switch rather easily from a posture of defiance or arrogance to a posture of humility and back again. commenting on debates on nanotechnology, the french philosopher jean - pierre dupuy notes that scientists often oscillate between two opposed attitudes : on the one hand, vainglory, an excessive and often indecent pride ; and on the other, when it becomes necessary to silence critics, a false humility that consists of denying that one has done anything that departs from the usual business of normal science. as a philosopher, dupuy says he is less disturbed by a science that claims to be the equal of god than by a false humility that actually denies the essential distinction between life and non - life (ibid.). it seems to me that it might be helpful to consider the two contrasting postures, arrogance and humility, as different registers from the same rhetorical repertoire, which scientists can play according to the demands of the situation.6 if the situation demands that critics be silenced, scientists will indeed play the register of humility. when, in a recent interview, drew endy was asked if the creation of new life forms should not be left to god, he played the register of humility with aplomb:i do nt view [my research ] projects as creating life, but rather [as ] construction projects. for me as an engineer, there is a big difference between the words creation and construction. creation implies i have unlimited power, perfect understanding of the universe, and the ability to manipulate matter at a godlike level. that s not what i have. i have an imperfect understanding, a budget, limited resources, and i can only manipulate things quite crudely. in that context, with those constraints, i m a more humble constructor. i do nt view [my research ] projects as creating life, but rather [as ] construction projects. for me as an engineer, there is a big difference between the words creation and construction. creation implies i have unlimited power, perfect understanding of the universe, and the ability to manipulate matter at a godlike level. that s not what i have. i have an imperfect understanding, a budget, limited resources, and i can only manipulate things quite crudely. in that context, with those constraints, i m a more humble constructor. given the seemingly religious character of the playing god argument, we might expect theologians to be able to enlighten us about its precise status from a doctrinal point of view. in this regard ted peters, professor of systematic theology at berkeley, california, has a surprise for us in store. he claims that playing god is by no means a theological term ; on the contrary, it articulates a secular rather than a religious vision (: 2 and 13 ;). he also sees no principled objections of a religious nature against making new life forms : what venter is doing is an extremely complicated form of animal breeding. the ethics of genomics group, the panel of bioethicists and theologians installed by venter, also rejected quasi - religious objections against far - reaching human interference with life processes : too often, concern about playing god has become a way of forestalling rather than fostering discussion about morally responsible manipulation of life. (, 2088). one notion that can not be simply rejected out of hand is that the members of venter s ethical panel may have been chosen partly because of their liberal views. a liberal theologian like ted peters would not pretend to speak on behalf of all believers. in this connection the expositions of the dutch theologian frits de lange on the doctrinal differences between orthodox and heterodox views are particularly illuminating, even though he confines his discussion to the various denominations within protestantism. de lange distinguishes between the restoration model of redemption, which is endorsed by orthodox protestants and which considers redemption as a return to the situation before the fall, and the liberal model of redemption, which sees redemption as the completion or perfection of creation. the latter model is characterized as follows:creation is not a separate act of god in the beginning, but an ongoing, dynamic process in history (creatio continua). sinfulness in this connection is just the blockage that occurs when salvation is frustrated by people. creation is not a separate act of god in the beginning, but an ongoing, dynamic process in history (creatio continua). sinfulness in this connection is just the blockage that occurs when salvation is frustrated by people. it is the future that supplies the norm, not the past.. in this view the relationship between creator and creature is such that man can be promoted to the position of creator next to god ; he is, in the words of the lutheran theologian philip hefner, created co - creator.7 de lange points out that such religious beliefs lead to a largely unrestrained optimism with regard to the possibilities of genetic technologies : [ted ] peters often goes so far as to suggest that almost anything that can be done technologically is also morally acceptable, as long as it is good for people. orthodox protestants would rather be guided by the idea that man is inclined to all evil and incapable of doing any good. de lange summarizes the two positions as follows:whoever considers creation as creatio continua and man in this connection as created co - creator [... ] will subscribe to liberal and progressive bioethics. whoever emphasizes the weight of sinfulness and accentuates the discontinuity between human history and god s culmination, on the other hand, will defend a more restrictive ethics and rather point to the risks and hazards involved in tampering with hereditary structures. (ibid.). whoever considers creation as creatio continua and man in this connection as created co - creator [... ] will subscribe to liberal and progressive bioethics. whoever emphasizes the weight of sinfulness and accentuates the discontinuity between human history and god s culmination, on the other hand, will defend a more restrictive ethics and rather point to the risks and hazards involved in tampering with hereditary structures. (ibid.). mit professor george church, for one, provides a slightly secularized version of the theological doctrine that man can act as a created co - creator:we seem to be designed by nature to be good designers. in that sense we re part of some huge recursive design, but we re not doing something we re not designed (and microevolved) to do. engineering is one of the main things that humans do well. [... ] it s just what we do and it s natural. we seem to be designed by nature to be good designers. in that sense we re part of some huge recursive design, but we re not doing something we re not designed (and microevolved) to do. [... ] it s just what we do and it s natural. in other words, nature has designed man to be a designer, just as in the eyes of the liberal theologians, the creator has created man to be a co - creator. in both cases, man s creations, including synthetic life forms, will be considered natural and acceptable. to avoid unwelcome associations with dr frankenstein s pursuits and to escape the charge of playing god, many current practitioners of synthetic biology plead humility and deny that their activities resemble anything that might come close to creating or making life. thus mit professor george church elaborates on the purportedly humble comparison of synthetic biologists with simple engineers, even if he does not seem entirely willing to give up the coveted epithet of intelligent designers:were acting as engineers, possibly as intelligent designers. the religiously - inclined would not put humans in the same league with the intelligent designer, or god. as creative as we become, and as industrious and as good as we are at designing and manufacturing living things, which we ve been doing since the stone age no matter how good we get at that, it s like calling a candle a supernova. a candle is not a supernova ; it s not even in the same league. and we, as intelligent designers, are not in the same league as the intelligent design forces that started the whole shebang. we re not designing sub - atomic particles from scratch ; we re not designing galaxies. we re really not even designing the basic idea of life ; we re just manipulating it. (; my italics). the religiously - inclined would not put humans in the same league with the intelligent designer, or god. as creative as we become, and as industrious and as good as we are at designing and manufacturing living things, which we ve been doing since the stone age no matter how good we get at that, it s like calling a candle a supernova. a candle is not a supernova ; it s not even in the same league. and we, as intelligent designers, are not in the same league as the intelligent design forces that started the whole shebang. we re not designing sub - atomic particles from scratch ; we re not designing galaxies. we re really not even designing the basic idea of life ; we re just manipulating it. (; my italics). to which one might reply that in this sense victor frankenstein was not designing life (the idea of life) from scratch either : the dissecting room and the slaughterhouse furnished many of my materials (...) (: 55). if the construction of artificial life forms only deserves to be called creation of life when it is created literally out of nothing (creatio ex nihilo), then we can be pretty sure that this elusive aim will never be achieved. belatedly, venter also takes a more modest stance. while he used to compare himself with frankenstein, he now insists that he is not in the business of creating life, but is merely engaged in modifying life to come up with new life forms. perhaps with the cover page of newsweek in mind, he now dismisses any suggestion that he is trying to play god as media sensationalism (ibid.). his collaborator hamilton smith once gave a less timid and less evasive reply to the charge that he was playing god : we do nt play. equally defiant is the response of james watson, the doyen of molecular biology, to the same allegation : if scientists do nt play god, who else is going to?. for the etc group, such declarations simply expose the incurable hubris from which many molecular and synthetic biologists suffer. it seems that synthetic biologists can switch rather easily from a posture of defiance or arrogance to a posture of humility and back again. commenting on debates on nanotechnology, the french philosopher jean - pierre dupuy notes that scientists often oscillate between two opposed attitudes : on the one hand, vainglory, an excessive and often indecent pride ; and on the other, when it becomes necessary to silence critics, a false humility that consists of denying that one has done anything that departs from the usual business of normal science. as a philosopher, dupuy says he is less disturbed by a science that claims to be the equal of god than by a false humility that actually denies the essential distinction between life and non - life (ibid.). it seems to me that it might be helpful to consider the two contrasting postures, arrogance and humility, as different registers from the same rhetorical repertoire, which scientists can play according to the demands of the situation.6 if the situation demands that critics be silenced, scientists will indeed play the register of humility. when, in a recent interview, drew endy was asked if the creation of new life forms should not be left to god, he played the register of humility with aplomb:i do nt view [my research ] projects as creating life, but rather [as ] construction projects. for me as an engineer, there is a big difference between the words creation and construction. creation implies i have unlimited power, perfect understanding of the universe, and the ability to manipulate matter at a godlike level. that s not what i have. i have an imperfect understanding, a budget, limited resources, and i can only manipulate things quite crudely. in that context, with those constraints, i m a more humble constructor. i do nt view [my research ] projects as creating life, but rather [as ] construction projects. for me as an engineer, there is a big difference between the words creation and construction. creation implies i have unlimited power, perfect understanding of the universe, and the ability to manipulate matter at a godlike level. that s not what i have. i have an imperfect understanding, a budget, limited resources, and i can only manipulate things quite crudely. in that context, with those constraints, i m a more humble constructor. given the seemingly religious character of the playing god argument, we might expect theologians to be able to enlighten us about its precise status from a doctrinal point of view. in this regard ted peters, professor of systematic theology at berkeley, california, has a surprise for us in store. he claims that playing god is by no means a theological term ; on the contrary, it articulates a secular rather than a religious vision (: 2 and 13 ;). he also sees no principled objections of a religious nature against making new life forms : what venter is doing is an extremely complicated form of animal breeding. the ethics of genomics group, the panel of bioethicists and theologians installed by venter, also rejected quasi - religious objections against far - reaching human interference with life processes : too often, concern about playing god has become a way of forestalling rather than fostering discussion about morally responsible manipulation of life. (, 2088). one notion that can not be simply rejected out of hand is that the members of venter s ethical panel may have been chosen partly because of their liberal views. needless to say, there are also more orthodox views in religious circles. a liberal theologian like ted peters would not pretend to speak on behalf of all believers. in this connection the expositions of the dutch theologian frits de lange on the doctrinal differences between orthodox and heterodox views are particularly illuminating, even though he confines his discussion to the various denominations within protestantism. de lange distinguishes between the restoration model of redemption, which is endorsed by orthodox protestants and which considers redemption as a return to the situation before the fall, and the liberal model of redemption, which sees redemption as the completion or perfection of creation. the latter model is characterized as follows:creation is not a separate act of god in the beginning, but an ongoing, dynamic process in history (creatio continua). sinfulness in this connection is just the blockage that occurs when salvation is frustrated by people. creation is not a separate act of god in the beginning, but an ongoing, dynamic process in history (creatio continua). sinfulness in this connection is just the blockage that occurs when salvation is frustrated by people. it is the future that supplies the norm, not the past.. in this view the relationship between creator and creature is such that man can be promoted to the position of creator next to god ; he is, in the words of the lutheran theologian philip hefner, created co - creator.7 de lange points out that such religious beliefs lead to a largely unrestrained optimism with regard to the possibilities of genetic technologies : [ted ] peters often goes so far as to suggest that almost anything that can be done technologically is also morally acceptable, as long as it is good for people. orthodox protestants would rather be guided by the idea that man is inclined to all evil and incapable of doing any good. de lange summarizes the two positions as follows:whoever considers creation as creatio continua and man in this connection as created co - creator [... ] will subscribe to liberal and progressive bioethics. whoever emphasizes the weight of sinfulness and accentuates the discontinuity between human history and god s culmination, on the other hand, will defend a more restrictive ethics and rather point to the risks and hazards involved in tampering with hereditary structures. (ibid.). whoever considers creation as creatio continua and man in this connection as created co - creator [... ] will subscribe to liberal and progressive bioethics. whoever emphasizes the weight of sinfulness and accentuates the discontinuity between human history and god s culmination, on the other hand, will defend a more restrictive ethics and rather point to the risks and hazards involved in tampering with hereditary structures. (ibid.). mit professor george church, for one, provides a slightly secularized version of the theological doctrine that man can act as a created co - creator:we seem to be designed by nature to be good designers. in that sense we re part of some huge recursive design, but we re not doing something we re not designed (and microevolved) to do. engineering is one of the main things that humans do well. [... ] it s just what we do and it s natural. we seem to be designed by nature to be good designers. in that sense we re part of some huge recursive design, but we re not doing something we re not designed (and microevolved) to do. [... ] it s just what we do and it s natural. in other words, nature has designed man to be a designer, just as in the eyes of the liberal theologians, the creator has created man to be a co - creator. in both cases, man s creations, including synthetic life forms, will be considered natural and acceptable. venter s bioethical panel also busied itself with the challenges that synthetic biology raises with regard to received cultural views on life. in an article published in science magazine in december 1999, the panel stated that the attempt to create a minimal genome (and by implication the rise of synthetic biology) can be interpreted as the culmination of a long - standing reductionist research agenda about the meaning and origin of life. this (interim) last step, more than any previous steps, would place the question what is life? at the centre of the debate. as might be expected, the panel of liberally - minded bioethicists and theologians did not object to the attempt to create a minimal genome as such. wrong conclusions that researchers, the general public or the press could draw from such an attempt:there is a serious danger that the identification and synthesis of minimal genomes will be presented by scientists, depicted in the press [reference omitted ], or perceived by the public as proving that life is reducible to or nothing more than dna. but life need not be understood solely in terms of what technology permits scientists to discover. this may threaten the view that life is special. (: 2088 ; my italics). there is a serious danger that the identification and synthesis of minimal genomes will be presented by scientists, depicted in the press [reference omitted ], or perceived by the public as proving that life is reducible to or nothing more than dna. but life need not be understood solely in terms of what technology permits scientists to discover. this may threaten the view that life is special. (: 2088 ; my italics). the bioethicists and theologians seem to be dispensing far too easily with the issue here. in the near future we may be confronted with situations in which various new life forms will be routinely assembled by synthetic biologists as they see fit ; while, according to the panel, we should keep saying to ourselves that these biological syntheses do not have any real cultural significance and that life remains special and is certainly not reducible to mere dna. as if the only thing we need to guard against is that things are presented this way. a very sanctimonious thought indeed ! meanwhile, arthur caplan, a prominent member of venter s panel, has distanced himself somewhat from the panel s appeasing statement. while creating new life may not be playing god, says caplan, it has revolutionary implications for how we see ourselves. when we can synthesize life, it makes the notion of a living being less special.. now that our view of life and our self - image are directly affected by the new technological potential to make artificial life forms, it seems that the question is no longer seen as just a matter of drawing the the scientific journal nature entered the debate on the meaning of life with an editorial published on 28 june 2007 under the heading meanings of life. as usual, the editorial was unsigned, but in this particular case we know that the publicist philip ball is responsible for its content, because an earlier version is posted on his personal blogspot under the title what is life ? ball s comment was triggered by the response of the etc group to venter s recent scientific feats, in particular pat mooney s previously quoted statement : for the first time, god has competition. the editorial in nature argues that synthetic biology would do us an immense service if it were to free us once and for all from the view precisely because such a difference does not exist, the editorial argues, the concomitant notion of creating life, to which the western cultural tradition, from the myths of the golem and the homunculus to the frankenstein story attaches such great importance, is close to meaningless (: 1032). even scientists trying to set up criteria for what constitutes life are accused of exhibiting a vitalist deviation. we have to rid ourselves of the idea, the editorial insists, that life is a precise scientific concept. instead, the contextual contingency and the ambiguity of the term neither is life a solitary phenomenon, the editorial further explains, because cells come together in colonies and organisms in ecosystems. synthetic biology s view of life as a molecular process lacking moral thresholds at the level of the cell is a powerful one (: 1032). the intervention of nature in the debate on the meaning of life is remarkable for several reasons. ironically, the editorial draws precisely the conclusion that, according to venter s ethical panel, should not be drawn from the new scientific and technological developments, viz., that life is not special. however, the editorial also employs post - modern qualifications such as ambiguous, contingent and contextual, which suggest a positive characterization of the meaning of life after all (just as the remark that life is not a solitary affair can be read with a positive twist). furthermore, the question needs to be asked whether the rejection of thresholds and qualitative transitions is warranted on purely natural scientific grounds or whether it stems from a preconceived dogma that only gradual transitions exist.8 one might wonder if nature s editorial board would be willing to reject the rather unique and fairly liberal british legislation that regulates embryonic stem cell research on the same grounds. the uk legislation allows stem cell research on embryos (or rather pre - embryos) until the age of 14 days. proponents justify this age limit by pointing out that this is the moment when the so - called primitive streak appears and cells start to differentiate (: 115). one could say that in this case an ethical commission (the warnock commission) has given a moral meaning to a in the concluding pages of frankenstein s footsteps, jon turney notes that the story of frankenstein has outlived its former usefulness and nowadays tends to foster a sterile polarization of the debate on new advances in the life sciences. the same could perhaps be said of the standard objection of playing god, which, according to some, has been reduced to a facile journalistic clich or an alarmist slogan. however, it would not be acceptable in a liberal democracy to ban allusions to the frankenstein theme or the playing god argument in public debates. if participants think that these kinds of arguments help to convey their concerns or promote their cause, they can not be prevented from using them. still, it may come across as a little weird for secular organizations like the etc group to accuse synthetic biologists of assuming the role of god (and only slightly less weird to compare them with dr frankenstein). indeed, as the christian philosopher gordon graham notes,... if, as the secular world believes, there is no god, how could there be any danger of human beings illegitimately abrogating to themselves his function? (: 145).9 the dutch physicist - cum - theologian willem b. drees also notes that even non - believers often find playing god a useful metaphor in criticizing new technologies (: 651). a possible answer to the paradox of playing god without god may lie in ted peters hint that the god of playing god is not necessarily the god of the bible, but rather deified nature (: 383). it is the presumed sacredness of nature that the modern life sciences threaten to profane. also to modern secular minds, there appear to be lines that may not be crossed and boundaries that may not be breached (the phrase used by pat mooney in his comment on venter s patent), even in the absence of a god or gods who have the authority to institute such lines and boundaries in the first place. overstepping these boundaries may be construed as inviting unknown and unprecedented risks. to accuse scientists of playing god may thus be just another way of alerting the wider public to the recklessness of their pursuits in the relentless quest for profit and glory. as georgiana kirkham writes, [i]n secular formulations, such phrases [i.e. playing god and interfering in nature s plan ] can act as metaphors for mistaking a considerable amount of power, knowledge and foresight for omnipotence and omniscience, and as metaphors for humans letting their power and knowledge exceed their caution (: 176). you need not be a religious believer to recognize the dangers of hubris and the wisdom of the proverb there are also christian believers who are critical of developments in the life sciences but eschew any allusion to the argument of playing god. one example is the franciscan philosopher keith douglass warner o.f.m., who deliberately refrains from using this argument in his attempt to develop a catholic ethics on agricultural biotechnology:religious ethics can play a more constructive role in the debate on agricultural biotechnology by addressing [the ] patent regime rather than by raising questions about playing god through genetically engineering germplasm, questions that are hard to answer and unlikely to be resolved in industrial societies. the catholic social teaching tradition and its principle of the universal destination of goods fundamentally conflicts with the negative right conferred by gene patents. (: 316). religious ethics can play a more constructive role in the debate on agricultural biotechnology by addressing [the ] patent regime rather than by raising questions about playing god through genetically engineering germplasm, questions that are hard to answer and unlikely to be resolved in industrial societies. the catholic social teaching tradition and its principle of the universal destination of goods fundamentally conflicts with the negative right conferred by gene patents. (: 316). thus warner arrives at a very critical position with regard to biotech patents on the basis of the traditional social teaching of the roman catholic church. one could read this article as an implicit message from a catholic priest to a secular organization like the etc group : stop raising questions about synthetic biologists playing god, but concentrate your critical effort on questioning the legitimacy of their patents ! (by the way, in his public lectures, venter is conspicuously silent about the role of patents in synthetic biology.) it is by no means certain that the playing god argument or references to frankenstein will always help the groups that question the activities of life scientists. although most scientists are quick to deny that they are attempting to play god or following in frankenstein s footsteps, sometimes the more intrepid among them adopt a more defiant attitude. we have seen that at one point venter invited a comparison of his team s work with that of frankenstein by claiming shelley would have loved this!. and james watson s famous retort if scientists do nt play god, who else is going to? may be another disarming counter - strike. the option of using quasi - religious arguments, for one purpose or another, is open to all parties in the debate. so what, finally, about the question of the meaning of life ? the answer does not lie first and foremost in a rigorous definition. indeed, the whole quest for a scientifically robust definition that has been triggered by the rise of synthetic biology is a red herring (cf. : are we allowed to create new life at will ? or should some kind of religious awe prevent us from emulating frankenstein ? these metaphysical questions are indeed difficult to deal with. some synthetic biologists evade the issue by denying that their constructions of artificial biological systems amount to creating life at all. venter s ethical panel superficially suggested that the new biological syntheses would not affect our cultural understanding of the meaning of life. it can hardly be denied, however, that the debate on what is life?, which has been revitalized by the emergence of synthetic biology, is being largely conducted from a rather narrow anthropocentric perspective. in other words, the question about the meaning of life affects us mainly insofar as it concerns our own life. thus arthur caplan was primarily concerned about the potentially revolutionary implications of synthetic biology for how we see ourselves. the editorial in nature on the meaning of life made particular use of the advances of synthetic biology to score points against religious views on the moral sanctity of human embryos. conversely, when the vatican broached the subject of synthetic biology during the good friday meditations of 2006, the endeavour to modify the very grammar of life was exclusively interpreted in the light of the attack on the family allegedly perpetrated by modern secular society. whenever the subject of synthetic biology is raised in the media, the debate almost inevitably becomes focused on possible interventions in human nature and/or the consequences for human beings. the ethical debate surrounding the birth of dolly the sheep in 1996 is an earlier example of this narrow interest : it turned largely on the prospect of cloning humans. the same anthropocentric focus can be found in the following quotation from leon kass, a conservative bioethicist and former chair of the us president s council on bioethics, in which he sketches the effects of the modern life sciences:all of the natural boundaries are up for grabs. all of the boundaries that have defined us as human beings, boundaries between a human being and an animal on one side and between a human being and a super human being or a god on the other. these are the questions of the 21st century, and nothing could be more important. (kass quoted in). all of the boundaries that have defined us as human beings, boundaries between a human being and an animal on one side and between a human being and a super human being or a god on the other. these are the questions of the 21st century, and nothing could be more important. (kass quoted in). the blurring of the boundary between life and death, or life and non - life, is regarded as important only insofar as it concerns one of the boundaries that have defined us as human beings. kass s statement was quoted with approval in a newspaper article by the dutch molecular biophysicist cees dekker, who is also a committed christian. dekker writes that doom - mongering with regard to future science and technology is not opportune and that he is fascinated by the possibilities of synthetic biology., we may hazard a prediction that as long as synthetic biology confines itself to constructing microbial biochemical factories, hardly anyone will lose any sleep over the fact that this achievement actually comes down to creating life. the same ambiguity or hidden double standard characterizes mary shelley s novel. it is true that frankenstein overstepped the mark by indulging in the unhallowed arts of bestowing animation on lifeless matter, but his real transgression was that he sought to create a human being !	the emergent new science of synthetic biology is challenging entrenched distinctions between, amongst others, life and non - life, the natural and the artificial, the evolved and the designed, and even the material and the informational. whenever such culturally sanctioned boundaries are breached, researchers are inevitably accused of playing god or treading in frankenstein s footsteps. bioethicists, theologians and editors of scientific journals feel obliged to provide an authoritative answer to the ambiguous question of the meaning of life, both as a scientific definition and as an explication with wider existential connotations. this article analyses the arguments mooted in the emerging societal debates on synthetic biology and the way its practitioners respond to criticism, mostly by assuming a defiant posture or professing humility. it explores the relationship between the playing god theme and the frankenstein motif and examines the doctrinal status of the playing god argument. one particularly interesting finding is that liberal theologians generally deny the religious character of the playing god argument a response which fits in with the curious fact that this argument is used mainly by secular organizations. synthetic biology, it is therefore maintained, does not offend so much the god of the bible as a deified nature. while syntheses of artificial life forms cause some vague uneasiness that life may lose its special meaning, most concerns turn out to be narrowly anthropocentric. as long as synthetic biology creates only new microbial life and does not directly affect human life, it will in all likelihood be considered acceptable.
one of the most obvious cutaneous manifestations of tuberous sclerosis (tsc) is facial angiofibromas, which start appearing at 3 to 4 years of age. the number and size of angiofibromas varies markedly between patients, but they have the potential to become extremely unsightly. typically, facial angiofibromas do not improve spontaneously and are frequently treated for the disfigurement. current treatments for facial angiofibromas include vascular lasers, ablative lasers and physically destructive techniques such as shave excision and electrodessication. despite this proactive and costly approach, oral sirolimus has already been used for the renal and neurological manifestations of tsc. in recent years it has been administered topically in varying concentrations for treatment of angiofibromas associated with tsc with good results. this is possibly the first report of the use of topical sirolimus in an indian patient. the article further discusses the problems encountered with the use of the drug in the indian scenario. a 17-year - old female, a known case of tsc, presented to the outpatient department for the cosmetic treatment of disfiguring facial angiofibromas since 5 years of age. dermatological examination revealed several other cutaneous manifestations characteristic of the disease apart from angiofibromas, namely ash leaf macules, shagreen plaque on the back and confetti like hypopigmented macules on the trunk. she had undergone multiple sessions of electrodesiccation earlier with recurrence of lesions. in order to avoid an invasive procedure and its inherent complications, it was planned to start the patient on topical sirolimus. currently sirolimus is available in the international market in two formulations : rapamune oral solution (60 mg per 60 ml in an amber colored bottle) and rapamune tablet available in 1 mg and 2 mg strengths.. oral solution available commercially has been used topically as it is, or as a compounded product. since there is no oral solution of sirolimus available in india, crushed tablets of sirolimus (rapacan-1, biocon, india) were compounded in white soft paraffin using basic pharmacy equipment. prior to starting treatment, the complete blood count, hepatic and renal parameters of the patient were done and they were found to be normal. since there are no available reports of tolerability of sirolimus in the indian setting, it was thought prudent to start sirolimus in a low concentration of 0.1% and further increase the concentration according to the tolerability of the patient. also, starting at a lower concentration would lower the cost of the medication for the patient, ensuring better patient compliance. the patient was instructed to apply the ointment twice daily after washing and drying the face completely. after using the 0.1% preparation for 3 months, there was a definite decrease in the erythema as well as the size of the angiofibromas [figures 1 and 2 ]. encouraged by the results ; in an attempt to accelerate the response, the concentration of sirolimus was increased to 1% which was used for a period of a month with a definite decrease in the size as well as number of angiofibromas with substantial decrease in the erythema [figures 1 and 2 ]. (a) pretreatment image, (b) after 3 months of topical 0.1% sirolimus ointment twice daily, (c) after 1 month of topical 1% sirolimus ointment twice daily (a) pretreatment image, (b) after 3 months of topical 0.1% sirolimus ointment twice daily, (c) after 1 month of topical 1% sirolimus ointment twice daily the patient did not experience any cutaneous adverse effects throughout the treatment with topical sirolimus. as this report is being written, the patient is currently on treatment with 1% sirolimus ointment. the complete blood count, renal and hepatic profile of the patient was monitored every 2 months and was found to be normal. it was not possible to measure the systemic absorption of the sirolimus on account of cost constraints. tsc is an autosomal dominant genodermatosis characterized by development of hamartomatous tumors in multiple organs including brain, skin, kidney, heart and lungs. facial angiofibromas are the most visible and unsightly of all the cutaneous manifestations of tsc and they are seen in up to 80% of the cases. they often result in a negative psychological impact on pediatric patients and stigmatization of their families. current treatment modalities include vascular lasers, ablative lasers and other destructive techniques such as shave excision, cryosurgery, dermabrasion, photodynamic therapy and electrodesiccation. despite proactive and costly approaches, there is no sustained efficacy in the long term and the risks of complications like scarring continue to exist. sedation utilized for these procedures poses a risk because of the underlying seizure disorders in these patients, so does anti epileptic loading and prolonged intubation. sirolimus is a lipophilic macrocyclic lactone which was first isolated from a soil bacterium streptomyces hygroscopicus in rapa nui (easter island) in 1965, hence the old name rapamycin. sirolimus belongs to a novel class of anticancer drugs known as mtor (mammalian target of rapamycin) inhibitors. sirolimus and the related mtor inhibitor everolimus have been used as a targeted therapy for the renal and neurological manifestations of tsc. a patient of tsc who was receiving oral sirolimus after undergoing renal transplantation had pronounced regression of her cutaneous angiofibromas and this triggered its use as a topical agent, in an attempt to minimize systemic toxicity. in patients with tsc, the mtor is aberrantly activated in fibroblast like cells located within the dermal layer of the skin. these cells produce an epidermal growth factor, epiregulin, which stimulates epidermal cell proliferation. hence, epidermal cells are produced at a faster rate than the ability to slough the dead cells from the skin surface. this overproduction of skin cells in conjunction with angiogenesis results in initial appearance and continued progression of facial angiofibromas over time. angiofibromas of tsc shows prominent vascular component owing to increased expression of angiogenic factors like vascular endothelial growth factor (vegf) and mtor overactivation that promotes angiogenesis. sirolimus binds with high specificity to mtor and this binding results in inhibition of mtor activity and ultimately downregulation of cell growth. it also inhibits progression from g1 phase to s phase, suppresses t lymphocyte and antibody production, and inhibits keratinocytic proliferation and neutrophilic inflammatory activity also, inhibition of the mtor pathway decreases output of vegf by inhibiting hypoxia sirolimus has a molecular weight of 914.2 grams / mol, allowing for it to be absorbed through the superficial layers of the epidermis to the deep dermal layer implicated in the development of facial angiofibromas. growing tumors which are probably due to greater proliferative component during early stages of life, could be more sensitive to the inhibitory action of sirolimus on mtor and hence it is justifiable to initiate treatment as soon as the angiofibromas start to appear in early childhood. various investigators have used different concentrations of topical sirolimus for varying duration for the management of facial angiofibromas [table 1 ]. use of topical sirolimus in the treatment of facial angiofibromas of tuberous sclerosis hence, topical sirolimus is an effective treatment for angiofibromas especially in young children with flatter lesions, with the preparation formulated in petrolatum being well tolerated with no adverse effects. it is also cost effective if compared to hospital admission for laser therapy under general anesthesia with due risks. the treatment considerations that would probably be more relevant in the indian sitting are : high cost of the medication when the concentration of the medication was increased to 1%, the expenditure of the patient can be more than rs 200 per day which is very high for economically strained conditions existing in indiait is practical to use commercially available oral solution of sirolimus, since compounding pharmacies are not always readily accessible and the stability and efficacy of the compounded preparation can not be ensured. since oral solution was not available a topical preparation using crushed tablets of sirolimus was used in this casecutaneous irritation or burning sensation has been mentioned in the literature as the most common side effect after topical sirolimus. it has been advised to co - prescribe topical hydrocortisone 1% or desonide 0.05% lotion to counteract irritation and ensure compliance. in this case, the patient tolerated the preparation very wellthough topically applied sirolimus has minimal systemic absorption, it should have been monitored using chromatographic and immunoassay methodologies, which was not possible in a resource - constrained settingthe duration of treatment and the chances of recurrence are not defined. high cost of the medication when the concentration of the medication was increased to 1%, the expenditure of the patient can be more than rs 200 per day which is very high for economically strained conditions existing in india it is practical to use commercially available oral solution of sirolimus, since compounding pharmacies are not always readily accessible and the stability and efficacy of the compounded preparation can not be ensured. since oral solution was not available a topical preparation using crushed tablets of sirolimus was used in this case cutaneous irritation or burning sensation has been mentioned in the literature as the most common side effect after topical sirolimus. it has been advised to co - prescribe topical hydrocortisone 1% or desonide 0.05% lotion to counteract irritation and ensure compliance. in this case, the patient tolerated the preparation very well though topically applied sirolimus has minimal systemic absorption, it should have been monitored using chromatographic and immunoassay methodologies, which was not possible in a resource - constrained setting the duration of treatment and the chances of recurrence are not defined. thus, randomized controlled trials are necessary to enable us to confirm the efficacy of this treatment, its long term safety, the optimal dosage and possibility of reappearance once the drug is withdrawn. also, given the small number of cases described to date, the most suitable presentation of the product has not yet been determined. this is possibly the first case report of use of topical sirolimus in an indian patient.	facial angiofibromas are the most visible and unsightly of all the cutaneous manifestations of tuberous sclerosis (tsc). a 17-year - old female, a known case of tsc, presented for the treatment of cosmetically disfiguring facial angiofibromas. she was started on twice daily application of 0.1% sirolimus ointment prepared from crushed tablets of sirolimus compounded in white soft paraffin. after 3 months of use, there was visible decrease in the erythema and the size of the angiofibromas. in an attempt to accelerate the response, the concentration was further increased to 1% sirolimus which was used for a month, resulting in a decrease not only in the size and redness but also in the number of the angiofibromas. the patient did not experience any cutaneous or systemic complications related to therapy. sirolimus belongs to a novel class of anticancer drugs known as mtor (mammalian target of rapamycin) inhibitors. sirolimus has been used as a targeted therapy for the renal and neurological manifestations of tsc. topical preparation of sirolimus is not commercially available till date and hence preparations from crushed tablets or oral solution of sirolimus have been used with beneficial effects in treatment of angiofibromas especially in younger patients with flatter lesions. randomized controlled trials are necessary to enable us to confirm the efficacy, long - term safety, the optimal dosage and possibility of reappearance once the drug is withdrawn. this is possibly the first case report of the use of topical sirolimus in india.
airway inflammation plays an important role in various respiratory lung diseases, including recurrent wheezing, asthma, cystic fibrosis (cf) and chronic obstructive pulmonary disease (copd). several attempts have been made therefore to detect and monitor inflammatory changes and mediators using non - invasive methods. analysis of exhaled breath condensate (ebc), a novel and a non - invasive method for studying the composition of airway lining fluid, has the potential for assessing airway inflammation. this study helps to validate the analysis of ebc by measuring hydrogen peroxide (h2o2) concentration in healthy non - smokers, smokers, diseased, and also comparing the response to treatment. elevated levels of h2o2 have been found in a number of respiratory disorders, thus h2o2 is considered to be a possible biomarker of airway inflammation. in this hospital - based study, conducted between july 2010 and september 2010, 100 randomly selected subjects were analyzed with ebc h2o2. sputum positive tuberculosis patients, pregnant women, children less than 12 yrs and immunocompromised patients were excluded from the study. ebc was collected and analyzed using ecocheck - ecosreen (jaeger, hoechberg, germany) device in all 100 subjects. the subjects were instructed to clean the oral cavity with water and then breathe through a mouth piece and a 2 way non - breathing valve, which also serve as a saliva trap. they were asked to breath at a normal frequency and tidal volume wearing a nose clip for a period of 15 min. about 1 - 3 ml of ebc was collected at 2 to 4c [figure 1 ]. of the 100 cases studied, 23 were healthy individuals with risk factors, like smoking, exposure to air pollution and urbanization. the values of h2o2 in smokers were 200 - 2220 nmol / l and in non - smokers values were 340 - 760 nmol / l [table 1, figure 2 ]. in 10 smokers the standard deviation was 643.135 and in 13 non - smokers standard deviation was 217.279i with significant p value of 0.045 (p<0.05) [table 2 ]. in people residing in rural areas values were from 20 - 140 h2o2 concentrations were correlated with pack years. in majority of subjects, as the pack years increased, the h2o2 levels were also found to be increased. however in some of the subjects varied h2o2 levels were observed irrespective of pack years. for instance, one subject with 8 pack years had the h2o2 level as high as 2220 nmol / l, whereas in two subjects with 15 pack years, the values were 180 and 340 nmol / l [table 3 ]. h2o2 concentration in cases of healthy subjects analysis in healthy subjects statistical analysis of h2o2 concentration in healthy subjects values of h2o2 in relation to pack years in patients who are known copd presented with acute exacerbations (as per anthonisen criteria and gold criteria), the predicted fev1% varied from 32 - 65% (4816%) with h2o2 levels of 540 - 3040 nmol / l. these patients were treated with bronchodilators and corticosteroids as per treatment protocol. following treatment, the predicted fev1% varied from 35 - 71%(5318%) and the concentrations of h2o2 were reduced to 240 - 480 nmol / l [table 4, figure 3 ]. before treatment standard deviation was 770.076 and following treatment standard deviation was 94.571 with p value of 0.022 [table 5 ]. h2o2 concentration and predicted fev1% in copd analysis report in copd statistical analysis of h2o2 concentration in copd in cases of acute exacerbations of bronchial asthma, the values of h2o2 were 400 - 1140 nmol / l and following treatment the values were reduced to 100 - 320 nmol / l [table 6, figures 4 and 5 ] with a significant p value of 0.002 [table 7 ]. in these patients predicted fev1% varied from 18 - 62% (4022%) and following treatment the predicted fev1% drastically improved to 68 - 89% (7810). h2o2 concentration and predicted fev1% in bronchial asthma analysis report in bronchial asthma analysis report in bronchial asthma statistical analysis of h2o2 concentration in asthma in other conditions like bronchiectasis, values of h2o2 were 300 - 340 nmol / l and 200 - 280 nmol / l [table 8 ], in pneumonia 1060 - 11800 nmol / l and 540 - 700 nmol / l [table 9 ], and in patients with interstitial lung diseases 220 - 720 nmol / l and 210 - 510 nmol / l [table 10 ] before and after treatment, respectively. the p values of the above three conditions could not be calculated as the sample size was small. spirometry was also performed in all these patients but the lung function tests could not be correlated with h2o2 in all these patients as the sample size was small. a variety of inflammatory markers present in ebc have been investigated as possible biomarkers of disease activity. there is increasing evidence that exhaled markers may reflect biochemical changes in airway lining fluid. lung is constantly exposed to oxygen, so highly susceptible to oxidative stress in the form of reactive oxygen species (super oxide ion, hydroxyl radical, and hydrogen peroxide). these reactive oxygen species produced by active inflammatory cells like neutrophils, macrophages, activated eosinophils, epithelial cells, and endothelial cells. thus, measurement of concentration of reactive oxygen species in exhaled breath condensate can reflect the underlying inflammation. in the present study, contents of exhaled breath condensate exhaled breath condensate was measured in cigarette smokers versus healthy control subjects. cigarette smokers had a 5-fold higher mean expired breath h2o2 level than non - smokers. in another study that attempted to correlate exhaled breath h2o2 with h2o2 generated from the alveolar lining fluid, exhaled h2o2 was 5 10 times lower than h2o2 produced in the alveolar lining fluid. this difference was attributed to the presence of antioxidants in the lining fluid of the lower respiratory tract. the above study showed that level of h2o2 in exhaled breath condensate of smokers is increased half an hour after combustion of one cigarette. in the present study, the levels of h2o2 were elevated in healthy smokers and also in healthy non - smokers who are residing in urban area compared to those of rural area. the h2o2 values in healthy individuals with risk factors are more than 180 nmol / l, whereas the healthy individuals residing in rural areas with minimal risk factors had values of h2o2 varied from 20 - 140 nmol / l. hence, the level of h2o2 below 200 nmol / l can be considered as normal reference value as per our study and needs further studies to support our observation in india. dekhuijzen and coworkers demonstrated increased h2o2 in exhaled breath condensate of patients with stable copd relative to healthy controls with a further increase noted during an acute exacerbation.[1316 ] the effect of corticosteroids on the level of hydrogen peroxide studied by van beurden. levels of h2o2 also correlated with eosinophils differential counts in induced sputum. in the present study, h2o2 was increased in all stable copd patients with further increase during acute exacerbations with reduced predicted fev1%. these patients with exacerbations after treatment with bronchodilators, corticosteroids (both inhalational and parenteral) showed reduction in h2o2 levels with the improvement in predicted fev1%. they concluded that exhaled h2o2 may be useful to assess the degree of airway inflammation and oxidative stress in asthmatic patients and significant negative correlation among exhaled h2o2 and fev1. in the present study, the h2o2 levels were elevated during acute exacerbation with decreased predicted fev1% and reduced h2o2 levels with significant improvement in predicted fev1% in all cases following treatment. bronchiectasis, a suppurative lung disease, is characterized by significant pulmonary oxidant stress that can be measured using exhaled breath h2o2. in a study by loukides and coworkers, patients with bronchiectasis displayed exhaled h2o2 levels higher than normal controls, and a negative correlation between the h2o2 levels and fev1 was documented. in the studied cases of bronchiectasis, h2o2 was raised significantly with reduction in the levels following treatment. in the pilot study by mikuls., patients with rheumatoid arthritis with interstitial lung diseases had increased levels of exhaled h2o2 compared with controls, suggesting that ebc h2o2 is a potentially useful biomarker. in the present study, in patients with interstitial lung disease, pneumonia h2o2 estimated by ebc were found to be raised and showed decreased values following treatment. the measurement of the h2o2 marker in exhaled breath condensate can be used routinely for i) early prediction of the ongoing inflammatory process in healthy individuals who are exposed to risk factors, and for educating them in future, ii) early tool of assessing exacerbation of the lung condition and to reduce the morbidity, iii) as a marker in assessing the inflammatory response to treatment. hence, the detection of h2o2 in ebc can be used for routine clinical practice and research activities. this type of facility is a rare modality available in india, as per our knowledge. the drawbacks of this facility are that the establishment of the unit is quite expensive and sensors need to be changed for every new patient, which are to be imported and of high cost. measurement of h2o2 in ebc sample can be used as a method of measuring oxidative destruction in the lung and inflammation of the airways. even in healthy individuals with risk factors, elevated h2o2 levels in ebc is a general marker for airway inflammation and can be used as an early predictor of the ongoing inflammatory process. this measurement can be carried out easily and its application in inflammatory airway diseases has been extensively studied. most of the clinical studies reported higher levels of h2o2 in healthy individuals with risk factors and diseased conditions compared to normal subjects without risk factors and also the levels of h2o2 decreased following treatment.	objectives : to study the ongoing inflammatory process of lung in healthy individuals with risk factors and comparing with that of a known diseased condition. to study the inflammatory response to treatment.background:morbidity and mortality of respiratory diseases are raising in trend due to increased smokers, urbanization and air pollution, the diagnosis of these conditions during early stage and management can improve patient 's lifestyle and morbidity.materials and methods : one hundred subjects were studied from july 2010 to september 2010 ; the level of hydrogen peroxide concentration in exhaled breath condensate was measured using ecocheck.results:of the 100 subjects studied, 23 were healthy individuals with risk factors (smoking, exposure to air pollution, and urbanization) ; the values of hydrogen peroxide in smokers were 200 - 2220 nmol / l and in non - smokers 340 - 760 nmol / l. in people residing in rural areas values were 20 - 140 nmol / l in non - smokers and 180 nmol / l in smokers. in chronic obstructive pulmonary disease cases, during acute exacerbations values were 540 - 3040 nmol / l and 240 - 480 nmol / l following treatment. in acute exacerbations of bronchial asthma, values were 400 - 1140 nmol / l and 100 - 320 nmol / l following treatment. in cases of bronchiectasis, values were 300 - 340 nmol / l and 200 - 280 nmol / l following treatment. in diagnosed pneumonia cases values were 1060 - 11800 nmol / l and 540 - 700 nmol / l following treatment. in interstitial lung diseases, values ranged from 220 - 720 nmol / l and 210 - 510 nmol / l following treatment.conclusion:exhaled breath condensate provides a non - invasive means of sampling the lower respiratory tract. collection of exhaled breath condensate might be useful to detect the oxidative destruction of the lung as well as early inflammation of the airways in a healthy individual with risk factors and comparing the inflammatory response to treatment.
atr - x syndrome, formally called alpha - thalassaemia x - linked mental retardation syndrome results from an abnormality in the atrx gene. clinical diagnosis is based on the presence of mental retardation in combination with alpha - thalassaemia and a characteristic and recognizable facial gestalt consisting of small head circumference, upsweep of the frontal hair, telecanthus, epicanthic folds or hypertelorism, small triangular nose, retracted columella, midface hypoplasia and tented upper lip which results in a consistently open mouth1. we report here two brothers with severe developmental delay, characteristic facial anomalies, and alpha - thalassaemia. clinical diagnosis of atr - x syndrome was not suspected until the second brother was evaluated for developmental delay. presence of atr - x syndrome was confirmed by mutation analysis of atrx gene, which showed a novel substitution in exon 31 of the atrx gene. case 1 : patient 1 was male, delivered at term by lower segment caesarian section with a breech presentation. antenatally there was intra uterine growth retardation (iugr) and a single umbilical artery was found. he was evaluated at the age of 5 months with complaints of delayed head holding. metabolic screening by mass tandem spectrometry and gas chromatography mass spectrometry was carried out and found normal. ophthalmic evaluation and eeg the cause of delay was not recognized then and at the age of five years he was re - evaluated for delay. he could neither recognize his parents, nor could he speak and was noted to have microcephaly (head circumference 42 cm,-4 sd) and severe hypotonia. dysmorphic features consisted of a cowlick, bilateral epicanthic folds, depressed nasal bridge and bilateral camptodactyly of the upper limbs (fig. facial features were similar to the elder sib consisting of a widow 's peak or upsweep of the frontal hair, hypertelorism, low - set ears, flat nasal bridge, small nose, tented upper lip and everted lower lip (fig. he had multiple caf - au - lait spots on his back, scrotum and left thigh. these spots were less than 6 in number and size was also less than 1 cm. mri brain findings included a thin corpus callosum, mildly dilated ventricular system and white matter changes suggestive of periventricular leukomalacia. complete blood count showed anaemia and low mean corpuscular volume (mcv) and mean corpuscular haemoglobin (mch). peripheral smear suggested microcytosis, anisocytosis, tear drop cells. serum iron and tibc was normal. hplc for haemoglobins was normal, with a slightly raised hbf level of 4.2 per cent corresponding to his age. faint band in the region of hba2, prominent band in the region of hb a and fast moving band were suggestive of the presence of hb h. three generation pedigree. staining of red blood using brilliant cresyl blue was done which showed hbh inclusions in 25 per cent of cells examined. diagnosis of atr - x was then suspected, mutation study was undertaken for confirmation. molecular study of atrx gene : deletion / duplication analysis of the alpha - thalassaemia gene was first performed using multiple ligation probe amplification (mlpa) using mrc holland kit, the netherlands. atrx gene analysis in younger child was then done by the methods described earlier using denaturating hplc technique4. a hemizygous substitution in exon 31 c.6718c > t (p.l2240f) of the atrx gene was found. x - inactivation study was done in mother, which showed that she had skewed x- inactivation. clinical features of both patients were consistent with atr - x syndrome. some facial features present in atr - x syndrome are also found in coffin - lowry syndrome. however, the absence of genital anomalies, presence of pudgy tapering digits and the carrier manifestations in coffin - lowry distinguish it from atr - x syndrome3. the facial features may have a similarity with smith lemli opitz syndrome as was reported by stanek. because of overlap of clinical features with other syndromes and history of perinatal asphyxia, the diagnosis of atr - x syndrome in this case was not suspected until presentation of the second sibling. this highlights the importance of correct interpretation of dysmorphic features and combining clinical data with additional investigations and pedigree analysis. the combination of mental retardation and alpha- thalassaemia could be confused with atr-16 syndrome, which involves a gene deletion on chromosome 16. this can be differentiated from atr - x by the absence of typical dysmorphic features. most cases will have characteristic and recognizable facial gestalt during infancy which is probably due to facial hypotonia3. the characteristic facial features seen in both cases are found in 94 per cent of the patients with atr - x syndrome. the occurrence of hepatosplenomegaly in patient 1 is usually not reported as a feature of atr - x syndrome. in this case as patient 2 did not have the same finding, we are unsure whether this should be included as a clinical feature. as per our knowledge, the caf - au - lait spots observed in patient 2 have not been reported before in cases of atr - x syndrome. the gene contains two major domains one phd domain (zinc finger domain) at n- terminus and the other helicase domain at the c- terminus. the gene is involved in the chromatin remodeling in combination with other chromatin associated proteins5. alpha- thalassaemia is caused as the atrx gene is involved in the regulation of the alpha - thalassaemia gene expression. although the causative mutations are scattered all over the gene, 43 per cent of mutations are seen in phd- like domain and 41 per cent in helicase domains6. our patient had mutation in exon 31, which is a novel mutation not reported earlier. carrier status for atr - x syndrome can be identified by mutation detection, which is the most sensitive method. however, in case the mutation is unknown, x inactivation studies can be opted for. unaffected carriers will display skewed x - inactivation, like the proband 's mother in the present case. it should however, be noted that this is only a supportive test and a skewed x inactivation could also signify the presence of other x - linked syndrome or may simply be a variation of the normal pattern, which is found in about 10 per cent of women3. on the other hand, non - skewed x - inactivation could also result in phenotypic features of atr - x syndrome in females and hence cause (moderate) mental retardation7. once a mutation is identified, prenatal diagnosis can be offered for future pregnancies and relevant family members could be tested for their carrier status. management of patients with atr - x is based on a multidisciplinary approach focusing on developmental skills and secondary problems related to the syndrome. the presence of anaemia due to alpha - thalassaemia does not require any treatment in general3. in conclusion, this report described the presence of atr - x syndrome in two brothers displaying many of the typical features associated with this syndrome. a new c.6718c > t mutation has been identified in the family, which has further increased the number of causative mutations within the spectrum of atr - x syndrome.	atr - x syndrome is an x - linked mental retardation syndrome characterized by mental retardation, alpha thalassaemia and distinct facial features which include microcephaly, frontal hair upsweep, epicanthic folds, small triangular nose, midface hypoplasia and carp - shaped mouth. here we report two brothers with clinical features of atr - x syndrome, in whom a novel missense (c > t) mutation was identified in exon 31 of the atrx gene.
deep venous thrombosis (dvt), characterized by a blood clot which forms in deep veins (leg or pelvis) in the body, is a major medical problem worldwide. according to the statistical data, the annual incidence of dvt was about 0.1% and the mortality of dvt cases was 6%, which makes it one of the most common and severe cardiovascular disease. moreover, dvt, especially happened in iliofemoral vein, could frequently cause postthrombotic syndrome (pts) and pulmonary embolism (pe), which will result in high mortality and severe impairment of our normal life. therefore, searching for the efficient therapy could relieve the burden brought by the disease. historically, the standard care for the patients with dvt has been anticoagulation treatment with heparin and coumadin. however, this form of therapy does not effectively treat the existing thrombus and will consequently lead to obstruction of the venous outflow and destruction of the valve function. optimal therapies which address the existing thrombus include surgical thrombectomy, systemic thrombolytic therapy, and catheter - directed thrombolysis (cdt). among them, cdt is the most attractive method because it can effectively achieve the patency of lumen and remove the thrombus lining the venous valves. besides, several agents including streptokinase, urokinase, and recombinant tissue - type plasminogen activator (rt - pa) have been suggested to be efficacious in clinical practice in the past 30 years. among them, the second - generation pa urokinase is the dominant thrombolytic agent for cdt due to its consistency, predictability, and low costing. in china, cdt with urokinase is also widely used, and the dosage is relatively lower than the report in western countries. until now, no report has focused on the safety and efficacy of low dosage urokinase for cdt. here, we performed a retrospective review on the safety and efficacy of low dosage urokinase for cdt in the chinese population, and our results suggested low - dose urokinase with cdt is an efficacious and safe therapeutic approach for dvt patients. this study was approved by the institutional review board of the second affiliated hospital of soochow university and all participants provided written informed consent for the procedure. inclusion criteria consisted mainly age range 1880 years, suffered from acute dvt extending to the high femoral or iliac vein, symptom duration of less than 2 weeks, verified by ultrasound or digital subtraction angiography (dsa), good functional status, life expectancy of 1-year or more and low risk of bleeding. the following exclusion criteria were applied : isolated infrapopliteal thrombosis, contraindications to contrast media or thrombolytic agents, stroke within 3 months, gastrointestinal bleeding or trauma within 3 weeks, uncontrollable hypertension, bacterial endocarditis, anemia, and renal failure. from july 2009 to december 2012, a total of 702 patients with dvt were registered for the study. in the initial phase, in addition, 14 patients who refused to perform the cdt therapy were also excluded. thus, altogether 427 patients with high femoral or iliac vein thrombosis were eligible for intervention with cdt in the study and electronic hospital records were interrogated with regard to patient demographics, co - morbidities, risk factors for dvt [table 1 ]. demographic data of the patients (n=427) recent major surgery was defined as surgery experienced 3090 days before the onset of dvt ; the scope for a classification of immobilization was 430 days before the onset of dvt. first of all, lower limb venography was performed under dsa (dsa, ge innova 3100, usa) by injecting contrast media via dorsal metatarsal vein to determine the extent of the thrombus and the approach to place vena cava filter. based on the angiographic results, permanent or temporary filter (optease retrievable vena cava filters, trapease vena cava filters, cordis, usa and aegisy vena cava filters, lifetech, china) was inserted percutaneously via either contralateral femoral vein or internal jugular vein. next, with the patient in the prone position, venous access was achieved with a 4f micropuncture needle set through the ipsilateral small saphenous vein. other appropriate venous access, such as popliteal vein, anterior or posterior tibial vein, the calf or inguinal veins, then a 4f or 5f vascular sheath was carefully inserted into the vein in which all subsequent catheter and wire exchanges were performed. a 4f or 5f unifuse infusion catheter (length 2040 cm, unifuse infusion catheter, angiodynamics, ny, usa) was then gently placed with the tip embedded in the proximal extent of the thrombus. urokinase (uk, lizhu pharmacy corp, zhuhai, china) was first injected at a bolus dose of 200,000300,000 u and followed by continuously infusions of 400,0001,000,000 u / d pumped through the catheter. the dosage of urokinase was adjusted according to the level of fibrinogen measured by daily analysis of blood coagulation function. if the fibrinogen level dropped below 100 mg / dl, we immediately ceased the use of urokinase. venographic controls were performed every 24 h to follow lysis, and the catheter was repositioned until > 90% of thrombi was lysed. when there was no residual thrombus or the venography assessment indicated unchanged thrombus after 24 h, the infusion catheter was removed. contemporary low molecular weight heparin (lmwh, aventis intercontinental, france) was administered subcutaneously at 4000 u/12 h with a target of 1.21.7-fold level of activated partial thromboplastin time in comparison to reference values (target 4060 s). warfarin was started prior to hospital discharge and given in accordance with local routines based on international guidelines. all the patients were recommended to wear compression stockings (class ii 3040 mmhg) as standard adjunctive treatment. clinical follow - up was scheduled in the outpatient department by venography or duplex ultrasound after 6 months, 1 year, and 2 years thereafter. early efficacy of thrombolysis was assessed by venography using a contrast injection through the introducer and perfusion catheter. a thrombus score was evaluated for seven deep vein segment, including inferior cava vein, the common iliac vein, the external iliac vein, the common femoral vein, the proximal and distal segments of superficial femoral veins, and the popliteal vein. the score was 0 when patent vein, 1 when partially occluded, and 2 when completely occluded. the difference between the pre- and post - lysis thrombus scores divided by the prelysis score resulted in the percentage of thrombolysis, which was classified into three groups : grade i 50% ; grade ii = 5090%, and grade iii = complete thrombolysis. lysis grades ii and iii (50%) were considered as successful outcomes (marked lysis). long - term outcomes were assessed during follow - up after 6 months, 1 year, and 2 years. iliofemoral patency was defined as regained when the following findings were present : flow in the pelvic and femoral vein, compressibility of the femoral vein, and no functional venous obstruction. pts was diagnosed using the villalta scale, consisting of five patient - rated leg symptoms (pain, cramps, heaviness, paresthesia, and pruritus) and six physician - rated clinical signs (pretibial edema, skin induration, hyperpigmentation, redness, venous ectasia, and pain on calf compression). each sign / symptom is rated as 0 (none), 1 (mild), 2 (moderate), or 3 (severe), and the scores are summed up to produce a total score whereby a score 90% of thrombi was lysed. when there was no residual thrombus or the venography assessment indicated unchanged thrombus after 24 h, the infusion catheter was removed. contemporary low molecular weight heparin (lmwh, aventis intercontinental, france) was administered subcutaneously at 4000 u/12 h with a target of 1.21.7-fold level of activated partial thromboplastin time in comparison to reference values (target 4060 s). warfarin was started prior to hospital discharge and given in accordance with local routines based on international guidelines. all the patients were recommended to wear compression stockings (class ii 3040 mmhg) as standard adjunctive treatment. clinical follow - up was scheduled in the outpatient department by venography or duplex ultrasound after 6 months, 1 year, and 2 years thereafter. early efficacy of thrombolysis was assessed by venography using a contrast injection through the introducer and perfusion catheter. a thrombus score was evaluated for seven deep vein segment, including inferior cava vein, the common iliac vein, the external iliac vein, the common femoral vein, the proximal and distal segments of superficial femoral veins, and the popliteal vein. the score was 0 when patent vein, 1 when partially occluded, and 2 when completely occluded. the difference between the pre- and post - lysis thrombus scores divided by the prelysis score resulted in the percentage of thrombolysis, which was classified into three groups : grade i 50% ; grade ii = 5090%, and grade iii = complete thrombolysis. lysis grades ii and iii (50%) were considered as successful outcomes (marked lysis). long - term outcomes were assessed during follow - up after 6 months, 1 year, and 2 years. iliofemoral patency was defined as regained when the following findings were present : flow in the pelvic and femoral vein, compressibility of the femoral vein, and no functional venous obstruction. pts was diagnosed using the villalta scale, consisting of five patient - rated leg symptoms (pain, cramps, heaviness, paresthesia, and pruritus) and six physician - rated clinical signs (pretibial edema, skin induration, hyperpigmentation, redness, venous ectasia, and pain on calf compression). each sign / symptom is rated as 0 (none), 1 (mild), 2 (moderate), or 3 (severe), and the scores are summed up to produce a total score whereby a score 24 weeks varied between 70% and 90%. in this study the duration of symptoms within different grades was as follows : class i : 7.25 5.79 days ; class ii : 6.42 5.35 days, and class iii : 5.03 3.79 days. the longer symptom duration could lead to worse lysis grade (p < 0.05). however, no significant differences were observed in different grades on the period of treatment [table 4 ]. the incidence of bleeding is lower than that reported by mewissen. in a multicenter register study of 473 treated limbs (11%), and by manninen notably, only one symptomatic pe (0.002%) was found in our study while mewissen. reported a 1% incidence of pe, and grossman and mcpherson showed a 0.9% incidence in 214 patients. furthermore, we encountered minor bleeding complication in 31 patients (7.2%), and 20 of them were presented in the puncture site. we found that there did not exist significant differences on the dosage between minor bleeding and uncomplicated patient and believed it might be associated with puncture technology. thus, cautions should be taken to avoid inadvertent puncture of adjacent vessels such as the popliteal artery or the common femoral artery during the needle access to the vein. what 's more, the dosage of urokinase was same (all 8.0 10 u / d) in 7 patients presented major bleeding, which had a higher average daily infusion than those with minor bleeding (p < 0.05). this result indicates that the higher dose has a significant correlation with major bleeding, which is in accordance with two previous studies. lacking of data about patients who received high - dose urokinase resulted in an inability to compare the results of patients treated with a different dose of urokinase. another limitation of this study is its retrospective nature and differences from randomized controlled trials. furthermore, adjunctive techniques to cdt were not employed due to higher cost. in conclusion, our results showed that cdt with a low - dose infusion of urokinase was safe and effective for the patients with dvt. however, considering the application of more aggressive endovascular treatments, including adjunctive pharmacomechanical techniques with a fewer dosage of thrombolytic agent and anticoagulation agent, it is likely to accelerate clot lysis and shorten procedural time. these changes could not only lead to further improvements on patency of vessel and clinical outcomes but also decrease risk of complications. thus, the optimal dosage of thrombolytic agent and adjunctive techniques with cdt might be performed in the further study.	background : catheter - directed thrombolysis (cdt) has been a mainstay in treating deep venous thrombosis (dvt). however, the optimal dosage of a thrombolytic agent is still controversial. the goal of this study was to evaluate the safety and efficacy of low dosage urokinase with cdt for dvt.methods:a retrospective analysis was performed using data from a total of 427 patients with dvt treated with cdt in our single center between july 2009 and december 2012. early efficacy of thrombolysis was assessed with a thrombus score based on daily venography. the therapeutic safety was evaluated by adverse events. a venography or duplex ultrasound was performed to assess the outcome at 6 months, 1 year and 2 years postoperatively.results:the mean total dose of 3.34 (standard deviation [sd ] 1.38) million units of urokinase was administered during a mean of 5.18 (sd 2.28) days. prior to discharge, grade iii (complete lysis) was achieved in 154 (36%) patients ; grade ii (5099% lysis) in 222 (52%) ; and grade i (50% lysis) in 51 (12%). the major complications included one intracranial hemorrhage, one hematochezia, five gross hematuria, and one pulmonary embolism. moreover, no death occurred in the study.conclusions:treatment of low - dose catheter - directed thrombosis is an efficacious and safe therapeutic approach in patients with dvt offering good long - term outcomes and minimal complications.
foot drop is the dropping of the forefoot due to weakness and it is characterized by the inability to dorsiflex the ankle. there are various aetiologies for foot drop ; it can be caused by injury to the peroneal nerve, neuromuscular disease, trauma to the l5 nerve root (disc prolapse, direct trauma or iatrogenic), degenerative diseases like poliomyelitis, spinal cord tumours, stroke, intracranial tumours and genetic, such as charcot marie tooth disease and hereditary neuropathy. a 89 year old gentleman presented with sudden onset of low back pain which came on suddenly when he was standing. his mobility deteriorated gradually over the next two weeks until he developed bilateral complete foot drop. he was awaiting a right hemi colectomy for carcinoma of the caecum. in the past he also had a nodular melanoma on his nose with node metastases 7 years ago. he also had past medical history of temporal arteritis, hypertension, left hip arthroplasty and left inguinal hernia repair. mri demonstrating synovial cyst on examination he had straight leg raise of 45 degrees bilaterally. he had 1/5 power of bilateral ankle dorsiflexion and 3/5 power of dorsiflexion of big toe. mri demonstrating synovial cyst mri of the whole spine revealed that he had multiple metastatic deposits throughout his spine with complete collapse of l3 vertebral body but intact disc space. significant spinal canal stenosis at l4/5 level was seen due to a degenerative posterior disc bulge as well as a 2 x 1 x 1 cm size well capsulated, well defined lesion seeming to arise fromthe ligamentum flavum contributing to the canal narrowing.the appearances were thought to be either due to underlying haematoma from ligamentum flavum or a haemorrhagic synovial cyst. he underwent emergency l4/5 lumbar laminectomy. a juxta articular synovial cyst containing straw coloured fluid histology showed dense collagen bearing a small cyst lined by reactive synovium and containing some fibrin but no evidence of neoplasia is seen. however this turned out to be from a completely different pathology in the form of a synovial cyst, even though he did have spinal metastases. synovial cysts are relatively uncommon in the spine but occur most frequently at the l4 - 5 level. the majority of these cysts are lined with synovial cells and contain a straw - coloured fluid. there have been several previous reports of symptomatic hemorrhagic synovial cysts in the lumbar spine. most cases are associated with facet joint osteoarthritis and degenerative spondylolisthesis, but there is no correlation with age, gender, or degree of disc degeneration. as in this case, most patients present with progressive low back and lower extremity radicular symptoms resulting from compression of the thecal sac or nerve roots. patients may complain of focal tenderness over the facet joints with exacerbation of pain during extension. other common findings not present in the current case, include motor and sensory deficits and positive straight leg raising test. the recommended treatment of symptomatic synovial cysts is excision through laminectomy. in this case, a wide decompression was required to facilitate removal of the cyst. other reported treatment options include computed tomography guided fine needle aspiration and percutaneous injection of hyaluronidase (1). symptomatic lumbar facet cysts should be included in the differential diagnosis for patients presenting with progressive back pain and radicular symptoms. patients with significant facet degeneration and instability are predisposed to the development of facet cyst (2). haemorrhage into a lumbar synovial cyst causing an acute cauda equina syndrome has been reported (3). normally synovial cysts cause a gradual onset of symptoms, however these two cases highlight that this can present acutely if haemorrhage occurs into it.	an 89 year old gentleman awaiting surgery for carcinoma of caecum presented with sudden back pain and developed foot drop two weeks later. mri revealed multiple spinal metastases with a cyst in the canal at l4/5 causing spinal canal stenosis. surgery revealed a juxta articular synovial cyst with haemorrhage in it. we discuss the presentation and management of juxtarticular cysts with a review of the literature.
tympanostomy tube (t - tube) insertion is the most common procedure performed in children. the most common complication of t - tube insertion is the development of postoperative otorrhea, which typically has an incidence of 15 - 19%, but can range from 3.4 to 74%.1,2) post - tympanostomy tube otorrhea (ptto) is defined as active drainage through an existing t - tube. based on a recent meta - analysis by hochman,.,2) the following classification has been proposed to describe ptto : 1) early ptto occurs less than 2 weeks postoperatively. 2) late ptto occurs more than 2 weeks postoperatively and is usually caused by pathologies similar to those responsible for acute otitis media or from external contamination. early ptto seen at the first postoperative visit may be due to the operative procedure, underlying disease process, or both. mupirocin calcium ointment is a new topical antimicrobial agent that has broad - spectrum antimicrobial activity against many gram - positive organisms, including methicillin- and vancomycin - resistant strains of staphylococcus aureus (s. aureus).3) it has a unique mechanism of action in which the drug binds to isoleucyl transfer rna synthetase.4) mupirocin ointment is 97% effective at reducing s. aureus.4) this study evaluated the clinical effectiveness of topical mupirocin ointment in reducing early ptto. this study included 98 ears (67 patients ; 36 males, 31 females ; mean age 32.9 years) that had t - tubes inserted because of otitis media between october 2008 and november 2009 (table 1). all patients undergoing t - tube insertion against chronic middle ear effusion or atelectatic otitis media were included during this period. informed consent was obtained from the patients or the families of children enrolled in the study. local or general anesthesia was selected case by case. as local anesthesia, xylocaine spray was primarily applied. in specific cases, infiltration anesthesia with a dental syringe a consistent operating technique was applied in all patients, with the myringotomy performed in the anterior inferior quadrant and the effusion aspirated. the type of middle - ear content was described as mucoid, serous, or none. a paparella type - i polyethylene tube coated with mupirocin was inserted through the tympanostomy. all patients were seen by day 14 postoperatively, with a majority seen between days 7 and 14. the attending surgeon assessed early ptto at the follow - up appointment. oral antibiotics, analgesics, and otic drops were prescribed and no further symptoms persisted 3 days later. most commonly, t - tubes were inserted in patients younger than age 5 years. after 40 years of age, middle - ear effusion (mee) and atelectatic otitis media occurred equally (fig. 1). serous content was the most common finding, followed in order by mucoid, no fluid. oral antibiotics, analgesics, and otic drops were prescribed and no further symptoms persisted 3 days later. most commonly, t - tubes were inserted in patients younger than age 5 years. after 40 years of age, middle - ear effusion (mee) and atelectatic otitis media occurred equally (fig. serous content was the most common finding, followed in order by mucoid, no fluid. gates,.1) proposed a comprehensive list of extrinsic and intrinsic risk factors for ptto. the extrinsic factors included tube type, ear canal sterilization, type of tympanostomy tube placed, perioperative antibiotic use, frequency of water contamination, environmental exposure to upper respiratory infections (i.e., in a daycare environment), and pacifier use. intrinsic factors included patient age ; type of effusion ; prior treatment for acute otitis media ; bacteriology of the mee at the time of tympanostomy tube placement ; the status of the middle - ear content at the time of the tympanostomy tube placement ; status of the nose, adenoids, and paranasal sinuses ; underlying environmental allergy ; eustachian tube dysfunction ; presence of nasopharyngeal reflux ; and gastroesophageal reflux. notably, scott and strunk5) found that canal preparation with povidone iodine and 70% isopropyl alcohol successfully sterilized only 33% of ears that contained bacteria and did not reduce the incidence of early ptto. giebink,.6) reported no significant difference in early ptto, when either 70% alcohol or povidone iodine was used to prepare the external auditory canal. furthermore, kinsella,.7) suggested that it is reasonable to assume that early ptto is determined more by the preexisting condition of the middle ear than by external contamination at the time of surgery, because no difference in ptto rates were observed when comparing touch and no - touch techniques. a more frequently used method for prophylaxis against early ptto is the use of ototopical drops at the time of surgery. one study attempted to distinguish between the chemical effects of the prophylactic antibiotic drops versus the mechanical effects of normal saline instillation and found that ptto rates were lower in the antibiotic group.8) however, gross,.9) looked at early ptto rates in a prospective, blinded, controlled trial comparing saline irrigation of the middle - ear space at the time of tympanostomy tube placement versus the use of otic drops postoperatively for 3 days. they found that the rate and degree of drainage were both reduced statistically in the saline irrigation group, which is in contrast to the findings mentioned above. a meta - analysis of 9 randomized studies suggested that prophylaxis using topical antibiotic drops may reduce the incidence of ptto by half.2) in a recent study conducted in our country, korea, the common pathogens of ptto were coagulase - negative staphylococcus, pseudomonas, and methicillin - resistant s. aureus (mrsa).10) it is very difficult to treat mrsa otorrhea, which tends to worsen rapidly. furukawa,.3) used mupirocin ointment and ofloxacin otic ear drops to treat mrsa otorrhea in chronic otitis media and reported complete control of the otorrhea in the mupirocin group without ototoxicity, but not in the ofloxacin otic drop group. this patient had a problem during the procedure ; the skin of the external auditory canal was damaged during anesthesia. acute external otitis developed 1 week later and was treated with oral antibiotics and analgesics ; no further symptoms were observed 3 days later. debruyne,.11) found a greater incidence of early ptto in children younger than 2 years of age, with an incidence of 20% in this age group compared to a 7.5% incidence of early ptto in children older than 6 years. none of our patients younger than 2 years of age developed early ptto and our data showed no age - related differences. many studies have established that ptto rates are higher in children with mucoid or purulent mees at the time of tympanostomy tube placement as opposed to dry ears or those with serous effusion.12) however, no difference related to mucoid or serous effusion was seen in our study. chronic mee and retracted otitis media are not the result of infection, but are infection - prone states. therefore, we postulate that early ptto is the result of a wound complication induced by the tube insertion procedure. the most effective method to prevent wound complications is to maintain a clean wound and through postoperative wound care. however, a clean wound is practically impossible in middle - ear surgery. therefore, effective postoperative management, dressings, and local antibiotics, are important. golz,.13) retrospectively investigated the use of postoperative systemic antibiotic prophylaxis and found some statistically significant differences. they followed this with a prospective study investigating a one - time preoperative prophylactic dose of augmentin (glaxosmithkline beecham, philadelphia, pa, usa) and found a significant reduction in early ptto. if the probability of early ptto developing is high, as in acute otitis media, purulent discharge, or a traumatic procedure, then more frequent aural cleaning should be performed and systemic and local antibiotics should be used. early ptto is the most common postoperative complication of t - tube insertion. to prevent this complication our data suggested that coating the t - tube with mupirocin ointment could be effective for preventing early ptto.	background and objectivesthe most common complication of tympanostomy tube (t - tube) insertion is the development of postoperative otorrhea. post - tympanostomy tube otorrhea (ptto) is defined as active drainage through an existing t - tube. many surgeons routinely use topical antibiotics as prophylaxis against early ptto. mupirocin calcium ointment is a topical antimicrobial agent with broad - spectrum antimicrobial activity against many gram - positive organisms. this study evaluated the clinical effectiveness of topical mupirocin ointment in reducing early ptto.subjects and methodsthe study included 98 ears (67 patients, mean age 32.9 years) that had a t - tube inserted because of chronic middle ear effusion or atelectatic otitis media. a paparella type - i polyethylene - tube coated with mupirocin was inserted through the tympanostomy. patients were instructed not to use otic drops or any other medications. all patients were seen by day 14 postoperatively.resultsearly ptto occurred in one case (1.5%). no early ptto was seen with a middle ear effusion. nineteen children were treated under general anesthesia ; none developed early ptto.conclusionsinsertion of a t - tube coated with mupirocin ointment could be effective at preventing early ptto.
current theories of declarative memory, drawing on the canonical memory systems framework, suggest two complementary memory systems [13 ] : a hippocampal - based system that specializes in rapid acquisition of specific events (episodic memory) and a neocortical system that slowly learns through statistical regularities (semantic memory). according to these theories, semantic memory is represented by neocortical structures but is acquired only through a slow consolidation [15 ] or transformation processes. the initial acquisition of declarative memories (semantic or episodic) critically depends on the hippocampal memory system, which continues to support them until slow consolidation processes allow neocortical networks to represent memory independently. contrary to this view, recent exciting findings from rats [7, 8 ] suggest that when new information is associated with previously known, well - integrated, knowledge (schema [911 ]) rapid neocortical consolidation occurs. similarly, we recently reported rapid acquisition of arbitrary associations through a mechanism dubbed fast mapping (fm) in amnesic patients due to medial temporal lobe (mtl) damage [12, 13 ] but see [14, 15 ]. if true, this suggests that under some conditions the neocortex is capable of rapid leaning - induced plasticity independently of the hippocampus or with minimal hippocampal support. it is a process by which children infer by exclusion the meaning of new words and which supports subsequent memory for these novel associations even after a single exposure. in their experiment, carey and bratlett showed 3 - 4-year - old children two trays, one of them being red and the other olive and asked the children to bring the chromium tray, not the red one, the chromium one. the children retrieved the olive tray, correctly inferring that the word chromium refers to this unknown color ; moreover, when children were asked after a week to select the chromium one from among six color chips they did so with success such that memory for this new word was retained by the children over a period of at least a week. since this pioneering study, fm has been studied extensively and has been described as critically supporting at least the initial stages of language development and the rapid acquisition of vocabulary in very young children [1619 ]. (1) it involves incidental rather than intentional learning, there is no reference for a later test and no effort is made to memorize the new associates. disjunctive syllogism, a cognitive reasoning process of eliminating a familiar item before inducing the association between the unfamiliar item and the novel label (a or b, not a, therefore b), is believed by some to support this process [15, 20 ]. (3) new information is learned in the context of old information that supports the discovery of the associative relationship and potentially its rapid integration into existing knowledge structures [21, 22 ]. (4) the new information does not overlap with previous associations, avoiding forgetting through neocortical catastrophic interference. although primarily investigated in children, it has been suggested that fm serves as a general learning mechanism, not solely dedicated for word learning and as such should be accessible to adults [23, 24 ]. indeed, fm has been found to also be available in adulthood [12, 14, 20, 24, 25 ]. moreover, this mechanism has been found to exist in other mammals [26, 27 ] and even birds, suggesting it is more than a juvenile language learning apparatus. surprisingly, despite its centrality for human knowledge acquisition, there have been very few empirical studies of the cognitive neuroscience of fm. some theories have postulated that fm is supported by the same neurocognitive systems that support declarative memory. thus, it has been suggested that as in adult declarative learning, only initial acquisition through fm is mtl - dependent [3033 ]. later conceptual rehearsal and storage of word meanings depend on neocortical regions that support gradual and slow storage of context - free information. furthermore, declarative / procedural distinctions have been suggested similar to the memory systems approach. according to these models, the acquisition of a mental lexicon depends on declarative memory and the mtl, whereas grammar involves procedural memory and is supported by the prefrontal cortex and basal ganglia. however, the findings showing that fm efficiently functions in very young children in whom episodic memory and the hippocampal system are not yet fully developed [34, 35 ] led us to hypothesize that fm may be a learning mechanism that is independent of the mtl memory system. to test this, in the first empirical study on this topic, we studied adult amnesiacs with extensive damage to the hippocampus using an adapted fm paradigm. on each incidental learning fm trial in sharon. 's study participants saw two items, one familiar and one unfamiliar (e.g., a dog and a tenrec) and had to answer a perceptual question about one of them (e.g., does the tenrec have quills ?). a surprise forced choice memory recognition test for the unfamiliar items was administered after 10 minutes and again after a week. patients performed as well as controls on associative recognition both after 10 minutes and after one week demonstrating learning and retaining the learning of arbitrary associations after only two short exposures to the picture - label pairs. by contrast, patients were markedly impaired compared with controls on a matched explicit encoding (ee) task. importantly, controls performed much better on ee than fm so that task difficulty or depth of processing can not account for the data. moreover, preliminary data suggested that the information acquired was declarative in nature ; that is, memories were reportable (i.e., conscious) and could be used flexibly, two defining features of declarative memory [3639 ]. these data suggest that fm learning might enable rapid acquisition of arbitrary, declarative, and semantic information independently of episodic memory and the hippocampus (see also but see [14, 15 ] for conflicting findings which we discuss later on). data from two patients who had extensive anterior temporal lobe (atl) and who failed to learn associations through fm provide first clues as to the neocortical substrates that may support fm. more recently, we tested short - term (30 minutes) and long - term (24 hours) retrieval of associations acquired either through fm or through ee using fmri in healthy controls. connectivity analysis revealed that both short- and long - term items studies through fm drove atl - centered networks coupled with posterior lateral cortical regions, with little evidence of overnight systems changes. by contrast, there were widespread time - dependent changes in networks supporting retrieval of ee items, driven by vmpfc - hippocampal interactions, in line with our predictions. however, contrary to our predictions, hippocampal activity was also part of some of the fm networks, suggesting that, whether or not the hippocampus is needed for this kind of learning, healthy controls under high levels of interference may recruit the hippocampus during fm (cf.). either way, these data suggest that, contrary to canonical models of declarative memory, the neocortex may in fact be capable of the rapid plasticity that is required for the acquisition of explicit arbitrary associations and that the atl is critical for this learning. these surprising findings could have important implications for our conception of neocortical plasticity. in the present study we continue to investigate the neuroanatomical basis of fm learning using fmri and multivoxel pattern analysis (mvpa) techniques. data from focal lesion patients can indicate which structures are critical for a particular brain function but do not tell us what other networks might be involved in supporting that function. in addition, brain lesions might change the manner in which an individual performs a task. thus, investigating large - scale networks in healthy brains while they learn associations through fm is important for understanding the functional neuroanatomy of this early learning mechanism in the adult healthy brain. two groups of healthy adults participated in an fmri study, using the subsequent memory design. in the magnet, participants either incidentally encoded picture - label associations through fm or were explicitly instructed to remember these associations (ee). memory for the associations was tested outside the magnet after 15 minutes and after a week. memory performance outside the magnet was then used to investigate the differences in neural networks associated with successful and unsuccessful encoding in fm and ee, respectively. note that investigating within - condition subsequent memory effects alleviates some of the problems associated with the unavoidable differences between the ee and fm conditions. these differences, such as the incidental nature of encoding, active discovery of the association, and the semantic context within which items appear, are inherent to fm but are characteristic to both successful and unsuccessful encoding of fm associates. discovering the brain structures that predict fm - induced successful memory would reveal its possible promotion of rapid neocortical learning. a growing number of studies have demonstrated that machine - learning techniques can be used to extract new information from the neuroimaging data [4247 ]. within the declarative memory field, these techniques have been used to decode specific memories and subsequent memory success and to characterize the information used by participants when they make source memory judgments. the mvpa 's advantage in this study is its superior sensitivity to cognitive states and relating brain activity to behavior on a trial - by - trial basis. we investigated large - scale networks using multivariate analysis methods to address the following questions. (1) can successful versus unsuccessful encoding in fm and ee, respectively, be distinguished from bold response ? (2) what is the relative contribution of the hippocampus and atl to classification success in fm and ee, respectively ? (3) what other neural structures interact with the hippocampus and the atl to allow memory for associations in fm and ee, respectively ? brain decoding refers to decoding stimuli, mental states, behaviors, and other variables of interest from the brain scan data (and thereby showing the data contain information about them) and in our case distinguishing subsequently successful and unsuccessful encoding events. question 2 involves a mixture of brain decoding and brain mapping approaches investigating how the information is mapped onto the activity patterns in particular brain regions. to that end we investigated the decoding success of imaging data when only voxels from the hippocampal region or only voxels from the atl were used. based on our patient data, we expected the loss in decoding success when only hippocampal data are used would be smaller for ee than fm. conversely the loss in decoding success when only atl data are used would be smaller for fm than for ee. question 3 integrates a more conventional brain mapping approach and multivariate analysis that is not limited to our target regions but rather investigates which large - scale networks contribute to each of the encoding conditions. based on previous studies, we expected the ee condition to involve mtl and midline neocortical structures in the frontal and parietal areas. conversely, if fm reflects direct semantic acquisition, lateral and anterior temporal neocortical areas should emerge. forty healthy volunteers were recruited for the study and were randomly assigned to either the fm or the ee conditions. fifteen participants were excluded from the final data analysis due to the following reasons : three were excluded due to technical problems (1 participant from the ee version and 2 from fm version) and an additional 4 subjects were excluded due to unreliable imaging data (movement artifacts ; 1 participant from the ee version and 3 from fm version). moreover, eight participants were excluded because their memory score was not significantly higher than chance (1 participant from the ee version and 7 from fm version). note that, in order to allow for sufficient data to be collected in this study, 62 novel stimuli were used in each condition whereas fm has never been tried with such a large number of stimuli before ; most studies use 1 - 2 associations, and our previous study used 16. in anticipation of this inherent difficulty and based on pilot data, we took the approach of testing participants twice and only included participants scoring above chance across both tests. this minimized the probability of inclusion of guessed items as ones that are remembered and increased our confidence that we only included participants who successfully performed the difficult task. thus, for the statistical analysis, 25 participants were included : thirteen participants who performed the fm paradigm and twelve who performed the ee paradigm. of these participants, 15 were males and 10 females ; their mean age was 26.64 (sd = 3.41). no significant difference was found in the gender distribution between paradigms [(1) = 0.03, ns ] (7 males and 6 females in the fm task and 7 males and 5 females in the ee task), while a marginal difference was found in the age of the participants [t(23) = 2.03, p = 0.054 ] in the fm (m = 25.38, sd = 2.36) and ee paradigms (m = 28.09, sd = 3.93) despite the randomized placement. the fmri versions of the fm and ee paradigms resembled the paradigms used by sharon., 2011 with amnesic patients ; however, they were slightly modified to better fit administration in the magnet. the two paradigms were matched so as to share as many visual and motor features as possible (figure 1(a)) and differed in the unique characteristics of fm. thus, the fm paradigm in contrast to the ee paradigm included incidental learning such that the instructions included no reference to learning or memory processes and disjunctive syllogism such that a familiar picture was presented alongside the novel picture. in each paradigm, novel and familiar target trials (either fm or ee trials) eye - movement experiments have indicated that participants employ an inferential it is not a, therefore it is b strategy when learning through fm in similar paradigms and that direct comparisons with known semantic associates are critical for this kind of learning. novel, familiar and baseline trials were intentionally pseudorandomized across trials. only the novel target trials were chosen for the subsequent memory analysis. the familiar target trials were inserted to prevent participants from automatically choosing the novel picture as the target without examining the pictures and labels and to add credibility to the instructions that described fm encoding as a perceptual task to make sure that encoding was incidental. each trial, whether fm, ee, or baseline, was composed of the following subtrial stages : (1) a question / statement presented both visually and auditorily (3 seconds). (3) participants were given instructions to respond to the question while the pictures remained on screen (1.5 seconds). (4) subjects received relevant on - screen feedback for their response (0.5 seconds). (5) a red fixation cross was presented for either 2 seconds in half of the events or 6 seconds in the other half. thus, each event lasted either 9 or 13 s, a mean of 11 s per event. in the fm task participants were informed they would perform a task designed to examine differences in the way people make perceptual decisions about objects of different levels of familiarity. the stimuli were two pictures of a novel and a familiar animal / fruit / vegetable / flower and the question presented was a perceptual question regarding one of these pictures, the target picture (e.g., does the chayote have leaves ?). the target and lure pictures of every trial always differed on the queried dimension such that there was only one possible correct response. the participants were instructed to press either the right button on the response box in order to answer yes and the left button if their answer was no. no mention was made about a later memory test. in the ee trials, one picture, either novel or familiar, was presented alongside a scrambled picture and participants were explicitly instructed to remember the item for a later test (e.g., try to remember the tenrec). because the fm task involves a visual search and a motor response to a question regarding the stimuli presented, in order to equate the two tasks the participants in the ee task were also requested to look for an x on the screen and, as in the fm paradigm, to press the right (to answer yes, if they noticed the x) or left (to answer no if they did not see an x) response buttons on the response box in order to answer the question. finally, in the baseline trials, the participants were presented with two scrambled pictures (the original pictures from the fm paradigms were scrambled) and were asked is the picture on the right brighter ? again, participants were instructed to answer using the response box similarly to the fm and ee trials. a total of 124 events were designed for each experiment, whether fm or ee. these events were administered in 3 runs such that the first two runs included 40 events and lasted 8 minutes and 2 seconds each and the last run included 44 events and lasted 8 minutes and 52 seconds. the events were organized in 5 sequences of 8 events in the first two runs and an additional sequence of 4 events in the third run. the events were pseudorandomly assigned such that each sequence of 8 events contained 4 novel fm / ee target trials, 2 familiar fm / ee target trials and 2 base line trials (accordingly, the last sequence of 4 events in the 3rd run contained 2 novel fm / ee target trials, 1 familiar fm / ee target trial and 1 base line trial). every run began with 12 seconds of a presentation of either a reminder of the instructions (on the first run) or a blank screen (the second and third runs). the images acquired during these 12 seconds were intended to allow global image intensity to reach equilibrium, and they were later excluded from data analysis. between every 8 events a blank screen appeared for duration of 6 seconds. memory for associations between novel target pictures and novel labels was tested outside the scanner, as described below, using a 4-alternative forced choice recognition in which the label appeared in the center and four pictures around them. the target picture in the test was one of the novel target pictures presented in the experiment, while the lures were themselves novel pictures presented as targets in other trials in the experiment. the recognition test was designed such so as not to allow a familiarity effect to account for the findings. participants had to select the correct picture to go with the label (figure 1(b)). this memory test was administered twice, once about 15 minutes after exiting the magnet and a second time after a week. we only included participants scoring above chance on the first recognition test and above chance across both tests, as tested by the binomial probability to remember the same stimuli in two consecutive tests (binomial test, p value used for cutoff 0.05). items reported by the participants to have been previously familiar (as tested by a yes / no question for each item while items marked as familiar required indicating the name of the item) were omitted from the analysis but this was exceedingly rare. imaging was performed on a ge 3 t signa hdx mr system with an 8-channel head coil located at the wohl institute for advanced imaging in tel aviv sourasky medical center. the scanning session included t1-weighted anatomical 3d sequence spoiled gradient (spgr) echo sequences (tr = 9.14 ms, te = 3.6 ms, flip angle = 13) obtained with high - resolution 1 mm thick contiguous slices and a 256 256 matrix. in addition t2-weighted functional axial images (tr = 2000 ms, te = 40 ms, flip angle = 90) were acquired in 32 contiguous slices aligned parallel to the ac - pc plane, of 5 mm thickness with no interslice gap, a field of view of 20 cm, and a 64 64 acquisition matrix. the functional images covered the whole cerebrum and yielded 3 3 5 mm voxels. in the first 2 runs, 241 images were acquired during each run and in the third run, 266 images were acquired. the functional images were corrected for differences in slice acquisition timing by resampling all slices in time to match the middle slice. this was followed by a realignment of the time series of images to the first image of the run performed after acquisition of the anatomical image. the data were then spatially normalized to mni space, detrended, and scaled into the same range. the scaling was done by performing a runwise normalization and computing standard deviation and mean for z - scoring based on the volumes corresponding to baseline periods in the experiment. the multivoxel pattern analysis was used in this study for both brain decoding and brain mapping, taking the advantage of the information contained in the activity patterns across the entire brain volume, from multiple voxels. for brain decoding, our features were voxels and the classes were the subsequent memory performance of the participant in the different encoding paradigms. thus, the trained classifier was essentially a model of the relationship between brain activity during the encoding process and the later recognition performance. the classifier 's prediction accuracy was used as a measurement for model quality. to answer questions 1 and 2, we were interested in recognition success versus recognition failure for both ee and fm. trial - level classifier data were obtained by selecting volumes that corresponded to the expected peak of the hemodynamic response function after the appearance of the pictures (i.e., corresponding to 68 s postassociative stimulus onset). these were labeled according to the subsequent memory test results, either as recognition success or as recognition failure for either fm or ee encoding conditions. all data sets were counterbalanced, with an equal number of recognition success and recognition failure points selected randomly for each experimental run. a linear support vector machine was used as an underlying classifier for all conditions. classification results were evaluated using 3-fold cross - validation for within - subject scans (according to the number of the experimental runs) and leave - one - out cross - validation for cross - subject experiments. in all analyses, the accuracy of prediction was based only on test data that were completely independent of the training data. considering the high dimensionality of input data, the multivariate classification analysis was preceded by feature selection procedure. most relevant voxels (features) from the high - dimensional set of available voxels. including feature selection procedure into classification process was shown to improve the classification rate (see, e.g.,). the feature selection process was performed on training dataset. in order to decide which voxels should be included in the multivariate classification analysis, each voxel was scored separately using criteria of prediction accuracy, based on f - value accepted from anova statistics for differences between recognition success versus recognition failure conditions for this voxel. then, 100 voxels with the highest f - values were selected for further classification procedure. this selection criterion was empirically determined taking into account the additional improvement in classification accuracy and the cost in computational resources. increasing this criterion to 500 resulted in only minimal improvement in classification, and from 500 to 1000 there was no improvement at all. a process of feature selection was separately performed, anew for each classification experiment in n - fold cross - validation scheme. in all cases, the prediction accuracy was evaluated for both within - subject and cross - subject cases for each encoding condition (ee, fm), using the optimal spatial and temporal aspects of the input data. in the within - subject case, the accuracy value was produced for each participant individually, and then the average accuracy was calculated. in the cross - subject case, the accuracy was produced on the union of all participants ' datasets, using the leave - one - out cross - validation method. the accuracy was calculated as an average over all cross - validation folds. to test the individual contributions of the hippocampus and the atl to classification in each condition, we replicated the classification procedures described above using either only the data from hippocampal or atl (ba38/ba21) voxels. both hippocampal and atl roi analyses were carried out using anatomical templates constructed from the wfu pickatlas 2.5.2. as a control region we examined the same for the putamen, which was not expected to be differentially involved in fm or ee encoding. unlike the contrasts classifications, discovering the brain areas associated with each paradigm, fm or ee (question 3), requires constructing brain maps. in machine - learning terms, brain mapping is a process of highlighting voxels contributing most strongly and reliably to the classifier 's success. it may be achieved by determining which voxels are being selected by a classifier and also how their classification weights affect the classifier prediction. the major issue with this straightforward approach is that a group of voxels appearing in a conjunction of all cross - validation fold sets is relatively small (as a result of the initial information redundancy) and can not be used as a completely reliable source for brain mapping. searchlight classifiers, work by going voxel by voxel and judging by a multivariate method to what degree the information is in a localized neighborhood of the voxel. we used a relatively large neighborhood of 4-voxel radius (257 voxels) in the experiments, which is still smaller than the volume of subcortical structures that could be of importance (hippocampus, thalamus, and caudate) but is large enough to benefit from the power of multivariate analyses. smaller radii of 2 and 3 voxels produced spatially very similar information maps but inferior classification performance, suggesting that informative voxels were relatively evenly distributed across the searchlight neighborhoods we report. this is also indicated by the distribution of voxels selected by the feature selection procedure for the whole brain classification. the information checking can be done in different ways ; in this paper, we used the same kind of classifier as in our previous experiments (svm with a linear kernel) but with the data vector projected only onto the searchlight neighborhood. this rate was used for judging the neighborhood contribution to the differentiation between two experimental conditions, with neighborhoods performing significantly better than a chance level included into the maps. the software used for these experiments was developed using python programming language and based on pymvpa library. overall, participants in the fm paradigm answered the yes / no perceptual question correctly, thus correctly inferring that the novel target pictures were the referents of the novel labels, in 85.48% (sd = 6.34%) of the trials. because in a previous pilot study no difference was found between analysis with or without consideration of the erroneous study trials, items were analyzed irrespective of selection accuracy ; the determining factor was subsequent memory of the item - label association outside the mri. in the ee paradigm, participants correctly identified the x in 95.77% (sd = 3.33%) of the trials ; however, this manipulation was intended to equate the ee paradigm to the fm one in terms of visual search, simple perceptual decision making, and motor response and is not hypothesized to be relevant for subsequent memory for the picture - label association. deeper encoding, in healthy controls the ee task invariably leads to better memory performance (see below). participants in the fm task recognized a mean of 35.28% (sd = 8.25%) of the associations between novel labels and novel pictures. participants in the ee task recognized a mean of 43.67% (sd = 8.66%) of the associations. performance in both tasks was found to be significantly higher than chance (25%) [t(12) = 4.50, p 0.001), reflecting the better classification of the fm subsequent memory effect. interestingly, there was a significant roi by group interaction (f(1.33, 30.71) = 18.12, p 0.1), although contrary to our prediction putamen activity led to above - chance classification in the fm condition. our predictions regarding the respective contributions of the atl and the hippocampus to encoding through fm and ee were only partially fulfilled. as we hypothesized, activity in the atl could be used to classify subsequent memory following fm just as well as activity in the rest of the brain, whereas using the atl alone for prediction of ee subsequent memory effect resulted in significant classification decrease and chance performance of the classifier. in addition, using the hippocampus for classification resulted in significant decrease in classification efficiency for fm and improved classification performance for ee. however, even for fm activity in the hippocampus provided sufficient information for above - chance classification. this was unexpected given our previous findings with hippocampal amnesics, but consistent with our [13, 40 ] and others ' prior studies with healthy controls. it may be that in healthy individuals the intact hippocampi automatically participate in encoding of environmental stimuli. the use of a very long list of stimuli may also be a contributing factor for hippocampal involvement in fm in the present study due to increased interference. nonetheless, the basic pattern of relative hippocampal / atl involvement in fm and ee was consistent with the idea that fm enables more rapid and direct neocortical integration [13, 21, 40 ]. next we sought to examine which large - scale networks beyond the hippocampus and atl contribute to prediction of subsequent memory in each of the conditions and whether these networks significantly differ from one another. the results from the region specific classification analyses indicate that, in all likelihood, subsequent memory effects of fm and ee rely on both overlapping and distinct neural networks. next we sought to examine which large - scale networks beyond the hippocampus and atl contribute to prediction of subsequent memory in each of the conditions and whether these networks significantly differ from one another. the results from the region specific classification analyses indicate that, in all likelihood, subsequent memory effects of fm and ee rely on both overlapping and distinct neural networks. the results presented below were obtained with a searchlight algorithm (see section 2) representing a two - way classification between successful versus failed recognition conditions. the overall pattern is that the fm model displays a more distributed pattern that involves many more brain regions than the ee model (tables 2 and 3 and figure 5). examining the specific patterns, it is apparent that ee is associated with regions in the medial temporal lobe (mtl) including the hippocampus, supporting the finding from the classification roi analysis. ee is also associated with extensive bilateral ventral medial prefrontal cortices (vmpfc), right lateral prefrontal cortex (dorsal and ventral), anterior cingulate, and right posterior lateral temporal neocortex (table 2, figure 5(a)). by contrast, fm was associated with bilateral anterior temporal lobe (atl ; ba38), again confirming the classification roi analysis. fm memory success was also prominently associated with more posterior lateral and inferior temporal neocortical regions and posterior inferior occipital cortices. frontal lobe involvement included orbitofrontal, dorsolateral, and ventrolateral pfc, but no vmpfc (table 3, figure 5(b)). this pattern converges with the importance maps identified by the feature selection for the whole brain classification analysis and with the roi analyses results but extends it to additional structures whose activity contains information about subsequent memory. the extent to which this pattern is driven by overall voxelwise levels of activity that is typically studied using univariate analyses could not be formally assessed because these analyses lacked sufficient statistical power for a subsequent memory test of the kind performed here. the present paper investigated subsequent memory effects for semantic encoding through fm contrasted with standard intentional encoding in healthy individuals. previous studies with amnesic patients have indicated that fm encoding allows for hippocampal - independent acquisition of novel arbitrary associations and that a neocortical structure that may be crucial for this type of learning is the atl [12, 13 ]. here we demonstrate that it is possible to classify with great accuracy encoding - related neural states that lead to subsequent successful associative recognition from those associated with failed recognition. moreover, in partial agreement with the patient data, the hippocampus contained relatively more information about subsequent memory status for the ee condition, although subsequent memory accuracy following fm could also be classified from hippocampal activity. by contrast, the atl contained more subsequent memory information for fm than the hippocampus, and ee subsequent memory performance could not be classified based on atl activity at all. finally a searchlight algorithm approach demonstrated very distinct networks associated with successful compared with unsuccessful memory encoding process. fm successful encoding was primarily related to atl, inferior - posterior lateral temporal and occipital neocortex and ventrolateral prefrontal cortex. by contrast, ee successful encoding was related to mtl activity (including the hippocampus), midline structures of the prefrontal and parietal lobes, and lateral temporal / temporoparietal junction cortex, in line with previous neuroimaging studies of episodic encoding. much of our semantic knowledge, including word meanings and conceptual knowledge, is acquired during childhood and forms the basis for later memory. the developmental literature points to fast mapping as a key mechanism in this process, supporting the initial stages of concept formation [24, 57 ] and the lexicon [1619 ]. we suspected that learning through fm involves rapid and direct changes to neocortical structures, as also suggested by a handful of neurophysiological studies. these include structures that are implicated in semantic memory such as the lateral and anterior temporal lobes (atl), posterior temporal neocortex, and ventrolateral prefrontal cortex (pfc) [36, 5862 ]. the results of testing two patients with left atl damage who failed to learn through fm lent initial support to this hypothesis. a recent imaging study during retrieval also suggested the atl serves as a hub for posterior neocortical networks that represent associations generated by fm almost immediately ; these networks were only weakly present during retrieval of associations generated by ee, and only after a night 's sleep, presumably following network reorganization. the region of interest classification analysis demonstrated that a high proportion of the information about subsequent memory performance during fm encoding could be exclusively derived from voxels in the atl. by comparison, ee classification success was reduced to chance when only the atl was used. moreover, a whole brain searchlight algorithm with no a priori hypothesis about the atl further demonstrated its important role as part of a network that predicts the formation of associative memory during fm. bilateral atl cortical areas appeared as part of the extensive neocortical network that supports fm, while atl did not appear in the ee searchlight analysis, suggesting it does not play an immediate central role during such learning. the neural representation of semantics involves broadly distributed circuits that reflect distinct conceptual categories and their associated properties, such as perceptual, motor, functional, and affective features [60, 6264 ]. according to semantic hub models [58, 60, 65 ] an amodal, domain - general hub is required to bind together these discrete property regions, a function served by the atl. according to the semantic hub model, the activity in the atl observed in the present study reflects the binding together of properties represented in posterior neocortex to allow fine - grained distinctions between perceptually similar stimuli, a type of neocortical pattern separation. subsequent successful associative recognition following fm may depend on the creation of efficient atl - mediated amodal representations of coactivated areas in posterior neocortex. a similar model suggests that the atl serves as an intermediary convergence zone system for triggering word form retrieval given conceptual knowledge, and vice versa for unique entities [66, 67 ]. retrieval of novel items (e.g., the word tenrec) after a single encounter is similar to retrieval of unique entities, that is, specific and precise knowledge including proper names, for stimuli that are concrete and unique. atl activity in the present study would thus reflect the formation of an index that associates a novel word form with novel conceptual knowledge. if, as we suspect, the atl is critical for direct neocortical acquisition of names of unfamiliar animals, fruit, flowers, and so forth, then it would be compatible with both the semantic hub model and the convergence zone ' account. either way, it would be expected that, in addition to the atl activity, posterior temporal and occipital neocortical regions would be coactivated when such associations are being laid down, as described below. in addition to the atl that proved important for fm in both the classification and the searchlight analyses, fm successful memory classification also relied on posterior and frontal neocortical regions. these patterns appeared to be more distributed and widespread than the ones predictive of ee successful recognition and to involve more lateral and inferior neocortical regions. we propose that this widespread neocortical activity may be a signature of direct semantic acquisition. the activated neocortical networks identified by the searchlight algorithm may reflect activation of existing knowledge structures that support acquisition of novel associations. fm successful encoding depended on extensive cortical regions in the middle occipital gyrus and in the fusiform gyrus bilaterally. these areas constitute part of the visual ventral stream and have been described in previous studies as correlated with different stages of object recognition [6971 ]. according to zannino., ventral occipitotemporal activations are the most consistent finding in neuroimaging studies of semantic processing. its activation during semantic tasks has been a basis for the theory that claims that semantic knowledge is mediated in the brain by the same areas by which it is encoded. interestingly, in addition to structures associated with the visual stream, the searchlight algorithm also identified extensive activation in secondary and tertiary auditory cortical regions. caudal area 22 in the left superior temporal gyrus is considered to be a major component of wernicke 's area and support processing of lexical semantics. it is important to note that while posterior regions are likely where the actual representations of the features that make up novel lexical semantic entries reside, by no means do the patterns picked up by the classifier reflect specific item identities. our task and the spatial and temporal specificity of fmri could not possibly capture that information. moreover, such information even if captured, could not be diagnostic of the broad categories of remembered versus forgotten. instead, what the classifier might tap onto is a pattern within higher - level unimodal associative cortices that reflects successful generation of unique codes in lower level perceptual regions. thus, successful encoding through fm is predicted by coactivation of modality - specific perceptual representations of items and their associated auditory information, coupled with activation of the atl amodal hub in which higher - level semantic knowledge is represented. object - label associates that engage these regions to a greater extent are more likely to be effectively processed and for relations among their features to be created. some experimental evidence that neocortically represented features of semantic representations acquired through fm may significantly overlap was recently presented by merhav.. they demonstrated that, in both healthy controls and patients with amnesia using ab - ac interference, overlapping paired - associates learned through fm suffer from catastrophic interference, a hallmark of independent neocortical learning, while associates studied through ee are immune to such interference presumably by virtue of their hippocampal component. these patterns also are consistent with the prominence of linguistic processing in memory through fast mapping. the posterior regions the classifier identified have been implicated in language processing as parts of both the dorsal stream, along the superior longitudinal fasciculus, and the ventral streams, along the inferior frontooccipital and uncinate fasciculi. successful fast mapping, as implemented in our task, requires both parsing of linguistic information to identify the target stimulus and holistic representations of lexical semantics that are mediated by dorsal and ventral streams, respectively. previous studies of rapid perceptual learning of word forms have similarly identified changes in perceptual lexical processing associated with dorsal stream regions [75, 76 ]. by contrast, the combination of lexical semantics and lexical phonology acquired through fm engages both dorsal and ventral streams. the ee task, on the other hand, could be decoded primarily by right - sided lateral cortical regions, medial prefrontal and medial temporal structures that are structures associated with visual episodic memory rather than a linguistic network. one region that we had not expected to be predictive of successful memory following fm is the putamen, which classified at above chance levels for the fm but not ee conditions. the dorsal and ventral striatum are differentially connected to discrete prefrontal cortical regions in segregated corticostriatal circuits. the putamen plays a critical role within the so - called motor circuit while the caudate forms part of the oculomotor, dorsolateral, and ventral / orbital circuits. this is why we assumed it would not play a significant role in vocabulary learning through fm. we were similarly surprised ; however, to find putamen was part of functional networks that support retrieval of fm (and not ee) associations in our recent study of retrieval, suggesting this is more than a coincidence or some leakage of information from other structures. one possibility is that the role of the putamen and in particular the left putamen in language production (initiation and execution of speech) as part of its connectivity with the supplementary motor somehow supports the novel lexical entries acquired through fm. interestingly the left sma also features prominently in the searchlight results for the fm, which may be part of the same network. one characteristic of fm that may determine the way new information is acquired is that novel information appears in the context of already known items. fm learning is thought to depend on preexisting categorical or conceptual knowledge [22, 79 ]. previous studies of amnesia suggest that anchoring new knowledge onto existing knowledge may allow patients to acquire a new language and lexicon and even patient hm acquired new facts when these were anchored to information he knew. we have suggested that conditions in which the associations are completely novel (i.e., both item and label have never been encountered before) but are clearly embedded within existing conceptual frameworks are conducive for rapid modification of connection between neocortical nodes [12, 13 ]. this is consistent with much of what we know about prior knowledge effects on new learning [9, 82 ] and also with some evidence on fm. recent work using similar stimuli to ours, but nonsense words, demonstrated that encoding through fm leads to immediate lexical integration, as indexed by lexical competition effects on the processing of neighboring existing words. it also led to semantic priming effects after a day, compared to ee that led to stronger declarative memory but no evidence for either lexical or semantic integration. importantly, both effects required that a competing item be present, suggesting that activation of existing semantic networks is an inherent part of the process of integration of novel associations. it has also been demonstrated that the more transparent the distinction between target items and competing items in the environment is, the more likely the correct mapping would occur and be maintained cross - situationally. therefore, it appears that variations in levels and richness of knowledge of the competing items affect mapping and learning of new items, such that the more one knows about the competing items the more likely the novel associations can be formed. the activation predictive of later memory of novel items through fast mapping could reflect both the activation of old associations and the integration of new information, which are indistinguishable under this model. similar processes have been shown to be central in other domains of memory including reconsolidation of conditioned associations and updating of episodic declarative memory. recently, tse and colleagues [7, 8 ] elegantly demonstrated that when novel flavor - location pairs are studied in the context of old well - established schematic flavor - location setting, neocortical consolidation is greatly accelerated in rats. while this is consistent with our claim that the neocortex is capable of rapid consolidation of novel arbitrary associations, we note that initial acquisition in that study was still reliant on the hippocampus. importantly, our data indicate that the atl is a critical epicenter for the formation of novel associations through fm when novel associations are embedded in previous conceptual knowledge. tse and colleagues [7, 8 ] on the other hand present evidence that the prelimbic cortex, a homolog of the human medial pfc, was key for schema - based hippocampal - dependent rapid consolidation. as we discuss below, the medial pfc appears critical during acquisition and transformation of hippocampally dependent episodic memory into independent neocortical semantic memories [6, 85, 86 ] particularly when novel memories are embedded in schematic knowledge. this contrasts with fm that depends of prior conceptual knowledge, rather than schema, and in which learning is mediated by the atl. while fm subsequent memory could be primarily classified by activations in atl, pfc, and widespread posterior and inferior neocortical structures, the ee roi classification and searchlight results demonstrated a very different pattern. successful subsequent performance following ee could be predicted quite efficiently based on hippocampal voxels alone. the atl - only model on the other hand was associated with significantly greater reduction in classification success and in fact led to chance performance of the classifier. that pattern was the reverse of what was observed for the fm, forming a this pattern is consistent with the idea that the hippocampus critically supports acquisition of novel associations under standard encoding conditions and replicates numerous other studies of subsequent memory effects in episodic memory tasks [41, 8789 ]. the findings from the roi classification analysis were later confirmed by the searchlight algorithm with no a priori delineation of structures, which also identified the hippocampus and parahippocampal gyrus as key regions in a limited collection of regions that together predicted subsequent memory following intentional encoding. the other major contributor identified by the searchlight was the medial aspect of the prefrontal cortex bilaterally as well as dorsolateral and ventrolateral pfc. within the memory literature, ventral medial prefrontal cortex (vmpfc) has mostly been identified as a key structure in retrieval processes of episodic [90, 91 ] and semantic [62, 92 ] memory. the vmpfc is intimately connected with limbic and paralimbic declarative memory structures including the hippocampus, parahippocampus, and retrosplenial cortex [93, 94 ]. although it has been linked primarily to motivation and reward processing [95, 96 ], damage to vmpfc can also lead to memory syndromes. specifically, lesions to that region can lead to confabulation (false memories) that may arise from deficits in automatic memory monitoring (felt rightness [97, 98 ]. deficits may reflect failed automatic biasing of relevant posterior neocortical representations and consequent erroneous retrieval of strong (but irrelevant) attractor schematic knowledge [92, 99, 100 ] cf. for a detailed cognitive model). the idea that vmpfc interacts with posterior neocortical structures also figures prominently in theories that suggest this neocortical structure is key to the process of systems consolidation or reorganization as episodic memories are transformed from being hippocampally bound to being hippocampal - independent [6, 8, 85, 86 ]. some have even suggested that when new associations are highly congruent with existing schema, the vmpfc may take over the role of binding together these associations independently of the mtl. either way, increased activity in medial prefrontal cortex during successful intentional memory encoding may reflect the use of specific encoding strategies and recruitment of attentional and executive resources that together with the mtl provide the substrate that allows for later memory. it may contribute to successful concept interference resolution as our stimulus set involved labels and items that were similar to each other. associating novel information to existing knowledge at encoding through vmpfc connections may have supported pattern separation performed by the hippocampus and allowed for later accurate associative recognition. the discussion so far highlighted the preferential role of anterior, later, and posterior neocortical regions in encoding associations through fm compared with the hippocampal - vmpfc axis that appear to be central for encoding through ee. however, hippocampal voxels contained enough information to allow above - chance classification in fm in the present study. moreover, the finding that fm can support learning independently of the hippocampus in patients with amnesia is by no means unanimous [14, 15 ]. the study by warren and duff deviated significantly from standard fm tasks, which may have led to the difference in findings. for example, the participants in that study were provided with explicit memory instructions, while fm typically involves incidental learning. moreover, items were intensively trained prior to associative learning, possibly interfering with the novel item to novel label mapping. indeed, it appears that the ee and fm conditions in the warren and duff study likely did not tap different encoding processes, as suggested by the finding that control participants performed equally well on the two conditions, failing to show the typical ee advantage reported in this study and others. the study by smith. more closely resembled the studies by sharon. and merhav. it was suggested that specific aspects of task administration affected the memory performance or that fm may support learning in the context of less severe amnesia. one possibility that may account for the differing results from amnesic patients and the involvement of the hippocampus in fm in our neuroimaging study is that the paradigms we use involve higher levels of interference than typical developmental fast mapping occurring either naturally or in the lab. a typical lab experiment of fm involves very few items that are very distinct and allow for very distinctive interaction with the items. we use much longer lists (sixteen items in the patient studies and over 60 here) of highly similar items. smith. also suggested that learning through fm might not be robust, and the study by merhav. coutanche and thompson - schill made similar observations with respect to fm 's failure to support free recall, which contrasted with its superior ability to integrate new information into existing semantic networks. within the developmental literature it has been suggested that associations acquired through fm initially have a hypothesis status [83, 102 ] such that if contradictory evidence is encountered, the system re - sets and all associations are erased. this erasure susceptible state is presumably maintained until novel meanings are refined and confirmed by the child, to prevent perpetuation of foundational errors [83, 102 ]. in healthy adults this interference susceptibility may induce automatic hippocampal activation that could contribute to fm learning but obviously to a lesser extent and in parallel to the more prominent neocortical structures. it appears that although both fm and ee lead to the acquisition of declarative memory as reflected in the postscan recognition performance, they do so by recruiting very distinct neuronal networks that can be efficiently distinguished using the machine - learning classifier svm. thus, although healthy individuals possess the neural machinery that should allow for encoding information using the mtl memory system, under conditions that promote fm, they do not rely on the mtl to the same extent. an alternate explanation of this finding is that the fm and ee conditions differ on some perceptual and motor aspects and that the classifier may have picked up on features associated with these differences. we can not completely rule out that possibility ; however, we note that the classification was performed within each condition and that subsequently remembered and subsequently forgotten items did not differ on any of these features, other than on the final outcome of memory. we also note that the regions associated with each condition (i.e., separating successful and unsuccessful subsequent memory within each condition) are consistent with the hypothetical areas of each memory system and our previous patient lesion studies. this suggests the information was extracted from functionally relevant areas to each memory system rather than those that are only activated by peripheral aspects of the task demands. either way, the finding that learning declarative information can be supported directly by the neocortex is surprising and challenges certain aspects of current theories of fm [30, 32, 103 ]. it also suggests there are exceptions to current more general theories of declarative memory that preclude the possibility of direct neocortical plasticity [3, 104, 105 ].	neocortical structures typically only support slow acquisition of declarative memory ; however, learning through fast mapping may facilitate rapid learning - induced cortical plasticity and hippocampal - independent integration of novel associations into existing semantic networks. during fast mapping the meaning of new words and concepts is inferred, and durable novel associations are incidentally formed, a process thought to support early childhood 's exuberant learning. the anterior temporal lobe, a cortical semantic memory hub, may critically support such learning. we investigated encoding of semantic associations through fast mapping using fmri and multivoxel pattern analysis. subsequent memory performance following fast mapping was more efficiently predicted using anterior temporal lobe than hippocampal voxels, while standard explicit encoding was best predicted by hippocampal activity. searchlight algorithms revealed additional activity patterns that predicted successful fast mapping semantic learning located in lateral occipitotemporal and parietotemporal neocortex and ventrolateral prefrontal cortex. by contrast, successful explicit encoding could be classified by activity in medial and dorsolateral prefrontal and parahippocampal cortices. we propose that fast mapping promotes incidental rapid integration of new associations into existing neocortical semantic networks by activating related, nonoverlapping conceptual knowledge. in healthy adults, this is better captured by unique anterior and lateral temporal lobe activity patterns, while hippocampal involvement is less predictive of this kind of learning.
however, there continues to be a substantial gap between the avalanche of genomic data, which are abundant but not reliable, and our limited biological knowledge, which can only be discovered through careful, small - scale techniques. this disparity has been exacerbated with the development and popularity of next - generation technologies, such as rna - seq, which enable genome - wide measurements at unprecedented resolution and cost (1). a paucity of biological knowledge (i.e. experimentally validated gene function) limits the coverage and accuracy of computational methods that require prior knowledge to learn novel biology, even when large - scale genomic data are available (2). thus, these methods are limited to performing well on processes and pathways that are already well characterized in an organism. imp (integrated multi - species prediction) was originally developed to address the growing need to interpret and analyze results from genome - wide studies and generate hypotheses for experimental follow - up in the context of integrated functional gene networks, even when prior knowledge is limited in an organism or for a specific biological context (3). imp is an exploratory tool that provides a high - quality, interactive interface for functional prediction and interrogation. for example, biologists can overlay their genes from a high - throughput experiment onto imp 's functional gene networks, expanding or contracting the network and identifying enriched, unifying functional themes. alternatively, researchers can generate specific functional hypotheses by querying imp 's collection of gene - pathway predictions, identifying candidate genes for a biological context of interest. in all of these analyses, imp systematically applies a previously developed network - based method that identifies functionally similar homologs to transfer annotations (i.e. gene - pathway membership) between organisms. this more specific homology detection method extends beyond simple annotation transfer by sequence similarity and enables accurate gene pathway predictions, even for processes that have few or no experimental annotations in an organism (2). there are several successful web servers that allow researchers to analyze their gene sets in the context of gene networks (46), however, they are either constrained by the availability of prior knowledge in an organism and biological process of interest or limited to sequence - based transfers of input data (7,8). imp is unique in its systematic incorporation of a functional genomics - based homology transfer of prior knowledge (9) in all of its analyses, improving the accuracy and coverage of functional interrogation (2). imp has been continuously maintained and developed since the original publication and here we describe major updates to the server. we have extensively updated the gene networks and functional predictions across all seven organisms, adding publicly available gene expression experiments from the subsequent years, and updating the already included data sources. additionally, we extend imp 's functional coverage to include human diseases, allowing biologists to analyze disease contexts and predictions in human and across model organisms. human disease gene knowledge is transferred to other organisms and predictions are made using each organism 's gene network. by exploring disease gene predictions across the model organisms, biologists can find candidate genes to serve as targets for follow - up in human and in potential animal models for their disease of interest. additionally, we have created a flexible tool that furthers the original goal of the web server : to enable biologists to analyze their experimental results in the context of massive - scale integrated data compendia. we developed a prediction platform that allows biologists to bring their larger experimental result (for example, a list of hundreds of genes identified as over - expressed in a microarray experiment) and run a sophisticated machine - learning method for classification. this tool can be used to answer many pertinent questions, for example, identifying additional candidate disease genes from a microarray experiment, or additional players in a biological process of interest. such an analysis might otherwise be infeasible due to biologists limited computational resources or expertise. the software is maintained and executed on imp 's servers and only requires a list of genes from the user. genome - wide results are available by email, if provided, or directly on the web server. imp is a flexible tool that can be used to answer diverse biological questions posed in the form of a biological context (a process or a disease), a single gene, or a set of genes of interest. these questions can be broad and exploratory, for example, determining the shared pathways among a set of genes that are co - expressed in an mrna experiment. alternatively, researchers can generate specific experimentally testable hypotheses, such as identifying functional partners of a gene of interest or possible pathways that the gene participates in. as an exploratory tool, imp provides interactive visualizations of gene - gene functional relationships, gene - process predictions and cross - organism network alignments. imp is both a collection of gene - pathway predictions that users can query for specific targeted results and a suite of user - driven tools that can be customized for broad discovery. all of imp 's diverse analyses leverage an organism 's functional gene network, which integrates thousands of genome - wide experiments from an array of public data sources (1013) and describes whether genes participate in similar biological processes. these networks are constructed using previously described methods (2,6,14) and have been extensively updated in the subsequent years since imp was originally released. we use a new expert - curated set of gene ontology (go) terms (15) to define the gold standard for learning gene gene functional relationships and have updated the standard to include the latest experimental annotations. imp networks now integrate 3540 data sets, a 49% increase in the number of data sets from imp 's original release (3), and include over 70 000 experimental conditions. in addition to adding gene expression experiments from the last three years, imp networks have been updated with the most recent releases of popular functional genomic databases. for example, biogrid (10) has been updated to include 196 909 additional protein protein interactions, an increase of 78% from the original networks. biologists can query imp with a gene set or biological context of interest to retrieve putative gene - pathway assignments. we have extended imp 's biological contexts to include human diseases, in addition to go biological processes. imp applies the same machine - learning method for predicting genes to biological processes (2,3) as it does to diseases, which uses a functional network as input to a support vector machine (svm) to classify genes (figure 1). we showed previously that this method is accurate and competitive among state - of - the - art methods in predicting genes to biological processes (2,3). disease gene predictions are inferred directly in human from disease genes curated by online mendelian inheritance in man (omim) (16) and using the human functional network and in the six model organisms. the disease predictions inferred in the other organisms leverage biological knowledge from human by transferring omim knowledge using our previously described method to identify the appropriate homologs for gene annotation transfer (2,9). these human - transferred gene - disease annotations are then used as training data for prediction with the organisms functional network, and the subsequent gene predictions are specific to that organism. by applying a model organism 's functional network to predict disease genes, imp can help biologists address an important challenge in the study of human disease : identifying the best model system for a given disease and the appropriate orthologs for a disease of interest. (a) imp constructs a functional network for each of seven organisms by integrating heterogeneous genomic data. (b) disease - gene annotations from human are transferred to the functionally similar homologs in other organisms. (c) additional disease genes are predicted using the human - transferred disease genes in the organism - specific functional networks. using imp, users search by disease ontology (do) (17) term or by gene to retrieve gene - disease predictions. omim disease genes are mapped to do, using the mapping provided by do, to leverage the unified naming and hierarchical structure of the ontology. figure 2 shows queries for hypertrophic cardiomyopathy (hcm) in both human (figure 2a) and mouse (figure 2b). many of the top genes in the human query are known to be involved in the disease (highlighted rows), and the others are potential disease candidates. for example, the second novel gene prediction is trim63, which encodes an e3 ubiquitin ligase and plays a role in the atrophy of skeletal and cardiac muscle (18,19). the gene has recently been suggested (independent of imp) as a candidate for hcm with several mutations observed in patients with the disease (20). hypertrophic cardiomyopathy in human returns a list of genes predicted to be involved in the disease, sorted by probability. imp applies known hypertrophic cardiomyopathy genes in human (from omim) to predict additional genes from the human functional network. (b) the same disease query can be performed in mouse, returning predicted mouse genes. these predictions were learned using human disease genes transferred to mouse with the mouse functional network. these gene predictions, which leverage human disease knowledge transferred to mouse, are potentially informative as a mouse model for the disease. in fact, the most confidently predicted gene, csrp3, was a target in the first model for dilated cardiomyopathy with hypertrophy in a genetically manipulatable organism. csrp3-deficient mice reproduce the same morphologic and clinical features of the disease as in human (21). the csrp3 mouse model serves as a valuable resource for understanding the pathophysiology of heart failure and for identifying potential therapies for the disease (22,23). thus, in these example use cases, imp independently, and in a data - driven predictive fashion, identifies a candidate human gene for hcn and a mouse gene that is already a model for understanding human hcm. many biological questions can not be posed as a predefined gene set, such as a go biological process or omim disease, or expressed as a small gene set (i.e. < 50 genes), requiring more advanced and flexible data - mining techniques. for example, a researcher with results from a genetic screen may be interested in identifying additional candidate genes. alternatively, a biologist may want to combine her private experimental result with public gene pathway annotations to make customized predictions. most biologists lack the computational resources or expertise to implement and support the necessary machine learning software and data compendia for such an analysis. with imp 2.0, we provide a flexible platform for researchers to run state - of - the - art machine learning methods and pose customized, sophisticated biological questions. users provide a gold standard, in the form of a set of relevant genes, or use imp provided gene sets, which include go biological process and do terms. imp uses the same previously described and validated method for predicting go function (2,3), which applies a svm with features constructed from the organism of interest 's functional gene network for classification. the svm classifies all genes in the genome based on its pattern of functional relationships with the provided genes of interest, up - weighting the parts of the network that are informative for membership in the gene set. this method has been previously shown to be accurate in predicting genes to biological processes and phenotypes, with corresponding estimates of prediction performance (2,24). a user starts an analysis by specifying an organism and her genes of interest, either manually, from a user - saved gene set, or pre - defined by imp. pre - defined gene sets can be from go or do, and can include annotations transferred from other organisms by selecting the corresponding checkbox. figure 3a shows the input for an analysis of five user - provided breast cancer genes. these genes are treated as positive examples for classification, with random negative gene examples selected by imp for classification. the researcher runs the analysis on imp 's servers using the human functional gene network (figure 3b). each gene in the genome is assigned a probability based on its five - fold cross - validated svm result, and results are sent by email, if provided, or viewed directly on the server though a result - specific url (figure 3c). performance is evaluated as the area under the receiver - operator curve (auc) and provided with the genome - wide prediction results. as we continue to update imp 's collection of functional networks in the future, the prediction performance of this tool is expected to improve even further, and we encourage biologists to rerun their analyses. with these features, imp enables biologists to both pose complex biological questions and easily run sophisticated machine - learning tools to help answer them. genes can be pasted, selected from a saved gene set, or chosen from a pre - defined set. (b) imp applies an svm with the provided gene set as positive examples and predicts additional genome - wide genes likely to be functionally related. (c) the output is a list of genome - wide genes, ranked by their probability of functional relationship with the provided gene set. this result can be emailed to the user or accessed directly on the web server. imp is a flexible, user - friendly web server that serves as an intuitive and accessible resource for molecular biologists who want to leverage heterogeneous biological big data collections to explore predictions of gene function and disease association in human and model organisms. the described updates add substantial value to imp as a unique resource and suite of analysis tools for biological researchers. in the future, we plan to continue to add additional organisms (arabidopsis thaliana) and additional data sources for our functional gene networks. we continue to develop additional tools that leverage our cross - organism collection of networks and predictions with the goal of making complex tools and analyses accessible to biological researchers. national science foundation (nsf) career [dbi-0546275 ] ; national institutes of health [r01 gm071966, r01 hg005998, t32 hg003284 ] ; national institute of general medical sciences (nigms) center of excellence [p50 gm071508 ].	imp (integrative multi - species prediction), originally released in 2012, is an interactive web server that enables molecular biologists to interpret experimental results and to generate hypotheses in the context of a large cross - organism compendium of functional predictions and networks. the system provides biologists with a framework to analyze their candidate gene sets in the context of functional networks, expanding or refining their sets using functional relationships predicted from integrated high - throughput data. imp 2.0 integrates updated prior knowledge and data collections from the last three years in the seven supported organisms (homo sapiens, mus musculus, rattus norvegicus, drosophila melanogaster, danio rerio, caenorhabditis elegans, and saccharomyces cerevisiae) and extends function prediction coverage to include human disease. imp identifies homologs with conserved functional roles for disease knowledge transfer, allowing biologists to analyze disease contexts and predictions across all organisms. additionally, imp 2.0 implements a new flexible platform for experts to generate custom hypotheses about biological processes or diseases, making sophisticated data - driven methods easily accessible to researchers. imp does not require any registration or installation and is freely available for use at http://imp.princeton.edu.
colchicine is an alkaloid found in the plant colchicurn autumnal, first recommended for the relief of articular pain in the 6 century a.d., and now most commonly used to treat gout, familial mediterranean fever, behet 's disease, atrial fibrillation following cardiac tissue ablation, and pericarditis. an overdose of colchicine inhibits cell division, and thus the most affected organs are those that have a high rate of cell turnover, such as the gastrointestinal tract, bone marrow, and hair follicles. colchicine poisoning typically shows three phases : initially, gastrointestinal symptoms predominate ; in the second phase, multiorgan failure may occur ; and if the patient survives, the third phase of recovery follows, during which the patient often presents with hair loss. we present a case of a 17-year - old girl with a personal history of depression, several suicide attempts, alimentary behavioural disorder, and a recently diagnosed pericarditis, for which she was taking colchicine (1 mg / day). she was admitted to the psychiatric department of our hospital after recovering from a suicide attempt, apparently taking 40 pills of colchicine (40 mg), which led to severe pancreatitis and bicytopenia. one week after poisoning, a sudden onset of hair loss was observed [figure 1 ]. positive hair pull test (+ + +) and trichoscopy demonstrated the presence of anagen hairs with pigmented long roots covered by the root sheaths [figure 2a ]. dermoscopy of the scalp showed no signs of trichotillomania such as broken hairs, black dots, flame hair, v - sign, or follicular hemorrhages [figure 2b ]. the diagnosis of anagen effluvium following acute colchicine poisoning was made. hair loss on day 7 after colchicine poisoning (a) trichoscopy of anagen hairs collected with hair pull test. (b) dermoscopy of the scalp where no signs of trichotillomania such as broken hairs, black dots, flame hair, v - sign, or follicular hemorrhages can be observed as occurs with exposure to toxic chemicals, hair loss due to colchicine poisoning presents as anagen effluvium. hair loss usually begins 714 days after the exposure and gradually recovers after 36 months, as the follicular ostia remains intact. pharmacotherapy or specific treatment is not usually required because the follicle resumes its normal activity after withdrawal of the antimitotic factors. anagen effluvium should be differentiated from telogen effluvium, androgenetic alopecia, and trichotillomania. in this case, we focused on the differential diagnosis between trichotillomania and anagen effluvium because of the personal psychiatric history of the patient. there are few reports in the literature describing hair loss following acute colchicine poisoning, and none of them are recent. to our knowledge, this is the first report describing the dermoscopic and trichoscopic findings in colchicine poisoning alopecia. physicians should bear in mind this infrequent cause of anagen effluvium and try to avoid prescribing colchicine to psychiatric patients due to its potentially dangerous side effects. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed.	we present a case of a 17-year - old girl admitted to the psychiatric department recovering from a suicide attempt with colchicine. one week after poisoning, a sudden onset of hair loss was observed. positive hair pull test and trichoscopy demonstrated the presence of anagen hairs with pigmented long roots covered by the root sheaths. colchicine poisoning is an uncommon, but potentially life - threatening toxicologic emergency. an overdose of colchicine inhibits cell division, and thus the most affected organs are those which have a high rate of cell turnover. hair loss resulting from colchicine poisoning presents as anagen effluvium, as it occurs with an exposure to toxic chemicals. pharmacotherapy or specific treatment is not usually required, since the follicle resumes its normal activity after withdrawal of the antimitotic factors.
cancer - related facial pain accounts for approximately 1 - 13% of patients with clinical trigeminal neuralgia symptoms4,16). direct tumorous invasion of the trigeminal nerve or tumors in close proximity to the trigeminal nerve can cause facial pain similar to the pain experienced by patients with trigeminal neuralgia4). it can be associated with shock - like pains which are abrupt in onset and termination, and limited to certain distributions of the trigeminal nerve11). the frequency and intensity of the pain increase with the progressive growth of the intracranial lesions and with more severe nerve compression. what is worse, most cases of this type of cancer pain are refractory to medication and respond only to surgical measures. surgical resection has been reported as a modality for pain relief13) ; however, some patients are not candidates for surgery due to medical comorbidities or prohibitive tumor characteristics4,13,16). in such cases, pain surgery becomes necessary for symptomatic relief as an alternative mode of management. since the primary pain pathway has been ascertained to be the spinothalamic tract in the spinal level28) for the body and the trigeminothalamic tract above the spinal cord level for the face, head and neck, various surgical procedures have been performed and ablative procedures still have a role to play in the treatment of cancer pain20). however, if the pain occurs in the upper extremities, upper body, or even in the face, it becomes necessary to interrupt the primary pain pathway at a level above the spinal cord. stereotactic ablation of the thalamic sensory nucleus is widely practiced but this does not provide consistent results and neurosurgeons still require definite solutions to overcome unknown aspects17,20). interruption of the limbic pathways, particularly the cingulate gyrus, reduces the associated suffering but does not abolish pain perception18,30). there are clearly advantages and disadvantages between the two surgical approaches for obtaining high analgesic levels. the former has a little shorter trajectory toward the target and the outcome is good in terms of pain control20). moreover, recent magnetic resonance image (mri)-based electrophysiologic localization techniques can minimize the occurrence of any associated complications such as oculomotor disorders or injury to the reticular formation. that is why the mesencephalotomy procedure targeting the spinothalamic and trigeminothalamic pathways in the midbrain level is frequently performed. the author reports a case of successful management using stereotactic mesencephalotomy for a patient with facial cancer - related trigeminal pain, without any complications and, as far as we know, this is the first case documented domestically. a 72-year - old man presented with a 3-year history of intractable left - sided facial pain. his pain was characterized by stabbing, burning, and dysesthesia, and involved the v1, v2, and v3 distributions of the trigeminal nerve. in the early stage, he was misdiagnosed with trigeminal neuralgia and underwent gamma knife radiosurgery at an outside hospital. when the procedure and pharmacologic measures failed to provide pain alleviation, he was reexamined with paranasal sinus mri and his diagnosis was confirmed with a biopsy of the nose and neck. at that time, his final diagnosis was inoperable hard palate cancer with intracranial extension, stage ivb (t4bn1m0). since then, he has received concurrent chemoradiation therapy as another treatment option. during the treatment, his cancer - related facial pain became aggravated and refractory to the maximum dose of opioids including oxycontin, durogesic patches, and so forth. he was then referred to the neurosurgery department for surgical treatment of the intractable pain. when we checked his neurologic status, left hemifacial pain corresponding to a visual analogue scale (vas) score of 9 combined with mild hypoethesia (decreased to 80 percent compared with the right side) and left masseter muscle atrophy with decreased mastication were identified. after careful consideration of the factors, including the factors involved in multiple clinical disciplines, the surgical option of a mesencephalotomy was discussed with the patient and his caregivers and they decided to proceed with it as a last resort due to the severity of his symptoms and the refractoriness to medical treatment. on the morning of the operation, the patient 's head was fixed parallel to the line between the infraorbital margin and the upper margin of the external auditory meatus in a leksell stereotactic frame (elekta instruments, atlanta, ga, usa), and coronal and axial t2-weighted mri images were then obtained. the surgical target based on the mr image was chosen 5 mm behind the posterior commissure, 6 mm lateral to, and 5 mm below the intercommissural plane (fig. the surgery was carried out under local anesthesia. when the underlying dura and pia were opened with bipolar cautery, three straight microelectrodes (fhc inc., bowdoin, me, usa) were inserted into the center, 2 mm medial, and 2 mm lateral to the target, for electrophysiologic mapping. after stimulation with the electrode, typical tingling and a warm sensation in the left upper arm and face were noted in the center tract (6 mm lateral to the right of the midline). after confirming the final target based on the electrophysiologic mapping findings, a lesion was made using a 2 mm diameter tip monopolar straight electrode with a 4 mm uninsulated length (leibinger gmbh, freiburg, germany) and raising the temperature of the electrode to 80 for 60 seconds. and the lesion was increased in length by 2 mm by moving the electrode along the dorsolateral direction and by repeating the heating (fig. a navigation - guided ommaya reservoir was inserted for intraventricular injection of morphine to aid in the control of the remaining cancer pain. on the first day after the operation, the left hemifacial pain had dramatically improved according to the vas score, which dropped from 9 to 1, and the hypoesthesia on the left face showed a noticeable decrease of 50 percent compared with the right side. besides, there was no evidence of any new gaze paresis or left - sided extremity dysesthesia. however, the pain slightly recurred with a vas score of 3 a week after the surgery and the pain progressively increased, with the vas increasing to 5 at postoperative two months. until a recent date, the pain has been relatively well controlled, staying at vas 4 or 5, by intermittent intraventricular morphine injections and oral opioids ; also, there have been no neurologic deficits detected. they reported pain relief that lasted for at least 18 years for facial dysesthesia27). since then, various authors have developed this technique, making some minor revisions involving changes in the approaches or anatomical targets, for relieving medically refractory pain1,7,12,14). different and more accurate approaches through the posterior parietal lobe or an anterior frontal approach and a more medially placed target or a more laterally positioned lesion have been attempted and they have shown good results, with significantly reduced morbidities. the integrated data show that the anatomical target points of several authors were 5 mm behind the posterior commissure, 5 - 10 mm lateral to, and 5 mm below the intercommissural plane15,21,22,24,28). the original stereotactic target was the lateral spinothalamic and trigeminothalamic tracts, avoiding the medial lemniscus. in some later procedures, several authors included a lesion in the dorsomedial nucleus of the thalamus in order to interrupt its connections to the pre - frontal area. their rationale was that interrupting the spinothalamic lesion treated the pain transmission, but the dorsomedial lesion helped the " suffering " component of the intractable pain, which is projected via the spinoreticular system to limbic structures26). in 1954, spiegel.24,25) demonstrated that pain was transmitted via the spinoreticular pathways, which had been recognized as being involved with pain transmission. they advocated that the mesencephalotomy lesion extend medially to include spinoreticular areas, especially in patients with a strong emotional component to the chronic pain syndrome31). in accordance with the previous work, shieff and nashold23) demonstrated that chronic pain is invariably associated with emotional distress and the interruption of the extralemniscal pathway alleviated the emotional aspect of the pain. consequently, if psychological generation of the suffering accounts for a considerable portion of the pain syndrome, the patient may benefit from the interruption of the spinoreticulothalamic pathways as part of the management of the suffering of the patient14). however, in the case reported here, we did not consider spinoreticular lesion involvement because the suffering was not a significant part of his cancer pain and he was not indicated to receive psychotropic medication or counseling. various postoperative disturbances can happen despite the accuracy of modern mri - guided stereotactic targeting, which has an accuracy within 1 mm, as recently described3). several authors demonstrated that dysesthesia correlated with damage to the medial lemniscus5,8). in 1969, nashold.15) verified that high - frequency stimulation in the central grey area medial to the spinothalamic tract produced unpleasant sensations involving the midline structures, as well as a strong negative or fearful emotional outburst. significantly disabling ocular mobility shieff and nashold22,23) as well as amano2) recommended a target at the level of the inferior colliculus, 5 mm below the intercommissural plane, to avoid that complication. gybels and sweet10) reported that oculomotor dysfunction was seen in 13 - 20% of patients, even with intraoperative stimulation control and directing the lesion to the safer more caudal target. besides, it has become evident that after interruption of the pain pathways for chronic pain of benign origin, the pain almost invariably returns, so mesencephalotomy is rarely indicated for pain other than cancer pain and thalamic syndrome9). pain recurrence that appears within the first month of the intervention is due to insufficient coagulation while late pain recurrence depends on either factors of nervous system regeneration or on the polimodal function of the central nervous system22). in consideration of these limitations, it is necessary to select patients carefully, since the procedure is not without danger. only for patients with cancer pain involving the head, neck, or upper extremities, it may be particularly helpful when all non - invasive modalities have failed and there is good correlation between the site of cancer involvement and the generation of pain. the pain can be elicited by everyday stimuli such as eating, shaving, talking and brushing teeth. it can be classified as either primary or idiopathic tn and secondary or symptomatic tn29). while primary or idiopathic tn has no clear cause, secondary tn has a presenting cause that can include etiologies such as tumor, multiple sclerosis or neurovascular compression. there have been few studies in the scientific literature dedicated to the treatment of tumor - related facial pain with chemoradiotherapy, radiosurgery or open surgery. pollock.19) reported a series of 8 patients with malignant tumor - related facial pain treated with gamma knife radiosurgery. all 8 patients with malignant tumors initially achieved pain relief, but 50% subsequently experienced a relapse of pain such as our patients did. our primary goal was to provide immediate pain relief and improve functional status and quality of life in patients near the end of life. stereotactic mesencephalotomy with the use of high - resolution mri and electrophysiologic localization is a valuable procedure, and as far as we know, this operation was attempted domestically for the first time. if performed meticulously and precisely, it may be beneficial in cancer pain as well as in central pain, with the avoidance of any related complications.	cancer - related facial pain refractory to pharmacologic management or nondestructive means is a major indication for destructive pain surgery. stereotactic mesencephalotomy can be a valuable procedure in the management of cancer pain involving the upper extremities or the face, with the assistance of magnetic resonance imaging (mri) and electrophysiologic mapping. a 72-year - old man presented with a 3-year history of intractable left - sided facial pain. when pharmacologic and nondestructive measures failed to provide pain alleviation, he was reexamined and diagnosed with inoperable hard palate cancer with intracranial extension. during the concurrent chemoradiation treatment, his cancer - related facial pain was aggravated and became medically intractable. after careful consideration, mri - based stereotactic mesencephalotomy was performed at a point 5 mm behind the posterior commissure, 6 mm lateral to and 5 mm below the intercommissural plane using a 2-mm electrode, with the temperature of the electrode raised to 80 for 60 seconds. up until now, the pain has been relatively well - controlled by intermittent intraventricular morphine injection and oral opioids, with the pain level remaining at visual analogue scale 4 or 5. stereotactic mesencephalotomy with the use of high - resolution mri and electrophysiologic localization is a valuable procedure in patients with cancer - related facial pain.
a 29-year - old japanese man presented with a reddish nodule, of approximately 15 mm in diameter, on the nose (figure 1). dermatoscopic examination of the nodule revealed a yellowish - white network, yellowish - orange globules at its center, and a pinkish structureless peripheral area (figure 2a). on histopathological examination, we observed an exophytic lesion composed of dilated, follicular infundibular structures connected with multiple sebaceous lobules and surrounded by fibrous connective tissue with thick collagen bundles (figure 3a). moreover, the surrounding stroma was fibrotic, with a high number of small vessels (figure 3c). fsch, first described by kimura. in 1991, is characterized by a relatively rare cutaneous hamartoma composed of follicular, sebaceous, and mesenchymal components. fsch clinically manifests as a solitary skin - colored sessile or pedunculated nodule that is most commonly located on the central part of face, particularly on the nose or the paranasal area. fsch shares several similar histopathological features with sebaceous trichofolliculoma (stf) and may be considered a variant of stf with a marked sebaceous component. however, certain histopathologic features of fsch are believed to be distinct, and thus, this condition can be differentiated from stf. described that the prominent mesenchymal component and double cleft formation between fibroepithelial units and the altered stroma are distinguishing features of fsch. in contrast, stf has rudimentary hair follicles and hair shafts connecting to the infundibular cyst wall, and lacks the distinctive mesenchymal component observed in fsch. on dermatoscopy, we detected a yellowish - white network and yellowish - orange dots / globules at its center, with a pinkish structureless peripheral area (figure 2a). the whitish - yellow network observed on histopathological examination represented the elongation of the rete ridges in addition to dermal sebaceous components (figures 2b and 3b). moreover, whitish - yellow clods were identified at the center of the nodule, indicating the presence of exophytic lesions consisting of sebaceous lobules connected to the dilated infundibular cystic structures via sebaceous ducts (figure 2b). furthermore, at the center of the nodule, the yellowish - orange globules represented conglomerations of sebaceous glands located in the superficial dermis (figure 2c). the color of the pinkish structureless peripheral area appeared similar to that of dermal dilated blood vessels. based on the clinical findings, the differential diagnoses would include melanocytic nevus, poroma, and sebaceoma. however, additional features observed via dermatoscopy could help exclude these disorders, including the lack of residual pigmentation around the hair follicles suggesting melanocytic nevus, the absence of glomerular or hairpin vessels and a whitish - pink network suggesting poroma, and the presence of a yellowish structureless area and arborizing vessels suggesting sebaceoma. to our knowledge, only one case presenting with the dermatoscopic features of fsch has been reported in japan. in that report, the dermatoscopic features of fsch included a yellowish - orange area and whitish - yellow globules. although fsch can only be diagnosed based on the microscopic features, certain characteristic dermatoscopic features observed in the present case may also be useful to distinguish among several differential diagnoses. moreover, we believe that the awareness of this entity and the use of dermatoscopy can facilitate the diagnosis of fsch. in conclusion, we described the dermatoscopic features of fsch, such as a whitish - yellow network, yellowish - orange dots / globules, and whitish - yellow clods. however, additional dermatoscopic findings are needed to elucidate whether the features described here are characteristic findings in fsch.	a 29-year - old japanese man presented to our institution with a nodule on his nose that had increased in size since childhood. physical examination indicated the presence of an elastic, firm, pedunculated red nodule measuring 15 mm in size. dermatoscopic examination of the nodule indicated a yellowish - white network, yellowish - orange dots / globules at its center, and a pinkish - white structureless peripheral area. histopathological examination of an excisional biopsy specimen showed a dilated infundibulocystic structure with sebaceous lobules proliferating radially, surrounded by fibrous stroma. moreover, mature adipocytes and small vessels were noted in the stroma. based on these histopatho - logic findings, the patient was diagnosed with folliculosebaceous cystic hamartoma.
a 40-year - old caucasian male presented to the emergency department complaining of intermittent painless frank haematuria over the preceding six weeks. past medical history was significant for hypercholesterolemia, type 2 diabetes mellitus and hereditary non - polyposis colon cancer (hnpcc). he had undergone a prophylactic total colectomy six years previously. his father had resection of colorectal cancer on two occassions, and was subsequently diagnosed with hnpcc. none of the index patient 's father 's siblings underwent genetic testing, but reportedly there were cases of colorectal cancer amongst them. genetic testing of the index patient and his six siblings showed two of the five to have lynch gene positivity. a first cousin of the index patient is lynch gene positive and has undergone a prophylactic total colectomy. he was a non - smoker and consumed approximately ten units of alcohol a week. computed tomography urogram showed thickening in the posterior wall of the bladder on the right side at the level of the seminal vesicles (figure 1). specifically thickened bland urothelium, which focally had papillary architecture and elsewhere had an inverted growth pattern, with no visible muscularis propria and no evidence of invasion was seen. figure 1transverse section of computed tomography abdomen and pelvis showing thickening of the right aspect of the posterior bladder wall at the level of the seminal vesicles. transverse section of computed tomography abdomen and pelvis showing thickening of the right aspect of the posterior bladder wall at the level of the seminal vesicles. hnpcc, also known as lynch syndrome, is an autosomal dominant cancer susceptibility disorder responsible for 35% of colorectal cancers. it is caused by germline mutations in four mismatch repair (mmr) genes ; nearly 90% in mlh1 and msh2, with the remaining 10% in msh6 and pms2. carriers of the abnormal gene have a risk of up to 70% of developing colorectal cancer by the age of 70. while most of these mutations are hereditary, sporadic cases are known to exist. these include, in addition to colorectal cancer ; endometrial, ovarian, stomach, pancreas, biliary tract, small bowel, brain, renal pelvic and ureteric tumours, sebaceous gland adenomas and keratocanthomas. while an association between hnpcc and bladder tumours is not as well established as the association with the above listed tumours, there are reports in the literature that suggest an association. one such case study from japan showed a possible association between lynch syndrome and two cases of bladder cancer. a study from the netherlands showed a correlation between lynch syndrome and urothelial bladder tumours, which was more significant when one specific mmr gene mutation in contrast analysis of the swedish family cancer database, showed that lynch syndrome patients have an increased risk of cancers in the ureter, but not in the urinary bladder. further large - scale evaluation is necessary to establish a link between hnpcc and bladder tumours. punlmp represents an indolent pathology which has a low propensity to recur, a negligible risk of progression and never results in tumour - related death. punlmp 's should be treated similarly to low grade, non - invasive urothelial carcinoma.	a 40-year - old caucasian male presented to the emergency department complaining of intermittent painless frank haematuria. past medical history was significant for hereditary non - polyposis colon cancer (hnpcc) and a prophylactic total colectomy. computed tomography urogram showed thickening in the posterior wall of the bladder. cystoscopy showed a small bladder mass. histology showed a papillary urothelial neoplasm of low malignant potential. hnpcc, also known as lynch syndrome, is an autosomal dominant disorder responsible for 35% of colorectal cancers. there are certain cancers known to be associated with hnpcc ; colorectal cancer, endometrial, ovarian, stomach, pancreas, biliary tract, small bowel, brain, renal pelvic and ureteric tumours, sebaceous gland adenomas and keratocanthomas. an association with bladder tumours is not well established.
there is a one - in - four chance that a drug used from any pharmacy has an active ingredient derived from a plant. indeed, the international consumer market for medicinal herbs and botanicals is estimated to be at about us $ 18 billion. hence, in our technological age, plants continue to play a significant role both medically and economically. even the most ancient written records of human civilization tell of humans using plants in everyday life. for centuries plants examples of medicine that contains plant derivatives include aspirin, used for pain relief and inflammation reduction ; physostigmine and pilocarpine, used for glaucoma control ; quinidine, which has saved the lives of many heart attack victims. the principal goal of this study was to determine if extracts from selected medicinal and nonmedicinal plants were cytotoxic ; often, the difference between a therapeutic and a toxic extract or compound is simply the dose level. our hope is that these survey data can be used as early indicators of some plants that may have therapeutic activity. moerman has done extensive screening studies on a variety of medicinal plants. from his investigation the four principal families, asteraceae, labiatae, ranunculaceae, and pinaceae, represent the first, third, fourth, and fifth families with the most medicinal species. it was hoped that our data might show some trends of toxicity within medicinally rich families. prokaryote cells included staphylococcus aureus, a gram - positive cocci responsible for infections of the skin and respiratory tract, food poisoning, and toxic shock ; salmonella choleraesuis, a gram - negative facultative aerobe responsible for food poisoning ; pseudomonas aeruginosa, a gram - negative rod that causes infections in wounds. for the eukaryotic system, hela cells, an epithelial carcinoma of the cervix, were used. the samples were filtered in glass - fiber filters fitted with coarse pore discs, and rotary evaporated down to 20 ml of extract on a buchi re111 rotary evaporator. twenty - four hours before the assay, each of the three bacteria were grown in a culture tube with 5 ml of tryptic soy broth without dextrose and incubated at 35c. (14.5 cm) petri dishes were previously prepared with a coat of muller hinton medium (agar). the cultures were checked on a spectrophotometer to ensure the proper growth (20% transmittance at 600 nm). six 1.4 cm circles of filter paper were then coated in plant extract, three with 20 l and three with 30 l, and placed on the plate. a disk with 20 l of water was added to the plate for a negative control and to s. aureus, s. choleraesuis, 10 l of ampicillin (bbl sensi - disc (becton dickinson)) was added as a positive control. the plates were then collected the next day and the zones of inhibition were measured. hela cells were maintained and assayed in mem with modification (sigma m-0894) supplemented with 10% fetal bovine serum, 1 mm sodium pyruvate, 1x mem - nonessential amino acids (sigma m-7145), 2 mm l - glutamine, and gentamicin at 50 g / ml. each extract was dried down and 2 mg / ml solutions were made using 10 mm tris buffer at ph 7.4. 150 l of a solution of suspended hela cells diluted with 15 ml of -mem is added to each well of a 96 well plate and incubated overnight at 37c and 5% co2. the next day 75 l, 50 l, 25 l with 25 l of -mem, 12.5 l with 37.5 l of -mem, or 6 l with 44 l of -mem of the 2 mg / ml extracts was added to 9 wells as a control.. the perchloric acid is removed, and the cells were stained in 4% sulforhodamine b in 1% acetic acid and then washed in 1% acetic acid. the dye was allowed to dry and 150 l of 10 mm tris base unbuffered was then added to each well, and the absorbance of each well was read using a spectrophotometer at 570 nm. the percent viability was calculated as the ratio of absorbance of the treated sample over the average of the controls. of the 55 plants tested, only four, pinus monticola, abies procera, salvia vaseyi, and salvia apiana, inhibited the growth of s. aureus. however, the zones of inhibition were quite small, only about 1 cm each. this is understandable because the zone of inhibition is directly proportional to the concentration of the biologically active agent and its diffusibility, so the possibility of active compounds not showing a positive response could be expected if the active ingredients did not diffuse. due to the screening nature of this procedure and small sample size, the quantitative analysis of the size of the rings of inhibition was quite subjective. some of the extracts were so toxic to the hela cells that very low doses of 0.0l and 0.001 mg / ml were studied in order to establish an lc-50. the lc-50 s were calculated from least squares regression using the linest function on microsoft excel 2000 over the dose response range or the whole data set in the case of nontoxic extracts to get a rough quantitative value in order to assess cytotoxicity. tris buffer, the control, gave an average 92% viability with no dose response. the first type was a clear dose - response over the full range of concentrations. type two followed a steep dose - response over the initial range of concentrations while the lower concentrations did not. of the 46 medicinal plant extracts, 54% were active, 26% were mildly active, and 20% were not active against hela cells. this strongly suggests that there may be some connection between plants known from indigenous cultures to have medicinal properties compared to empirically determined cytotoxicity. our eight non - medicinal plants also tended to be bioactive, with 50% active, 13% mildl, and 37% not active. only four samples showed antibacterial activity, which was only in s. aureus, and all these extracts were from medicinal plants. asteraceae, the sunflower family and one with the highest medicinal activity rating in moerman 's paper, was the only family from which we had a fairly large sample, 15 medicinal plants. extracts from asteraceae tended to be quite active and followed the general trends of medicinal plant bioactivity as stated above with 54% active, 29% mildly active, and 17% not active. the mint family, labiateae, also tended to be cytotoxic with 86% of the plants showing bioactivity. because only seven plants were tested, more data should be collected from this family before a general conclusion additional work is needed to determine which plant parts tend to have the highest bioactivity. the least active of our five medicinal families was ranunculaceae with two out of six plant extracts (33%) showing mild activity. overall these data clearly suggest that non - medicinal as well as so - called medicinal plants should be used in general cytotoxicity screening evaluations. in fact, de oliveira maria. also found significant bioactivity in 12 species of amazonian plants which were non - medicinal. though this work proved to be insightful, future studies should be undertaken in order to get a clearer picture of the evolutionary relationship of bioactivity and medicinal ranking of plants. from the literature, it appears that only three plants from our group, ambrosia ambrosioides [5, 6 ], gutierrezia microcephala, and atriplex confertifolia have had extensive research on their cytotoxicity. hence, there is a great deal of toxicology work yet to be done on the remainder of the plants shown to be bioactive in our investigation.	this study investigated the cytotoxicity of 55 species of plants. each plant was rated as medicinal, or nonmedicinal based on the existing literature. about 79% of the medicinal plants showed some cytotoxicity, while 75% of the nonmedicinal plants showed bioactivity. it appears that asteraceae, labiatae, pinaceae, and chenopodiaceae were particularly active against human cervical cancer cells. based on the literature, only three of the 55 plants have been significantly investigated for cytotoxicity. it is clear that there is much toxicological work yet to be done with both medicinal and nonmedicinal plants.
mucormycosis is a very aggressive invasive fungal disease caused by zygomycetes of the order mucorales. granulocytopenia, immunosuppression, diabetes and penetrating trauma are the most prevalent predisposing diseases associated with mucormycosis. severe infection of the facial sinuses, which may extend into the brain, is the most common presentation. cerebral mucormycosis is a devastating infection and associated with a high case - fatality rate, especially in immunocompromised patients.,, herein, we present a patient with ventriculitis due to infection with rhizopus arrhizus (rhizopus oryzae) that was initially clinically diagnosed as intracerebral aspergillosis but failed under empiric voriconazole therapy. after identification of r. arrhizus, the patient was successfully treated with intrathecal amphotericin b and systemic combination therapy of high - dose liposomal amphotericin and posaconazole. a 52-year - old man presented to the emergency department with acute onset of confusion, dysarthria and tendency to fall in july 2012. the patient was on immunosuppressive therapy because of a combined heart lung transplantation in june 2011. laboratory data on hospital admission (day 0), were normal besides of low hemoglobin (reference range in brackets) : wbc 6.4 gpt / l (4.411.3), platelets 22710/l (150360), hemoglobin 7.8 mmol / l (8.710.9) and c - reactive protein (crp)<2.0 mg / l (< 7,5). mri on admission showed ballooning of the left ventricle, midline shift over 11 mm and contrast enhancement in the anterior horn of the left ventricle with obstruction of the interventricular foramen (of monro) (fig. initial cerebrospinal fluid (csf) examination on day 0 showed a cell count of 377 cells/l (05) and a protein level of 340 mg / l (150400) ; no organisms were observed on gram stain. furthermore, 16s and 18s ribosomal rna gene targeting pcr performed on csf was negative. likewise aspergillus the patient was started on meropenem, vancomycin and voriconazole on day 0, but did not respond to this treatment. therefore, for further diagnosis and therapy, ventricle neuro - endoscopy was performed on day 12 after admission. calcofluor - white stain of the ventricular biopsy showed ribbon - like hyphae (fig. pas staining showed numerous ribbon - like, haphazardly branched fungal hyphae with pas positive thick walls and little or no septation (fig. species identification was performed on histological tissue biopsy and microbiological cultures by molecular diagnostic technique based on 18s - pcr (primers and protocol used for amplification and sequencing according to, length of resulting sequence 339 bp, 100% identity to acc. km527239.1) which revealed r. arrhizus (r. oryzae). empiric therapy of meropenem, vancomycin and voriconazole was stopped and therapy with intravenous liposomal amphotericin b (5 mg / kg / day with stepwise increase to 10 mg / kg / day), intrathecal amphotericin b (0,5 mg / day) and posaconazole (1200 mg / day) was initiated on day 14. intrathecal amphotericin b was terminated due to neurologic side effects including fluctuating level of consciousness on day 21. therapy with intravenous liposomal amphotericin b and high - dose posaconazole was continued until day 35, respectively day 55 after admission. thereafter suppressive therapy with posaconazole (600 mg / day) has been continued until now. except for hemianopsia and deficits in minute motor activity he presented the last time for follow up in our outpatient clinic in may 2015. overall, rhizopus species are the most common cause of mucor - mycosis in humans. zygomycetes such as rhizopus species can invade the cns by either direct extension or hematogenous spread. an european guideline for the diagnosis and management of mucormycosis was published recently. for diagnosis, direct microscopy of clinical specimens and culture is strongly recommended. colonies of rhizopus are characterized by rapid growth, coarse and floccose aerial mycelia, similar to the structures visualized during ventricle neuroendoscopy in our patient. to best of our knowlegde this is the first report showing pictures of colonies of r. arrhizus (r. oryzae) within the ventricle of a patient. histopathological examination may allow differentiation between hyphae of aspergillus or morphologically related fungi, and hyphae of mucorales, which is important for treatment decisions as latter are intrinsically non - susceptible to voriconazole and echinocandins. hyphae of mucorales have a variable width (625 m), are non - septate or pauci - septate and have an irregular, ribbon - like appearance. interestingly, repeatedly performed csf cultures showed no growth of r. arrhizus in our patient, only the tissue biopsy sampled during ventricle neuroendoscopy was positive finally. this emphasizes the importance of performing different modes of diagnostics including tissue biopsy. in our case, the failure of empiric voriconazole and the negative csf - galactomannan prompted us to invasive diagnostics that revealed the correct diagnosis. liposomal amphotericin b is the drug of choice, dose should be at least 5 mg / kg / day. in the case of cns infection 10 mg / kg / day is recommended for the initial 28 days. for salvage therapy posaconazole 200 mg four times daily is strongly recommended, the clinical significance of combination therapy however is uncertain according to the guideline. however, in a recent report on 32 patients with mainly hematological diseases the analysis suggested that a combined antifungal treatment with liposomal amphotericin b and posaconazole may be considered in patients with very aggressive forms of invasive mucormycosis. due to rapid deterioration of general condition and worsening of neurologic symptoms combination therapy of liposomal amphotericin b and posaconazole was performed in our patient. because of unreliable absorption rates of oral posaconazole suspension and no possibility for therapeutic drug monitoring of posaconazole we decided to administer high - dose posaconazole (1200 mg / daily) in our patient. this treatment regime was well tolerated by our patient and a probably devastating surgical focus control was not required.	a 52-year - old heart lung transplant patient presented to the emergency department with acute onset of neurologic symptoms. mri showed ballooning of the left ventricle, midline shift and contrast enhancement in the anterior horn of the left ventricle. ventricle neuroendoscopy revealed whitish, floccose aerial structures within the left ventricle. brain biopsy cultures grew rhizopus arrhizus. therapy with liposomale amphotericin b and posaconazole was performed. except for hemianopsia and deficits in minute motor activity, the patient completely recovered.
asia has the opportunity to be a global leader in combating the growing epidemic of chronic kidney disease (ckd). this opportunity should be based on our knowledge of the strengths (and weaknesses) of the asian medical and scientific communities and the environments in which they work. nearly 50% of the global population of nearly 7 billion persons live in the asian pacific region (1). six of the 10 most populous nations in the world are in asia, including the 2 largest (china 1.3 + billion, india 1.1 + billion) along with indonesia, pakistan, bangladesh and japan. population densities range from some of the world 's highest (macau, singapore, hong kong, maldives, and bangladesh - all over 1,000 persons per km) to some of the lowest (mongolia and australia - both less than 3 persons per km). asia has both strengths and weaknesses relating to this large population. in terms of annual gross domestic product (gdp) 3 of the world 's 4 wealthiest nations are in asia (us$gdp japan 4.3 10, china 3.3 10, india 1.1 10), and the gdp per capita is over us$30 10 in singapore, hong kong, brunei, australia, japan, and taiwan ; ranking them in the 22 highest incomes per person in the world (2). however we also have some of the poorest of people, with gdp per capita being less than us$2,000 p.a. in afghanistan, the solomons and papua new guinea, and total economies of gdp less than us$500 million in east timor, the solomon and tonga (2). though the large population of asia brings its own challenges, the world 's pharmaceutical industry has recognised its strength, with increasing numbers of prospective therapeutic trials being carried out in asia. regrettably however, a recent survey found that of 13,152 commercially sponsored trial sites in the world, asia had only 2 trial sites per million population, compared with north america 191 and europe 86 (3). this strongly suggests that asian populations are not being adequately recruited into clinical trials, and hence properly represented in efficacy and side - effect databases. we must move to accelerate the trend towards using asian populations in sponsored clinical trials. scientifically in addition to this, a growing number of publications from institutions in western countries have authors born in asia or descended from asian parentage. this scientific basis is already enhancing the two - way communication between asian and the rest of the world and is likely to result in asian leadership over the next decades. the cultural diversity within the asian pacific region is arguably greater than any other area in the world. underpinning the fascinating variety of religions and languages are important and unique behaviours and values which, though fundamentally similar, differ in many ways. but in terms of ckd, the influences of everyday behaviour can have enormous impacts on options for patients and their carers, influencing fundamental decisions such as live and deceased donor transplantation, self - care or dependent - care, healthcare funding processes, as well as solutions to clinical problems. the solutions on ckd problems, varying according to these values, should provide a basis for combating ckd world - wide. a particularly intriguing issue in the asian pacific region is the striking variance in incidence and prevalence of endstage kidney disease (eskd) as recognised by rates of dialysis and transplantation. in countries with the economic power to offer dialysis to virtually all citizens, japan (4) and taiwan (5) have the world 's highest incidence and prevalence of eskd with those of korea rapidly accelerating (6), while australia has amongst the lowest. this enormous disparity has been partially related to the impact of diabetes and ageing in asia (7) but also seems to involve other factors (8 - 13). one study of ckd in our region concluded that the reason for the high rates seen in more wealthy asian areas was a combination of increasing incidence of type 2 diabetes and ageing with an underlying propensity for ckd to progress more quickly in non - european asian populations (7). the widespread social and dietary deprivation of the first half of the last century, combined with the rapid improvements over the last 50 yr may be responsible for this, according to the theory relating malnutrition to low birth weight, fewer pancreatic islets and fewer nephrons ; hence later life type 2 diabetes and progression of ckd (8). it is not quite that simple. no doubt important ethnic (genetic) influences play a role, as well as environmental factors such as nutrition, infection and exposure to toxins (8 - 13). internationally, but stemming mainly from western countries, have come classifications and recommended treatment regimens for a wide variety of ckd issues. perhaps this is most evident when we look at formulae for calculating a glomerular filtration rate (gfr) from plasma creatinine. the first widely applied formula, resulting in an estimate of creatinine clearance (crcl), was the cockroft - gault formula which was developed in a caucasian canadian group of 249 patients, using gender, weight and plasma creatinine (14). the next major step forward was the formula from the modification of diet in renal disease (mdrd) study, which recognised some ethnicity effects with a correction to be applied if the patient is black (15). critically, since there was no data on asian persons in the mdrd study, there was no correction available for the asians. the lack of specific gfr formulae for asian populations is not an insignificant issue, since it reflects not only on the diagnosis and management of those with established ckd, but also on any attempts to determine the incidence of ckd in our communities. gfr formulae applicable in japan (16) and china (17) have been published, and other countries are rapidly following this lead, either testing the validity of previous formula in their community as in korea (18), or attempting new constructs. a substantive south - asian formula is eagerly anticipated. while it is clear that asia is unfortunately following and perhaps overtaking the western world in the increase in metabolic syndrome and its related renal consequences, we must not lose sight of other diseases which are more common in asia than in the west (table 1). this allows concentration on diseases that are locally preventable, and the knowledge gained may have global relevance. in developing countries, poor water supplies, isolated communities and a hot climate contribute to a high incidence of kidney stones (19) and acute renal failure due to gastroenteritis, kidney stones and peri partum bleeding (20, 21). because of the preponderance of rural dwellers and heavily forested areas, a wide variety of reptile and insect bites cause acute renal failure (22 - 24). tropical infections are commonly complicated by renal disease (25 - 30), including haemorrhagic fever with renal syndrome, where korea has played a major role in defining the clinical aspects and relationship to the hantaviruses (28 - 30). the rapid transition to industrial societies has had adverse environmental effects, resulting in industrial poisoning, and in rural communities improper use of insecticides contributes to both acute and chronic renal failure (31 - 34). the ingestion of various fungi and fruit has been associated with renal injury, both acute and chronic (35 - 37). in addition, there are areas of asia where pockets of unusual disease occur ; their causes are likely to be environmental or genetic but they are as yet unknown. in thailand, renal tubular acidosis, hypokalaemic periodic paralysis and renal stone disease occur (38). north central sri lanka has an area where chronic tubulointerstitial nephritis is common, similar to aristolochic acid nephropathy, but without evidence of fungal ingestion. renal tubular acidosis has been reported in areas of papua new guinea, possibly related to ovalocytosis (39, 40). consequent upon these regional diseases, asian pacific nephrology has a major opportunity to contribute to understanding their pathophysiology and management. less fortunately, however, most of asia seems prone not only to increased renal complications from the metabolic syndrome, but also it has been suggested that there are more common or severe examples of other diseases, particularly sle (41 - 43) and iga nephropathy (44, 45) than in most european communities. many have recognised its potential, and the benefits of a committed and collaborative approach. the asian pacific society of nephrology (apsn) of which the korean society of nephrology is a sponsor society is now 25 yr old and every asian national nephrology society in our region is affiliated. its journal, nephrology, is medline listed and in its 13th year. the next congress of the apsn is to be held in korea in 2010. in 2007 the asian forum on chronic kidney disease initiative (afckdi) held the first of what have become annual meetings to discuss the issues of collaborating ckd in our region. global bodies have quickly become involved with both the apcn and afckdi meetings, with isn comgan and the kidney disease improving global outcomes (kdigo) represented at all meetings.	asian pacific countries include those with the highest incidence of renal failure in the world, the richest and poorest economies and unparalleled diversity of economy, culture and geography. from this come many challenges, but also a strong basis for the introduction of strategies to combat renal diseases. with a rapidly developing scientific community, asia needs to accept the challenge of becoming a global leader in nephrology in the near future.
small non - protein - coding rna (ncrna) molecules are key players in controlling gene expression at multiple steps in all domains of life (amaral., 2008 ; httenhofer., 2005 ; mattick, 2004 ; tuck and tollervey, 2011). in the past years, it became evident that ncrnas represent a widespread class of regulatory molecules shaping cellular life (aalto and pasquinelli, 2012). the advantage of ncrna regulators is their almost immediate availability because they act on the rna level and thus do not need to be converted into a polypeptide in order to fulfill their cellular function. translation represents the last step in gene expression, and its regulation allows a swift and reversible adaption to changing environmental conditions (gebauer and hentze, 2004). the list of validated ncrnas regulating translation, such as micro rnas (huntzinger and izaurralde, 2011 ; krol., 2010) and small - interfering rnas (mello and conte, 2004), is growing steadily ; however, they almost exclusively target the mrna rather than the ribosome, the key enzyme of protein biosynthesis. this is unexpected given the central position the ribosome plays in cell metabolism and the assumption that the protoribosome originated in the rna world (crick, 1968 ; steitz and moore, 2003) and thus likely depended on regulatory input from nonproteinous cofactors such as small metabolites or short ncrnas. in contemporary biology, protein biosynthesis is a very energy - demanding process and therefore rigorously regulated in response to environmental changes. controlled translation regulation enables a cell or an organism to fine - tune its proteome in time and space. regulatory input is typically given by stress - induced modifications (e.g., phosphorylation) of essential initiation factors, by mrna - binding proteins that can sense environmental changes (e.g., iron regulatory proteins), or by the action of mrna - targeted micrornas (gebauer and hentze, 2004). the yeast s. cerevisiae is one of the few characterized eukaryal organisms known to lack components of the rna interference machinery and thus lives without microrna and small interfering rna (sirna) translation regulation (houseley and tollervey, 2008). here, we set out to functionally characterize an mrna exon - derived 18-residue - long ncrna candidate that was picked up in our recent genomic screen for ribosome - bound small rnas in s. cerevisiae (zywicki., 2012). we show that this 18-mer rna fragment is a functional ncrna capable of adjusting translation rates by interacting with polysomes under hyperosmotic growth conditions. to identify potential alternate mechanisms of translation regulation in s. cerevisiae and to address the question whether small ncrnas exist that directly target the ribosomes under specific growth conditions, we investigated the ribosome - associated rnome (size range 15500 nucleotides) (zywicki., 2012). in addition to known ribosome - bound ncrnas (trnas, 7sl rna), 20 mrna exon - derived fragments, sized between 18 and 70 nucleotides, copurified with ribosomes. the most abundant mrna fragment originates from the trm10 locus, which encodes a trna methyltransferase (jackman., 2003). the resulting trm10 mrna piece is 18 nucleotides long and is located 28 residues downstream of the translation start site (figure 1a). the 18-mer rna, and a putative 45-residue - long processing intermediate, was expressed in a stress - independent manner (figure 1b). by comparing the northern blot signals for the 18-mer rna in the pellet fraction of a 100,000 g centrifugation (p100) of cell lysates, which contains the ribosomes, with the corresponding supernatant (s100) (see supplemental information available online for details), about 80% of the signal was detected in the p100 fraction. this demonstrates that the vast majority of cellular 18-mer rna is associated with ribosomes in vivo (figure 1c). to gain insight into the in vivo function of this 18-mer ncrna candidate, the growth characteristics of a trm10 knockout strain (trm10) were investigated under nine different growth conditions. in line with previous reports (gustavsson and ronne, 2008 ; jackman., 2003), the lack of the trm10-encoded methyltransferase had no growth phenotype under most conditions (figures s1a s1f). however, under hyperosmotic stress conditions in the presence of elevated concentrations of nacl or sorbitol, the trm10 strain showed a slow growth phenotype (figures s1 g and s1h). to test if and how the trm10-derived 18-mer fragment plays a role in this phenomenon, polysome profiling and genetic analyses were performed. even though the total portion of ribosome - associated 18-mer rna remains constant in unstressed and high - salt - stressed cells (figure 1c), polysome profiling revealed significant differences. polysome profiling identified nontranslating 80s ribosomes in unstressed and polysomes in salt - stressed cells as main targets (figure 2a). more than 80% of 18-mer was associated with 80s ribosomes and 60s subunits in unstressed cells, and only a minor portion entered the actively translating polysome pool. however, the fractional distribution changed markedly upon salt addition. under these hyperosmotic conditions, the 18-mer rna relocates and almost 80% was present in the polysomes, whereas it was almost completely absent in the 80s ribosome fraction (figure 2a). although the data shown in figures 1c and 2a indicate a direct interaction between ribosomes and the 18-mer rna, they do not unequivocally exclude the possibility of an mrna - association mechanism. to clarify this, polysome profiling was performed in the presence of edta, conditions known to remove and dissociate translating polysomes from mrnas (del prete., 2007). if the 18-mer was mrna bound, it is expected to shift into the pool of free rna on top of the gradient, whereas it should remain in heavier fractions, when the 18-mer rna was ribosome associated. northern blot analysis revealed that the 18-mer rna sediments primarily in the 60s ribosomal subunit fraction and does not accumulate in the free rna pool (figure 2b). these data demonstrate that 18-mer rna targets the 60s subunit in vivo. to explore the role of the ribosome - bound 18-mer rna fragment during cell growth under stress conditions, genetic complementation experiments were performed. northern blot analyses showed that the trm10-derived 18-mer rna was expressed and associated with ribosomes also when the gene was transcribed from the plasmid (figure 1c). as expected, no northern blot signal for the 18-mer rna was evident in the trm10 strain thus demonstrating that this rna fragment derives from the trm10 gene. to monitor growth under stringent high - salt conditions, the strains were first grown to stationary phase in stress medium, diluted with fresh stress medium, and subsequently allowed to resume growth. complementation experiments under these harsh stress conditions demonstrated that the markedly reduced growth of the trm10 strain could be rescued by expressing the trm10 locus from a plasmid (figure 3a). to distinguish whether the absence of the trm10-encoded trna methyltransferase or the mrna - derived 18-mer rna fragment is responsible for the growth defects, we either introduced a uga stop codon or introduced synonymous mutations (m1, m2) within the mrna 18-mer region (figure 1a). these experiments showed that cells expressing a nontranslatable trm10 mrna had no growth defect at elevated salt concentrations (figure 3a), even though no active trna methyltransferase was produced (figures 3c and 3d). the reciprocal experiment, when two (m1) or three (m2) synonymous codons were introduced into the trm10 gene, showed the opposite effect. despite the fact that an active trna methyltransferase was expressed (figure 3d), the cells failed to resume growth in high - salt medium (figure 3b). these data reveal the lack of the mrna - derived ncrna candidate and not the mrna - encoded trna methyltransferase as the cause for the observed growth defects at high - salt concentrations. the m1 and m2 mutants could not recover growth thus demonstrating the sequence specific mode of action of the trm10 18-mer rna. in support of this, the m2 mutant version of the 18-mer rna was also largely absent from ribosomes (figure 1c). therefore, in order to promote growth under hyperosmotic conditions, the 18-mer rna needs to physically interact with ribosomes in vivo. the 18-mer rna acts specifically under hyperosmotic conditions, because its absence did not result in a growth phenotype during heat or cold shock (figure s2). to study the role of the trm10 18-mer fragment in vivo, we adapted a metabolic labeling approach using yeast spheroplasts (russell., 1991) by measuring the s - met incorporation into newly made proteins (figure 4a) introducing the 18-mer rna into the spheroplasts by electroporation resulted in an almost complete inhibition of protein biosynthesis, whereas a scrambled 18-mer rna control had no effect (figure 4b). gel electrophoresis and autoradiography indicated global translation inhibition, thus arguing for a general downregulation of protein synthesis in the presence of the 18-mer rna (figure 4c). quantification of the uptake efficiency of the synthetic 18-mer into spheroplasts indicated the presence of about 200,000 molecules per cell, thus roughly equaling the ribosome concentration (warner, 1999). also, in this assay, the inhibitory function of the 18-mer rna was sequence specific because rna strands containing two (m1) or three (m2) mutations (figure 1a) were unable to affect metabolic labeling of cellular proteins (figure 4b). additionally, also the secondary structure context of the 18-mer sequence within the introduced rna strand influenced the inhibitory potential (figure s3). these results described above indicate that the trm10 18-mer rna targets protein biosynthesis. to corroborate these findings, in vitro translation reactions addition of the 18-mer rna, but not the m2 mutant, clearly reduced in vitro protein synthesis to a similar extent as the known translation elongation inhibitor cycloheximide (figure 5a). translation inhibition by the 18-mer rna was dose dependent with an apparent ic50 of 2.5 m (figures 5b and 5c). this value is in a physiologically reasonable range considering the in vivo concentrations of the 18-mer rna (1.1 m ; figure s4) and of yeast ribosomes (8 m) (petelenz - kurdziel., 2011 ; warner, 1999). notably, the yeast 18-mer rna was also able to inhibit in vitro translation in a wheat germ system with a slightly increased ic50 of 7 m but affected mammalian and bacterial protein biosynthesis to a lesser extent (figure s5). to gain insight whether the 18-mer interferes with translation initiation or elongation, the s. cerevisiae in vitro translation assay was slightly modified. complete reactions were assembled in the absence of radiolabeled methionine and 18-mer rna at the regular temperature of 23c, thus allowing translation initiation and elongation. subsequently, the reactions were placed on ice and s methionine and 13 m synthetic 18-mer were added (figure 5d ; condition ii). at low temperature, translation initiation is massively inhibited (al - fageeh and smales, 2006 ; hofmann., 2012), whereas already initiated ribosomes can continue protein synthesis. under these conditions that prevent reinitiation, the 18-mer rna had no inhibitory effect on in vitro translation activity (figure 5d). on the other hand, when the 18-mer and s methionine were added in an experimental set - up that also monitors translation initiation (condition i, figure 5d), significant translation inhibition was observed. these findings thereby suggest the 18-mer rna to interfere with the initiation phase of protein biosynthesis. the lack of the 18-mer rna fragment results in a severely retarded growth under high - salt conditions (figures 3a and s1). likely, the 18-mer ncrna is needed to slow down the metabolic activity in yeast when the environmental conditions become unfavorable thus allowing the adjustment of gene expression. if this assumption is correct, one expects the trm10 strain to possess elevated translational activity, reflected by a greater polysome fraction compared to the wild - type (wt) strain in high - salt medium. to test this model, polysome profiling as well as the translational activity of untreated cells were investigated and compared to cells after salt - stress induction. indeed, the strain lacking the trm10 gene, and hence the 18-mer ncrna, shows on average a 2.1-fold higher polysome:80s ratio at elevated salt concentrations (figure 6a). although initially both strains had identical polysome profiles and metabolic activities under normal growth conditions, 20 min of high salt stress resulted in an accumulation of 80s ribosomes and a reduced polysomal fraction in the wt strain as compared to the trm10 strain, arguing for translation initiation to be affected by the 18-mer rna. the trm10 strain had a markedly elevated metabolic activity compared to the wt strain within the first 520 min (figure 6b). similarly, the strain expressing the m2 mutant variant of the 18-mer, an rna molecule that is unable to efficiently associate with ribosomes (figure 1c), also possessed an enhanced metabolic rate. these clear differences, however, disappeared rapidly after 45 min (figure 6b). the coordinated regulation of protein biosynthesis in response to intra- and extracellular signals is pivotal for the establishment of productive gene expression networks. translation control typically involves regulatory proteins or small ncrna molecules of the rna silencing machinery. with the notable exceptions of the bacterial tmrna (felden and gillet, 2011) and the signal recognition particle rna (present in all domains) (akopian., 2013), all functionally characterized ncrnas capable of regulating protein biosynthesis (e.g., mirnas and sirnas in eukarya, small antisense rnas in prokarya) target the mrna rather than the ribosome directly. here, we present evidence that an 18-mer rna fragment from the trm10 mrna in s. cerevisiae associates with ribosomes and regulates protein synthesis under hyperosmotic stress conditions. in vivo and in vitro data demonstrate that the trm10 18-mer rna represents a functional ncrna in s. cerevisiae where it attenuates protein biosynthesis under high - salt conditions. this small ncrna is remarkable in two ways, namely, that (1) it is a functionally characterized ncrna deriving from the coding region of an mrna, and (2) it belongs to an emerging class of ncrnas regulating translation by directly associating with the ribosome. association of the 18-mer rna with the 60s ribosomal particles and reduction of global protein synthesis is crucial for s. cerevisiae under hyperosmotic conditions. strains lacking the 18-mer rna or expressing a ribosome - binding - deficient mutant version thereof (m2 ; figure 1c) are significantly retarded in downregulating their metabolic activities (figure 6b). especially the time window between 5 and 45 min after salt - stress induction appears to be crucial for the fate of s. cerevisiae for adapting to the new environmental conditions. demonstrating that the transcriptome of yeast cells quickly responses to various stress stimuli within the first 1045 min, whereas, at later time points, it resembled the unstressed rnome. although the trm10 mrna follows this expression trend (orourke and herskowitz, 2004), the trm10-derived 18-mer rna fragment remains at constant levels independent of stress induction (figure 1b). also, the fraction of ribosome - bound 18-mer ncrna remains constant and does not change during hyperosmotic conditions (figure 1c). notably however, upon high salt stress the 18-mer relocates almost quantitatively from nontranslating 80s ribosomes and 60s ribosomal subunits to translating polysomes (figure 2a). because only the distribution but not the cellular abundance of the 18-mer rna changes upon stress induction allows a rapid response to environmental signals without the need for synthesizing new regulatory molecules. it is thus possible that the trm10 18-mer rna is involved in regulating this first wave of stress adaptation under hyperosmotic conditions by lowering the efficiency of protein biosynthesis and thus slowing down overall metabolic activity. from a mechanistic point of view, the 18-mer rna functions differently than known small ncrna translation regulators (such as mirnas, sirnas, or bacterial antisense rnas), because it directly binds to 60s ribosomal subunits and does not target mrnas (figure 2). the polysome profiles of cells exposed to hyperosmotic stress showed a decrease in the polysome / monosome ratio (figure 2a), which is indicative of inhibiting translation initiation (uesono and toh - e, 2002). in support of this, addition of the 18-mer rna to an in vitro translation reaction under conditions that allow elongation but prevent reinitiation does not affect protein production (figure 5d). even though the 18-mer rna is less abundant (27,000 molecules / cell ; figure s4) than the ribosome (200,000/cell), a dynamic relocation between initiating and elongating ribosomes is sufficient to yield global effects on translation (figure s6). although it might appear counterintuitive at the first glance that slowing down protein synthesis (figures 4 and 5) can in the end stimulate cell growth during stress (figure 3), very recent evidence suggests that the ribosome serves as a regulatory hub in proteostasis and stress response (reviewed in pechmann., 2013 ; sherman and qian, 2013). the picture that emerges from these recent findings is that slowing down translation, and thereby increasing overall accuracy of protein synthesis and protein folding, actually improves protein homeostasis and stress adaptation. it has been noted that repression of global translation seems faster than changes in regulatory signaling pathways (sherman and qian, 2013), such as the tor pathway that does not appear to be involved in translation inhibition at the onset of hyperosmotic stress (uesono and toh - e, 2002). the molecular entity that actually transmits the stress signal to the translating ribosomes, however, remained enigmatic. thus, the 18-mer ncrna characterized in this study fulfills the criteria for such a signaling molecule because it is already associated with the translation machinery and can rapidly shift between initiating and elongating ribosomes upon hyperosmotic stress induction to swiftly attenuate the rate of translation. this allows stress - specific adaptation programs to be established, which, in turn, enable cells to survive under challenging environmental conditions. the origin of the 18-mer rna (deriving by trm10 mrna processing or from an independent transcript unit) has yet to be determined. however, exon - derived rna fragments have been observed in numerous eukaryal deep - sequencing studies and are likely the result of an evolutionarily conserved mrna cleavage mechanism (fejes - toth,., 2009 ; mercer., the small rna transcriptome of s. cerevisiae has been reported to possess an abundant pool of 17- to 19-nucleotide - long rna molecules, which have been regarded as degradation products of mrna, trna, and rrna (drinnenberg., 2009). in the light of the accumulating evidence that some of these trna - derived (reviewed in gebetsberger and polacek, 2013) and mrna - derived (this study) fragments actually possess cellular functions, it appears more appropriate to refer to these rna pieces as processing rather than degradation products. the 18-mer rna described here, together with thousands of other candidates that were picked up in analogous genomic screens in various model organisms spanning all three domains of life (gebetsberger., 2012 ; our unpublished data), reveal the ribosome as a target for small regulatory ncrnas and suggest the existence of a so far largely unexplored mechanism of translation control. future work on the small ncrna interactomes of ribosomes in a variety of model systems will allow deeper insight into the conservation and functional repertoire of this emerging class of regulatory ncrna molecules. s. cerevisiae strain by4742 and all mutant cells derived from this strain were grown in sc - leu medium at 30c or at various stress conditions (see supplemental information for more details). to monitor growth under more stringent hyperosmotic conditions (the redilution assay), single colonies were picked and grown in sc - leu medium supplemented with nacl (final concentration [f.c. ] 0.7 m) or sorbitol (f.c. 1.025 m) for 72 hr. subsequently the cultures were rediluted to an od600 of 0.1 into fresh stress medium, and cell growth was monitored over 20 hr. to monitor the methylation status of mg9 of trna, primer extension analysis using the 5-p - end - labeled primer 5-caacgttggattttacc-3 was performed as previously described (jackman., 2003 ; polacek and barta, 1998) (see supplemental information for details). for metabolic labeling yeast spheroplasts (russell., 1991) were mixed with 10 pmol synthetic 18-mer rna (or 3-extended variants thereof) and introduced into the cell via electroporation. after electroporation 1 ml ypd/1 m sorbitol and 1 l s - methionine (1,000 ci / mmol, 10 mci / ml) were added, and the reaction was incubated for 1 hr at 30c. the extent of labeled proteins was monitored by tca precipitation and subsequent liquid scintillation counting or by sds page (see supplemental information). to monitor the translational activity of s. cerevisiae cells (wt, trm10, or the m2 mutant strain) after nacl stress induction, stationary phase cultures were diluted into fresh sc - leu medium to a final od600 of 0.3 and incubated at 30c in the presence of high - salt - stress conditions (f.c. : 0.7 m nacl). at indicated time points aliquots were taken, and translational activity was monitored for 10 min at 30c by the addition of 1 l s - methionine (1,000 ci / mmol, 10 mci / ml). after 10 min incubation, cells were pelleted and labeled proteins were precipitated by adding 500 l 20% tca followed by liquid scintillation counting. for in vitro translation, an s30 extract from s. cerevisiae was prepared (see supplemental experimental procedures for details) (hofbauer., 1982). for in vitro translation, 2.5 l creatine phosphokinase (10 mg / ml ; roche), 7.5 l cacl2 (20 mm), 25 l 10 translation cocktail (100 mm hepes / koh [ph 7.5 ], 10 mm mg[oac]2, 760 mm kcl, 4 mm gtp, 10 mm atp, 19 amino acid mix [500 m each ; methionine excluded ]), 5 l creatine phosphate (0.6 m ; roche), and 2.5 l mg(oac)2 (100 mm) were mixed with 150 l s30 and 16 l s - methionine (1,000 ci / mmol, 10 mci / ml). the resulting translation mix was aliquoted into 12 l portions and filled up to 15 l with sterile h2o, synthetic 18-mer rna (10200 pmol) or cycloheximide (7.5 g/ l), incubated at 23c for 30 min, and the products were separated by sds - page. to test whether translation initiation or elongation are inhibited by the 18-mer rna, complete reactions, but lacking s - methionine and the 18-mer rna, were assembled and preincubated for 10 min at 23c. subsequently the initiated samples were cooled down to 0c on ice for 5 min followed by the addition of s - methionine and 13 m of the 18-mer rna. translation elongation was then carried out for 30 min at 0c, stopped by tca precipitation, and the products were quantified by liquid scintillation counting (see supplemental experimental procedures for details). for polysome profiling, 150 a260 units of s. cerevisiae cell extracts (see supplemental information) were layered on top of a 10%40% sucrose gradient and centrifuged for 5 hr at 25,000 rpm in an sw28 swing - out rotor. polysomal, monosomal, and subunit fractions were isolated, and the ribosomal particles in the collected fractions were precipitated by adding 2.5 vol etoh. the ribosome - associated rna were purified by phenol / chlorophorm / isoamyl - alcohol (pci) extraction and finally used for northern blot analyses. for northern blotting, 15 g total rna extracted from wt or mutant cells under different stress conditions was separated by denaturating polyacrylamide gels (7 m urea) and subsequently electroblotted onto nylon membranes (amersham hybond n, ge healthcare) as described (gebetsberger., 2012). all membranes were hybridized to a p-5-end - labeled lna probe (exiqon) complementary to the trm10 18-mer rna sequence.	summarythe structural and functional repertoire of small non - protein - coding rnas (ncrnas) is central for establishing gene regulation networks in cells and organisms. here, we show that an mrna - derived 18-nucleotide - long ncrna is capable of downregulating translation in saccharomyces cerevisiae by targeting the ribosome. this 18-mer ncrna binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. although the 18-mer rna originates from the trm10 locus, which encodes a trna methyltransferase, genetic analyses revealed the 18-mer rna nucleotide sequence, rather than the mrna - encoded enzyme, as the translation regulator. our data reveal the ribosome as a target for a small regulatory ncrna and demonstrate the existence of a yet unkown mechanism of translation regulation. ribosome - targeted small ncrnas are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules.
shewanella species are gram - negative, non - fermentative, motile bacilli mainly found in seawater and other underwater environments (fresh water, stagnant water, lakes, rivers, sewage), as well as soil, fish, meat, poultry and dairy products [1, 2 ]. this species was initially known as achromobacter putrefaciens, followed by pseudomonas putrefaciens, and was reclassified into the novel genus shewanella in 1985, which contained approximately 30 shewanella spp.. among these, only s. algae and s. putrefaciens are known to cause human infection. human infection caused by shewanella spp. is rare and occurs in mainly immunocompromised hosts who have a history of contact with seawater or ingestion of raw seafood. infection is also associated with ulcerations on the lower extremities [1, 2, 3 ]. recently, we experienced a case of spontaneous bacterial peritonitis (sbp) with bacteremia caused by shewanella spp. in a patient with liver cirrhosis (lc) who had no history of exposure to seawater or raw seafood. through biochemical studies and 16s rrna polymerase chain reaction (pcr) with sequencing analysis polymicrobial infection by streptococcus mitis and escherichia coli was combined, which resulted in a fatal clinical course. a 57-year - old male was transferred to the emergency department after endoscopic hemostasis for 1,000 ml of massive hematemesis due to esophageal and cardiac variceal bleeding. the patient had no history of travel, no contact with seawater or fresh water, and did not consume raw fish within the last 6 months. on physical examination, the blood pressure was 100/40 mmhg, heart rate was 176 beats / min, respiration rate was 28/min and body temperature was 35. the mental state was alert but confused and icteric sclerae were observed. complete blood count showed white blood cells (wbc) counts of 12,960/mm (neutrophils, 78%), hemoglobin levels of 5.7 g / dl, and platelet counts of 46,000/mm. a coagulation test showed a prothrombin time (pt) of 22.8 seconds (33.8%, inr 2.04) and an activated partial thromboplastin time of 58.7 seconds. iu / l, total bilirubin / direct bilirubin 3.21/1.18 mg / dl, total protein / albumin 4.3/2.29 g / dl, creatine phosphokinase / lactate dehydrogenase 281/2,358 iu / l, blood urea nitrogen / creatinine 14.0/0.96 mg / dl, sodium / potassium 152/3.9 mmol / l. serum osmolarity was 349 mosm / kg, lactic acid was 119.5 mg / dl (1 - 13 mg / dl) and highly sensitive c - reactive protein (hscrp) was 29.34 mg / l. arterial blood gas analysis under 4 l of oxygen via nasal cannula revealed a ph of 7.19, paco2 of 15.6 mmhg, pao2 of 109.5 mmhg, hco3 of 5.9 mmol / l, and oxygen saturation of 97.3%, which represented metabolic acidosis with an anion gap of 35.1. chest x - ray was normal but, abdominal x - ray showed moderate ascites and paralytic ileus (fig. instead, we inserted a sengstaken blakemore (sb) tube. on the second hospital day (hd), the ascites fluid analysis revealed wbc counts of 5,800/mm (neutrophils, 93%), albumin levels of 0.43 g / dl and the serum - ascites albumin gradient of 2.24, which agreed with sbp. we initiated cefotaxime (6 g / day) and metronidazole (1,500 mg / day) and continued early goal - directed therapy (egdt) for sepsis. on the third hd, the patient was intubated and underwent mechanical ventilation. based on the follow - up chest x - ray (fig. 1b) and clinical course, we assessed the patient having the respiratory failure which was combined with pulmonary edema due to massive hydration and transfusion for gastrointestinal bleeding and aspiration related to hematemesis. on the third hd, the blood culture revealed growth of gram - positive cocci and gram - negative bacilli. we added gentamicin (160 mg / day) because we could not exclude the possibility of infective endocarditis. using the microscan system (siemens, inc., renton, wa, usa), shewanella spp. and s. mitis were isolated from both peripheral and central blood (4 of 4 bottles) and shewanella spp. and e. coli were isolated from ascites. s. mitis was susceptible to penicillin and e. coli was susceptible to all of the antibiotics including ampicillin, cefotaxime, levofloxacin and gentamicin. the shewanella isolate was sub - cultured on macconkey agar at 37, and growth was found after 16 hours of incubation. based on the api 20ne kit (biomrieux inc., marcy - l'etoile, france), the organism was identified as s. putrefaciens with 99.9% certainty. we performed additional biochemical tests, as well as 16s rrna pcr with sequencing analysis because it is known that an automatic bacterial culture system and biochemical identification system, such as id32e, id32gn, or api 20e could not distinguish s. algae from s. putrefaciens. the isolate was incubated on sheep blood agar and showed growth at 42 (but not 4) and on nutrient agar containing 6.5% nacl, which was compatible with s. algae. 16s rrna gene sequencing analysis was performed with the following primers : hda1 forward primer (5'-actcctacgggaggcagcagt-3 ') and hda2 reverse primer (5'-gtattaccgcggctgctggca-3 '). the isolate gene sequence showed 100% concordance with the s. algae strain qc39 (genbank accession number : jn384129). s. mitis was susceptible to penicillin, and s. algae was susceptible to piperacillin, cefotaxime, ceftazidime, cefepime, carbapenems, aminoglycosides and quinolones. a transthoracic echocardiogram showed no evidence of vegetation. because the patient had persistent fever, leukocytosis, and elevated hscrp with progressing pneumonia, we changed the antibiotics to piperacillin / tazobactam (12/1.5 g / day) and amikacin (500 mg / day) on the 9 hd, which could reinforce the antimicrobial activity for nosocomial pathogens such as pseudomonas. after then, fever subsided and clinical condition was improved with normalization of wbc and hscrp levels. follow - up culture of ascites on the 8 hd and blood on the 13 hd, showed no bacterial pathogens. however, hepatic failure was progressing with total bilirubin up to 30.84 md / dl and prolonged pt to 34.3 seconds (21.8%). hepatorenal syndrome was suspected and planned dialysis was proposed, but his family refused further treatment. a patient with alcoholic lc suffered sbp with bacteremia caused by s. algae, though he had no history of previous exposure to seawater and raw seafood, nor ulcerations on the extremities. the patient showed a fatal clinical course accompanied with co - infection by s. mitis and e. coli. as s. algae is known to be more virulent than s. putrefaciens and these two species seem to exhibit different pathogenicities in humans, correct identification is important. however, automatic bacterial identification systems fail to differentiate between s. algae and s. putrefaciens because the s. algae is not included in the databases of these systems. we performed species identification using the api 20ne kit, which resulted in s. putrefaciens with 99.9% certainty. however, we could ultimately confirm the organism as s. algae by 16s rrna pcr and gene sequence analysis., initially known as achromobacter putrefaciens, was reclassified under the genus pseudomonas with the name of pseudomonas putrefaciens in 1941. during the next three decades,, further phylogenetic studies resulted in a reclassification of these organisms into the family vibrionaceae, and the description of a novel genus, shewanella, named after james shewan in honor of his work in marine microbiology. in the early 1990s, it was reclassified as a new species, s. alga and s. putrefaciens, although dna homology between the two species was less than 10%. in 1997, the name of this new species was corrected to s. algae. recent results of 16s rrna gene sequence analyses led to a proposal for a new family, shewanellaceae. are found throughout the environment and human infection is rare but reports are increasing [1, 5 ]. there are several reports regarding hepatobiliary infection, spondylodiscitis, infective endocarditis and severe systemic infection such as sepsis [2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ]. in korea, three cases of systemic infection caused by s. algae and one case of endophthalmitis (which developed after trauma) have been reported [3, 6, 7, 8 ]. among the three cases of s. algae systemic infection, two were skin and soft tissue infections (ssti) with bacteremia. one patient was diagnosed with end - stage renal disease receiving hemodialysis and another was alcoholic lc. the other case was a spinal epidural abscess in a patient who had undergone distal pancreatectomy with cholecystectomy for an intraductal papillary mucinous tumor. to our knowledge, this is the first case of sbp with shewanella bacteremia in korea, particularly accompanied by polymicrobial infection. risk factors for shewanella human infection include hepatobiliary disease, malignancies, severe heart failure, renal failure, peripheral vascular diseases and chronic ulcerations on the lower extremities, poor hygiene, and low socioeconomic status [3, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ]. exposure to seawater or ingestion of raw seafood might be a predisposing factor of shewanella infection. we also reviewed global studies and found a total of 21 cases with bacteremia by shewanella spp.. among these, in only 10 cases, we could identify the history of exposure to seawater. six out of 10 (60%) were previously exposed to seawater or raw seafood, and those cases were presented as bacteremia with ssti. four cases, which had no history of exposure to seawater, were presented as primary bacteremia (n=2), ssti (n=1) and sbp (this case). in particular, hepatobiliary disease has been proposed as a risk factor for poor outcome [5, 9, 10, 11 ]. fifteen out of 21 (71.4%) developed in patients with hepatobiliary disease [5, 8, 9, 10, 11, 12, 13, 14, 15 ]. the mortality rate of shewanella bacteremia was higher in patients with hepatobiliary disease, at 53.3% (8/15) compared to 16.7% (1/6) in cases without hepatobiliary diseases. are usually susceptible to common antibiotics, including quinolones, carbapenems, aminoglycosides and erythromycin. susceptibility to ampicillin and cephalosporins is variable, with more isolates being susceptible to third- and fourth- than first- and second - generation cephalosporins, but resistant to penicillin. there is a report that 68% of s. putrefaciens are resistant to imipenem ; they recommended a combination of amikacin or gentamicin as an empirical therapy. in addition, the rapid emergence of resistance during imipenem treatment which was initially susceptible, was reported. however, in the majority of community - onset shewanella infections, resistance is not an issue because the clinical isolates are susceptible to commonly used empirical antibiotics [9, 12 ]. further studies of susceptibility patterns according to the geographical region and antibiotics commonly selected for empirical therapy are required. in our case, culture results for blood and ascites on admission showed polymicrobial infection with s. mitis, e. coli and s. algae. among these isolates, s. algae was identified from all four blood bottles (two aerobic and two anaerobic) and from the ascites, with clinical manifestations compatible with sbp. therefore, s. algae was considered to be the major pathogen of peritonitis and bacteremia. however, s. mitis and e. coli are also well known significant human pathogens that can cause bacteremia and sbp. we thought these organisms also played as significant pathogens and affected the outcome of this patient. in addition, there were several reasons for the poor outcome despite early administration of susceptible antibiotics and recovery from bacteremia. first, the patient suffered from alcoholic lc with child - pugh class c, which is known to be a predictive factor for death. second, this disease was accompanied by polymicrobial infection, which could be associated with a poorer outcome than monomicrobial infections. in conclusion, shewanella infection can occur without exposure to seawater nor leg ulcers in the immunocompromised patients and causes not only ssti but also severe systemic infections, especially in patients with hepatobiliary diseases. here, we report a fatal case of sbp with severe sepsis caused by s. algae, accompanied by s. mitis and e. coli coinfection. the patient died from hepatorenal syndrome and renal failure, presenting with progressive metabolic acidosis, despite early and appropriate antimicrobial therapy in combination. although it is an uncommon cause of disease in humans, we should be aware of the pathogenicity of this emerging bacterium.	human infection caused by shewanella algae is rare, which usually occurred after direct contact with seawater or ingestion of raw seafood in the immunocompromised host. there have been anecdotal reports about shewanella infections in human, but their pathogenic role and microbiologic data are limited. here, we report a fatal case of spontaneous bacterial peritonitis with bacteremia due to s. algae in a 57-year - old male with liver cirrhosis who had no history of exposure to seawater or raw seafood. polymicrobial infection with streptococcus mitis and escherichia coli was combined and the patient died in spite of early appropriate antimicrobial therapy and early goal - directed therapy for sepsis.
pediatric dentistry deals with consequences and adverse effects of treatment more than any other branch of dentistry in order to minimize harm the children. one of the most frequent dental problems is the lack of children 's cooperation which is mostly associated with their anxiety. pediatric dentistry has provided several pharmaceutical and nonpharmaceutical methods for controlling children 's behavior ; however, nonpharmaceutical and conscious sedative methods do not work for extremely uncooperative children, and general anesthesia is the best option for such children. dental treatment of patients requiring general anesthesia is often completed quickly, and patients may be discharged within a day ; that is why they should have a fast and safe recovery after surgery. thus, the duration of anesthesia and its complications are very important in the recovery room. agitation is one of the important issues that occur in the process of children 's recovery which has no clear reason. it depends on factors such as age, type of surgery, duration of anesthesia, pharmaceutical interference, pain, type of anesthetic agent, preoperative anxiety, early recovery, and their mood and temper. in addition to the type of treatment and underlying diseases, the anesthetic agent affects the time and complications of recovery ; therefore, it should be chosen carefully based on the patient 's conditions. safety, high effectiveness rate, fast recovery, and low complication rate are the significant characteristics of a proper anesthetic agent. propofol is a short - acting intravenous anesthetic agent which acts by increasing the function of g - aminobutyric acid type a receptor. its advantage is the quick effect, and its complications can be the pain during injection, cough, convulsion, seizure, hypotension, bradycardia, tachycardia, restlessness or agitation, nausea, and vomiting. isoflurane is one of the most common inhalant anesthetic agents used to maintain a deep anesthesia, and its required dose decreases with the patient 's age. hence, it should be used carefully and monitored continuously. of its complications are hypotension, tachycardia, and delayed recovery. bispectoral index (bis) is a noninvasive marker of the depth of anesthesia based on electroencephalography assessment. based on the depth of anesthesia, bis is scaled from 0 to 100. in conscious patients, the score is between 90 and 100 while it is zero for the inactivity of the cortical part of the brain. in order to maintain an individual in general anesthesia bis monitoring is a simple way to prevent unwanted increases in the concentration of the anesthetic agent, which leads to fast effect and short recovery. considering the increasing need for general anesthesia in pediatric dentistry and complications of various anesthetic agents, this study was conducted to determine the relation between the values ofbis during dental treatment and recovery conditions in children receiving either of two regimes of elective anesthesia with propofol and isoflurane. the null hypothesis was : there is no significant difference between bis scores of individuals who receive propofol and those who receive isoflurane. this study was approved under irct2013032212848n1 by system for clinical trials. in this single - blind clinical trial, a total of 57-healthy children american society of anesthesiologists i with age range of 4 to 7 years who had been referred for dental treatment (pulpotomy, stainless steel crown and extraction) under general anesthesia and needed some anesthesia time between 60 and 90 min were selected by convenience sampling, after obtaining written consents from their parents to enter the study. patients who had been exposed to sedative or analgesic medicines before surgery and seven patients whose time schedule did not match with that of the study were excluded. in this study, no medical complications were observed during the anesthesia administration. the patients were given codes, randomly assigned to two similar groups and their data were evaluated confidentially by a single - blind method. in both groups, the patients were intubated, and the anesthesia was induced by inhalation, using a mixture of oxygen, nitrous oxide, and isoflurane (oretim yeri ; minrad, inc.). after each patient had started sleeping, venipuncture and pure oxygen ventilation the anesthesia was preserved by a mixture of oxygen (50%), nitrous oxide (50%), and isoflurane (1%). in the second group, the anesthesia was preserved by a mixture of oxygen (50%), nitrous oxide (50%), and propofol (corden pharm spa, italy) intravenously at a dose of 100 ng / kg / min. a bis sensor (aspect medical system, usa) specially designed for children pulse oximetry and heart monitoring were used to measure vital signs and cardiac rhythm during anesthesia. their vital signs and bis score were recorded during operation every 10 min. in the end, when the anesthesia terminated, a member of the research team would move the patient to the recovery room and keep them under observation by a standard single - blind method. in the recovery room, each patient was evaluated for agitation every 10 min for 60 min using the pediatric anesthesia emergence delirium scale(from bajwa. having achieved the discharge requirements based on postanesthesia discharge scale [appendix 2 ], each patient would be discharged. the collected data were analyzed by repeated measure anova and t - tests at the significance level of = 0.05 using spss (version 18.0, chicago, usa) this study investigated 57 children with a mean age of 5.29, who required general anesthesia for dental procedures. the mean age of the children for whom propofol and isoflurane were used to preserve their deep anesthesia was 5.32 1.01 and 5.26 0.98, respectively. the independent t - test showed no statistically significant difference between the two groups (p = 0.82). the normality of data distribution in both groups was evaluated using kolmogorov - smirnov test, the results of which showed the normal distribution of data in both groups. the mean time of anesthesia was 78.44 26.9 and 79.64 30.5 min in isoflurane and propofol groups, respectively. the results of the independent t - test showed that the difference between the isoflurane and propofol groups was not statistically significant (p = 0.87). the mean discharge time for the propofol and isoflurane groups were 108.9 23.1 and 128.4 31.2, respectively, for which the independent t - test showed a statistically significant difference (p = 0.01). table 1 and figure 1 show the mean bis scores of the subjects in the two groups from 1 to 90 min of surgery. mean bis scores of the subjects in the two groups from min 1 to 90 of surgery mean bispectoral index scores of the subjects in the two groups from 1 to 90 min of surgery. the results of the repeated measure anova test showed that the difference between bis scores in each group at different measuring times was significant (p 0.05). pearson correlation coefficient showed that regardless of type of anesthetic agent, there was a negative correlation between age and bis scores (r = 0.64, p 0.500, p 0.250, p 0.380, p 0.325, p table 2 and figure 2 show the mean agitation score in both groups of the study during recovery. mean agitation scores in both groups of the study during recovery mean agitation scores in both groups of the study during recovery. repeated measures anova tests showed that the differences between agitation scores at the times of measurement were statistically significant (p 0.05). during the recovery period, the researchers had to use analgesics in only 5 cases (8.8%), of whom 2 patients (6.9%) were in the isoflurane and 3 (10.7%) in the propofol groups. in this study, the null hypothesis was not rejected ; no significant difference was observed between the bis scores of individuals who had received propofol and those who had received isoflurane (p > 0.05). mehmandoost and naghibi compared the effectiveness of isoflurane and propofol on consciousness levels of women undergoing a cesarean. the results of their study showed no significant difference between the two agents in terms of patients consciousness level, bis, and hemodynamic variables. from 90 patients in their study, only 7 patients showed recovery complications (8.9% and 6.7% in propofol and isoflurane groups, respectively) ; and no significant differences were observed between the two anesthetic agents. the results of the present study are consistent with those of mehmandoost and naghibi where there was no statistically significant difference between propofol and isoflurane in maintaining deep anesthesia ; both agents provided similar anesthesia depth. the results of their study showed that the need for anesthetic agents had a negative correlation with age but had no effect on bis scores. they concluded that bis could be used in communities with a wide age range for determining the depth of anesthesia. the results of the present study revealed a statistically significant (p < 0.001) correlation between bis scores and the patients age. they also indicated that bis scores have a negative correlation with age and, consequently, with the dose of anesthetic agent needed for maintaining deep anesthesia. in both studies, the older patients needed a less anesthetic agent dose, though the changes in bis scores varied by age in both studies. this variation could be due to the difference in the number of subjects in the sample groups of the two studies. the participants in the study of katoh. were between 18 and 85 years of age ; whereas, those in our study were 4 - 7-year - old. hence, it is not possible to compare the two studies ; further studies need to be conducted under similar conditions to verify the results. in a study, kaviani and karamzadeh evaluated the effect of two intravenous and inhalation anesthesia methods on the rate of postoperative agitation in children. their results showed no difference in the degree of agitation between their groups but indicated a negative correlation between the rate of agitation and age. the results of the present study showed that the agitation scores were higher in the propofol group, and the difference between the two anesthetic agents was statistically significant at the 10 min. it can thus be inferred that the type of anesthetic agent in this study had no effect on the rate of postoperative agitation. regardless of the type of anesthetic agent in the present study, a significant correlation was observed between age and agitation rate ; the younger patients showed higher agitation scores. thus, basedf on the results of this study as well as those of kaviani and karamzadeh ; age is an important factor in the outbreak of postoperative agitation. the incidence of recovery complications in various studies has been variable. in a study, investigated anesthesia complications in 6914 patients in 4 canadian hospitals and reported the incidence of complications at 8%, whereas hines. in a study of 18,473 patients in a training hospital noted incidences of postanesthesia recovery complications at 23.7%. in a study on 90 patients in 1987, edelist investigated the effects of two, propofol and thiopentone, anesthetic agents. his results revealed that, after regaining consciousness, recovery complications were detected in 38% and 47% of patients who had received propofol and thiopentone, respectively. such factors such as duration of anesthesia, anesthetic technique ; and even the organ which is being treated affect the incidence of recovery complications. these factors can justify the reasons for major differences between complications detected in different studies. in the present study, the recovery complications were detected in 31% and 39.3% of individuals who received isoflurane and propofol, respectively. the percentage of complications in the propofol group was the same as edelist, s study. the difference between the two used anesthetic agents was statistically significant ; nevertheless, the isoflurane group showed fewer complications in maintaining the depth of anesthesia than the propofol group. the results of the present study showed a significant correlation between age, agitation scores, bis scores, duration of anesthesia, and the incidence of recovery complications. the younger patients showed more complications and had higher agitation, bis scores, longer duration of anesthesia, and greater probability of complication occurrence. it is likely that younger children have more agitation after operations and are less able to endure the anesthetic agent. in addition the longer duration of anesthesia indicates the longer effect of the anesthetic agent, and the probability of a proportional increase in the anesthetic agent 's side effects, which may entail recovery complications. in addition, we recommend similar studies using other methods of measuring agitation to compare methods and to ensure their accuracy. both propofol and isoflurane produce similar depth of anesthesia, but isoflurane produces fewer recovery complications.in older patients and those with higher bis and agitation scores, the need for anesthetic agents decreases.the higher the agitation and bis scores and the longer the duration of anesthesia, the higher the probability of recovery complications occurrence. both propofol and isoflurane produce similar depth of anesthesia, but isoflurane produces fewer recovery complications. in older patients and those with higher bis and agitation scores, the need for anesthetic agents decreases. the higher the agitation and bis scores and the longer the duration of anesthesia, the higher the probability of recovery complications occurrence. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article.	background : this study was planned to determine the relationship between bispectoral index (bis) during dental treatment and recovery conditions in children undergoing two regimes of anesthesia of propofol and isoflurane.materials and methods : in this single - blind clinical trial study, 57 4 - 7-year - old healthy children who had been referred for dental treatment under general anesthesia between 60 and 90 min were selected by convenience sampling and assigned to two groups, after obtaining their parents written consent. the anesthesia was induced by inhalation. for the first group, the anesthesia was preserved by a mixture of oxygen (50%), nitrous oxide (50%), and isoflurane (1%). for the second group, the anesthesia was preserved by a mixture of oxygen (50%), nitrous oxide (50%), and propofol was administered intravenously at a dose of 100 ng / kg / min. the patients vital signs, bis, and agitation scores were recorded every 10 min. the data were analyzed by repeated measure anova and t - tests at a significance level of = 0.05 using spss version 20.results:the results of independent t - test for anesthesia time showed no statistically significant difference between isoflurane and propofol (p = 0.87). controlling age, the bis difference between the two anesthetic agents was not significant (p > 0.05) ; however, it was negatively correlated with the duration of anesthesia and the discharge time (p = 0.001, r = -0.308) and (p < 0.001, r = -0.55).conclusion : the same depth of anesthesia is produced by propofol and isoflurane, but lower recovery complications from anesthesia are observed with isoflurane.
the study was conducted at the family medicine clinics of the american university of beirut in lebanon, a tertiary academic medical center. the clinics serve a large population that includes the hospital employees and their dependents as well as community members. there are 43 physicians : 22 faculty members and 21 residents. on average, the faculty physician has three half - day clinics per week, and the resident has one half - day clinic per week. the clinics use a home - grown emr and are almost paperless ; yet, there is neither a patient portal nor a structured secure messaging system between patients and physicians. any email use between physicians and patients has been done through their own personal emails and was based on voluntary and individual motivation without any guidance or training. this is a cross - sectional paper - based survey of all the physicians at the family medicine clinics and 500 adult patients (> 18 years) who attended the clinic between march and may 2012. estimating that 50% of the patients visiting the clinic use email communication with their physicians with a margin of error of 5% and a confidence interval of 85%, the minimum number of patients required is 400. assuming that a quarter of respondents would submit incomplete questionnaires, the sample size was set at 500. patients are expected to pass by the triage nurse for assessment before meeting the physician. in the nursing triage station patients were selected by systemic sampling on alternate morning and afternoon sessions weekly to ensure the recruitment of patients of all the physicians. patients who were illiterate or unable to read or write due to a medical condition were excluded from the study, as these patients could not personally use email. verbal informed consent was obtained and patients were handed a questionnaire to fill out on their own. the physicians questionnaires were distributed at their offices in the clinic to be filled anonymously. different questionnaires were developed for the patients and the physicians based on the literature review. early versions of both questionnaires were piloted and the patients versions were translated into arabic. for patients, collected data included demographics, health status, frequency of visits to the clinic, access and use of the internet, and willingness to pay for email communication. both patients and physicians were asked whether they had ever communicated with each other, whether they thought email is useful for communication and for what reasons, and whether there were any barriers to or concerns about email communication. percentages were used to measure the various demographics, practices, attitudes, and barriers toward the use of email communication between patients and physicians. chi - square test (or fisher s exact test if appropriate) was used to compare groups based on the independent variables. the dependent variable included willingness to use email to communicate with the physician, and the independent variables included sex, age, level of education, frequency of annual visits, accessibility to internet sites, hours spent on the internet daily, and use of email. the study was conducted at the family medicine clinics of the american university of beirut in lebanon, a tertiary academic medical center. the clinics serve a large population that includes the hospital employees and their dependents as well as community members. there are 43 physicians : 22 faculty members and 21 residents. on average, the faculty physician has three half - day clinics per week, and the resident has one half - day clinic per week. the clinics use a home - grown emr and are almost paperless ; yet, there is neither a patient portal nor a structured secure messaging system between patients and physicians. any email use between physicians and patients has been done through their own personal emails and was based on voluntary and individual motivation without any guidance or training. this is a cross - sectional paper - based survey of all the physicians at the family medicine clinics and 500 adult patients (> 18 years) who attended the clinic between march and may 2012. estimating that 50% of the patients visiting the clinic use email communication with their physicians with a margin of error of 5% and a confidence interval of 85%, the minimum number of patients required is 400. assuming that a quarter of respondents would submit incomplete questionnaires, the sample size was set at 500. patients are expected to pass by the triage nurse for assessment before meeting the physician. in the nursing triage station patients were selected by systemic sampling on alternate morning and afternoon sessions weekly to ensure the recruitment of patients of all the physicians. patients who were illiterate or unable to read or write due to a medical condition were excluded from the study, as these patients could not personally use email. verbal informed consent was obtained and patients were handed a questionnaire to fill out on their own. the physicians questionnaires were distributed at their offices in the clinic to be filled anonymously. different questionnaires were developed for the patients and the physicians based on the literature review. early versions of both questionnaires were piloted and the patients versions were translated into arabic. for patients, collected data included demographics, health status, frequency of visits to the clinic, access and use of the internet, and willingness to pay for email communication. for physicians, both patients and physicians were asked whether they had ever communicated with each other, whether they thought email is useful for communication and for what reasons, and whether there were any barriers to or concerns about email communication. percentages were used to measure the various demographics, practices, attitudes, and barriers toward the use of email communication between patients and physicians. chi - square test (or fisher s exact test if appropriate) was used to compare groups based on the independent variables. the dependent variable included willingness to use email to communicate with the physician, and the independent variables included sex, age, level of education, frequency of annual visits, accessibility to internet sites, hours spent on the internet daily, and use of email. patients were approached until the criterion of the total number of participants (500) was met. the main reasons given for the non - response were lack of time, lack of interest, or not feeling well. the vast majority of patients surveyed were email users, highly educated, and healthy (as reflected by the absence of chronic illness and less frequent doctor visits). most of the patients (87%) had internet access and two - thirds had access to internet at home. patients demographics by email use status numbers do not sum to 500 because of missing values., 67.4% told that they would like to communicate with their physicians through email and 71.6% agreed that it would be useful. two - thirds and half of the patients considered email communication between physicians and patients safe and confidential, respectively. yet, only 19.2% of patients communicate with their physicians through email. almost half of the surveyed patients were not aware of the possibility of communicating with their physician using email. of those who were aware the majority of patients (69.6%) obtained the physician s email address from the university directory ; 15.7% had asked their physicians for their emails. the barriers to email use by patients included the delay to get a response (11 participants), the suboptimal quality of care offered through an email consultation (18 participants), and the lack of face - to - face interaction with their physicians (15 participants). one participant was concerned that patients would abuse this form of communication, whereas another participant thought that it was not part of lebanese culture. however, patients who communicated through email with their physicians were more likely than non - communicators to consider email communication as safe (p=0.000), confidential (p=0.013), and worth the cost of the consultation (p=0.004). using univariate analysis, it was found that males, younger patients, frequent clinic visitors, and those who spend less than half an hour daily on the internet were less likely to use emails to communicate (table 2). using binary logistic regression, two variables were found to be statistically significant predictors of participants who would like to communicate with their physicians through email : participant email use and daily hours of internet use. patients who use email as compared with non - users (odds ratio (or)=4.25 ; 95% confidence interval (ci)=1.8010.05, p=0.001) and those who used the internet for 2 h or more daily as compared with those who used it less than half an hour (or=3.01 ; 95% ci=1.326.87, p=0.008) were more likely to like to communicate through email with their physicians. predictive variables of patients who like to communicate with physicians through email fischer s exact test. a total of 39 physicians returned completed questionnaires with a total response rate of 91% : 86% (18/21) for residents and 95% (21/22) for the faculty. the most frequent duration of hours spent on email use daily was between 15 and 30 min, and one quarter of participants had internet access mainly at home, at work, and in cell phone. demographics of the family physicians (n=39) using a likert scale with grading from never, only 5.1% of physicians reported that they often send or receive emails to or from their patients, and a quarter reported that they never communicated with their patients through email. there was only a statistically significant relationship between physicians who send or receive emails to or from their patients and increasing age (p=0.003). also, 87.5% of physicians aged 3650, 62.5% of those aged 5165, and 17.4% of those aged 2535 reported some use of email with patients. the vast majority of physicians surveyed thought that email would be useful for communication with patients for health - related issues. physicians were more likely than patients to find email useful for communication with patients (87.2% vs. 78.3%, p=0.039). however, physicians and patients did not agree on the categories of services to be communicated through email (see table 4). in descending order, the preference of physicians regarding the usefulness of email communication with patients in certain categories of services was as follows : laboratory results (88.2%), medical questions / advice (70.6%), follow - up on certain medical conditions (64.7%), clarification of treatment plans (64.7%), providing health educational materials (52.9%), and requesting prescription refills (29.4%). physicians and patients attitudes toward usefulness of email communication among themselves and preferences for specific uses for the email communication pearson s chi - square test. significants of p value < 0.05. however, 82.1% of physician surveyed had concerns about using email for medical consultation. almost half of the physicians reported lack of security, lack of time, lack of reimbursement, and medicolegal issues concerning the use of email for medical consultation. further concerns listed by physicians included suboptimal quality of care (11 physicians), patients abuse of this form of communication (3 physicians), and administrative concerns such as lack of documentation of the email (1 physician). patients were approached until the criterion of the total number of participants (500) was met. the main reasons given for the non - response were lack of time, lack of interest, or not feeling well. the vast majority of patients surveyed were email users, highly educated, and healthy (as reflected by the absence of chronic illness and less frequent doctor visits). most of the patients (87%) had internet access and two - thirds had access to internet at home. patients demographics by email use status numbers do not sum to 500 because of missing values., 67.4% told that they would like to communicate with their physicians through email and 71.6% agreed that it would be useful. two - thirds and half of the patients considered email communication between physicians and patients safe and confidential, respectively. yet, only 19.2% of patients communicate with their physicians through email. almost half of the surveyed patients were not aware of the possibility of communicating with their physician using email. of those who were aware the majority of patients (69.6%) obtained the physician s email address from the university directory ; 15.7% had asked their physicians for their emails. the barriers to email use by patients included the delay to get a response (11 participants), the suboptimal quality of care offered through an email consultation (18 participants), and the lack of face - to - face interaction with their physicians (15 participants). one participant was concerned that patients would abuse this form of communication, whereas another participant thought that it was not part of lebanese culture. however, patients who communicated through email with their physicians were more likely than non - communicators to consider email communication as safe (p=0.000), confidential (p=0.013), and worth the cost of the consultation (p=0.004). using univariate analysis, it was found that males, younger patients, frequent clinic visitors, and those who spend less than half an hour daily on the internet were less likely to use emails to communicate (table 2). using binary logistic regression, two variables were found to be statistically significant predictors of participants who would like to communicate with their physicians through email : participant email use and daily hours of internet use. patients who use email as compared with non - users (odds ratio (or)=4.25 ; 95% confidence interval (ci)=1.8010.05, p=0.001) and those who used the internet for 2 h or more daily as compared with those who used it less than half an hour (or=3.01 ; 95% ci=1.326.87, p=0.008) were more likely to like to communicate through email with their physicians. predictive variables of patients who like to communicate with physicians through email fischer s exact test. a total of 39 physicians returned completed questionnaires with a total response rate of 91% : 86% (18/21) for residents and 95% (21/22) for the faculty. the most frequent duration of hours spent on email use daily was between 15 and 30 min, and one quarter of participants had internet access mainly at home, at work, and in cell phone. demographics of the family physicians (n=39) using a likert scale with grading from never, only 5.1% of physicians reported that they often send or receive emails to or from their patients, and a quarter reported that they never communicated with their patients through email. there was only a statistically significant relationship between physicians who send or receive emails to or from their patients and increasing age (p=0.003). also, 87.5% of physicians aged 3650, 62.5% of those aged 5165, and 17.4% of those aged 2535 reported some use of email with patients. the vast majority of physicians surveyed thought that email would be useful for communication with patients for health - related issues. physicians were more likely than patients to find email useful for communication with patients (87.2% vs. 78.3%, p=0.039). however, physicians and patients did not agree on the categories of services to be communicated through email (see table 4). in descending order, the preference of physicians regarding the usefulness of email communication with patients in certain categories of services was as follows : laboratory results (88.2%), medical questions / advice (70.6%), follow - up on certain medical conditions (64.7%), clarification of treatment plans (64.7%), providing health educational materials (52.9%), and requesting prescription refills (29.4%). physicians and patients attitudes toward usefulness of email communication among themselves and preferences for specific uses for the email communication pearson s chi - square test. significants of p value < 0.05. however, 82.1% of physician surveyed had concerns about using email for medical consultation. almost half of the physicians reported lack of security, lack of time, lack of reimbursement, and medicolegal issues concerning the use of email for medical consultation. further concerns listed by physicians included suboptimal quality of care (11 physicians), patients abuse of this form of communication (3 physicians), and administrative concerns such as lack of documentation of the email (1 physician). this study shows that email communication between patients and physicians using their personal emails exists in developing countries, although it is currently infrequent. both physicians and patients showed interest in this mode of communication and considered it beneficial. yet, physicians are still reluctant to initiate email communication and offer their emails to the patients. moreover, patients and physicians expressed non - aligned preferences for the categories of services suitable for email communication. despite expressing their willingness, a small percentage of physicians (5.1%) frequently used email communication with patients, and only 19.2% of patients have ever communicated with their physicians through email. these findings support previous research in developed countries showing similarly infrequent email communication between patients and physicians, such as the usa, european union, australia and uk (5, 12, 18, 19). the barriers stated by physicians in this study were similar to that found by previous studies (2024) : the lack of security, increased workload, lack of time, lack of reimbursement, and medicolegal issues. in this study, patients who found email useful for communication were females, highly educated, younger, healthier, and had fewer annual visits. these are similar to the characteristics of patients who used email communication with their physicians in previous studies (12, 18). thus, email communication between physicians and patients is limited to a small percentage of patients who most probably benefit the least from such communication. this could contribute to the lack of scientific evidence found by five recent cochrane systematic reviews on the effect of email use on health promotion and disease prevention, appointments, and test result management (1317). in this study, this is common and accepted in developing countries, where emrs are scarce and there are no bounding legal laws for confidentiality and privacy similar to health insurance portability and accountability act of 1996 (hipaa) in the usa (2527). abiding by hipaa, it is expected that proper encryption of email content is assured or secure structured messaging is used to avoid breach of confidentiality. interestingly, patients considered email use as safe and confidential, especially those who use email communication with their physicians. physicians should be careful about the topics discussed in email communication, for example, sensitive issues such as sexuality, psychiatric illnesses, and sexually transmitted diseases. have shown that 21% of the users used email for urgent matters such as chest pain and suicidality (11). however, this is in contrast to the large body of published literature, showing that most email inquiries from patients were for non - acute issues, such as health - related questions, medical update, administrative issues, and lab test results (5, 2831). one of the strengths of this study is the simultaneous survey of both physicians and patients in the same health care setting, where cultural and clinical processes are common to both patients and physicians. the study is unique in exploring the use of email in countries that lack the legislation and clear standards for email communication between physicians and patients. another limitation of the study is the inability to generalize the findings to solo practitioners because our setting was that of a managed care or disciplines other than family medicine. although the general use of email communication was infrequent, a proportion of patients and physicians were still interested in this form of communication despite all the barriers. studies have shown that patients are more motivated to use online communication when their physicians are motivated (32). yet, physicians provided their email to only 18% of patients who were aware of the possibility of email communication. therefore, there is still room of improvement in the utilization of email communication among this subgroup of interested patients and physicians through better advertisement. for example, motivated physicians should be encouraged to advertise email communication and provide their email addresses on business cards, prescription forms, and brochures in the waiting room. to ensure that email communication is used effectively, physicians should establish their own policies and educate their patients about proper use, especially in the context of urgent medical matters. the american medical informatics association and the american medical association have published guidelines for physician patients and physicians have different perspectives on the value of different services suitable for email communication. physicians were more interested in sending information about laboratory results and clarification of treatment plans, whereas patients appreciated administrative requests such as prescription refills. have shown that while patients preferred email communication for medication refills (34), they appreciated a two - way communication on specific issues. for example, patients preferred using the telephone or direct personal communication when discussing a health issue (35) or getting treatment instructions (34), and they expressed their concerns about understanding the laboratory results communicated through email (36). this poses the question of whether email communication should be restricted to administrative requests and exclude medical care. as such, email communication for administrative issues this study shows that email communication between patients and physicians using their personal emails exists in developing countries, although it is currently infrequent. both physicians and patients showed interest in this mode of communication and considered it beneficial. yet, physicians are still reluctant to initiate email communication and offer their emails to the patients. moreover, patients and physicians expressed non - aligned preferences for the categories of services suitable for email communication. despite expressing their willingness, a small percentage of physicians (5.1%) frequently used email communication with patients, and only 19.2% of patients have ever communicated with their physicians through email. these findings support previous research in developed countries showing similarly infrequent email communication between patients and physicians, such as the usa, european union, australia and uk (5, 12, 18, 19). the barriers stated by physicians in this study were similar to that found by previous studies (2024) : the lack of security, increased workload, lack of time, lack of reimbursement, and medicolegal issues. in this study, patients who found email useful for communication were females, highly educated, younger, healthier, and had fewer annual visits. these are similar to the characteristics of patients who used email communication with their physicians in previous studies (12, 18). thus, email communication between physicians and patients is limited to a small percentage of patients who most probably benefit the least from such communication. this could contribute to the lack of scientific evidence found by five recent cochrane systematic reviews on the effect of email use on health promotion and disease prevention, appointments, and test result management (1317). this is common and accepted in developing countries, where emrs are scarce and there are no bounding legal laws for confidentiality and privacy similar to health insurance portability and accountability act of 1996 (hipaa) in the usa (2527). abiding by hipaa, it is expected that proper encryption of email content is assured or secure structured messaging is used to avoid breach of confidentiality. interestingly, patients considered email use as safe and confidential, especially those who use email communication with their physicians. physicians should be careful about the topics discussed in email communication, for example, sensitive issues such as sexuality, psychiatric illnesses, and sexually transmitted diseases. similarly, houston. have shown that 21% of the users used email for urgent matters such as chest pain and suicidality (11). however, this is in contrast to the large body of published literature, showing that most email inquiries from patients were for non - acute issues, such as health - related questions, medical update, administrative issues, and lab test results (5, 2831). one of the strengths of this study is the simultaneous survey of both physicians and patients in the same health care setting, where cultural and clinical processes are common to both patients and physicians. the study is unique in exploring the use of email in countries that lack the legislation and clear standards for email communication between physicians and patients. another limitation of the study is the inability to generalize the findings to solo practitioners because our setting was that of a managed care or disciplines other than family medicine. although the general use of email communication was infrequent, a proportion of patients and physicians were still interested in this form of communication despite all the barriers. studies have shown that patients are more motivated to use online communication when their physicians are motivated (32). yet, physicians provided their email to only 18% of patients who were aware of the possibility of email communication. therefore, there is still room of improvement in the utilization of email communication among this subgroup of interested patients and physicians through better advertisement. for example, motivated physicians should be encouraged to advertise email communication and provide their email addresses on business cards, prescription forms, and brochures in the waiting room. to ensure that email communication is used effectively, physicians should establish their own policies and educate their patients about proper use, especially in the context of urgent medical matters. the american medical informatics association and the american medical association have published guidelines for physician patients and physicians have different perspectives on the value of different services suitable for email communication. physicians were more interested in sending information about laboratory results and clarification of treatment plans, whereas patients appreciated administrative requests such as prescription refills. have shown that while patients preferred email communication for medication refills (34), they appreciated a two - way communication on specific issues. for example, patients preferred using the telephone or direct personal communication when discussing a health issue (35) or getting treatment instructions (34), and they expressed their concerns about understanding the laboratory results communicated through email (36). this poses the question of whether email communication should be restricted to administrative requests and exclude medical care. as such, email communication for administrative issues in an era of widespread use of the internet in health care, email communication between physicians and their patients is foreseeable, though patients and physicians have different perspectives of its use and importance. physicians are encouraged to establish appropriate personal policies for email communication with adequate announcement and a patient education plan. further rigorous research is needed to clarify the advantages and disadvantages of this form of communication, especially in developing countries. both authors contributed to the design of the study, collection of data and writing of the article. the authors have not received any funding or benefits from industry or elsewhere to conduct this study.	backgroundemail communication between physicians and patients could improve access to and delivery of health care. most of the literature studies about email communication between physicians and patients have been conducted in developing countries. therefore, this study aims to analyze the practices, attitudes, and barriers of both physicians and patients use of email within the same health care setting of a developing country.methodsa cross - sectional paper - based survey was conducted among 39 physicians and 500 patients at the family medicine clinics of the american university of beirut, a tertiary academic medical center.resultsmost of the surveyed patients and physicians reported that they would like to communicate through email and agreed that it is useful. however, only 19% of the patients have ever communicated with their physicians via email, and only 5.1% of physicians have often communicated with their patients via email. almost half of the patients surveyed were unaware of the possibility of this form of communication, and only 17% reported that their physician offered them his or her email address. in addition, physicians and patients did not agree on the services to be provided by email communication. for instance, almost half of the patients indicated consultation for an urgent medical matter as suitable for email communication.conclusionthe use of email communication in health care is still scarce. patients and physicians have different perspectives of its use and importance. further rigorous research is needed to clarify the advantages and disadvantages of this form of communication, especially in the developing world. interested physicians are encouraged to establish appropriate personal policies for email communication with adequate announcement and patient education plans.
fasting during ramadan is an islamic rule and, therefore, muslims fast a 29 - 30-day consecutive period per year. this islamic rule is excepted for patients and whom fasting may be harmful to them. ramadan is a month of islamic lunar calendar and, therefore, its duration varies in different seasons year to year. in fasting days, individuals do not eat anything from brightening to sunset. from sunset to brightening so, many studies have focused on the effect of ramadan fasting on metabolic changes and health outcomes in different groups of muslims population. studies reported that total cholesterol (tc), low - density lipoprotein (ldl), high - density lipoprotein (hdl) and blood glucose have been improved after ramadan compared to before ramadan among athletes. as smocking has been forbidden during fasting of ramadan, studies revealed a significant reduction in second - hand smoke levels in public places that might be related to mortality and morbidity. moreover, reported evidence illustrated that smoking is positively related to elevated tc and ldl, poor glycemic control and increased risk of type 2 diabetes prevalence, lower birth weight and increased short for gestational age rates, impaired function of dendritic cells and altered immune cell count. furthermore, a direct association between smoking and weight reduction was observed among older adults. conducted studies have assessed the impact of ramadan fasting on a different aspect of human metabolic and healthy such as an immune system, hormones secretion and gestation. the effect of ramadan on diseases (e.g., gastrointestinal diseases) has been also examined. although previous review studies have assessed the impact of ramadan on cardiovascular risk factors (i.e., body mass index [bmi ] and lipid profile), athlete performance, diabetes and transplantation, in this study we have appraised some on these review by focusing on limitations and also, we have reviewed more recently published study. moreover, several recent studies have been published in these regards (e.g., the impact of ramadan fasting on the immune system) that is not reviewed till now. in this article, we reviewed recently conducted studies in regarding the impact of ramadan fasting on weight, lipid profile, diabetes, gestation, immune system and kidney function. included papers were sorted by year of publication to determine the most recent review articles. therefore, we can choose the most recent studies and articles not reviewed till now regarding weight, lipid profile, diabetes, gestation, immune system and kidney function [table 1 ]. based on the results of a currently published meta - analysis included 21 articles, 531 men and 299 women, fasting during ramadan results in a moderate reduction in body weight in men but not in women (0.24, 95% confidence interval [ci ] = 0.36 to 0.12 ; p = 0.0001 and 0.04, 95% ci = 0.20 to 0.12 ; p = 0.62, respectively). the overall estimated effect size for both genders was also significant (0.17, 95% ci = 0.26 to 0.07 ; p = 0.001). moreover, within study heterogeneity was not significant (i = 0%, p = 0.82). a recent study reported that body weight, bmi and body fat had a significant reduction in 3 week of ramadan in comparison with 1 week before or after ramadan fasting (p 0.05). in overall, the results of foresaid study showed that ramadan fasting had no effect on intrauterine growth, pregnancy duration and anthropometrical measures of infants. similarly, another study conducted on 25 fasting (mean of fasting days : 23 6) and 27 nonfasting healthy women reported that there are no significant differences in femoral length, abdominal circumference, biparietal diameter, fetal weight, amount of amniotic fluid and fetal heart rate between two groups. the results of a prospective cohort study on 402 women (201 ramadan fasted and 201 nonfasted) demonstrated that time of delivery had no difference between fasted and nonfasted pregnant women and bmi is the most important factor which affect on time of delivery. moreover, the finding disclosed that ramadan fasted mothers had lower rate of cesarean delivery (28.4% in fasted and 39.3% in nonfasted women ; p = 0.027) and birth weight average (3094 467 g in fasted and 3202 473 g in nonfasted ; p = 0.024). researches regarding the effect of fasting during ramadan on offspring are not limited to childhood, and it is evaluated till adulthood. a recent population - based study compared the bmi and height of adults whose fetal period had been in ramadan with those whose fetal period had not been in ramadan. after adjusting for adulthood bmi, results showed that subjects with fetal period during ramadan were thinner and shorter than other muslims. similar differences were not observed between non - muslims who were not in the fetal period during ramadan and non - muslims who were in the fetal period during ramadan. animal studies reported that the expression of immunoglobulin a (iga) in the intestinal mucosa, monocyte killing, natural killer - cell activity and macrophage activity were increased during fasting. in a human study, 35 healthy men recruited and pre - ramadan igs concentrations and immune system activity were compared with post - ramadan. findings illustrated that although igg concentration was decreased in post - ramadan period in comparison to pre - ramadan, it was not out of the normal range. the reduction in concentration of salivary iga was also observed, but the lymphocyte number was elevated. another study conducted on 50 subjects reported that the concentration of interleukin 6 (il-6), il-1 and tumor necrosis factor- (tnf-), and the number of total leukocytes, granulocytes, lymphocytes and monocytes were significantly decreased during ramadan in comparison to pre - ramadan. based on the results of a currently published meta - analysis included 21 articles, 531 men and 299 women, fasting during ramadan results in a moderate reduction in body weight in men but not in women (0.24, 95% confidence interval [ci ] = 0.36 to 0.12 ; p = 0.0001 and 0.04, 95% ci = 0.20 to 0.12 ; p = 0.62, respectively). the overall estimated effect size for both genders was also significant (0.17, 95% ci = 0.26 to 0.07 ; p = 0.001). moreover, within study heterogeneity was not significant (i = 0%, p = 0.82). a recent study reported that body weight, bmi and body fat had a significant reduction in 3 week of ramadan in comparison with 1 week before or after ramadan fasting (p 0.05). in overall, the results of foresaid study showed that ramadan fasting had no effect on intrauterine growth, pregnancy duration and anthropometrical measures of infants. similarly, another study conducted on 25 fasting (mean of fasting days : 23 6) and 27 nonfasting healthy women reported that there are no significant differences in femoral length, abdominal circumference, biparietal diameter, fetal weight, amount of amniotic fluid and fetal heart rate between two groups. the results of a prospective cohort study on 402 women (201 ramadan fasted and 201 nonfasted) demonstrated that time of delivery had no difference between fasted and nonfasted pregnant women and bmi is the most important factor which affect on time of delivery. moreover, the finding disclosed that ramadan fasted mothers had lower rate of cesarean delivery (28.4% in fasted and 39.3% in nonfasted women ; p = 0.027) and birth weight average (3094 467 g in fasted and 3202 473 g in nonfasted ; p = 0.024). researches regarding the effect of fasting during ramadan on offspring are not limited to childhood, and it is evaluated till adulthood. a recent population - based study compared the bmi and height of adults whose fetal period had been in ramadan with those whose fetal period had not been in ramadan. after adjusting for adulthood bmi, results showed that subjects with fetal period during ramadan were thinner and shorter than other muslims. similar differences were not observed between non - muslims who were not in the fetal period during ramadan and non - muslims who were in the fetal period during ramadan. animal studies reported that the expression of immunoglobulin a (iga) in the intestinal mucosa, monocyte killing, natural killer - cell activity and macrophage activity were increased during fasting. in a human study, 35 healthy men recruited and pre - ramadan igs concentrations and immune system activity were compared with post - ramadan. findings illustrated that although igg concentration was decreased in post - ramadan period in comparison to pre - ramadan, it was not out of the normal range. the reduction in concentration of salivary iga was also observed, but the lymphocyte number was elevated. another study conducted on 50 subjects reported that the concentration of interleukin 6 (il-6), il-1 and tumor necrosis factor- (tnf-), and the number of total leukocytes, granulocytes, lymphocytes and monocytes were significantly decreased during ramadan in comparison to pre - ramadan. ramadan fasting has no impact on kidney function and urine component in healthy subjects but the effect of fasting on renal function should be assessed among nonhealthy individuals. one study conducted on 31 chronic kidney diseases patients evaluated the renal function at 3 times : 1-month before ramadan, during ramadan and 1-month after ramadan. in comparison to before ramadan period, estimated glomerular filtration rate (egfr) was improved, and proteinuria and urinary sodium were decreased during and after ramadan. the effect of ramadan fasting on kidney transplant patients has been assessed in recent studies. one study compared the post - ramadan measurements of renal function with pre - ramadan values. findings illustrated that creatinine, urea, uric acid, and sodium, potassium and hco3 content of urine had no significant difference between two periods. these results are approved by another study conducted on 43 fasters and 37 nonfasters kidney transplant recipients. the general conclusion of these two study demonstrated that fasting has not adverse effect on kidney transplant recipients. one study evaluated the effect of ramadan fasting on calculus formation among 57 men (37 calcium calculus formers). results showed that in comparison to nonfasting period, a reduction in calcium, phosphate, magnesium and urine volume was observed but the concentration of uric acid, citrate, phosphate, sodium, and potassium was increased. these findings do not support the hypothesis that ramadan fasting is a risk factor for calculus formation. a is that during ramadan, men should continue their usual outdoor activities while most muslim women are housekeeper and, therefore, their activity level is lower than men. in general, two possible reasons were suggested for weight reduction in ramadan : decrease in calorie intake and subsequently body fat reduction, and negative fluid balance. moreover, the correlation between the decrease of body weight and a decrease in meal frequency in people with type 2 diabetes was found, which leads to a decrease in calorie intake. although a statistical significant weight lost was observed during ramadan, foresaid reduction (170 g) is not physiologically valuable, because it is too small for a 1-month period. one study reported that the lost weight during ramadan was regained and reduced bmi was returned to pre - ramadan values. a possible reason of this finding may be that individuals would return to their pre - ramadan lifestyle in which dietary habits result in weight regain. it is suggested to evaluate the sustainability of the reduced weight in ramadan during the following months to see whether or not this reduced weight is maintained during the following months. the results of a meta - analysis were reported in weight and lipid profile section. it should be kept in mind that the design of conducted studies regarding fasting during ramadan is similar to before - after intervention studies except that the control of confounder is weaker in ramadan - related studies because they are categorized as observational study. hence, the results of this meta - analysis may be affected by confounders of included studies. moreover, the authors of this meta - analysis did not discuss regarding confounder adjustments, and it seems that different studies with different confounder adjustments were pooled. these differences may be a source of heterogeneity as well as cause of attention of attenuated the effect of ramadan fasting on weigh and lipid profile. so, the results were not reliable. as mentioned regarding the effect of ramadan fasting on pregnancy, the risk of low birth weight was not statistically higher in fasting mothers. appropriate nutritional status has an important role in these findings, so that fasting in well - nourished women had no adverse effect on offspring. although published studies shows that ramadan fasting had no serious adverse effect on offspring, it is strongly recommended that pregnant women avoid fasting because the most of current studies did not adjusted the impact of several confounder variables such as pregnancy duration, nutritional status before, during and after the ramadan, maternal age, socioeconomic status and other potential covariates. studies reported that ramadan fasting may decrease the concentration of il-6, il-1, tnf- and number of leukocytes and monocytes. elevated concentration of proinflammatory cytokines (e.g., il-6, il-1 and tnf-) is known as a risk factor for cardiovascular diseases, insulin resistance and cancers. suggested mechanism is that ramadan fasting results in oxidative stress reduction, and therefore, lower level of reactive oxygen species. on the other hand, body fat decreased during ramadan in this study, and therefore, proinflammatory cytokines secretion would be decreased. this reviewed studies approved the safety of ramadan fasting for chronic kidney diseases patients. suggested mechanism for this improvement in renal function is a reduction in blood pressure and body weight and decline in dietary protein and creatinine intake. although conducted studies regarding ramadan fasting and health outcomes were designed similar to before - after intervention studies, confounders were not controlled as well as possible. it is suggested that researchers should collect sufficient data for estimating confounders. several studies reported that ramadan fasting has health protective effects ; however these effects were attenuated during post - ramadan period. dieticians should provide a guideline for maintaining health protective effects of ramadan fasting during post - ramadan period. in conclusion, although studies showed that ramadan fasting has health protective effects, patient individuals should consult their medical team for fasting during ramadan.	background : fasting during ramadan is an islamic rule. although previous review studies have assessed the impact of ramadan on cardiovascular risk factors, athlete performance, diabetes and transplantation, in this study we have appraised some on these reviews by focusing on limitations and also, we have reviewed more recently published study and several recent studies, which are not reviewed till now.materials and methods : in this article, we reviewed recently conducted studies in regarding the impact of ramadan fasting on weight, lipid profile, diabetes, immune system and gestation. medline (http://www.pubmed.com) was searched by using ramadan as keyword and the most recent articles in mentioned topics since 2009 until february 2014 were selected.results:although weight has been decreased during ramadan in the most studies, weight regain is prevalent during the following months. meta - analysis of pre - ramadan lipid profile in comparison to post - ramadan values had been showed that total cholesterol and triglyceride were decreased in men and high - density lipoprotein was increased among women. in regarding diabetes and fasting, diabetic patients should be aware that medical, nutritional and physical activity consulting is necessary for individuals with diabetes who want to fast during ramadan. although published studies show that ramadan fasting had no serious adverse effect on offspring, it is strongly recommended that pregnant women avoid fasting because of the limitations of studies. the effect of fasting during ramadan on the immune system is favorable. ramadan fasting has no impact on kidney function and urine component.conclusion:studies showed that ramadan fasting has health protective effects. more precise studies should be conducted for more reliable conclusion.
ebolavirus (ebov) is a member of the family filoviridae in the order mononegavirales (mnv), and causes a lethal hemorrhagic fever in both humans and non - human primates (peters, 2005). five species of ebov have been defined to date on the basis of genetic divergence : zaire ebolavirus (zebov), sudan ebolavirus (sebov), tai forest ebolavirus (tfebov), reston ebolavirus (rebov), and bundibugyo ebolavirus (bebov). zebov, sebov, tfebov, and bebov cause clinical symptoms in humans and non - human primates, while rebov causes disease only in non - human primates, and not in humans. at present, there are no licensed vaccines or effective therapies for ebov infection. recently, tetherin / bst-2 was identified as a cellular factor that inhibits the release of a wide variety of enveloped viruses, including retroviruses, herpesviruses, and lassa virus, and the production of virus - like particles (vlps) of filoviruses and nipa virus (jouvenet. tetherin / bst-2 may function as a host innate antiviral system against a wide variety of viruses, as tetherin / bst-2 is broadly induced by treatment with type i interferons (ifns) in various cell types (ishikawa. electron microscopic studies have indicated that ebov is morphologically pleomorphic, with u - shaped, figure 6-shaped, or circular configurations, or as elongated filamentous forms of varying length (up to 14000 nm). the virions are usually 80 nm in diameter and 8001000 nm in length and enveloped with a lipid bilayer (envelope) that is derived from the host cell, anchoring a glycoprotein that projects spikes 710 nm in length from its surface (sanchez., 2007). the genome is approximately 19 kb in length and encodes the viral proteins in the order np vp35vp40gp / sgp vp30vp24l (figure 1). the extragenic sequence at the 3 end, which is called the leader, of ebov is short, ranging from 50 to 70 bases in length, while the length of the 5 end sequence, which is called the trailer, varies between species, ranging from 25 to 677 bases (25 bases for rebov and 677 bases for zebov). the extreme 3 and 5 end sequences are conserved and potentially form stem - loop structures (geisbert and jahrling, 1995 ; sanchez., 2007). these sequences contain the encapsidation signals as well as the replication origin and transcription promoter. the np and vp30 proteins are the major and minor viral nucleoproteins, respectively, which are phosphorylated, and interact strongly with the genomic rna molecule to form the viral nucleocapsid along with vp35 and l (mhlberger., 1999). the l and vp35 proteins form the viral polymerase complex, which transcribes and replicates the viral genome. the l protein has the rna - dependent rna polymerase activity of the complex, and possesses motifs linked to rna binding, phosphodiester bonding, and ribonucleotide triphosphate binding. vp35 is thought to play an essential role as a cofactor that affects the mode of viral transcription and replication. vp35 also functions as an antagonist against the type i ifn signaling pathway (basler., 2000). the gp gene contains a translational stop codon in the middle, thus preventing synthesis of full - length glycoprotein. twenty percent of the mrna was shown to be edited, containing one additional non - template a in a stretch of seven consecutive a residues. the edited mrna species encode full - length gp, whereas the primary gene product is a smaller secreted glycoprotein (sgp), which is produced in large amounts. the gp precursor (pregp), is synthesized as type i membrane protein in the endoplasmic reticulum (er). pregp is cleaved by the cellular proprotein convertase furin into the n - terminal fragment gp1 and the c - terminal fragment gp2 which are linked by a disulfide bond forming the gp1, 2 complex in the trans - golgi network (tgn ; volchkov., 1998). the trimeric spike of gp1, 2 is the only envelope glycoprotein of the virion and is assumed to be responsible for binding to cellular receptors and for fusion of the envelope with the cellular membrane in the course of viral entry into the host cell. recent report suggested that sgp can substitute for gp1 and present on virion as a structural protein, although sgp had been considered as a non - structural protein (iwasa., 2011). sgp may contribute to disease progression, as it has been reported that large amounts of sgp are present in the blood of acutely infected patients (volchkov. the vp40 and vp24 proteins are viral matrix proteins and are associated with the virion envelope. vp40 is the most abundant protein in the virion and plays a key role in virus assembly and budding as viral matrix protein. expression of vp40 in mammalian cells is sufficient to generate extracellular vlps, which resemble authentic virions (harty., vp24 has been reported to function as an antagonist of the type i ifn signaling pathway, along with vp35 (reid., tetherin / bst-2 (also known as cd317 or hm1.24) has been identified as an effective cellular factor that prevents human immunodeficiency virus (hiv)-1 release in the absence of the viral accessory protein vpu (neil., 2008 ; van damme., 2008). tetherin / bst-2 consists of four domains, i.e., an n - terminal cytoplasmic tail (ct), a single transmembrane domain, an extracellular domain, and a putative c - terminal glycosylphosphatidylinositol (gpi) anchor, and therefore both ends of this molecule are associated with the plasma membrane (figure 2a). this molecule is localized to lipid rafts at the cell surface and membranes of the tgn and recycling compartments (kupzig., 2003 ; blasius., 2006 ; rollason., 2007 ; evans., 2010) in addition, tetherin / bst-2 has two putative n - linked glycosylation sites in the extracellular domain and forms a homodimer by intermolecular disulfide bonds (ohtomo., 1999 ; tetherin / bst-2 has two putative n - linked glycosylation sites in the extracellular domain and forms a homodimer by intermolecular disulfide bonds. both ends of tetherin / bst-2 are associated with the plasma membrane via the n - terminal transmembrane domain and c - terminal gpi anchor. tetherin / bst-2 appears to inhibit the release of progeny viruses by directly tethering virions to cells, briefly by anchoring one end of the molecule on the cell membrane and the other end on the viral envelope. previous studies showed that tetherin / bst-2 is constitutively expressed in terminally differentiated b cells, bone marrow stromal cells, and plasmacytoid dendritic cells, and is also broadly induced by treatment with type i ifn in various cell types (ishikawa. in fact, the upstream region of tetherin / bst-2 contains a tandem repeat of il-6 response elements, stat3 binding site, and the ifn response elements irf-1/2 and isgf3 (ohtomo., 1999). therefore, tetherin / bst-2 may be involved in antiviral host defense as a mechanism of innate immunity. recent analysis for in vivo expression of tetherin / bst-2 showed that tetherin / bst-2 was expressed to varying degrees in most organs and a number of specialized cell types, including hepatocytes, pneumocytes, ducts of major salivary glands, pancreas and kidney, paneth cells, epithelia, leydig cells, plasma cells, bone marrow stromal cells, monocytes, and vascular endothelium, without ifn stimulation (erikson., 2011). we have shown that tetherin / bst-2 also efficiently inhibits the egress of vlps of marburgvirus and lassa virus and retains vlps on the cell surface (sakuma., 2009a). furthermore, tetherin / bst-2 has also been reported to inhibit the release of retroviruses other than hiv-1, kaposi s sarcoma - associated herpesvirus (kshv), and the production of vlps of ebov and nipa virus (jouvenet., 2009 ; kaletsky., 2009 ; mansouri., the n - linked glycosylation of tetherin / bst-2 is dispensable for the antiviral activity, while both n - terminal transmembrane domain and c - terminal gpi anchor are required for the activity (neil., 2008 ; andrew., 2009 ; sakuma. interestingly, tetherin / bst-2 mutant with complete loss of dimerization activity (the cysteine mutant) still showed apparent inhibitory activity for the production of lassa and marburg vlps, although its activity was reduced (sakuma. nevertheless, these observations suggested that dimerization of tetherin / bst-2 is important but not essential for its antiviral activity. a recent study showed that tetherin / bst-2 inhibits hiv-1 release by directly tethering virions to cells, briefly by anchoring one end of the molecule on the cell membrane and the other end on the viral envelope (perez - caballero. progeny virions released from cells could also be directly tethered to each other by tetherin / bst-2. it is likely that tethering of virion by tetherin dimer is stronger than that by tetherin monomer because of stronger association with the membrane. therefore, tetherin / bst-2 appears to inhibit release of a wide variety of enveloped viruses from host cells by a similar mechanism. human immunodeficiency virus-1 vpu is known to antagonize the antiviral function of tetherin / bst-2 (neil., 2008 ; van damme., 2008 moreover, recent studies have shown that hiv-2 and simian immunodeficiency virus (siv) env, siv nef, kshv k5, and ebov gp also function as antagonists of tetherin / bst-2, suggesting that tetherin / bst-2 plays an important role in host defense against viral infection and these viruses have evolutionarily acquired viral - encoded antagonists to counteract the antiviral function of tetherin / bst-2 (gupta., 2009b ; jia., 2009 ; kaletsky., 2009 ; le tortorec and neil, 2009 ; mansouri., 2009 ; zhang., 2009). (2009) demonstrated that ebov gp interacts directly with tetherin / bst-2 and abrogates the inhibition of vp40-induced vlp release by tetherin / bst-2. in addition, analysis using infectious virus showed that the expression of tetherin / bst-2 had no effect on zebov replication and spread (radoshitzky., 2010). one of the major functions of hiv-1 vpu for counteraction of antiviral action of tetherin / bst-2 is downregulation of the surface expression of tetherin / bst-2. several studies demonstrated that vpu directs the degradation of human bst-2 via a -trcp - dependent mechanism. vpu acts as an adapter molecule linking tetherin / bst-2 to the -trcp / scf e3 ubiquitin ligase complex to induce the trafficking to late endosomes, lysosomes, and proteasomes, and subsequent lysosomal and/or proteasomal degradation of tetherin / bst-2. vpu is thought to remove tetherin / bst-2 from the cell surface by these pathways (douglas., 2009 ; mitchell., recent report suggests that -trcp - independent mechanism is also involved in the downregulation of cell surface expression of tetherin / bst-2 by vpu, since mutations in the -trcp - binding motif of vpu did not completely abrogate its antagonism of tetherin / bst-2. 2011) have reported that vpu inhibited both the anterograde transport of newly synthesized tetherin / bst-2 and the recycling of tetherin / bst-2 to the cell surface and trapped trafficking tetherin / bst-2 molecules at the tgn. vpu interacts with tetherin / bst-2 through species - specific determinants in their respective transmembrane domains (gupta. hiv-1 vpu specifically antagonizes the tetherin / bst-2s from human, chimpanzee, and gorilla, which are susceptible to hiv-1 infection, but not those from african green monkey, rhesus macaque, and mouse, which are not susceptible to this virus (mcnatt. in contrast, ebola gp counteracted tetherin / bst-2 from different primate species, including rhesus macaque and african green monkey (khl., 2011). ebola gp does not seem to require a specific tetherin / bst-2 sequence for its activity (lopez., 2010). it has been reported that tetherin / bst-2 interacts with the gp2 subunit of ebov gp, although the antogonism of tetherin / bst-2 function by gp2 have not examined (khl., 2011). vpu reduces cell surface expression of tetherin / bst-2, while ebola gp appears to counteract tetherin / bst-2 without removing it from the cell surface, suggesting that both proteins employ different mechanisms to counteract tetherin / bst-2 (lopez., 2010 ; khl., ebola gp and tetherin / bst-2 colocalize in intracellular compartment, but not on the plasma membrane (khl., 2011). the sequestration of tetherin / bst-2 in the specific intracellular compartment may be one of the mechanisms of antagonism by ebola gp. so far, the mechanism by which ebov gp antagonizes tetherin / bst-2 remains unclear. further investigations are required to understand the mechanism by which ebov gp counteracts the antiviral function of tetherin / bst-2. tetherin / bst-2 inhibits the production of a wide variety of enveloped viruses. on the other hand, several viruses have evolved viral - encoded antagonists to counteract antiviral action of tetherin / bst-2. however, it has been reported that high - level expression of tetherin / bst-2 inhibits zebov production even in the presence of gp (khl., 2011). furthermore, it may be possible to identify tetherin / bst-2 mutants that are not counteracted by ebov gp. therefore, regulation of the progeny ebov release may be possible by in vivo induction or exogenous expression of tetherin / bst-2. tetherin / bst-2 has great potential for the development of novel antiviral therapeutic strategies against ebov infection. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	ebolavirus (ebov) is an enveloped, non - segmented, negative - stranded rna virus, which consists of five species : zaire ebolavirus, sudan ebolavirus, tai forest ebolavirus, bundibugyo ebolavirus, and reston ebolavirus. ebov causes a lethal hemorrhagic fever in both humans and non - human primates. the ebov rna genome encodes seven viral proteins : np, vp35, vp40, gp, vp30, vp24, and l. vp40 is a matrix protein and is essential for virus assembly and release from host cells. expression of vp40 in mammalian cells is sufficient to generate extracellular virus - like particles, which resemble authentic virions. tetherin / bst-2, which was identified as an effective cellular factor that prevents human immunodeficiency virus-1 release in the absence of viral accessory protein vpu, has been reported to inhibit zebov vp40-induced vlp release. tetherin / bst-2 appears to inhibit virus release by physically tethering viral particles to the cell surface via its n - terminal transmembrane domain and c - terminal glycosylphosphatidylinositol anchor. replication of zebov is not inhibited by tetherin / bst-2 expression, although tetherin / bst-2 was expected to inhibit ebov release as well as vlp release. recently, it was reported that viral glycoprotein of ebov, gp, antagonizes the antiviral effect of tetherin / bst-2. however, the mechanism by which gp antagonizes the antiviral activity of tetherin / bst-2 and whether gp of the other ebov species function as antagonists of tetherin / bst-2 remain unclear.
the translational research investigating underlying disparities in acute myocardial infarction patients ' health status (triumph) study is a prospective ami registry at 24 u.s. hospitals (6). in this analysis, patients with known dm (self - reported or documented in the chart) or those on glucose - lowering drugs on admission were included. patients were considered to have clinical a1c if measured during hospitalization or obtained in the preceding 3 months. patients consenting to triumph laboratory assessments also had a1c measured separately (laboratory a1c) ; these results were not available to treating clinicians. standard a1c cut points were used (good, suboptimal, and poor control for a1c 9%, respectively) (7). gti was defined as increase in the dose of an oral antihyperglycemic agent, addition of a new antihyperglycemic agent, or 20% increase in daily insulin dose on discharge versus admission (8). changing one oral agent to another was not considered gti. hierarchical poisson regression models (controlling for clustering by hospital) were constructed to identify independent predictors of gti. candidate variables included demographics, factors associated with gti in bivariate analysis, or those considered a priori as clinically important (bmi, admission glucose, mean fasting glucose, clinical a1c, intravenous insulin infusion, and admission dm medications). to evaluate whether physicians are more likely to prescribe gti in patients with worse glycemic control when a1c is clinically available (versus when a1c levels are not known), we performed a secondary analysis in which gti rates were compared between patients with clinical a1c versus laboratory a1c only within each glucose control subgroup (good, suboptimal, and poor). the translational research investigating underlying disparities in acute myocardial infarction patients ' health status (triumph) study is a prospective ami registry at 24 u.s. hospitals (6). in this analysis, patients with known dm (self - reported or documented in the chart) or those on glucose - lowering drugs on admission were included. patients were considered to have clinical a1c if measured during hospitalization or obtained in the preceding 3 months. patients consenting to triumph laboratory assessments also had a1c measured separately (laboratory a1c) ; these results were not available to treating clinicians. standard a1c cut points were used (good, suboptimal, and poor control for a1c 9%, respectively) (7). gti was defined as increase in the dose of an oral antihyperglycemic agent, addition of a new antihyperglycemic agent, or 20% increase in daily insulin dose on discharge versus admission (8). changing one oral agent to another was not considered gti. hierarchical poisson regression models (controlling for clustering by hospital) were constructed to identify independent predictors of gti. candidate variables included demographics, factors associated with gti in bivariate analysis, or those considered a priori as clinically important (bmi, admission glucose, mean fasting glucose, clinical a1c, intravenous insulin infusion, and admission dm medications). to evaluate whether physicians are more likely to prescribe gti in patients with worse glycemic control when a1c is clinically available (versus when a1c levels are not known), we performed a secondary analysis in which gti rates were compared between patients with clinical a1c versus laboratory a1c only within each glucose control subgroup (good, suboptimal, and poor). between 2005 and 2008, triumph enrolled 1,274 ami patients with dm on admission (6% type 1, 87% type 2, and 7% unknown type). clinical a1c assessment was performed in 886 patients (70%), and an additional 263 individuals had laboratory a1c measured. of these 1,149 patients with known a1c levels, glycemic control was good in 419 (37%), suboptimal in 415 (36%), and poor in 315 patients (27%). overall, 396 of 1,274 patients (31%) had gti at hospital discharge (33% new oral medication, 37% new insulin, 5% new oral medication and insulin, 9% oral medication up - titration, and 15% insulin increase). gti was more frequent in patients with versus without clinical a1c assessment (34 vs. 24%, p 9%, respectively ; p 9%, vs. a1c 9% ; interaction p = 0.30). between 2005 and 2008, triumph enrolled 1,274 ami patients with dm on admission (6% type 1, 87% type 2, and 7% unknown type). clinical a1c assessment was performed in 886 patients (70%), and an additional 263 individuals had laboratory a1c measured. of these 1,149 patients with known a1c levels, glycemic control was good in 419 (37%), suboptimal in 415 (36%), and poor in 315 patients (27%). overall, 396 of 1,274 patients (31%) had gti at hospital discharge (33% new oral medication, 37% new insulin, 5% new oral medication and insulin, 9% oral medication up - titration, and 15% insulin increase). gti was more frequent in patients with versus without clinical a1c assessment (34 vs. 24%, p 9%, respectively ; p 9%, vs. a1c 9% ; interaction p = 0.30). although guidelines recommend a1c assessment for all hospitalized patients with dm (if not recently measured) (5), we found that only 70% of ami patients with dm had a1c levels measured clinically. when assessed, nearly two - thirds of patients with dm had suboptimal or poor long - term glycemic control, but only a minority underwent intensification of their glucose - lowering therapy by hospital discharge. of note, clinical a1c assessment was strongly and independently associated with gti during ami hospitalization, especially in patients with suboptimal and poor glucose control. although this analysis shows modest improvement in a1c assessment compared with earlier data (8), nearly one in three patients with dm still do not have a1c checked during ami hospitalization. the observed association between clinical a1c availability and higher rates of both gti and nonpharmacologic measures (8) in patients with suboptimal and poor glucose control suggests that presence of clinical a1c may lead to important therapeutic interventions for dm management. in addition, many physicians relegate dm evaluation to the outpatient setting, but chronic dm management is not consistently addressed after hospital discharge post - ami (8,9). although randomized trials have not demonstrated reductions in cardiovascular events with intensive glucose control (10,11), better glucose control does reduce microvascular complications of dm, and optimization of a1c levels continues to be recommended by professional societies (5,12). incorporating routine a1c assessment as part of in - hospital care for ami patients with dm represents an opportunity to emphasize individualized, patient - centered dm management during ami hospitalization that may improve transition to the outpatient setting, and potentially reduce long - term dm - related complications. only patients with established dm and ami were included in this study, so its implications can not be extrapolated to patients with prediabetic states or newly diagnosed dm, and generalizability of findings to other hospitalized patients is unknown. information about gti during the immediate postdischarge period was not available, and some patients may have received gti during early outpatient follow - up. furthermore, the impact of therapeutic intensification on clinical outcomes remains unclear, as our study was not designed to address this question. nearly two - thirds of hospitalized ami patients with dm have suboptimal or poor long - term glycemic control, but only a minority receives intensification of glucose - lowering therapy at discharge. inpatient a1c assessment is strongly associated with higher rates of gti, particularly when glycemic control is suboptimal or poor. future studies should evaluate whether clinical outcomes are affected by intensification of glucose - lowering therapy after ami in patients with poor glucose control. although this analysis shows modest improvement in a1c assessment compared with earlier data (8), nearly one in three patients with dm still do not have a1c checked during ami hospitalization. the observed association between clinical a1c availability and higher rates of both gti and nonpharmacologic measures (8) in patients with suboptimal and poor glucose control suggests that presence of clinical a1c may lead to important therapeutic interventions for dm management. in addition, many physicians relegate dm evaluation to the outpatient setting, but chronic dm management is not consistently addressed after hospital discharge post - ami (8,9). although randomized trials have not demonstrated reductions in cardiovascular events with intensive glucose control (10,11), better glucose control does reduce microvascular complications of dm, and optimization of a1c levels continues to be recommended by professional societies (5,12). incorporating routine a1c assessment as part of in - hospital care for ami patients with dm represents an opportunity to emphasize individualized, patient - centered dm management during ami hospitalization that may improve transition to the outpatient setting, and potentially reduce long - term dm - related complications. only patients with established dm and ami were included in this study, so its implications can not be extrapolated to patients with prediabetic states or newly diagnosed dm, and generalizability of findings to other hospitalized patients is unknown. information about gti during the immediate postdischarge period was not available, and some patients may have received gti during early outpatient follow - up. furthermore, the impact of therapeutic intensification on clinical outcomes remains unclear, as our study was not designed to address this question. nearly two - thirds of hospitalized ami patients with dm have suboptimal or poor long - term glycemic control, but only a minority receives intensification of glucose - lowering therapy at discharge. inpatient a1c assessment is strongly associated with higher rates of gti, particularly when glycemic control is suboptimal or poor. future studies should evaluate whether clinical outcomes are affected by intensification of glucose - lowering therapy after ami in patients with poor glucose control.	objectiveto evaluate the relationship between a1c and glucose therapy intensification (gti) in patients with diabetes mellitus (dm) hospitalized for acute myocardial infarction (ami).research design and methodsa1c was measured as part of routine care (clinical a1c) or in the core laboratory (laboratory a1c, results unavailable to clinicians). gti predictors were identified using hierarchical poisson regression.resultsof 1,274 patients, 886 (70%) had clinical a1c and an additional 263 had laboratory a1c measured. overall, a1c was 9% in 315 patients (27%). gti occurred in 31% of patients and was more frequent in those with clinical a1c both before (34 vs. 24%, p < 0.001) and after multivariable adjustment (relative risk 1.34 [95% ci 1.121.62 ] vs. no clinical a1c).conclusionslong - term glucose control is poor in most ami patients with dm, but only a minority of patients undergo gti at discharge. inpatient a1c assessment is strongly associated with intensification of glucose - lowering therapy.
the human gastrointestinal tract (git) is the body 's largest interface with the environment and is a dynamic barrier that harbours a complex microbial community. the intestinal epithelium allows the uptake of nutrients, secretes water and electrolytes, and simultaneously acts as a barrier to exclude pathogens and toxins. humans and their symbiotic bacteria have co - evolved and their mutual interactions are essential for human health and well - being. there is increasing experimental evidence for the role played by intestinal bacteria in modulating development of the host immune system and the barrier properties of the intestinal epithelium. they are also frequently used as probiotics in foods, cultured milks, and various pharmaceutical preparations [46 ]. the presence of lactobacilli is important for maintenance of the intestinal microbial ecosystem and for providing protection against pathogen infection [79 ]. they are dominant in the proximal small intestine, a nutrient rich environment, whereas in the faecal microbiota they are present at most ~0.01%0.6% and this proportion varies significantly between individuals [11, 12 ]. they have the ability to adhere and interact with the epithelium and the mucosal layers, while surviving the hostile conditions of the luminal environment and the competing microbiota. these properties add to their potential to be used as probiotics that fit the parameters set by the operating standards in 2002 (fao / who : guidelines for the evaluation of probiotics in food). however, studies have shown that different strains of lactobacilli can evoke different responses in the host and therefore, the results from one strain can not be generalised to others. adherence of lactobacilli to the intestinal epithelium is an important characteristic as it promotes persistence time and colonisation, stimulates microbe - host interactions through immunomodulation, and provides protection to the intestinal barrier by various mechanisms including antagonistic activities against pathogens. bacterial cell surface components (adhesins, polysaccharides, and proteins) play major roles in the adherence of lactobacilli to the intestinal epithelium, interactions that might lead to pathogen exclusion and immunomodulation of host cells [15, 16 ]. the adhesive properties of lactobacilli are directly linked to their surface properties which are influenced by the structure and composition of their cell wall. several studies implicate cell surface components, either individually or collectively, in microbe - host interactions [17, 18 ]. lactobacilli show great diversity in cell surface architecture and are known to modify their surface properties in response to environmental changes [19, 20 ]. different macromolecules constituting the cell wall of lactobacilli have been shown to contribute to maintaining bacterial cell integrity during environmental stress. the cell surface architecture of lactobacilli and their ability to express certain surface components, or to secrete specific compounds that act directly on the host cells, may thus influence the physicochemical properties of the bacterial cell and strain - specific properties. this paper will focus on cell surface components of lactobacilli that influence host response and impart strain - specific characteristics to lactobacilli. the cell envelope of lactobacilli, like that of all lactic acid bacteria, is composed of the bilipidic plasma membrane with embedded proteins encompassed by the cell wall. the bacterial cell wall consists of a thick multilayered sacculus made of peptidoglycan (pg), decorated with teichoic acids (wall teichoic acids (wta) and/or lipoteichoic acids (lta)), exopolysaccharides (eps), proteinaceous filaments called pili, and proteins that are anchored to the cell wall through different mechanisms (figure 1). some species of lactobacilli display an additional paracrystalline layer of proteins surrounding the pg layer, referred to as the s - layer. these macromolecules together may play crucial roles in determining species and strain - specific characteristics of lactobacilli by influencing host - microbe interactions and microbial adaptations to the changing host environment. pg is the largest component of the bacterial cell wall and is an essential polymer in lactobacilli that determines the shape and preserves the integrity of the bacterial cell. the pg layer has been described as a fisherman 's net, functioning both as a container for and a sieve to the bacteria. the elastic nature of pg helps withstand stretching forces caused by bacterial turgor pressure, excludes large molecules from entering the bacterial cell, and at the same time restricts secretion of large proteins. proteins with theoretical molecular mass as large as 49.4 kda and 82.1 kda have been reported to be secreted by lactobacillus rhamnosus gg and lactobacillus plantarum, respectively [25, 26 ]. some large proteins are unable to diffuse through the cell wall and are dependent on the cell wall expansion process to be dragged to the outer surface of the thick pg layer before being passively released into the external milieu [24, 27 ]. the threads of this net are polymers of covalently linked alternating residues of n - acetyl - glucosamine (glcnac) and -1 - 4-linked n - acetyl - muramic acid (murnac). the glycan strands are held together by crosslinking pentapeptide side chains providing elasticity to the net. the pentapeptide side chain is made of alternating l- and d - amino acids and this attaches to the d - lactyl carboxyl group of murnac. considerable variations occur in the basic compositions of the glycan strands and pentapeptides which impart strain - specific characteristics to the bacteria [28, 29 ]. following biosynthesis, assembly, and incorporation of the pg subunits, modifications in the glcnac and murnac structures can occur and affect interactions between host and lactobacilli. these modifications include removal of acetyl groups from cell wall pg, 6-o - acetylation of cell wall murnac residues, and the substitution of c6 of murnac by teichoic and teichuronic acids. these modifications can affect the physiology of the bacterial cell wall by increased sensitivity to autolysis, resistance to lysozyme, and hydrophobicity of the cell envelope which in turn affects recognition by host receptors and bacterial adhesion [33, 34 ]. teichoic acids (tas) are the second major component of cell walls of lactobacilli and account for up to half of the cell wall dry weight. they are anionic polymers made of repeating units of glycerol- or ribitol - phosphate, covalently linked to pg as wta or attached to the cytoplasmic membrane through their lipid anchors as lta [3638 ]. a fraction of lta can be found free in the cell wall or may be released into the extracellular medium through deacetylation of the lipid anchor, where they are recognised as ligands by receptors present on intestinal epithelial cells. ltas contribute to the anionic character of the cell wall and provide hydrophobicity, which in turn influences the adhesiveness of the cell wall. the overall structure of ta is a chain made of phosphodiester - bound glycerol or ribitol residues hooked through a terminal linkage unit on the c6 of the murnac residue of a growing pg chain. the structure of the linkage unit is well conserved and is made of a disaccharide n - acetylmannosaminyl (14) glucosamine followed by glycerol phosphate. the variety of ta can occur in the nature of the sugars and number of phosphate residues. their size and physicochemical properties depend on several factors such as species or strain, stage or rate of growth, availability of phosphate, acidity of medium, and carbon source, and so forth. although all lactobacilli have ta in their cell walls, not all lactobacillus cell walls contain wta and some species appear to contain only lta. ta can function as a reservoir for phosphates and also as a scavenger of cations (mg in particular) [40, 41 ]. tas can also help in creating a ph gradient across the cell wall and are also known to be involved in phage adsorption and autolysin activity. glycosylated tas have been reported to be essential for the adsorption of some l. plantarum phages, and studies with l. delbrueckii subsp. cell wall polysaccharides are neutral polysaccharides that can either form a thick outer capsule closely associated with the cell wall and often be covalently bound to murnac of pg (referred to as capsular polysaccharide ; cps) or be loosely associated with it (wall polysaccharides ; wps) or be released into the extracellular medium (eps) [44, 45 ]., eps usually refers to extracellular polysaccharides that can be attached to the cell wall or released into the surrounding medium. the complex variations in the composition of eps, which differs in the nature of the sugar monomers along with their linkages, distribution, and substitution, add to the structural variety of the lactobacillus cell wall [46, 47 ]. eps is generally composed of heteropolysaccharides consisting of different sugar moieties such as glucose, galactose, rhamnose, glcnac, and n - acetylgalactosamine. residues of glucuronic acid, phosphate, acetyl, and pyruvate groups may also be present in some strains of lactobacilli. in addition to the heteropolymeric eps molecules, some strains of lactobacilli are capable of synthesising homopolysaccharides such as glucans or fructans from sucrose. studies with l. rhamnosus gg identified two different classes of eps : long galactose rich molecules and short glucose / mannose rich eps molecules. some polysaccharide chains can also be present as glycoproteins, providing anchorage to s - layer proteins, creating an extra level to the complexity of the bacterial cell wall architecture. specific contributions of eps to cell wall functionality are unclear, although their general role is to mediate interactions of lactobacilli with environmental components and promote bacterial adhesion and biofilm formation to inert or living surfaces [51, 52 ]. pili are multisubunit protein polymeric structures that have been functionally analysed and characterised only in l. rhamnosus gg [53, 54 ], although they have been identified at the genome level in some lactobacilli. these nonflagellar appendages are an assembly of multiple pilin subunits that are covalently coupled to each other by the transpeptidase activity of the pilin - specific sortase [53, 55 ]. the resulting isopeptide bonds are formed between the threonine of an lpxtg - like motif and the lysine of ypkn pilin motif in the pilin subunits. after assembly, the pilins are attached to the cell wall by a membrane bound transpeptidase, the housekeeping sortase. the roles of pili in bacterial adhesion, invasion, aggregation, formation of biofilms, and modulation of immunity are well established [58, 59 ] but the receptors in the host that recognise these pili are still unknown and their function in signalling host response is unclear. the presence of flagella is an unusual feature found in lactobacilli and at present, at least twelve motile species of lactobacilli have been recognised. the bacterial flagellum comprises of polymers of protein called flagellin, which is suggested to act as a ligand and mediate activation of signalling pathways and modulation of host immune cells. the cell surface proteins in lactobacilli are either anchored to the cell wall by various mechanisms or secreted from the bacterial cell into the surrounding medium, where they reassociate with the cell wall through electrostatic interactions. cell surface proteins include the s - layer proteins which constitute the major cellular proteins that surround the cell. examples of cell surface proteins include the 43 kda collagen binding s - layer protein from l. crispatus and cell surface proteins of 15 kda and two proteins of 45 and 58 kda from l. acidophilus crl639 that are involved in binding to fibronectin and collagen. covalently anchored proteins are further subcategorised into n- or c - terminally anchored proteins, lipid - anchored proteins (lipoproteins), and lpxtg - anchored proteins. the n - terminally anchored proteins represent the largest group of cell - surface - anchored proteins in lactobacilli and are mainly involved in cell - envelope metabolism, extracellular transport and signal transduction, competence, and protein turnover [35, 63 ]. many c - terminally anchored proteins, linked to the cell membrane through c - terminal transmembrane domains, are encoded by lactobacilli, but the function of several of these proteins remains unclear. the lipid - anchored proteins constitute the second largest group of predicted membrane - anchored proteins in lactobacilli, and are involved in transport, adhesion, antibiotic resistance, sensory processes, homeostasis of the cell envelope and secretion, folding and translocation of proteins [35, 64 ]. the c - region of the signal peptide of these lipoproteins contains the lipobox motif [l-(a / s)-(a / g)-c ]. lipidation followed by cleavage at the n - terminal of the cys - residue in the lipobox results in the covalent binding of the lipoprotein to the cell membrane through a thioether linkage. lpxtg - anchored proteins or sortase - dependent proteins (sdp) are covalently attached to the pg and reportedly play a crucial role in lactobacilli - host interactions. these proteins typically contain a cleavage site, an lpxtg motif, located in the c - terminal region of the mature domain, followed by a stretch of hydrophobic residues and a positively charged tail. the lpxtg motif is recognised by the sortase (srta) enzyme, which cleaves between the t and g residues and then covalently links the threonine carboxyl group to an amino group of the pg cross - bridges. although srta recognises the sequence lpxtg, another sortase, called srtb in s. aureus, has been reported to recognise and process proteins bearing the sequence npqtn. recent studies involving cross - linked protein products of srta and srtb indicate that different types of sortases may be able to attach proteins to distinct positions within the cell wall. some proteins can also be found anchored to other cell wall proteins through protein - protein interactions, while others are known to reassociate with the cell wall after being secreted, through electrostatic interactions. many species of lactobacilli display a surface coating made of a crystalline, two - dimensional array of protein or glycoprotein subunits assembled in lattices with different symmetries, also referred to as the s - layer. lactobacilli s - layer proteins represent up to 10 to 15% of total cell wall proteins. these proteins are highly basic, with stable tertiary structures ranging from 40 to 60 kda. s - layer proteins are the most prominent glycoproteins in prokaryotes, and although in lactobacilli most s - layer proteins appear to be nonglycosylated, some lactobacilli have glycosylated s - layer proteins that have been identified. s - layer proteins are relevant to cell wall polysaccharide pyruvylation and are noncovalently bound to the underlying pg cell wall, generally through secondary polymers such as lta, wta, and neutral polysaccharides. properties such as adhesion, aggregation, and pathogen inhibition have been related with the occurrence of particular types of s - layers, although s - layer functions in lactobacilli are not just species but also strain specific. studies indicate that there is a correlation between the different structural and chemical characteristics of the s - layer proteins with the surface properties of lactobacilli [50, 70 ]. there is ample evidence of s - layer proteins influencing the development of microbial communities as biofilms and therefore, it is likely that s - layer proteins have a role in the interaction of lactobacilli with other microorganisms. lactobacilli have enzymes with binding domains that help to keep them anchored to the bacterial cell surface. for example, extracellular enzymes such as autolysins display a stretch of 20 amino acids that have conserved multiple tandem repeats of aromatic residues and glycines that anchor to the bacterial cell surface by binding to the choline residues of wta and lta. the lysm domain (lysine motif) is found in many extracellular enzymes that are suggested to have a pg binding function and are involved in cell wall metabolism. wxl domain - containing proteins were identified in lactobacilli based on in silico analysis and are suggested to interact with the pg layer through their protein c terminus. this domain has also been reported to mediate noncovalent binding between the bacterial cell wall of enterococcus faecalis and other gram - positive bacteria. sh3b domains have been identified in some lactobacilli and are proposed to be involved in cell wall turnover. they have been suggested to recognise specific sequences within the peptide cross - bridges of the pg, thus targeting and binding to the cell wall. a putative domain composed of three -helices at the c- or n - terminal of an extracellular protein has been reported in some lactobacilli (l. plantarum, l. johnsonii, l. casei, l. brevis, l. helveticus, and l. gasseri) and is suggested to be involved in cell wall degradation through binding to the pg. lactobacilli encounter several environmental stress factors during their transit through the git such as low ph, bile salts, and oxidative and osmotic stress, along with starvation stress. stress responses of lactobacilli rely on the coordinated expression or suppression of genes that act in concert to improve stress tolerance. these genes can alter cellular processes such as cell division, membrane composition, transport systems, housekeeping, and dna metabolism and are regulated by factors that can control several genes and sometimes even other regulators. lactobacilli respond to stress in specific ways dependent on the strain, species, and the type of stress. the coordination of these stress responses is achieved by the network of regulators that allow the bacterial cell to react and adapt to different stressors. survival under acidic conditions is achieved by adapting to low ph through a mechanism called acid tolerance response (atr). studies with acid- and bile - resistant variants of l. acidophilus suggest that an inducible pre - existing system co - exists with a de novo protein synthesis mechanism, which together protect against acid stress. bile acids are conjugated to glycine or taurine in the liver and enter the intestine where the amino acid may be hydrolysed by bile salt hydrolases (bsh) expressed by bacteria, including lactobacilli. in l. plantarum, the capacity to tolerate taurodeoxycholic acid (tdca) has been attributed to the expression of tdca hydrolase, but other studies have shown that bsh activity and resistance to bile are unrelated properties in lactobacilli [79, 80 ]. many resistance mechanisms resulting in alteration of lactobacilli cell surface structures are common for bile and acid stress. the macromolecules composing the bacterial cell envelope (cell wall and cell membrane) contribute to maintaining the cell integrity under these stress situations. for instance, bile salts and cholesterol have been shown to induce changes in the lipid cell membrane of l. reuteri while low ph causes alterations in the fatty acid composition of an oral strain of l. casei. screenings of acid responses and bile salt responses in lactobacilli have identified genes involved in pg biosynthesis and cell envelope functions. gene expression analysis of l. acidophilus identified a high number of genes involved in pg and cell surface protein (e.g., srta) biosynthesis that are differentially expressed after bile exposure. in l. reuteri, the response to acidic conditions involves the clpl chaperone, an atpase with chaperone activity and a putative cell wall - altering esterase. these enzymes are also reported to be induced by bile exposure, further implying common resistance mechanisms for acid and bile stress [83, 84 ]. other cell surface structures (lta, wta, and eps) have also been suggested to play roles in proper functioning of cell integrity in acidic conditions and in the presence of bile. eps biosynthesis also reportedly involves suppression of genes after bile exposure as noted in l. acidophilus and l. reuteri, although the role of eps in bile and acid resistance is still unclear [82, 84 ]. in addition to acid and bile stress, the survival capacity of lactobacilli to oxidative and osmotic stress in the git is important. oxidative stress that can adversely affect cell fitness is caused by exposure to reactive oxygen species (ros) resulting from partial oxygen reduction to superoxide anion radicals (o2), hydroxyl radicals polyunsaturated fatty acids are sensitive to ros attack and the resulting peroxidation of membrane lipids and protein alteration affect cell membrane permeability and osmoregulation. to minimise the damage caused by ros, lactobacilli counteract ros generation with the help of enzymes such as catalase, nadh oxidase / peroxidise, and superoxide dismutase (sod) or nonenzymatic compounds such as ascorbate, glutathione, and mn. stress handling mechanisms range from preventing formation of ros, elimination of ros, and defence against oxidative damage to repair of oxidative damage. the fatty acid composition of the cell membrane of l. helveticus has been shown to change underoxidative stress and this was reported to be due to an increased activity of the o2-consuming fatty acid desaturase system which reduces the free radical damage in the cell. interestingly, bile stress has also been shown to induce oxidative stress, and studies indicate that the expression of glutathione reductase is influenced by bile treatment. lactobacilli are often exposed to changes in the osmolarity of their environment which can compromise essential cell functions. changes in solute concentrations in the environment cause changes in cell turgor pressure which lead to changes in cell volume. to maintain turgor pressure and retain water in the cell, lactobacilli accumulate compatible solutes under hyper - osmotic conditions and release them under hypo - osmotic conditions. in l. acidophilus, disruption of the cell division enzyme cdpa similar effects were shown by the slpa mutant of l. acidophilus, which was more sensitive to osmotic stress while being more resistant to bile. according to these studies, certain components of the cell wall remain uncleaved or cross - linked resulting in an immature structure of the cell wall in the mutant thus altering its phenotype [89, 90 ]. studies with l. alimentarius showed that when grown under sublethal doses of nacl, an increased tolerance was observed towards hyper - osmotic conditions or an increased atr against organic acids. similar cross - protection was observed when the cells were exposed to sublethal doses of these acids implying that common mechanisms were involved. the capacity to adapt to a specific nutritional environment is important to lactobacilli and ensures their residence time and survival in the git. starvation is one of the most common stresses faced by lactobacilli and bacterial growth leading to nutrient exhaustion, accumulation of fermentation end product (e.g., lactic acid), and subsequent starvation contributes to this stress. nutrient starvation in lactobacilli has been mainly studied by limiting the supply of carbohydrate, phosphate, and nitrogen. lactobacilli adapt to these nutritional limitations by either downregulating nucleic acid and protein synthesis and/or protein degradation and amino acid synthesis. moreover, extreme environmental stress conditions can indirectly provoke starvation by decreasing the activity of transporters resulting in reduced availability of essential nutrients that might be present in the extracellular environment. nutrient starvation leads to growth arrest, and different lactobacilli have developed different strategies to survive starvation. modification of cell morphology and cell division at the entry of the stationary phase, resulting in diminished cell size, has been reported in lactobacilli under these conditions. starvation resistance mechanisms in lactobacilli are diverse as they occupy different niches and do not encounter the same starvation conditions. it is well established that bacteria become more resistant to various types of stresses and develop a general stress - resistant state on entering the stationary phase. amino acid catabolism, in particular arginine degradation, plays a role in the enhanced survival of l. sakei during stationary phase. in l. acidophilus, 16 proteins were reported to be synthesised as a response to starvation, of which 7 were induced by stationary phase while the others in response to low ph. in l. lactis, glucose starvation was shown to induce resistance to many stresses (heat, low ph, and oxidative and osmotic stress). similarly in l. bulgaricus, lactose starvation increased resistance to heat, acid, and bile stress. although studies indicate a small overlap between stress - specific and starvation regulator genes and many proteins can be commonly induced by more than one stress, only a few proteins are common to all stresses. the human git represents the first line of defence against bacteria, viruses, fungi, and parasites that can act as pathogens. thus, the epithelium is important for the maintenance of git homeostasis in the presence of commensal microorganisms while preventing pathogen invasion. lactobacilli interact with the intestinal epithelium through several mechanisms that help modulate the immune response of the host, preserve barrier integrity, and maintain microbial balance through exclusion of pathogens by direct antimicrobial activity (production of bacteriocins or inhibitors), competitive exclusion (competing for binding sites), and/or stimulating anti - inflammatory immune responses (table 1). adherence of bacteria to the git mucosa is an important factor for colonisation and leads to direct interactions that can result in competitive exclusion of pathogens and the modulation of host response. adhesive mechanisms of human pathogenic bacteria have been studied extensively through the use of in vitro model systems. human colorectal adenocarcinoma cell lines such as caco-2 or ht-29 cells, immobilised intestinal mucus and extracellular matrices, quantitative measurements, microscopic enumeration, and immunological detection methods have been used for assessing adhesive mechanisms [120, 121 ]. however, knowledge of the bacterial cell surface molecules mediating adhesion to the git mucosa is still limited. genomics - based approaches have revealed several bacterial cell - surface - associated proteins that bind to mucus and intestinal cells. lactobacilli adhesins have been grouped into mucus binding proteins ; sortase - dependent proteins ; s - layer proteins ; proteins mediating adhesion to extracellular matrix (ecm) components of the intestinal epithelial cells ; nonprotein adhesins (lta and eps). intestinal epithelial cells form a barrier between the host and the content of the lumen and are covered by a protective layer of mucus. the mucus layer exists in a dynamic equilibrium, balanced between production, degradation, and physical erosion. it provides bacteria with only a short residence time in the git upon adhesion, thereby protecting the host against pathogens and undesirable bacterial colonisation. however, the mucus layer also provides a habitat for commensal bacteria, such as lactobacilli. adherence of lactobacilli to mucus has been experimentally validated in vitro using adhesion assays with probiotic - pretreated intestinal mucus glycoproteins, as well as in vivo by microscopic analysis of biopsy samples. lactobacillus adhesion to mucus involves mucus binding proteins (mubs) which in addition to the same domain organisation typical of cell surface proteins (the n- terminal signal peptide and c terminal lpxtg anchoring motif) share a mucus binding domain. mubs are encoded by lactobacillales - specific clusters of orthologous protein coding genes (lacog) and contain one or more mub repeats. proteins containing mub repeats are abundant in lactobacilli that inhabit the git, suggesting that mub repeat is a functional unit that may be an evolutionary adaptation for survival in the git. a database search using the sequence from the extracellular mub domain of l. reuteri and l. acidophilus, and the lectin - like mannose - specific adhesin (msa) of l. plantarum, resulted in the identification of proteins containing multiple mub domains in several species of lactic acid bacteria (lab), further suggesting that this domain is a lab - specific functional unit. studies with l. fermentum bcs87 have helped identify and characterise a 32 kda surface - associated protein (32-mmubp) that is suggested to mediate adhesion to mucus. the mub domain consists of a series of amino acid residues, varying in size from 100 to 200 residues per domain. studies have shown that mub and mub - like proteins contribute to mucus binding and autoaggregation, but high genetic heterogeneity among strains results in strain - specific diversity in adhesion to mucus. some lactobacilli (e.g., l. rhamnosus gg) have fimbriae (also called pili) that reportedly enhance adhesion to mucus glycoproteins of the host cells with subsequent colonisation of the git. studies with l. rhamnosus gg have shown a mucus binding factor (mbf) with a presumed ancillary involvement in pilus - mediated mucosal adhesion. however, fimbriae of some gram - positive pathogens were shown to induce pro - inflammatory responses, while capsular polysaccharide of l. rhamnosus gg was found to shield fimbriae, possibly suppressing pro - inflammatory responses. such role and possible positive effects of l. rhamnosus gg fimbriae, a subgroup of surface proteins that contains the lpxtg motif at their c terminal is recognised by srta. srta cleaves those proteins and anchors the resulting product to pg, thus incorporating these srta - dependent proteins on the microbial surface. although many sortase - dependent proteins are encoded by lactobacilli, the majority have no assigned function. of the functionally characterised proteins belonging to this family, three correspond to the mucus adhesins of l. reuteri (mub), l. plantarum (msa), and l. acidophilus (mub). lspa, of l. salivarius ucc118, is the fourth characterised sortase - dependent protein that also binds mucus and is known to mediate adhesion of this species to intestinal epithelial cells [109, 128 ]. recent studies with l. casei bl23 sortases and srta mutants suggest that srta might be involved in adhesion of this strain to caco-2 and ht29 cells. although most sortase - dependent proteins of lactobacilli seem to have mucus - binding capacity, not all of them have affinity to mucus components and the function of putative lactobacilli sortase - dependent proteins remains unclear. domain analysis and phylogenetic profiling of the extracellular proteins of l. plantarum involved in adhesion reported 10 of the 12 identified proteins to contain the lpxtg motif. of these 12 identified proteins, the role of msa from l. plantarum in adhesion has been experimentally validated, but the roles of the other in silico identified putative adhesins are speculative and need in vitro and in vivo validation. s - layer proteins form the outermost interacting surface in different species of lactobacilli and have been shown to act as adhesins to epithelial cells and components like mucus and extracellular matrix proteins. the role in adhesion of s - layer proteins of l. acidophilus (slpa), l. crispatus (cbsa), and l. brevis (slpa) has been experimentally validated [63, 110, 129 ]. the removal of the s - layer that reduced bacterial aggregation in l. acidophilus, l. kefir, and l. crispatus suggests their functional involvement in this process [112, 113 ]. there is considerable evidence that aggregation directly influences the development of structured microbial communities as biofilms, and the removal of the s - layer completely abolishes coaggregation, thus suggesting that it is mediated by s - layer proteins. studies also suggest that s - layer proteins with lectin - like activity interact with glycoproteins and polysaccharides and thus influence interactions of lactobacilli with other microorganisms. immunoblotting assays show direct interaction between l. kefir s - layer proteins and salmonella surface adhesins. pretreatment of salmonella with purified s - layer proteins has been shown to protect two human intestinal epithelial cell lines, parental caco-2 and the tc-7 clone, from salmonella invasion, but the protective effect was not observed when salmonella was pretreated with nonaggregative strains. these observations strengthen the theory that coaggregation prevents invasion by salmonella and protects epithelial cell damage. in l. kefir, the s - layer also influenced hemagglutinating, but not adhesion to caco-2 cells, unlike the s - layer of some strains of l. acidophilus that are involved in both caco-2 adhesion and aggregation [100, 112, 113 ]. in l. crispatus, the removal of the s - layer did not affect autoaggregation or hemagglutinating, suggesting that the s - layer may not be the only structure involved in these processes and that other covalently bound proteins or molecules such as lta or lectin - like molecules can mediate adhesion to intestinal epithelial cells. the extracellular matrix (ecm) is a complex structure surrounding intestinal epithelial cells and is composed of various proteins such as laminin, fibronectin, and collagen. some lactobacilli can bind to these proteins, thus competing with pathogens that have ligands for the same binding sites. examples of ecm binding adhesins are the fibronectin - binding protein (fbpa) of l. acidophilus and the collagen - binding protein (cnbp) of l. reuteri [17, 130 ]. pfam domain analysis of cnbp predicted a bacterial extracellular solute - binding domain (pf00497) that was also detected in mucus adhesion promoting protein (mapa), which was found to be a homologue for cnbp. although mapa reportedly mediates the binding of l. reuteri to caco-2 cells and mucus, database analysis detected no mucus binding proteins, suggesting a role for the extracellular solute - binding domain of mapa in adhesion. lactobacillus adhesion to the git has also been shown to involve surface - associated nonprotein factors such as the ltas and eps. ltas contribute to the anionic character of the cell wall and provide hydrophobicity, which in turn influences the adhesiveness of the cell envelope. eps may contribute to the physicochemical properties of the cell surface by shielding other cell surface adhesins, acting as ligands mediating adhesion and coaggregation [131, 132 ]. in l. acidophilus bg2fo4, carbohydrates on the bacterial cell wall were reported to be partly responsible for adhesion of this strain to caco-2 cells and to mucus secreted by the mucus producing human adenocarcinoma cell line ht29-mtx cells. in l. johnsonii, lta has been reported to mediate adhesion to caco-2 cells and in l. acidophilus, different types of exopolysaccharides have been shown to influence adhesion to ecm components. two peculiar examples of cytoplasmic - localised proteins that act as surface - translocated adhesins in lactobacilli are elongation factor tu (ef - tu) and the heat shock protein groel of l. johnsonii. ef - tu is involved in protein biosynthesis in the cytoplasm but has been reported as surface translocated in many lactobacilli. in l. johnsonii, surface translocated ef - tu fulfills an alternative role of mediating adhesion to intestinal epithelial cells and mucins. but when localised at the bacterial surface, it mediates adhesion to human intestinal cells and mucins [115, 116 ]. no domains or motifs have been found in either protein to account for their translocation across membranes. a cell - surface - associated enzyme gapdh of l. plantarum la318 has been found to mediate adherence to human colonic cells supposedly by recognising the sugar chains on the mucus and acting as a lectin - like protein. gapdh is surface localised although it lacks the conventional n - terminal signal sequence or a membrane anchoring motif. the role of lactobacilli in maintaining the intestinal barrier function is achieved by various mechanisms such as inducing mucus production, modulation of cytoskeletal, and tight junction protein phosphorylation, which can enhance tight junction function, immune response, and preventing apoptosis of the intestinal epithelial cells. enhancement of epithelial barrier integrity by lactobacilli has been observed in both in vitro and in vivo models. for example, l. brevis strengthens epithelial barrier function in healthy rats as assessed by mannitol permeability, with mannitol being used as a probe to study colonic wall permeability. administration of l. plantarum and l. reuteri to rats with methotrexate - induced enterocolitis improves bowel barrier function. l. plantarum has also been shown to increase epithelial barrier integrity using transepithelial electrical resistance assays as a measure of the integrity of tight junctions between intestinal epithelial cells with caco-2 cell line as a model. studies with interleukin-10 gene - deficient (il-10) mice indicate that most of them develop chronic enterocolitis, as il-10 has been suggested as an essential immunoregulator in the git and is a potent suppressor of macrophage and t - cell functions. studies with human intestinal epithelial ht29 cells show that the lipid moiety of lta from l. johnsonii and l. acidophilus inhibits e.coli and lipopolysaccharide- (lps-) induced il-8 production (il-8 is a chemokine and is a potent promoter of angiogenesis) by epithelial cells thus identifying important bacterial cell surface factors that confer beneficial effects on the git. recent studies with l. rhamnosus gg using caco-2 epithelial cells validate that the lipid chains of lta are needed for il-8 mrna expression and that d - alanine substituents are also important for il-8 induction in caco-2 cells. the intestinal epithelial barrier is also affected by alterations in mucus and chloride secretion by epithelial cells. mucin forms a physicochemical protective barrier for the underlying intestinal epithelial cells and assists in the prevention of mechanical, chemical, enzymatic, and microbial damage to the intestinal barrier and also restricts microbial invasion following adherence. in vitro experiments with selected lactobacillus strains have shown that adherence of enteropathogenic e. coli to human intestinal epithelial cells is inhibited by induction of intestinal mucin gene expression. mucin is known to inhibit bacterial translocation, and studies with l. casei lgg showed increased expression levels of mucin genes in a caco-2 cell model. expression of mucin genes, induced by lactobacilli, has been shown to be dependent on direct cell contact between l. plantarum and intestinal epithelial cells. in addition to mucus production, modulation of tight junction protein expression in epithelial cells is an important factor in preserving epithelial barrier integrity. tight junction proteins are dynamic structures that bind together epithelial cells at their apical junctions and help maintain barrier integrity. (zo-1), a tight junction protein, and f - actin, a structural component of the epithelial cell cytoskeleton, are known to play important roles in maintaining cytoskeleton architecture of epithelial cells thus preserving barrier integrity. l. acidophilus has been shown to prevent disruption of the distribution of zo-1 and occludin by e. coli and enhance cytoskeletal and tight junction protein structures such as occludin and actinin in intestinal epithelial cells. lactobacilli also improved barrier function in rats by increasing occludin expression and maintaining epithelial tight junctions [138, 139 ]. the adherence ability of lactobacilli enables them to compete with pathogenic bacteria for receptors that are expressed on intestinal epithelial cells, thus shielding them from damage caused by pathogenic bacteria and preserving barrier integrity [22, 140 ]. l. rhamnosus r0011 and l. acidophilus r0052 inhibit infection of intestinal cells caused by exposure to e. coli by reducing bacterial adhesion and cytoskeletal rearrangements. studies with specific lactobacilli strains show that direct cell contact is needed to induce expression of opioid and cannabinoid receptors in intestinal epithelium mediating analgesic functions in the git implying involvement of cell - surface - related effector molecules. antiapoptotic effect of l. rhamnosus gg in intestinal epithelial cells is also dependent on direct cell contact. the activation of the antiapoptotic akt / protein kinase b and inhibition of the activation of proapoptotic p38/mitogen - activated protein kinase by cytokines were suggested to prevent apoptosis in the intestinal epithelial cells. lactobacilli are able to modulate immune responses of the host by interaction with the git mucosa. bacterial surfaces exhibit characteristic features known as microbe - associated molecular patterns (mamp), which are usually cell wall components, such as lps, pg, lta, and wta, but can also be lipids, lipoproteins, proteins, and nucleic acids [142, 143 ]. mamps are recognised by various pattern recognition receptors (prr) that are expressed by many cell types including immune cells, intestinal epithelial cells, and nonimmune cells. recognition of these mamps by prrs induces a signalling cascade that can result in the production of cytokines, chemokines, and other effector molecules thus activating the innate immune response in the host. prrs include toll - like receptors (tlr), nucleotide oligomerization domain (nod)-like receptors (nlr), and c - type lectin receptors (clr). of these, tlrs and nlrs are well - characterised receptors of the host immune system that are known to interact with bacterial cell surface components like the lta and pg. tlr signalling pathways involve the recruitment of adaptors such as myeloid differentiation primary response gene 88 (myd88), which in turn activates the mitogen - activated protein kinase (mapk) pathway and the nuclear factor b (nf-b) pathway signalling cascades. activation and translocation of nf-b result in the transcription of numerous genes that regulate inflammatory responses. genes regulated by nf-b include those encoding cytokines such as interleukins (ils) and tumour necrosis factors (tnfs). these changes in cytokine production can result in dendritic cell (dc) maturation and activation, which in turn modulates the activation and differentiation of t cells [145, 146 ]. the specific interactions of mamps with prrs and the subsequent induction of signalling pathways depend on the microorganism and the reactivity of the host, which together play major roles in maintaining the functionality and homeostasis of the intestinal epithelial barrier. lactobacilli cell wall components such as lta and lipoproteins are recognised by tlr2 in combination with tlr6, leading to activation of nf-b. the two lipid chains of lta have to be exposed to mediate the interaction with the lipid - binding pocket of tlr2 implying that ltas may not be key prr ligands for intestinal epithelial cells. wta and lta also bind to macrophage scavenger receptors such as sra, a type i macrophage scavenger receptor that recognises ltas, thus contributing to immune signalling. lta and s - layer protein a (slpa) interact with dc - specific intercellular adhesion molecule - grabbing nonintegrin (dc - sign) on dc to induce cytokine release and t - cell maturation. activation of dc - sign by some strains of lactobacilli affects maturation of dcs, which reduces their capacity to induce il-10-producing regulatory t - cell responses against pathogens. glycosylation of slpa might be necessary for its interaction with dc - sign but needs to be validated as dc - sign is known to interact with glycosylated ligands of pathogens influencing host response to microorganisms. eps and other cell wall polysaccharides can be recognised by c - type lectin receptors (clr) that are present on macrophages and dc. in l. casei shirota, suppression of pro - inflammatory responses in macrophages is mediated by eps thus indicating an immune suppressive role of cell wall polysaccharides. the ability of lactobacilli to induce host cytokine responses in immune cells can be strikingly different depending on both species and strain. studies of dc responses to 42 l. plantarum strains indicate that cytokines produced can vary from strain to strain, and different strains of the same species can have distinct pro - inflammatory and anti - inflammatory profiles, suggesting that multiple factors can influence immune phenotype. studies with l. ruminis show that some species of lactobacilli display flagella which act as mamps that are recognised by the tlr5 of the host and are suggested to activate the nf-b pathway signalling in epithelial and immune cells of the host. an understanding of the roles played by bacterial mamps (lta, wta, pg, and eps) in strain - specific effects observed in lactobacilli is still developing. although mamps have a similar basic structure in conserved classes of bacterial macromolecules, different microorganisms can display subtle structural variations between mamps located on their cell walls. these variations can mean that a macromolecule from one species or strain can act as an agonist for a prr, whereas a similar macromolecule from another species or strain acts as an antagonist for the same prr. studies indicate that adherence characteristics (a major factor in the colonising potential of commensal bacteria) are influenced by cell wall structure and show pronounced variation among strains. strain specificity is undoubtedly linked to the variability and biochemical complexity of lactobacilli ligands and mamps as seen in the substitution levels of tas, the variable backbone alditol compositions of the wta, and the modifications of the pg of the cell wall. these modifications in the structure of pg can affect the physiology of the bacterial cell wall by increased sensitivity to autolysis, resistance to lysozyme, and hydrophobicity of the cell envelope which in turn affects recognition by host receptors and bacterial adhesion [33, 34 ]. for example, l. salivarius str. ls33 protects against chemically induced colitis in mice through the interaction of muramyl dipeptides present in its pg with nlr of the intestinal epithelial cells. ncfm, as variation in the pg composition of this strain blocks the nucleotide binding domain and leucine - rich repeat containing family (nlr) signalling pathway, which activates the mapk and nf-b pathways thereby hindering the activation of host defence mechanisms. another example of strain - specific characteristics imparted by variation in pg composition is observed in several lactobacilli, where resistance to vancomycin (a glycopeptides antibiotic) was shown to be the result of a replacement of the c - terminal d - alanine residue of murnac - pentapeptide by d - lactate. this illustrates the importance of the variable biochemistry of mamps such as pg to strain or species specificity. in addition, milieu - dependent switching between the multiple variants of cell wall polymers and/or tas adds to strain variation in lactobacilli. studies with mutants of lactobacillus strains that produce alternative lta variants suggest that modifications to the lta backbone can alter cytokine induction capacity thus increasing anti - inflammatory immune modulation [156, 157 ]. studies with dairy - isolated strains of l. delbrueckii showed anti - inflammatory effects in vitro, but the extent of these effects varied between strains. an interesting observation is that l. delbrueckii subspecies bulgaricus 1489 shows poor adherence to caco-2 epithelial cells implying lack of surface factors in this strain that may be involved in adherence. the high diversity of cell surface components found in lactobacilli adds to strain variation and is reflected in the ecological versatility observed in lactobacilli. chain length variation, subcellular localisation, and interactions of these polymers most likely contribute to strain - specific characteristics and are still being validated experimentally [50, 160 ]. the cell wall is a dynamic entity and plays an essential role in many aspects of the physiology and functioning of lactobacilli. it is where interaction with the bacterial environment occurs, which influences communication and adaptation to host - derived factors encountered in the git. environmental stressors have been shown to affect the cell surface architecture by influencing pg biosynthesis, expression of eps and cell surface proteins, and lta decoration with d - alanine residues. lactobacilli display considerable variation in their cell surface properties, through adaptations which undoubtedly are important for the functioning and survival of these bacteria in the git. the increasing possibilities of genomics - based approaches and mutant analyses have resulted in the identification of several effector molecules of lactobacilli. these effector molecules are proposed to be involved in direct interactions with host epithelial or immune cells and many of these effector molecules are components of the cell wall itself. considering the complexity of host - lactobacilli interactions involving host - cell signalling and regulation pathways, it seems unlikely that single - effector molecules regulate the entire host response. these molecules probably have an expanded repertoire in addition to playing crucial roles as building blocks of the bacterial cell wall. knowledge of the molecular mechanisms underlying the physiological characteristics of lactobacilli, and identification and validation of effector molecules complemented with parallel studies for their corresponding receptors in the host cells, can strengthen the concept of strain specificity and contributes to the development of strains with enhanced health benefits.	lactobacillus species can exert health promoting effects in the gastrointestinal tract (git) through many mechanisms, which include pathogen inhibition, maintenance of microbial balance, immunomodulation, and enhancement of the epithelial barrier function. different species of the genus lactobacillus can evoke different responses in the host, and not all strains of the same species can be considered beneficial. strain variations may be related to diversity of the cell surface architecture of lactobacilli and the bacteria 's ability to express certain surface components or secrete specific compounds in response to the host environment. lactobacilli are known to modify their surface structures in response to stress factors such as bile and low ph, and these adaptations may help their survival in the face of harsh environmental conditions encountered in the git. in recent years, multiple cell surface - associated molecules have been implicated in the adherence of lactobacilli to the git lining, immunomodulation, and protective effects on intestinal epithelial barrier function. identification of the relevant bacterial ligands and their host receptors is imperative for a better understanding of the mechanisms through which lactobacilli exert their beneficial effects on human health.
a 53-year - old female patient, 161 cm and 46 kg was diagnosed with mechanical ileus and admitted for adhesiolysis. she underwent total gastrectomy for cancer of stomach 2 months ago and had received adjuvant chemotherapy three weeks ago ; 2nd time with s-1 (ts-1, jeil pharmaceutical co., ltd, seoul, korea) 60 mg bid, for 14 days plus cisplatin (cispuran, dong - a pharmaceutical co., ltd, seoul, korea) 60 mg / m on day 1. there was no previous history of cardiopulmonary disease, allergies, tuberculosis exposure, or smoking. on her visit to emergency room, vital signs of the patient were as follows : body temperature, 37.2 ; heart rate, 106 beats / minute with a regular rhythm ; spo2, 100% (on room air) ; and blood pressure, 116/86 mmhg. laboratory findings on er were : white blood cell (wbc) count, 3.3 103/mm ; hemoglobin (hb), 12.8 g / dl ; platelet count, 184 103/mm ; c - reactive proteins (crp), 0.56 mg / dl ; pt (inr), 1.05 (normal, 0.8 - 1.3) ; and activated ptt, 20.9 sec (normal, 25.0 - 35.0). a preoperative chest x - ray, pulmonary function test and electrocardiogram were within the normal range. she was premedicated by oral midazolam (3.75 mg). after the patient was taken to the operating room, she was on ecg standard leads ii, noninvasive monitors for blood pressure, heart rate, arterial oxygen saturation, capnogram, and bispectral index. the patient underwent 3 minutes of denitrogenation with 100% oxygen through a facemask following which 2% propofol and remifentanil with the master target controlled infusion was given (orchestra base primea ; fresenius - mcm gmbh, germany) after administration of lidocaine (40 mg). after confirming loss of consciousness, rocuronium (0.5 mg / kg) was administered. endotracheal intubation was then performed without any complications. after verifying that patient continued to have clear breath sounds in both lung fields, the tube was fixed at a depth of 20 cm at the level of incisor teeth. the etco2 was monitored by capnogram and maintained at 35 - 40 mmhg. during the operation, 700 ml of hartmann solution was administered. after discontinuing the anesthetics a mixture of glycopyrrolate (0.4 mg) and pyridostigmine (15 mg) were administered for muscle recovery and the endotracheal tube was carefully extubated. she was awake fully and had no complaints of any discomfort or respiratory disturbance except for the surgical site pain, and she was therefore transported to the post anesthetic care unit. oxygen at 5 l / min was administered through a venturi mask when the pulse oximetry indicated a spo2 of 97%. fifteen minutes after extubation, the pulse oximeter indicated spo2 of 70%. a sudden onset of dyspnea and hemoptysis developed. the patient was promptly intubated and approximately 50 ml of fresh blood was suctioned through the endotracheal tube. the apparent bleeding was localized to originate from right upper lobe in the beginning, but the bleeding continued to become bilaterally with no identifiable bleeding focus (fig. arterial blood gas analysis after endotracheal intubation and mechanical ventilator applied were : arterial oxygen tension (pao2), 55 mmhg ; arterial carbon dioxide tension (paco2), 47 mmhg ; ph, 7.31 ; hb, 10.5 g / dl. the patient was transferred to the intensive care unit (icu) due to her unstable vital signs. the vital signs measured upon arrival showed a blood pressure of 80/50 mmhg, heart rate of 162/min and spo2 of 90% and swan - ganz catheter was inserted. the blood test results revealed the following : hb, 8.7 g / dl ; hematocrit, 26% ; platelet count, 57,000/mm ; wbc count, 900/mm ; absolute neutrophil, 640/mm. the vasopressor drugs such as dopamine, norepinephrine and vasopressin were infused to stabilize the blood pressure and hydrocortisone (300 mg) was injected. five units of packed rbcs, 6 units of fresh frozen plasmas and 10 units of platelets were transfused for 7 hours. a bronchoscope was performed again at the icu by pulmonologist after the vital signs were stable. the bronchoscopy revealed presence of alveolar hemorrhage in both lungs and no focal bleeding sources again. five days after the intubation, the patient sufficiently recovered and subsequently mechanical ventilation was discontinued. on the postoperative day 6 bilateral infiltration resolved and confirmed by high resolution computed tomography (hrct) scan images (fig. 3). the patient became symptom - free and was discharged from the hospital 20 days after the surgery. s-1 was excluded from the chemotherapy regimen in this patient and no recurrences of symptoms were observed after discharge. the term ' diffuse alveolar hemorrhage ' refers to a distinct form of pulmonary hemorrhage that originates from the pulmonary microcirculation including alveolar capillaries, arterioles, and venules. it is also known by names such as intrapulmonary hemorrhage, diffuse pulmonary hemorrhage, pulmonary alveolar hemorrhage, pulmonary capillary hemorrhage, alveolar bleeding, or microvascular pulmonary hemorrhage. dyspnea, cough, hemoptysis, and new alveolar infiltrates in conjunction with bloody bal establish the diagnosis of dah. dah can occur in association with various drugs and a wide variety of clinical disorders, many of which have overlapping features of glomerulonephritis, immune complex, and antiglomerular basement membrane disease. since the serological investigations and renal ultrasound were unremarkable for immune or renal disease in our case, together with the careful exclusion of these causes, several elements led to the suspicion that her lung damage was an expression of drug - induced adverse reaction. drug - induced lung injury may represent three mechanisms such as an immune or hypersensitivity reaction, an injury to the alveolar capillary basement membrane, or a coagulation defect. in this case we suspect that the drug may have a role to play in causing dah in this patient. the patient 's manifestation of dah in the setting of total gastrectomy and adjuvant chemotherapy raises a question of the possible predisposition of drug toxicity by cytotoxic drugs. a majority of anti - neoplastic drugs have the potential to induce pulmonary toxicity, involving lung parenchyma, airways, pleura, and pulmonary circulation. s-1 is a recently approved oral anti - neoplastic agent containing prodrug 5-fu that has been a first line drug in the therapy of gastrointestinal cancers. the major adverse effects of s-1 documented and reported so far are hematological and gastrointestinal nature. there are a few reports of pulmonary complication of s-1 available. s-1 and other cytoxic agents, 5-fu, have been reported to cause the interstitial lung disease which may be resolved by corticosteroid [6 - 8 ]. thus, increasing the probability of dah to be caused by s-1, although the exact component of the drug that is implicated was not identified by drug lymphocyte stimulation test (dlst). as far as the pathogenesis is concerned, a positive dlst result on 5-fu does not always automatically cause lung injuries. it seems that the direct toxic effect of the drugs on the parenchymal structures is responsible for the lung injuries rather than that of immunologically - driven mechanisms. for these experiences it becomes extremely important to obtain a full treatment history in all patients who manifest diffuse pulmonary infiltrates. despite these findings, the timing of the drug administration in relationship to the development of this complication points to its role in the causation of the event. however, it is reported that the pulmonary effects of cytotoxic drugs, such as bleomycin or mitomycin, occurs several months following completion of therapy. the use of oxygen in high concentrations during an unrelated surgery in a patient who was treated previously with bleomycin, and, possibly, other cytotoxic drugs, can result in an acute pulmonary syndrome years after the initial drug exposure. another drug that we could not exclude our suspicion, according to the timing of administration, is propofol, though such toxicity has not been reported previously in the literature. the patient underwent total gastrectomy 3 months ago with same anesthetic method and total infused dose of propofol was twice more than that of this instance. in the endotoxic rat model, pretreatment and simultaneous treatment with propofol provided protection against acute lung injury by inhibiting the tgf - beta1-smad2 dependent pathway. transbronchial lung biopsy was intentionally not performed in our patient because of the characteristic finding of dah (i.e. progressively diffuse hemorrhage) in the beginning. also, the need for mechanical ventilation and the severity of respiratory failure precluded us from obtaining the biopsy at that point of time. performing a surgical lung biopsy, although useful in confirming dah, is generally not suitable to identify the underlying causes. consequently, the first goal in the management of patients with dah is to achieve or preserve stability of the respiratory status. a possible management protocol should include supplemental oxygen, bronchodilators, reversal of any coagulopathy, intubation, protective strategies for the less involved lung, and mechanical ventilation. if therapy targets the autoimmune destruction of alveolar capillary membrane and the coexisting autoimmune condition, the application of corticosteroids and immunosuppressive agents should be considered. this case report describes the unexpected hemoptysis following surgical procedures in patients who underwent recent chemotherapy. this uncommon complication should be considered in the differential diagnosis of unknown etiology of hypoxia during peri - operative periods. the prevalence of this form of pulmonary hemorrhage in patients receiving s-1 chemotherapy is unclear at this time. the objective of this write - up is to prime the clinicians on this occurrence and to increase the awareness of the fact that s-1 has the potential to cause lung injury when it is included in chemotherapy. furthermore, the patients ' medication history should be taken into a careful consideration for perioperative evaluation especially in patients with pulmonary complications.	a 53-year - old woman who had undergone total gastrectomy and received adjuvant chemotherapy two months ago underwent adhesiolysis of the small bowel. she presented with sudden desaturation and dyspnea of unknown etiology at postanesthetic care unit. following et intubation, the endotracheal tube suction revealed massive hemoptysis. bilateral lung infiltrated on her chest radiograph and bronchofibroscopic examination disclosed a diffuse hemorrhage on both lung fields without bleeding focus. these findings were consistent with diffuse alveolar hemorrhage (dah) syndrome. as per our knowledge and search, this is the first reported case of dah that occurred during the recovery period immediately after general anesthesia. dah is known to have a high mortality rate and an early detection followed by adequate treatment is essential.
allergic bronchopulmonary aspergillosis (abpa) is a hypersensitivity reaction to aspergillus species. in children, the reported prevalence of abpa complicating cf lung disease varies from 6 to 25% depending on the age and the diagnostic criteria used [13 ]. the clinical diagnosis is difficult because symptoms are nonspecific and resemble bacterial cf airway infection. different diagnostic criteria have been established based on a combination of clinical and radiological signs together with the following biochemical parameters, again none of them being very specific : elevated ige (> 5001000 iu / l or a 4-fold rise), eosinophilia, specific ige for a. fumigatus or recombinant antigens (or positive skin prick test), and precipitins to a. fumigatus.. systemic corticosteroids are still the cornerstone of therapy starting at a dose of 1 - 2 mg / kg / day for 2 - 3 weeks, but the pace of tapering is highly variable and individualized. adjunctive antifungal agents like itraconazole (200400 mg / day for several months) may reduce corticosteroid need. however, few studies involving only a small number of cf patients have been published [3, 5 ]. in our cf center, abpa is usually treated with a combination of systemic steroids and itraconazole. in patients with frequent abpa flares or failure of steroid tapering, inhaled amphotericin b (iamb) because only scarce data exist [6, 7 ], we report our experience with this treatment in 7 cf children with a difficult - to - treat abpa course. inhaled amb is considered in case of one or more of the following : (1) insufficient response to initial therapy with corticosteroids and itraconazole (2) frequent abpa relapse during / after steroid withdrawal despite itraconazole treatment (3) failure to taper systemic steroids (4) intolerance for itraconazole. data were retrospectively collected on 7 cf patients treated for abpa with inhaled amb. in a first step amb deoxycholate (amb - d) (fungizone bristol - meyers squibb belgium) was administered as a test dose (20 mg in a concentration of 1 mg / ml ; nebulization for 1015 minutes with a pari turboboy nebulizer or aeroneb go). if tolerated, therapy was given 3 times a week. in case of intolerance for amb - d (cough, wheeze, and shortness of breath), amb - lipid complex (ablc) was used (abelcet wyeth 50 mg in a concentration of 5 mg / ml, 2 /week ; nebulization time 1015 minutes). choice of the dosage scheme was mainly based on doses used in other indications and the reported long half - life of iamb in the lung in animal models, especially when using a lipid formulation. because ablc has a 1/1 molar ratio for the active product versus the phospholipids (see table 2), the weekly dose was about the double compared to amb - d. treatment was considered a success if systemic steroids could be stopped without abpa relapse for at least 12 months after steroid withdrawal. prednisolone was started at a dose of 1 - 2 mg / kg / day. after 3 weeks, the patient was seen in clinic, and tapering was started in case of clinical improvement and fall in ige. if clinically indicated steroids were tapered with 5 mg every 2 weeks. however, final decision for speed of tapering was made by the treating cf physician. improvement of lung function is not used as an absolute criterion for treatment success in this small patient series because important comorbidities influence lung function evolutions. specific ige for different recombinant aspergillus antigens were measured (f1, f2, f3, and f4) (immunocap 1000, phadia ; cut - off > 0.7 ua / ml). patient 1 (ws 21 - 12 - 2001)this cf girl had a difficult respiratory course from young age with frequent infectious exacerbations treated with oral and iv antibiotics. abpa was first diagnosed at age 5 based on a respiratory exacerbation not improving with iv antibiotic treatment combined with a rise in ige to 1089 ku / l and positive rast to the recombinant aspergillus antigen f6 (46.5 ua / ml). treatment with oral steroids and itraconazole (sporanox janssen - cilag 1 100 mg) was started (figure 1). because of persisting atelectasis of the left upper lobe after 2 weeks of oral steroids and itraconazole, inhaled amb - d was added after which the atelectasis cleared. one year after steroid stop and 8 months after inhaled amb - d was stopped, abpa recurred and was successfully treated with the same combination of prednisolone and antifungal therapy (itraconazole and amb - d). she is now 9 month off steroids, and inhaled amb - d was changed to ablc and will be continued until at least 1 year free of abpa. this cf girl had a difficult respiratory course from young age with frequent infectious exacerbations treated with oral and iv antibiotics. abpa was first diagnosed at age 5 based on a respiratory exacerbation not improving with iv antibiotic treatment combined with a rise in ige to 1089 ku / l and positive rast to the recombinant aspergillus antigen f6 (46.5 ua / ml). treatment with oral steroids and itraconazole (sporanox janssen - cilag 1 100 mg) was started (figure 1). because of persisting atelectasis of the left upper lobe after 2 weeks of oral steroids and itraconazole, inhaled amb - d was added after which the atelectasis cleared. one year after steroid stop and 8 months after inhaled amb - d was stopped, abpa recurred and was successfully treated with the same combination of prednisolone and antifungal therapy (itraconazole and amb - d). she is now 9 month off steroids, and inhaled amb - d was changed to ablc and will be continued until at least 1 year free of abpa. patient 2 (bl 13 - 4 - 1998)from diagnosis, the respiratory course of this girl was unstable with frequent infections and lung infiltrates treated with iv antibiotics. because of severe gastro - esophageal reflux, a nissen fundoplication was performed at the age of 5 years. she developed asthmatic symptoms (in the context of familial atopy and asthma) for which she has been treated with inhaled steroids.she was diagnosed with a first episode of abpa based on a respiratory exacerbation not improving with iv antibiotics, ige rising from 230 ku / l to 500 ku / l, eosinophilia (1200/l), and a positive rast for recombinant aspergillus antigen f4 (between 9 and 43 ua / ml). aspergillus was cultured from sputum. she was treated with oral steroids and itraconazole (sporanox janssen - cilag 1 100 mg, later 2 100 mg) (figure 2). only slow clinical improvement was seen under oral steroids, and itraconazole was replaced by inhaled ablc. because of decreasing lung function and repeated scedosporium prolificans in sputum cultures, voriconazole (vfend pfizer 2 125 mg increased to 2 200 mg / d based on subtherapeutic blood levels) was added. sputum was initially not clear ; however, she finally became free of aspergillus (from march 2009).only under combined antifungal therapy, steroids could be stopped and lung function improved. the respiratory course of this girl was unstable with frequent infections and lung infiltrates treated with iv antibiotics. because of severe gastro - esophageal reflux, a nissen fundoplication was performed at the age of 5 years. she developed asthmatic symptoms (in the context of familial atopy and asthma) for which she has been treated with inhaled steroids. she was diagnosed with a first episode of abpa based on a respiratory exacerbation not improving with iv antibiotics, ige rising from 230 ku / l to 500 ku / l, eosinophilia (1200/l), and a positive rast for recombinant aspergillus antigen f4 (between 9 and 43 ua / ml). she was treated with oral steroids and itraconazole (sporanox janssen - cilag 1 100 mg, later 2 100 mg) (figure 2). only slow clinical improvement was seen under oral steroids, and itraconazole was replaced by inhaled ablc. because of decreasing lung function and repeated scedosporium prolificans in sputum cultures, voriconazole (vfend pfizer 2 125 mg increased to 2 200 mg / d based on subtherapeutic blood levels) was added. sputum was initially not clear ; however, she finally became free of aspergillus (from march 2009). only under combined antifungal therapy, patient 3 (bw 30 - 10 - 1995)this boy developed chronic lung infection with b. cepacia (multivorans) from the age of 8. he was diagnosed with abpa in the same year after a prolonged respiratory exacerbation being not resolved despite repeated antibiotic administration. ku / l ; specific ige for aspergillus recombinant antigens were positive only for f3 (1 ua / ml), not for f4 nor f6. he was treated with oral steroids and itraconazole (sporanox janssen - cilag 2 100 mg). one year later, he developed an abpa relapse with prolonged course and difficult steroid tapering (figure 3). after start of inhaled amb - d (later switched to abcl), steroids could be tapered and finally stopped. inhaled abcl was stopped after he was free of steroids and abpa relapse for 1 year. sputum cultures became negative for aspergillus since start of iamb.despite the resolution of the abpa, the overall evolution in this cf patient was unfavorable. he developed a chronic lung abscess in the right lung for which a right lower lobe resection was performed at the age of 12. however, chronic b. cepacia suppurative infection of the remaining right lung persisted with development of collapse and a functional right lung. this boy developed chronic lung infection with b. cepacia (multivorans) from the age of 8. he was diagnosed with abpa in the same year after a prolonged respiratory exacerbation being not resolved despite repeated antibiotic administration. ku / l ; specific ige for aspergillus recombinant antigens were positive only for f3 (1 ua / ml), not for f4 nor f6. he was treated with oral steroids and itraconazole (sporanox janssen - cilag 2 100 mg). one year later, he developed an abpa relapse with prolonged course and difficult steroid tapering (figure 3). after start of inhaled amb - d (later switched to abcl), steroids could be tapered and finally stopped. inhaled abcl was stopped after he was free of steroids and abpa relapse for 1 year. sputum cultures became negative for aspergillus since start of iamb. despite the resolution of the abpa, he developed a chronic lung abscess in the right lung for which a right lower lobe resection was performed at the age of 12. however, chronic b. cepacia suppurative infection of the remaining right lung persisted with development of collapse and a functional right lung. patient 4 (dk 13 - 1 - 1993)this girl developed severe obstructive lung disease and bronchiectasis despite intensive treatment and frequent courses of iv antibiotics.the first episode of abpa at the age of 10 (figure 4) was treated with oral steroids and itraconazole (sporanox janssen - cilag 2 100 mg). several attempts to taper the steroids failed. from the age of 11 (2004), mycobacterium avium - intracellulare (mac) infection has been documented. several combination treatments have been given with limited if any success (ethambutol, rifampicin, clarithromycin or azithromycin, iv amikacin, ciprofloxacin or levofloxacin, interferon gamma 1-b). lung function continued to decline.sputum cultures mainly grew candida and aspergillus species despite longstanding treatment with itraconazole. therefore, treatment with voriconazole (vfend pfizer 2 120 mg) was started. after 1 month of therapy, slight improvement of lung function and decreased cough were seen. posaconazole (noxafil schering - plough 2 400 mg) was given for a certain period but stopped because of reimbursement issue (see figure 3). ablc was only given for short periods of time but was disliked by the patient. voriconazole was successfully reintroduced at a higher dose (2 240 mg daily) based on subtherapeutic blood levels, but ige remains high (figure 4) and lung function fails to improve. this girl developed severe obstructive lung disease and bronchiectasis despite intensive treatment and frequent courses of iv antibiotics. the first episode of abpa at the age of 10 (figure 4) was treated with oral steroids and itraconazole (sporanox janssen - cilag 2 100 mg). several attempts to taper the steroids failed. from the age of 11 (2004), several combination treatments have been given with limited if any success (ethambutol, rifampicin, clarithromycin or azithromycin, iv amikacin, ciprofloxacin or levofloxacin, interferon gamma 1-b). therefore, treatment with voriconazole (vfend pfizer 2 120 mg) was started. after 1 month of therapy, slight improvement of lung function and decreased cough were seen. posaconazole (noxafil schering - plough 2 400 mg) was given for a certain period but stopped because of reimbursement issue (see figure 3). ablc was only given for short periods of time but was disliked by the patient. voriconazole was successfully reintroduced at a higher dose (2 240 mg daily) based on subtherapeutic blood levels, but ige remains high (figure 4) and lung function fails to improve. patient 5 (bp 25 - 7 - 90)abpa was diagnosed based on bilateral lung infiltrates not improving under adequate antibiotic therapy combined with raised ige (3295 ku / l) together with positive rast and precipitins for a. fumigates. she was treated with oral steroids and itraconazole (sporanox janssen - cilag 2 200 mg) but had frequent relapses over the years (figure 5).only after starting inhaled ablc, steroids could be tapered as she has been free of abpa relapse for almost 2 years although her ige levels remained high. abpa was diagnosed based on bilateral lung infiltrates not improving under adequate antibiotic therapy combined with raised ige (3295 ku / l) together with positive rast and precipitins for a. fumigates. she was treated with oral steroids and itraconazole (sporanox janssen - cilag 2 200 mg) but had frequent relapses over the years (figure 5). only after starting inhaled ablc, steroids could be tapered as she has been free of abpa relapse for almost 2 years although her ige levels remained high. patient 6 (vm 24 - 7 - 1989)childhood years were characterized by rather stable respiratory disease, but since adolescence, respiratory exacerbations became frequent. a nissen fundoplication was performed at the age of 13 because of severe gastro - oesophageal reflux. abpa was diagnosed at the age of 11 based on a respiratory exacerbation not improving with iv antibiotics and a high ige of 3890 she was treated successfully with oral steroids and itraconazole (sporanox janssen - cilag 2 100 mg later 2 200 mg) (figure 6). after a relapse 3 years later, tapering of steroids resulted in frequent abpa exacerbations. insisting on the importance of the antifungal therapy finally resulted in better adherence resulting not only in successful weaning (more than 2 years off steroids) of therapy but also improved lung function. childhood years were characterized by rather stable respiratory disease, but since adolescence, respiratory exacerbations became frequent. a nissen fundoplication was performed at the age of 13 because of severe gastro - oesophageal reflux. abpa was diagnosed at the age of 11 based on a respiratory exacerbation not improving with iv antibiotics and a high ige of 3890 she was treated successfully with oral steroids and itraconazole (sporanox janssen - cilag 2 100 mg later 2 200 mg) (figure 6). after a relapse 3 years later, tapering of steroids resulted in frequent abpa exacerbations. sputum cultures were intermittently positive for a. fumigatus, c. albicans and s. prolificans. after initial start of inhaled ablc insisting on the importance of the antifungal therapy finally resulted in better adherence resulting not only in successful weaning (more than 2 years off steroids) of therapy but also improved lung function. patient 7 (zb 13 - 10 - 89)diagnosis of abpa was first made at the age of 9 based on a respiratory exacerbation with a new lung infiltrate not clearing with antibiotics. ige was 881 ku / l, with positive specific rast for a. fumigatus as well as positive skin prick test. she was first treated with oral steroids only. at the time of the 3rd abpa episode, itraconazole (sporanox janssen - cilag 2 200 mg) was started, but steroid tapering was difficult.at the age of 17, inhaled ablc was started because of failure to stop the systemic steroids. she has been free of abpa since 2.5 years with more stable lung function evolution (figure 7). diagnosis of abpa was first made at the age of 9 based on a respiratory exacerbation with a new lung infiltrate not clearing with antibiotics. ige was 881 ku / l, with positive specific rast for a. fumigatus as well as positive skin prick test. she was first treated with oral steroids only. at the time of the 3rd abpa episode, itraconazole (sporanox janssen - cilag 2 200 mg) steroids were slowly tapered and successfully stopped after 8 months. she has been free of abpa since 2.5 years with more stable lung function evolution (figure 7). we report the use of inhaled amb in the treatment of 7 cf children with difficult - to - treat abpa. for some patients amb was used in combination with itraconazole or voriconazole. for 5 of the 7 patients treated with inhaled amb, treatment was considered a success : patients were weaned from systemic steroids without abpa relapse for at least 12 months. for 2 of these 5 patients, clinical evolution the patient that remained steroid dependent (patient 4) had complex lung disease with mac infection. in 4 out of 5 successes, lung function improved as well. the patient that had progressive lung function decline, despite successful abpa treatment, had b. cepacia lung infection. to reduce the burden of a. fumigatus in the respiratory tract it may be worthwhile to treat with antifungal medication [4, 11, 12 ]. other possible drug interactions include cyp3a4 inhibition leading to qt prolongation and decreased methylprednisolone metabolization. voriconazole, approved for the treatment of invasive aspergillosis, has a better oral bioavailability. it is however expensive, has a high potential for drug interactions, and has been associated with a number of adverse effects [13, 14 ], and no formal data exist on its absorption in cf. in the light of these concerns, data on clinical use mainly concern prevention and treatment of invasive aspergillus infections in neutropenic or lung transplant patients. amb has been the treatment of choice for most invasive fungal infections since its introduction in the 1950s. it has a broad spectrum of activity including aspergillus fumigatus, candida albicans, and cryptococcus neoformans. lipid formulations have been developed to reduce nephrotoxicity after parental administration while retaining the drug 's activity. clinically they differ mainly in risk for toxicity, dosing, and tissue concentration after parental administration. the nebulization of amb was first studied in a rat model of pulmonary aspergillus infection. prophylactic treatment before fungal inoculation with either formulation resulted in a significantly prolonged survival for all. mean concentrations of amb in the lungs were significantly higher and had a longer half - life with ablc compared to a similar dose of amb - d. the liposomal formulation had the longest half - life. in lung transplant patients, ablc and l - amb are better tolerated than conventional amb - d. the deoxycholate salt used in conventional amb - d acts as a detergent, impairing the surfactant function which is a potential toxic effect [17, 18 ]. in contrast, the liposomal carrier in l - amb exhibits a pulmonary surfactant - like function. few reports are found on the use of inhaled amb in the treatment of abpa. the use of nebulized amb - d in the treatment of acute abpa in a non - cf boy was reported. a dose of 2.5 mg was given 3 times daily, but duration of therapy was not mentioned. data on 5 cf patients treated weekly with l - amb was published in abstract form. duration of nebulization was reported as 150 minutes on average which makes this treatment burdensome. systemic steroids could be stopped in 4 of the 5 patients. in one patient, recently, a short report describes the successful use of nebulized amb (5 mg two times daily) in 3 cf children with abpa in combination with inhaled budesonide. based on pharmacokinetic data, we used a 2- or 3-time per week administration of inhaled amb. based on possible effects on pulmonary surfactant, we try however to avoid longstanding use of amb - d and switch to ablc. with amb - d, main adverse event was cough and wheeze induced by the nebulization. none of the patients had to stop the treatment with ablc because of side effects. prospective interventional trails will be needed in order to evaluate the place of inhaled antifungal medication in the treatment of abpa. we are aware that recently several case reports have been published on the use of omalizumab in abpa treatment in cf [2124 ]. even if this therapy proves to be efficient for this indication, omalizumab will not be suitable for all cf patients with abpa, merely because of side effects but also because of high cost and the necessary 2 weekly in - hospital administration. we report on the use of inhaled amb in the treatment of 7 cf patients with difficult - to - treat abpa. in 6 of the 7 patients treated, steroids could be stopped and patients remained free of relapse after several years of recurrent abpa episodes. based on this limited experience, inhaled amb may be considered as antifungal therapy in the context of abpa treatment in cf.	background. systemic steroids and adjunctive antifungal therapy are the cornerstone in treating allergic bronchopulmonary aspergillosis (abpa) in the context of cf. aim. evaluate the use of inhaled amphotericin b (iamb) as antifungal agent in this context. methods. report of 7 cf patients with recurrent or difficult to treat abpa and failure to taper systemic corticosteroids treated with amb deoxycholate (amb - d) (fungizone 25 mg 3 a week) or amb lipid complex (ablc) (abelcet 50 mg twice weekly). successful therapy was defined as steroid withdrawal without abpa relapse within 12 months. results. therapy was successful in 6 of 7 patients treated with iamb. in 5/6, lung function improved. the patient with treatment failure has concomitant mac lung infection. conclusion. inhaled amb may be an alternative to commonly used adjunctive antifungal therapy in the treatment of abpa. more data are needed on safety and efficacy.
the use of next - generation sequencing technologies (most commonly, whole exome sequencing (wes) and whole genome sequencing (wgs)) in research into the etiology of familial cancer syndromes has led to the identification of rare highly - penetrant genetic variants responsible for the increased rates of cancers in highly - selected families. at the same time, this technology has resulted in the identification of incidental and secondary findings with uncertain or known clinical utility. incidental findings are generally understood to comprise findings unrelated to the primary intent of a specific test that are stumbled upon in the course of analyzing research data ; they may be either secondary findings are defined as variants in genes that are not the primary focus of a specific test, but which are specifically, deliberately analyzed because they have been defined a priori as potentially medically actionable genetic loci (not necessarily related to the disorder under study) that are unavoidably interrogated when using diagnostic whole genome and whole exome sequencing. there is a growing belief in the genetics and ethics communities that investigators must at least consider disclosing such abnormalities to those being tested, since this information is potentially of great importance in their general medical care and that of their relatives. position statements from the american college of medical genetics and genomics (acmg) recommend that laboratories performing clinical sequencing : (a) obtain written informed consent regarding how these findings will be handled (after a discussion of the interpretive uncertainty, privacy and the potential impact on other family members), (b) seek out and report pathogenic variants that may predispose to a severe but preventable outcome to individuals being tested that are detected in specific classes or types of genes, (c) follow the same policy in children as in adults, and (d) offer parents of tested children the option to decline incidental and secondary findings disclosure. eurogentest and the european society of human genetics recently presented guidelines for diagnostic next - generation sequencing, including a rating system for diagnostic tests. the rating system provides information relevant to the coverage and diagnostic yield and aims to allow comparison of testing offered between different laboritories. the acquisition of next generation sequencing clinical data and its interpretation has resulted in an active, unresolved debate as to whether there is a similar obligation to screen for and report incidental and secondary findings to research study participants. this is based upon the idea that some specific results might be medically actionable, i.e., knowledge of their presence could significantly alter management and future health of the individual. the dominant view among genomic researchers, genomic health professionals and the public supports the return of all genomic research results (i.e., when a causative gene is identified as the basis for the disorder being studied, as a secondary finding or as an incidental finding) when there is perceived clinical utility and when the research result has been validated in a clia - certified laboratory, even when these stakeholders did not expect researchers to deliberately screen for incidental and secondary findings in the research setting. there exist only limited data (primarily from small studies) regarding adult participants interest in and intention to receive research genetic test results (rr), incidental findings (if) and secondary findings (sf) obtained from wes and wgs for use by themselves and their relatives. a study of motivation among adults to participating in wgs research (n=322) identified altruism and the expectation that the genetic research will improve the understanding of the etiology of disease, leading to the development of treatments for disease, to be the main motivating factors for research participation. adult participants enrolled in the national human genome research institute s (nhgri) clinseq study expressed nearly universal intention (294/311 ; 95%) to receive all types of genetic test results, including carrier status and results with no known clinical utility, in the hope that this information would help either themselves or their relatives improve their health outcomes. similar to previous reports, adults (n=35) undergoing personal wgs / wes indicated they would like to receive all wgs / wes results (94%), including the raw data (89%), while, at the same time, expressing worry about the emotional impact and the privacy of the results. on the other hand, in adults referred for clinical diagnostic sequencing, a greater number declined to consent to receipt of at least one category of secondary finding (e.g., a recessive trait, a cancer predisposition syndrome, an adult - onset disease predisposition, or an early - onset disease) for themselves (6/38 ; 16%) and for their children(7/162 ; 4%). in a population - based study of sarcoma patients, their spouses and selected family members (n=1200), evaluating attitudes towards genomic and incidental findings from genetic research, approximately 60% thought favorably about genetic testing for an inherited condition and virtually all the participants were receptive to receiving if where there was clinical utility. in another study, adult patients (13/19 ; 68%), who were clinically diagnosed with lynch syndrome (ls) and who previously received uninformative ls genetic results (i.e., high tumor microsatellite instability in absence of mismatch repair protein expression by immunohistochemistry, or family history suggestive of ls, or uninformative comprehensive testing of the ls - associated genes) indicated that they would like to undergo wes testing and receive all possible results from wes, even variants of unknown significance. findings related to parents motivations and intentions to receive genetic research results for their children are somewhat more varied. in one study, parents (25/25 ; 100%) were interested in disclosure if the genetic abnormality was the cause of their child s condition and if that condition was treatable. they were interested in disclosure of secondary variants only when the associated condition was treatable or preventable. however, fewer (10/25 ; 40%) wanted to learn about secondary variants for untreatable conditions. six parents did not want to learn any results, nine were ambivalent or placed restraints on the type of information being disclosed, and thirteen wanted to learn if they were carriers of an autosomal recessive trait. in an online survey of parents (n=219) interest in obtaining multiplex genetic testing of their children for diverse common adult - onset diseases, all enrolled participants were inclined to have their children tested despite the lack of evidence of benefit in children. finally, parental uptake of genetic testing of tp53, the tumor suppressor gene mutated in li - fraumeni syndrome, was high for children (159/172 families ; 92%), with 137/144 (95%) uptake in families for diagnostic testing (to learn if their family carried a pathogenic tp53 variant) and 22/28 (79%) for predictive testing (to learn if a family member carried the specific tp53 variant already known to exist in their family). we conducted a cross sectional analysis of study subjects responses to define the frequency with which adult clinical research participants consented to being offered clinically - validated, research genetic test results (rr) and incidental findings (if) among members of families at high genetic risk of cancer who were participants in a familial cancer research program. we developed the consents for each research study in 2012, before the distinction between incidental and secondary findings was clearly articulated in the literature. therefore we defined two groups of research findings within the consents : (1) primary genetic research results (rr) (i.e., both new genes relevant to the condition being studied and genetic modifiers), and (2) other genetic findings as incidental findings (if). we assumed that adult participants, who had enrolled in research studies designed to discover the underlying genetic basis of a rare hereditary syndrome, or improve cancer detection methods in rare cancer syndromes, would want to receive all types of genetic results, including clinically - validated, incidental genetic findings that were not the primary focus of our research. we conducted a cross sectional analysis of study subjects responses to define the frequency with which adult clinical research participants consented to being offered clinically - validated, research genetic test results (rr) and incidental findings (if) among members of families at high genetic risk of cancer who were participants in a familial cancer research program. we developed the consents for each research study in 2012, before the distinction between incidental and secondary findings was clearly articulated in the literature. therefore we defined two groups of research findings within the consents : (1) primary genetic research results (rr) (i.e., both new genes relevant to the condition being studied and genetic modifiers), and (2) other genetic findings as incidental findings (if). we assumed that adult participants, who had enrolled in research studies designed to discover the underlying genetic basis of a rare hereditary syndrome, or improve cancer detection methods in rare cancer syndromes, would want to receive all types of genetic results, including clinically - validated, incidental genetic findings that were not the primary focus of our research. the national cancer institute (nci), clinical genetic branch s familial cancer research program contained several studies actively accruing family members including : the li fraumeni syndrome study (lfs, nci protocol 11-c-0255 ; nct-01443468 ; http://lfs.cancer.gov), inherited bone marrow failure syndromes (ibmfs, nci protocol 02-c-0052 ; nct-00027274 ; http://marrowfailure.cancer.gov) and familial testicular cancer (ftc, nci protocol # 02-c-0178;nct-00034424 ; http://familial-testicular-cancer.gov./cgb.html). probands, spouses and their relatives (either affected or unaffected with the relevant syndrome or cancer, or other targeted disease) were participants in these institutional review board (irb)-approved longitudinal cohort studies at the nci, and all subjects provided written informed consent in accordance with health and human services regulation 45 cfr 46. the clinical genetics branch (cgb) integrated specific language soliciting the participants preferences for receipt of research and incidental genetic findings into these three consent documents beginning in january 2012 (text boxes 1 and 2). each study participant entered a field study cohort and subsets of the field cohort entered the clinical cohort and were evaluated at the nih warren magnuson clinical center. members of the study team obtained consent from participants after a detailed discussion of the study, including its aims, benefits and risks. participants were offered a tiered approach to indicating whether or not they wished to receive primary genetic rr or if. the participants were also provided the opportunity to decline future re - contact, thereby limiting their direct participation to the initial visit. however, our research participants rarely declined future re - contact and none of the participants in this analysis declined future re - contact. the consent document informed the participant that cgb s policy is to offer (but not require) return of rr and if which have clinical utility after verifying the genetic alteration in a clia laboratory. once those two conditions were met, the cgb research team would contact participants to inform them that a genetic finding that may be of clinical interest to them has been identified. participants are offered the option to decline disclosure during initial consent and again at time of re - contact. if they agree to learn more about the rr or if, they are offered the opportunity to obtain genetic education, counseling, clinical testing and disclosure. research consent was obtained either during a clinical visit to the nih clinical center or by telephone consent with study personnel. we obtained informed consent from 506 adult (18 years - old) participants enrolled in these three projects between january 2012 and march 2014. in the course of this study, we might identify a genetic change that is felt to alter the cancer risk associated with xxx in such a way that may potentially change clinical management. if such a finding is found and a clinical test for it is available, we will send you a letter to inform you of the finding. you can choose to 1) not receive this information at that time, or 2) receive the information but not have clinical testing done, or 3) receive the information and have clinical testing done to determine whether you have this change. please let us know your preference by initialing one of the following statements : _ [] _ _ _ _ i do not want to be contacted if genetic variants which could potentially alter cancer risk associated with xxx are discovered. _ [] _ _ _ _ i do want to be contacted if genetic variants which could potentially alter cancer risk associated with xxx are discovered. one research focus of this study is to look for changes in genetic material (dna) that could potentially alter cancer risk associated with xxx. in the process of looking for these changes, we might find changes that are not directly related to cancer risk or to xxx, but might be related to other illnesses. if we found changes that are known to cause a certain medical condition, or if we found changes that we think are of clinical utility, we will plan to contact you with the information, unless you prefer not to be contacted for such information. please let us know your preference by initialing one of the following statements : _ [] _ _ _ i do not want to be contacted if genetic changes with potential health implications unrelated to xxx or cancer risk are discovered. _ [] _ _ _ i do want to be contacted if genetic changes with potential health implications unrelated to xxx or cancer risk are discovered. if we find gene changes that are not known to be important at this time, we will not share that information with you. participants completed self - administered questionnaires that captured data on factors that might influence their preference regarding receipt of rf and if, including : age, race, education, marital status, children (yes / no), cancer affected status, number of cancers diagnosed, mutation status (carrier / non - carrier in a mutation - known family, unknown mutation status / untested). the study teams classified each family inheritance pattern [autosomal dominant (ad)/autosomal recessive (ar)/x - linked recessive (xl) or unknown ] after constructing a pedigree based on information from a family history questionnaire, completed by the proband or family contact, in addition to information from medical records and from other relatives. if consented participants had not completed the self - administered questionnaire, members of the study team reviewed the family pedigree to assess the demographics and covariates of individuals as reported in the family history questionnaire. we performed a cross - sectional analysis of the participants choice indicated on their study consent regarding receipt of rr and if discovered through research. descriptive statistics were used to summarize the participants choice regarding receipt of rr and if and participant characteristics. bivariate comparisons were planned, stratified by choice, with selected socio - demographic variables, affected status, variant status and whether the participant had children. the national cancer institute (nci), clinical genetic branch s familial cancer research program contained several studies actively accruing family members including : the li fraumeni syndrome study (lfs, nci protocol 11-c-0255 ; nct-01443468 ; http://lfs.cancer.gov), inherited bone marrow failure syndromes (ibmfs, nci protocol 02-c-0052 ; nct-00027274 ; http://marrowfailure.cancer.gov) and familial testicular cancer (ftc, nci protocol # 02-c-0178;nct-00034424 ; http://familial-testicular-cancer.gov./cgb.html). probands, spouses and their relatives (either affected or unaffected with the relevant syndrome or cancer, or other targeted disease) were participants in these institutional review board (irb)-approved longitudinal cohort studies at the nci, and all subjects provided written informed consent in accordance with health and human services regulation 45 cfr 46. the clinical genetics branch (cgb) integrated specific language soliciting the participants preferences for receipt of research and incidental genetic findings into these three consent documents beginning in january 2012 (text boxes 1 and 2). each study participant entered a field study cohort and subsets of the field cohort entered the clinical cohort and were evaluated at the nih warren magnuson clinical center. members of the study team obtained consent from participants after a detailed discussion of the study, including its aims, benefits and risks. participants were offered a tiered approach to indicating whether or not they wished to receive primary genetic rr or if. the participants were also provided the opportunity to decline future re - contact, thereby limiting their direct participation to the initial visit. however, our research participants rarely declined future re - contact and none of the participants in this analysis declined future re - contact. the consent document informed the participant that cgb s policy is to offer (but not require) return of rr and if which have clinical utility after verifying the genetic alteration in a clia laboratory. once those two conditions were met, the cgb research team would contact participants to inform them that a genetic finding that may be of clinical interest to them has been identified. participants are offered the option to decline disclosure during initial consent and again at time of re - contact. if they agree to learn more about the rr or if, they are offered the opportunity to obtain genetic education, counseling, clinical testing and disclosure. research consent was obtained either during a clinical visit to the nih clinical center or by telephone consent with study personnel. we obtained informed consent from 506 adult (18 years - old) participants enrolled in these three projects between january 2012 and march 2014. in the course of this study, we might identify a genetic change that is felt to alter the cancer risk associated with xxx in such a way that may potentially change clinical management. if such a finding is found and a clinical test for it is available, we will send you a letter to inform you of the finding. you can choose to 1) not receive this information at that time, or 2) receive the information but not have clinical testing done, or 3) receive the information and have clinical testing done to determine whether you have this change. please let us know your preference by initialing one of the following statements : _ [] _ _ _ _ i do not want to be contacted if genetic variants which could potentially alter cancer risk associated with xxx are discovered. _ [] _ _ _ _ i do want to be contacted if genetic variants which could potentially alter cancer risk associated with xxx are discovered. one research focus of this study is to look for changes in genetic material (dna) that could potentially alter cancer risk associated with xxx. in the process of looking for these changes, we might find changes that are not directly related to cancer risk or to xxx, but might be related to other illnesses. if we found changes that are known to cause a certain medical condition, or if we found changes that we think are of clinical utility, we will plan to contact you with the information, unless you prefer not to be contacted for such information. please let us know your preference by initialing one of the following statements : _ [] _ _ _ i do not want to be contacted if genetic changes with potential health implications unrelated to xxx or cancer risk are discovered. _ [] _ _ _ i do want to be contacted if genetic changes with potential health implications unrelated to xxx or cancer risk are discovered. if we find gene changes that are not known to be important at this time, we will not share that information with you. in the course of this study, we might identify a genetic change that is felt to alter the cancer risk associated with xxx in such a way that may potentially change clinical management. if such a finding is found and a clinical test for it is available, we will send you a letter to inform you of the finding. you can choose to 1) not receive this information at that time, or 2) receive the information but not have clinical testing done, or 3) receive the information and have clinical testing done to determine whether you have this change. please let us know your preference by initialing one of the following statements : _ [] _ _ _ _ i do not want to be contacted if genetic variants which could potentially alter cancer risk associated with xxx are discovered. _ [] _ _ _ _ i do want to be contacted if genetic variants which could potentially alter cancer risk associated with xxx are discovered. one research focus of this study is to look for changes in genetic material (dna) that could potentially alter cancer risk associated with xxx. in the process of looking for these changes, we might find changes that are not directly related to cancer risk or to xxx, but might be related to other illnesses. if we found changes that are known to cause a certain medical condition, or if we found changes that we think are of clinical utility, we will plan to contact you with the information, unless you prefer not to be contacted for such information. please let us know your preference by initialing one of the following statements : _ [] _ _ _ i do not want to be contacted if genetic changes with potential health implications unrelated to xxx or cancer risk are discovered. _ [] _ _ _ i do want to be contacted if genetic changes with potential health implications unrelated to xxx or cancer risk are discovered. you can choose to not receive the information when you are contacted. if we find gene changes that are not known to be important at this time, we will not share that information with you. participants completed self - administered questionnaires that captured data on factors that might influence their preference regarding receipt of rf and if, including : age, race, education, marital status, children (yes / no), cancer affected status, number of cancers diagnosed, mutation status (carrier / non - carrier in a mutation - known family, unknown mutation status / untested). the study teams classified each family inheritance pattern [autosomal dominant (ad)/autosomal recessive (ar)/x - linked recessive (xl) or unknown ] after constructing a pedigree based on information from a family history questionnaire, completed by the proband or family contact, in addition to information from medical records and from other relatives. if consented participants had not completed the self - administered questionnaire, members of the study team reviewed the family pedigree to assess the demographics and covariates of individuals as reported in the family history questionnaire. we performed a cross - sectional analysis of the participants choice indicated on their study consent regarding receipt of rr and if discovered through research. descriptive statistics were used to summarize the participants choice regarding receipt of rr and if and participant characteristics. bivariate comparisons were planned, stratified by choice, with selected socio - demographic variables, affected status, variant status and whether the participant had children. the study population was primarily white, well - educated and married with children (table 1). in addition, 74% of the individuals were unaffected with cancer and 32% were known or obligate mutation carriers of a known cancer susceptibility gene, the latter determined by pedigree analysis (table 2). of the 506 individuals who signed informed consent documents, only 16 (3%) indicated that they did not want to receive genetic rr and/or if (table 2). due to the small number of participants who declined to receive rr and/or if, no bivariate comparisons were conducted. participants who declined to receive both rr and if (n=7 ; table 3) include one who survived testicular cancer at age 25, was currently disease - free at age 49 years, and a familial testicular cancer (ftc) study participant. a second participant was a 67 year - old female who was aware of her fanconi anemia (fa) carrier status prior to study entry ; she had one child affected with fa. a third participant was the spouse of a known tp53 mutation carrier, who had no personal or family history suggestive of a hereditary cancer susceptibility syndrome. two others were parents of a child with diamond blackfan anemia (dba), an inherited bone marrow failure syndrome in which up to 50% of new cases are caused by de novo dominant germline mutations. the last was a sibling (phenotypically unaffected / untested) of a participant with dyskeratosis congenita (dc) who was phenotypically affected but without a mutation in any of the known dc genes. four participants declined receipt of rr only (table 3) ; one was the spouse of a known tp53 mutation carrier, without a personal or family history of cancer suggestive of a hereditary cancer syndrome ; two were unrelated participants who were aware of their fa carrier status prior to study entry and one participant who was a known tp53 mutation carrier. finally, of the participants (n=5) who declined receipt of if only (table 3), four were already aware of their mutation status (either true - positive or true - negative), and one was an individual who had the dc clinical phenotype but had not been tested for the known genes associated with dc. nearly all the research participants enrolled in the cgb s family research studies of rare, hereditary cancer syndromes consented to be offered disclosure of rr and if, if discovered. this finding is consistent with other highly - motivated persons who choose to enroll in a research study designed to discover the underlying genetic cause of disease in their families. of the few family members who did decline either rr or if, several already knew their personal underlying genetic risk or knew that they were not at risk (spouses of mutation - positive or mutation - negative family members). we can speculate that the known mutation carriers who declined rr or if already had sufficient information relative to their family s genetic risk, or were not interested in or prepared for additional information about themselves. one such individual was a cancer survivor in his late 40 s without offspring who perhaps felt that the information would nt be useful for personal healthcare decision - making. this analysis clearly demonstrates that the vast majority of individual participants in a family cancer research program are open to considering disclosure of both primary genetic rr and if. our results are similar to those observed in various adult study participant populations, in that they profess to be eager for the return of rr and if. similarly, genetics professionals largely support the return of rr and if when the findings have clinical utility for adult patients (85%), support the return of pediatric rr and if for adult - onset conditions (62%) and support disclosing carrier status of children (62%). the majority of genetics professionals also felt that individual patient preferences should guide whether and when to disclose results as well as the option to decline disclosure altogether. for individual patients, the timing of when the results are offered, within the context of their lives, may influence whether or not they are receptive to the return of results. to date, most of what we know about intentions to receive rr and if comes from highly - selected research participants and from small numbers of individuals undergoing clinical diagnostic genetic / genomic testing. we do not currently know whether or not these individuals are representative of the general population in their understanding and acceptance of the results derived from their use of genetic and genomic technologies. prior research suggests that research participants are interested in receiving individual research results, and believe that researchers have an obligation to return them, particularly if they are clinically actionable. however, it is unknown whether research participants and researchers interpret actionable in similar ways, but this is an understudied issue. the evolving legal obligations of the clinicians ordering the tests add to the complexity of the use of genomic technology in clinical care. failure to disclose if discovered in clinical genomic testing could potentially result in legal liability for the provider, for withholding information that might have been used to improve a health outcome. whether these standards will be applied to the research settings is actively being debated. the presidential bioethics commission strongly recommended that all informed consent documents related to wes and wgs data should clearly identify what the intent of the research is, enumerate the specific gene or genes are being targeted for analysis, indicate what uses will be made of the data, including with whom it can be shared, and describe the plan for how rr, if and sf will be managed. currently, disclosure is not mandatory, but each irb must decide for itself whether the proposed disclosure plan is equitable, given the specific study circumstances, and most research programs will likely require additional resources to support high - quality patient education, counseling and disclosure. previous research has demonstrated that there was variable uptake of genetic test results and genetic counseling after patients were notified of the availability of test results. even in families who were well - informed regarding the genetic risk associated with the disease in their family, the actual testing uptake was less than 50%. for individuals with scant information about genetic risk, the quantitative uptake of rr and if in our study population is unknown at present, but will be the subject of future analyses. there are other complexities in the return of genetic rr and if in a study population when compared with clinically identified genetic test results and if. the timing of receipt of genetic rr and if differs significantly when compared with clinically identified primary genetic rr and if. typically, when clinical wes or wgs is performed, the patient is notified within weeks to months about test results being available for disclosure. consequently, at the time of the disclosure both primary genetic test result and any if that are identified are available. although the interpretation of these results may be complex, and variants of unknown significance may be identified, the patient will have the opportunity to discuss the findings with their health care provider within a relatively short period of time. in contrast, when research - based wes or wgs is performed, frequently years elapse between initial consent of the individual and re - contacting them with results. in addition, the results may not all be available at any one given time. as research technologies advance, investigators will most likely re - test the original biospecimen or re - analyze data using new information and seek to re - contact research participants over time. strengths of this analysis include the large number of participants who provided informed consent relative to their preferences regarding receipt of rr and if. in addition, we employed a consistent, uniform informed consent process across all our studies, emphasized achieving high levels of comprehension of the risks and benefits involved in research participation, and facilitated pooling of data from three different protocols. finally, the eligibility evaluation prior to enrollment insured that the research participants and their families were truly at high genetic risk of cancer or closely related to a high - risk family member. one limitation of our findings is the inability to generalize these results beyond the family members who participated in our cancer susceptibility cohorts. additional limitations include a lack of access to all family members within each extended pedigree, which confines our findings to only those family members who chose to enroll and limits the generalization of these findings even within participating families. we also acknowledge that we have no measures of consent comprehension, reason why study participants chose to receive or not receive rr and if, or why a few participants made one selection but not the other. one might speculate that participants who did not indicate an intention to receive rr or if were undecided or may not have fully comprehended the question, and chose not to respond rather than seek clarification ; we have no data at present to support this possibility. in addition, the 260 participants categorized as mutation status untested or unknown are comprised of individuals who were non - bloodline (e.g., a spouse of mutation carrier in a family with a known mutation), individuals who had not been tested for a known familial mutation, and individuals from families in which an underlying genetic etiology of the syndrome has not yet been identified. the potential implications of rr are vastly different for these groups, yet the majority of them chose to receive rr as well as if. finally, the consent documents informed participants that it is the policy of cgb to offer (but not require) return of rr and if which have clinical utility, after verifying the genetic alteration in a clia laboratory. by including this statement in the consent, and not providing an option to consider other rr without clinical utility, we may have inadvertently communicated to participants that this is a normative practice and potentially biased participants toward opting to receive rr and if rather than declining. in this well - defined population of individuals from families at high genetic risk of cancer, adult research participants overwhelmingly indicated their preference to be offered disclosure of genetic rr and if. at this time, none of these 506 individuals have opted out of future re - contact, which provides us the opportunity to evaluate the rate of uptake of rr and if overtime. future research will seek to identify the underlying reasons for refusal of rr and if in the small numbers of those who declined rr and if, and to study the impact of receipt of rr and if in personal medical decision - making among individuals from families at high genetic risk of cancer. in this well - defined population of individuals from families at high genetic risk of cancer, adult research participants overwhelmingly indicated their preference to be offered disclosure of genetic rr and if. at this time, none of these 506 individuals have opted out of future re - contact, which provides us the opportunity to evaluate the rate of uptake of rr and if overtime. future research will seek to identify the underlying reasons for refusal of rr and if in the small numbers of those who declined rr and if, and to study the impact of receipt of rr and if in personal medical decision - making among individuals from families at high genetic risk of cancer.	purposeto define the frequency with which adult research participants consent to be offered clinically - validated research genetic test results (rr) and incidental findings (if).methodsconsents were obtained from 506 adults enrolled in one of three studies within nci s clinical genetics branch s familial cancer research program. a cross - sectional analysis was performed on the choices indicated on study consents regarding receipt of rr and if.resultsninety-seven percent opted to receive rr and if. participants who declined (n=16) included : 2 cancer survivors who were mutation positive (1=rr and 1=both), 8 who knew their primary mutation status (3=rr ; 4=if ; 1= both), 3 non - bloodline relatives (1=rr ; 2=both), 1 untested but with the syndromic phenotype (1=if), and 2 parents of an affected child (2=both). we speculate that these individuals either already had sufficient information, were not prepared to learn more, or felt that the information would nt change their personal healthcare decision-making.conclusionsadult research participants from families at high genetic risk of cancer overwhelmingly indicated their preference to receive both rr and if. future research will seek to identify the reasons for declining rr and if and to study the impact of receipt of rr and if on personal medical decision - making.
shoulder, by virtue of its anatomy and biomechanics, is one of the most unstable and frequently dislocated joints in the body.1 bankart2 (1920) published a paper stating that in acute dislocations the humeral head is forced anteriorly out of the glenoid cavity and tears not only the fibrocartilaginous labrum from almost the entire anterior half of the rim of glenoid cavity, but also the capsule and periosteum from anterior surface of the neck of scapula. the bankart lesion represents the most common form of labro - ligamentous injury in patients with traumatic dislocations of the shoulder. surgical treatment is by reattachment of the labro - ligamentous complex to the glenoid either arthoscopically or during an open procedure (bankart repair).3 several open and arthroscopic techniques have been described to address anterior shoulder instability. these procedures address both capsulo - ligamentous laxity and labral pathologies via a variety of instruments, suture passages, knot - tying techniques, and fixation devices. with the debate continuing regarding the indications for arthroscopic shoulder stabilization, several studies have shown favorable outcomes with regard to the arthroscopic method.46 moreover, with continuing criticisms with regard to the wide dissection, loss of external rotation, and postoperative pain associated with the open repair, the demand for arthroscopic surgery has increased over the past two decades. arthroscopic bankart repair for the treatment of instability of the shoulder has become increasingly popular as it is less invasive than open surgery and produces a better surgical outcome including range of movement and function.6 we report here a retrospective analysis of arthroscopic bankart repair in posttraumatic recurrent anterior dislocation of shoulder. sixty five non consecutive patients with posttraumatic anterior dislocation of shoulder were treated by arthroscopic repair from january 2005 to november 2008, out of them fifty patients who fulfilled the inclusion criterion were reviewed in the study. patients of both the sexes, in the age group of 15 - 50 years, with anterior instability of the glenohumeral joint, which interfered with activities of daily living or athletic activity and having at least two episodes of dislocations, a positive clinical apprehension test and radiological evidence of glenohumeral dislocation were taken up for the study. patients who were excluded from the study were those who were medically unfit for surgery or had either posterior, inferior, or multidirectional instability or habitual dislocation. those with previously failed arthroscopic or open surgery or with fractures involving > 30% of articular surface of gleniod or posterolateral humeral head (engaging hill sachs lesion), and having other unrelated conditions like rotator cuff tears were also not included in the study. patients who presented to the hospital with acute dislocation were managed by immediate close reduction under general anesthesia using the traction - countertraction technique. the reduction was confirmed radiologically and the shoulder joint was immobilized by using a shoulder immobilizer and the patients were asked to return for review in the sports injury center of the hospital. on followup visit at the sports injury center, the patients were subjected to a through history and clinical examination, which gave a good idea about the etiology, direction, and frequency of dislocation. this was supplemented by good quality radiographs in antero - posterior, lateral, axillary, and scapular y view to rule out any bony bankart or significant hill sachs lesion. during the study, 18 patients needed magnetic resonance imaging (mri), due to a discrepancy between the clinical history, examination findings, and the radiographs. mri detected a high grade hill sachs lesion in four patients and rotator cuff tear in three patients, which led to their exclusion. the clinical and radiological findings in all the patients were supported by a diagnostic arthroscopy performed before the procedure. in none of the study subjects were the findings contrary to what we had thought of preoperatively. the patients were explained regarding the procedure, its outcome, complications, and the prolonged rehabilitation protocol. this comparison was also done postoperatively giving us a baseline against which the results were evaluated. modified university of california los angeles (ucla)79 scale was used to evaluate the effectiveness of the arthroscopic bankart repair. the scale was used to evaluate the patient 's pain, function, active forward flexion, strength in forward flexion, and patient satisfaction. the maximum total score possible is 35, with a higher score indicating better shoulder function. we assigned a score of 34 - 35 points as excellent, 28 - 33 points as good, 21 - 27 as mild, and 20 or less as poor. the patients were followed up at 2 weeks, 1 month, and then at 6 monthly intervals. treatment failure was regarded as recurrent shoulder dislocation, any sensation of subluxation or instability preventing return to full activity or requiring a further stabilizing procedure. after induction of general anesthesia, a thorough clinical examination was performed to assess the magnitude and direction of instability. the patient was placed in a lateral decubitus position with arm position between 40 and 60 of abduction and 20 to 30 of forward flexion. peripheral pulses and pulse oximeter readings were evaluated to ensure that axillary structures were not compromised. the shoulder was prepared and draped in a sterile manner, and the bony landmarks were marked carefully to maintain orientation throughout the procedure. a standard posterior viewing portal was established approximately 2 cm inferior and 1 cm medial to the acromial angle. two anterior portals were established using outside - in technique with a spinal needle to establish the most appropriate placement of the cannulas. the antero - superior portal was made in the rotator interval just inferior to the anterior edge of the acromion, and the anterior midglenoid portal was made just over the superior border of the subscapularis tendon. a small cannula (internal diameter 5.5 mm) was inserted into the antero - superior portal, and a large (internal diameter 8.2 mm) threaded cannula was placed in the anterior midglenoid portal. complete diagnostic arthroscopy was done through the posterior and anterior portals, with assessment of the glenoid labrum, capsule, rotator cuff, and the humeral head for possible hill sachs lesions. the goal was to mobilize the labrum such that it could be shifted superiorly and laterally. the bio - suture tak is a 3 mm diameter by 14.5 mm long bio - absorbable push - in anchor. this suture anchor is molded from poly l - lactide - co - d, l - lactide (pldla), which is a noncrystalline, bio - absorbable copolymer. the anchor was loaded with no.2 fibrewire, which is a braided, nonabsorbable, polyblend suture. the first anchor was placed at the 5.00 oclock position for the left shoulder and 7.00 oclock for the right shoulder, care was taken to ensure that the suture anchors are placed on the glenoid face, centered about 2 - 3 mm from the edge of the glenoid cartilage. anchors placed more medial than this risk restoration of the glenoid labrum in a nonanatomic medialized position that will not restore the normal bumper effect of the anterior labrum. the suture anchor used required drilling a pilot hole or using a punch to create the pilot hole prior to impaction of the implant to a countersunk position in the bone. the strand of the suture anchor nearer the labrum was brought out through the antero - superior portal, and in turn through the labrum in a retrograde fashion using the suture passer and retrieved from the midglenoid portal. this suture limb remained as the post during suture tying and this would ensure that the knot rest on the capsular side of the glenoid labrum and not on the articular side. this technique would effectively push the labrum up toward the glenoid socket, restoring labral height and thereby recreating the labral bumper [figures 14 ]. the second and third suture anchors were done at the 4.00 and 3.00 oclock positions for the left shoulder and 8.00 and 9.00 oclock position for the right, in the same manner. the sutures were tied using the tennessee slider knot, which is easy to tie, has a low profile, and possesses good holding strength10 secured by a series of three reversing half - hitches on alternating posts. arthroscopic view showing probe under the torn anterior labrum of a bankart lesion arthroscopic view showing inserting the bioabsorbable suture anchor preloaded with nonabsorbable suture through the anterior - inferior portal arthroscopic view showing a curved, sharp suture - passing device is used to pierce the capsule and labrum lower than the anchor position, ensuring that the capsule and labrum will be shifted up to an anatomic position arthroscopic view showing a completed repair with anchors in positions, note the position of the knots on the capsular side of the repair and the restoration of an anterior bumper effect by the labrum when there was evidence of antero - inferior capsular laxity, the suture passer was passed through the perilabral capsule 1 cm anterior and 1 cm inferior to the bankart lesion to plicate the redundant capsule. this laxity is assessed by the ability to pass the arthroscope between the humeral head and the glenoid at the level of the anterior band of the inferior glenohumeral ligament. postoperatively, we focused on building up their confidence and resorted to a gradual mobilization protocol, conditioning the patient toward increasing level of physical activities. the patients were placed in a sling for 6 weeks. they were allowed to do pendular motion exercises for the first 3 weeks, followed by elevating the elbow to shoulder level (forward active flexion to 90) from the third to the sixth week. data analysis comparing the scores before and after surgery was performed using the wilcoxon sign rank test. there was no statistical relation between the age of the patient and return to sports. eighty percent of the patients were amateurs, indulging only in occasional sports like golf, football, badminton, and lawn tennis and 10 patients were professional sports person, 3 from kabaddi, 3 from basketball, and 4 from cricket. the average number of dislocations before surgery was 2.42 (range 2 - 5). the mean pre- and postoperative range of external rotation was 80.38 and 75.18, respectively. at the end of our study 86% patients (43 cases) had stability compared with the normal sided shoulder and were able to return to sports, three patients involved in professional sports were not able to return back to the game, two because of apprehension and one because of residual instability. four amateurs were not able to return to sports, two because of apprehension or limitation of motion and two because of residual instability. there were no cases of redislocation in our study, however, subluxation (grade i) was observed in three patients. the total ucla score improved from a mean and sd of 18.45 4.71 (range 9 - 28) preoperatively to 32.0 2.64 (range 14 - 35) postoperatively (p < 0.05) [table 2 ]. in one patient, there was breakage of a bio anchor (at 3 oclock) during insertion and we had to do with only two bio anchors (at 5 and 4 oclock) in that patient. but the postoperative scores, range of motion and stability in that patient were good.. however, there has been no concern over this individual 's range of movement [table 3 ]. final clinical results at twenty four months followup pre- and postoperative modified ucla scores complications encountered in the study anterior glenohumeral instability is the most common form of instability around the shoulder joint.1112 it usually affects young adults and most of the cases arise secondary to traumatic dislocations. rowe and zarins reported a rate of 95.6% traumatic origin to anterior dislocation in their study that included 500 patients.13 similarly, all patients in our study had recurrent anterior glenohumeral instability following initial traumatic anterior dislocation. detachment of the antero - inferior labrum (the bankart lesion) facilitates recurrent anterior instability. the socket - deepening effect of the glenoid labrum has been proved to be an important factor in maintaining stability.1415 reattaching the labrum onto the articular surface restores its socket - deepening bumper effect. this is accomplished using sutures and suture anchors, which can be done either open or arthroscopically.1516 capsular laxity is the other reason for glenohumeral instability. lack of diagnosing and treating variable capsular laxity accompanying bankart lesions may cause failure of repair.1718 for a perfect shoulder instability repair result, all the facts causing instability must be understood and treated appropriately. historically, arthroscopic repair for the treatment of the bankart lesion had been less satisfactory than the open technique.5 arthroscopic techniques described previously were using transglenoid sutures or bio - absorbable tacks19 in the past few years, newer techniques involving suture anchor fixation and capsular plication have evolved, with promising results. suture anchors are low - profile fixation devices that minimize articular surface damage of the humeral head, offering anatomic reconstruction of the glenoid labrum as well as the glenohumeral ligament complex. any redundant or loose capsule is also addressed during the same operation, allowing one to address any capsular laxity, restoring tension in the anterior - inferior glenohumeral ligament and stability to the glenohumeral joint. open method of bankart repair has several limiting factors, which renders it a less favorable option. it causes an increased blood loss during surgery, a prolonged period of stay in the hospital and a significant loss of range of motion. in the classic open bankart repair there is disruption of the subscapularis tendon, which may result in postoperative subscapularis insufficiency;20 in addition, there have been reported cases of postoperative subscapularis tendon rupture.2021 the arthroscopic bankart repair offers minimally invasive approach with less surgical trauma and blood loss, with improvements in operating time, perioperative morbidity, narcotic use, hospital stay, time loss from work, and decrease number of complications together with a lower cost of surgery.2224 postoperative recovery and rehabilitation is faster than open surgical techniques. patients are able to have a good range of motion functionally, especially external rotation, which allows them to return to their sports or high - demand jobs.2527 we have also shown that postoperative range of motion is not sacrificed for the sake of stability, with a mean of 80.38 of external rotation. we used the ucla system because it was one of the first shoulder outcome measures that was introduced, the test is easy to administer and clinicians who want to quickly and simply evaluate outcomes for a variety of diagnoses find ucla to be helpful.2830 we found a lot of research papers based on this score3631 and so a formal comparison between different studies could be performed. although it lacks formal validation, we have included it because of its historic standing and continued popularity. in one patient, there was breakage of a bio anchor (at 3 oclock) during insertion. bottoming out of the anchor in the tunnel should be avoided, one must insure that the hole is properly drilled and oriented and the drill guide is properly seated and does not move when the screw is being inserted and one should avoid impacting the anchor beyond the mark of the inserter.32 shane.33 observed a number of bio - absorbable suture anchors that break with screw in insertion and also reported that there was an inconsistency in the quality of the bio - absorbable material in the suture anchors. cole and provencher34 believed that insertion techniques are of utmost importance when using bio anchors. there were nine patients with limitation of shoulder movements. out of these nine there were no cases of dislocation observed in our study, however, three cases had mild laxity (grade i) of the joint, which was asymptomatic and not associated with any difficulty in lifting the arm up or limitation of the ability to throw. performed arthroscopic bankart repair, capsular plication, and if necessary thermal capsuloraphy in 53 patients with antero - inferior shoulder instability. after 2 years followup good and excellent results were 92%, and 7.5% of them had recurrence.27 mishra and fanton reported a failure rate of 7% with arthroscopic bankart repair combined with thermal treatment.35 sedeek. reached at a 92.5% successful rate after arthroscopic treatment of 40 shoulders.3 our results with instability in 6.0% patients was similar to the above mentioned studies. in a prospective study by karlsson. comparing arthroscopic and open methods, after a mean duration of 28 months, external rotation was 80 in open group and 90 in arthroscopic group postoperatively.36 gartsman.26 and synder.37 both reported a 5 degree decrease in external rotation and kim.24 reported 4 in their series. full details of the events in the postoperative period and the period of supervised physiotherapy were not always available. the study population was small and also the study did not take up any comparison between the open and the arthroscopic procedure. to conclude arthroscopic bankart repair with suture anchors for recurrent anterior glenohumeral instability	background : the bankart lesion represents the most common form of labro - ligamentous injury in patients with traumatic dislocations of the shoulder leading to shoulder instability. we report the clinical outcome of arthroscopic repair of bankart lesion in 50 patients.materials and methods : sixty five patients with posttraumatic anterior dislocation of shoulder were treated by arthroscopic repair from jan 2005 to nov 2008. fifty patients, with an average age of 26.83 years (range 18 - 45 years), were reviewed in the study. the average followup period was 27 months (range 24 - 36 months). university of california los angeles shoulder rating scale was used to determine the outcome after surgery. the recurrence rates, range of motion, as well as postoperative function and return to sporting activities were evaluated.results:thirty six patients (72.0%) had excellent results, whereas seven patients (14.0%) had good results. the mean pre- and postoperative range of external rotation was 80.38 and 75.18, respectively. eighty - six percent patients had stability compared with the normal sided shoulder and were able to return to sports. there were no cases of redislocation observed in this study ; however, three cases had mild laxity of the joint.conclusion:arthroscopic bankart repair with the use of suture anchors is a reliable treatment method, with good clinical outcomes, excellent postoperative shoulder motion and low recurrence rates.
the adenomatoid odontogenic tumor (aot) represents 3%7% of all odontogenic tumors and was once considered as a variant of ameloblastoma. microscopically, aot exhibits tubular characteristic and duct - like structures that led to the term the basal cells of oral epithelium were a potential source of origin. in the latest edition of who classification of odontogenic tumors in 2005, aot was classified into the first group of tumors (odontogenic epithelium without ectomesenchyme) instead of the second group (odontogenic epithelium with ectomesenchyme). because of the absence of ectomesenchyme in immunohistochemical staining, dysplastic dentin, aot is now considered the result of a metaplastic process rather than epithelial - ectomesenchyme interaction. in this paper, a rare odontogenic tumor with histopathologic features resembling aot along with hard tissue formation the armed forces institute of pathology (afip) in united states of america has described a similar neoplasm with recurrence potential and suggested the term adenoid ameloblastoma with dentinoid for these lesions. a 24-year - old indian female was referred by her general practitioner for evaluation of a maxillary swelling to department of oral medicine, faculty of dental sciences, institute of medical sciences, banaras hindu university. intraoral examination disclosed a nontender expansion of the left maxilla, covered by normal mucosa (figures 1(a) and 1(b)). an orthopantomogram revealed the presence of a significant unilocular radiolucent area with well - defined sclerotic borders, involving an embedded upper left permanent canine (figure 1(c)). a denta scan (64 slice ct scan) showed a well - defined tumour mass covering the complete left maxilla (figures 1(d) and 1(e)). according to the clinical and surgical findings enucleation of the lesion was performed, to completely extirpate the cystic lesion with extraction of upper left canine (figures 2(a), 2(b), and 2(c)). the differential diagnosis was of dentigerous cyst, calcifying odontogenic cyst, calcifying epithelial tumor, odontogenic keratocyst, and unicystic ameloblastoma. using local anesthesia, the surgical sample was fixed in formalin, embedded in paraffin, and stained with hematoxylin - eosin using the standard method. the tumor displayed a cystic pattern with characteristic features of a plexiform - type ameloblastoma, containing microcysts formation. sheets and cords of epithelial cells were observed, which demonstrated a loose arrangement similar to stellate reticulum, intermixed with focal areas showing a whorled appearance. reverse polarity of peripheral cells was prominent and tubular or duct - like structures lined by cuboidal cells were observed in some areas (figures 2(d) and 2(e)). there is a slight female over male incidence, almost 2 : 1, and appears most often in the second decade of life [57 ]. the sex and the age of the patient we described in this report are consistent with the literature. the lesions are typically asymptomatic, but may cause cortical expansion and displacement of the adjacent teeth. the origin of the aot is controversial. because of its predilection for tooth - bearing bone, it is thought to arise from odontogenic epithelium. the tumor has three clinico - pathologic variants, namely, intraosseous follicular, intraosseous extrafollicular, and peripheral. the follicular type (in 73% of all aot cases) is associated with an unerupted tooth ; whereas extrafollicular type (24%) has no relation with an impacted tooth. follicular and extrafollicular types are over two times more located in the maxilla than in the mandible and most of the tumors involve anterior aspect of the jaws. in our case, the tumor was an intrafollicular intraosseous type and also found in the anterior region of the maxilla. although larger lesions reported in the literature, the tumors are usually in the dimensions of 1.5 to 3 cm. radiographically, they usually appear unilocular, may contain fine calcifications, and irregular root resorption is rare [10, 11 ]. this appearance must be differentiated from various types of disease, such as calcifying odontogenic tumor or cysts. the differential diagnosis can also be made with ameloblastoma, ameloblastic fibroma, and ameloblastic fibro odontoma. the patient in the present report presented with no root resorption, but displacement of the adjacent teeth. radiographically, it was easily differentiated from dentigerous cyst, which usually occurs as a pericoronal radiolucency. the histological findings for aot are remarkably similar in the literature [68, 11 ]. the histological features of the tumor were described as a tumor of odontogenic epithelium with duct - like structures and with varying degree of inductive changes in the connective tissue. the tumor may be partly cystic and in some cases the solid lesion may be present only as masses in the wall of a large cyst. the tumor may contain pools of amyloid - like material and globular masses of calcified material. our case was consistent with the common features reported in the literature [10, 11 ].	the adenomatoid odontogenic tumour is a relatively uncommon lesion which mainly affects females in their second decade of life. it exhibits a predilection for the anterior region of the maxilla. the lesion is usually associated with the crown of an embedded tooth, most commonly the maxillary canine. in this paper, we present a case of adenomatoid odontogenic tumor affecting the left maxillary region in a 24-year - old female. the authors also discuss clinical, radiographic, histopathologic, and therapeutic features of the case.
multiple myeloma is a neoplastic plasma cell disorder characterized by clonal proliferation of malignant plasma cells in the bone marrow (bm) microenvironment, leading to bone destruction and marrow failure. the initial diagnostic workup in all patients with multiple myeloma includes a full skeleton x - ray survey to evaluate the lytic bone lesions. for detection of occult lesions, magnetic resonance imaging (mri) of the spine and pelvis is mandatory in all patients with a presumed diagnosis of smoldering multiple myeloma. in case of symptomatic multiple myeloma, several guidelines recommend mri only in certain circumstances, such as ambiguous plain radiographic findings or suspected cord compression, and for delineating the nature and extent of soft tissue masses. on the contrary, mri has been considered as a part of routine evaluation since unsuspected focal lesions and soft tissue plasmacytoma involving the spine and pelvis can be visualized and patterns of mri abnormality may have prognostic significance. thus, the role of a baseline mri in patients with symptomatic multiple myeloma, who have no related symptoms or signs, remains controversial. in this study, we evaluated the diagnostic and prognostic implications of spinal mri in patients who were newly diagnosed with multiple myeloma. we retrospectively analyzed all consecutive patients who were newly diagnosed with symptomatic multiple myeloma and underwent a whole - spine mri before initiation of antimyeloma therapy from 2004 - 2011 at chungnam national university hospital (daejeon, korea). multiple myeloma work - up included serum and urine protein electrophoresis, quantitation of serum immunoglobulin levels, 24-hour urinary protein excretion, serum 2 microglobulin and bm aspirations and biopsies for a morphologic interpretation. plain x - ray skeletal surveys were performed with digital radiographs, including posterior - anterior chest, a skull series, lateral view of the vertebral column, and anterior - posterior views of the pelvis, shoulders, and extremities. we classified the patients using the durie - salmon and international staging systems (iss). a series of mri sequences were performed to permit identification of the bm infiltration pattern and soft tissue masses, including sagittal fat - suppressed t2-weighted magnetic resonance (mr) images, sagittal t1-weighted mr images, axial t1- and t2-weighted mr images, and gd - enhanced axial and sagittal t1-weighted mr images of the whole spine. mri abnormalities not detected in digital radiographs were classified into two categories : malignant compression fractures and extramedullary plasmacytoma. malignant compression fractures were defined as compression fractures with a contour bulging of the involved masses. extramedullary plasmacytoma was classified into two categories, epidural extension and others, such as plasmacytoma in the rib or pelvis (fig. additionally, the patterns of bm infiltration were classified into the following five categories : homogenously diffuse infiltrative, micronodular, macronodular, mixed, and normal appearance (fig. 2). a kaplan - meier analysis was used to estimate overall survival with group comparisons completed using a log - rank test. overall survival was defined from the date of diagnosis until death from any cause, and survivors were censored at the time of last contact. we evaluated the correlation between the two factors, such as mri abnormalities and iss, using a test. basic statistical data were obtained using the spss ver. 17.0 (spss inc., chicago, il). statistical significance is represented by two - tailed p - values, with a cut - off value of 0.05. we retrospectively analyzed all consecutive patients who were newly diagnosed with symptomatic multiple myeloma and underwent a whole - spine mri before initiation of antimyeloma therapy from 2004 - 2011 at chungnam national university hospital (daejeon, korea). multiple myeloma work - up included serum and urine protein electrophoresis, quantitation of serum immunoglobulin levels, 24-hour urinary protein excretion, serum 2 microglobulin and bm aspirations and biopsies for a morphologic interpretation. plain x - ray skeletal surveys were performed with digital radiographs, including posterior - anterior chest, a skull series, lateral view of the vertebral column, and anterior - posterior views of the pelvis, shoulders, and extremities. we classified the patients using the durie - salmon and international staging systems (iss). a series of mri sequences were performed to permit identification of the bm infiltration pattern and soft tissue masses, including sagittal fat - suppressed t2-weighted magnetic resonance (mr) images, sagittal t1-weighted mr images, axial t1- and t2-weighted mr images, and gd - enhanced axial and sagittal t1-weighted mr images of the whole spine. mri abnormalities not detected in digital radiographs were classified into two categories : malignant compression fractures and extramedullary plasmacytoma. malignant compression fractures were defined as compression fractures with a contour bulging of the involved masses. extramedullary plasmacytoma was classified into two categories, epidural extension and others, such as plasmacytoma in the rib or pelvis (fig. additionally, the patterns of bm infiltration were classified into the following five categories : homogenously diffuse infiltrative, micronodular, macronodular, mixed, and normal appearance (fig. a kaplan - meier analysis was used to estimate overall survival with group comparisons completed using a log - rank test. overall survival was defined from the date of diagnosis until death from any cause, and survivors were censored at the time of last contact. we evaluated the correlation between the two factors, such as mri abnormalities and iss, using a test. basic statistical data were obtained using the spss ver. 17.0 (spss inc., chicago, il). statistical significance is represented by two - tailed p - values, with a cut - off value of 0.05. a total of 113 patients with a median age of 65 years (range, 40 to 89 years) were enrolled in the study, and the male to female ratio was similar. the median follow - up duration was 21 months (range, 1 to 90.5 months). based on the durie - salmon staging system, 61.1% of patients were stage iiia and 22.1% of patients were stage iiib ; whereas based on the iss, 40.7% were stage iii, followed by 38.9% in stage ii (table 1). malignant compression fractures were observed in 26 patients (23%), and epidural extensions of plamacytoma were observed in 15 patients (13.3%), while non - epidural extensions of plamacytoma were observed in seven (6.2%). malignant compression fracture combined with epidural extension was observed in six patients and non - epidural extension of plasmacytoma in two patients. two patients had both epidural and non - epidural extensions of plasmacytoma. in total, of the patients with malignant compression fracture, three (11.5%) had no related symptoms or signs. of the patients with epidural and non - epidural extensions, seven (46.7%) and four (57.1%), respectively, had no related symptoms or signs. malignant compression fracture and epidural extension of plasmacytoma was significantly related to the symptoms and signs, but non - epidural extension of plasmacytoma did not correlate to any of the related symptoms and signs (table 2). malignant compression fracture did not affect the overall survival (p=0.056) ; however, patients with extramedullary plasmacytoma had significantly poorer survival rates (p < 0.001) with either epidural extension (p=0.009) or nonepidural extension (p=0.028) of plasmacytoma (fig. 3). diffuse infiltrative pattern was found in 35 patients (31.0%), mixed pattern in 17 (15.0%) and normal pattern in seven (6.2%). however, all of the patients with normal pattern had < 33% bm plasma cells (table 5). thus, when patients were divided into two groups, mixed patterns showed significantly poorer survival rates than the other group (p=0.030) (fig. 4b). epidural extension and non - epidural extension of the plasmacytoma, iss, creatinine, total calcium, and lactate dehydrogenase were statistically significant in an univariate analysis. in a multivariate analysis using these prognostic factors, non - epidural extramedullary plasmacytoma and iss iii were significant prognostic indicators of poor outcome (hazard ratio [hr ], 3.49 ; 95% confidence interval [ci ], 1.126 to 10.819 ; p=0.03 and hr, 2.722 ; 95% ci, 1.109 to 6.681 ; p=0.029, respectively) (table 6). a total of 113 patients with a median age of 65 years (range, 40 to 89 years) were enrolled in the study, and the male to female ratio was similar. the median follow - up duration was 21 months (range, 1 to 90.5 months). based on the durie - salmon staging system, 61.1% of patients were stage iiia and 22.1% of patients were stage iiib ; whereas based on the iss, 40.7% were stage iii, followed by 38.9% in stage ii (table 1). malignant compression fractures were observed in 26 patients (23%), and epidural extensions of plamacytoma were observed in 15 patients (13.3%), while non - epidural extensions of plamacytoma were observed in seven (6.2%). malignant compression fracture combined with epidural extension was observed in six patients and non - epidural extension of plasmacytoma in two patients. two patients had both epidural and non - epidural extensions of plasmacytoma. in total, of the patients with malignant compression fracture, three (11.5%) had no related symptoms or signs. of the patients with epidural and non - epidural extensions, seven (46.7%) and four (57.1%), respectively, had no related symptoms or signs. malignant compression fracture and epidural extension of plasmacytoma was significantly related to the symptoms and signs, but non - epidural extension of plasmacytoma did not correlate to any of the related symptoms and signs (table 2). malignant compression fracture did not affect the overall survival (p=0.056) ; however, patients with extramedullary plasmacytoma had significantly poorer survival rates (p < 0.001) with either epidural extension (p=0.009) or nonepidural extension (p=0.028) of plasmacytoma (fig. diffuse infiltrative pattern was found in 35 patients (31.0%), mixed pattern in 17 (15.0%) and normal pattern in seven (6.2%). however, all of the patients with normal pattern had < 33% bm plasma cells (table 5). patterns of bm infiltration did not influence overall survival (p=0.244) (fig. thus, when patients were divided into two groups, mixed patterns showed significantly poorer survival rates than the other group (p=0.030) (fig. 4b). epidural extension and non - epidural extension of the plasmacytoma, iss, creatinine, total calcium, and lactate dehydrogenase were statistically significant in an univariate analysis. in a multivariate analysis using these prognostic factors, non - epidural extramedullary plasmacytoma and iss iii were significant prognostic indicators of poor outcome (hazard ratio [hr ], 3.49 ; 95% confidence interval [ci ], 1.126 to 10.819 ; p=0.03 and hr, 2.722 ; 95% ci, 1.109 to 6.681 ; p=0.029, respectively) (table 6). a skeletal survey in patients with multiple myeloma was performed conventionally by a plain whole body x - ray. however, plain x - rays had limitations since some areas were not well visualized or had low sensitivity, and analysis was observer - dependent. although the usefulness of novel imaging modalities, such as computed tomography (ct) or positron emission tomography (pet), has been reported, mri remains the standard imaging technique for detecting spine or pelvic lesions and for evaluating patterns of bm infiltration., reported that a whole - body, multi - detector ct led to a significantly lower detection rate than a whole - body mri in patients with multiple myeloma. furthermore, zamagni. and fonti., demonstrated that although pet - ct provides additional and valuable information for the assessment of myeloma bone disease, mri is superior with regard to the assessment of bm involvement in the spine and pelvis. in our study, a whole - spine mri could detect additional skeletal abnormalities that are not shown on digital radiographs in approximately 33% of patients. since some patients did not present any related symptoms and signs, we suggest that a whole - spine mri is needed as a baseline skeletal survey in all patients. in addition, baseline whole - spine mri has prognostic implications in the detection of focal lesions and patterns of bm infiltration. walker., reported that mri was a more powerful tool for detecting focal lesions than plain x - ray bone surveys since 52% of patients with normal metastatic bone survey had a focal lesion on mri, leading to the conclusion that mri can be routinely used for staging, prognosis, and response assessment in myeloma. the percentage of patients in the aforementioned report was higher than our current study because they included osteolytic lesions as a focal lesion. we classified patterns of bm infiltration in mri into five categories based on a previous description. in the literature, a micronodular pattern was described as a salt - and - pepper pattern, a macronodular pattern as a focal infiltrative pattern, and a mixed pattern as a combined or variegated pattern. the prognostic significance of diffuse and focal infiltration patterns has been demonstrated previously, whereby patients with a diffuse pattern of infiltration had features of more advanced disease and poorer overall survival [6,15 - 17 ]. in addition, walker., demonstrated that the number of focal lesions had independent prognostic implications. in our study, we did not find a correlation between infiltrative patterns and iss, and similar to the previous report, mixed patterns resulted in a poorer prognosis since they have both focal lesions and diffuse infiltration. it is clear that a large number of focal lesions or extents of involved lesions affect the prognosis. recently, dynamic contrast - enhanced mri was used to evaluate the prognosis of multiple myeloma, suggesting that functional mri techniques may in the near future for identifying patients who could benefit most from anti - angiogenic drugs. serial mri monitoring can be useful in assessing the response to treatment. in our study, we did not perform a follow - up mri in all patients with anti - myeloma therapy ; thus, we could not evaluate the changes in mri patterns or infiltration, although the data warrant a prospective study of serial mri monitoring. there was little selection bias in our study since we performed the whole - spine mri consecutively in all patients diagnosed with multiple myeloma from the beginning of enrollment in 2004 and treated patients using the same strategy at a single institution. in summary, this study demonstrates the importance of a baseline spinal mri in patients with multiple myeloma. spinal mri revealed bone lesions, as well as extramedullary plasmacytoma, which may not be detected in plain radiographs, even in patients with no relevant symptoms or signs. we conclude that spine mri at the time of diagnosis is beneficial not only to surveying skeletal lesions, but also assessing the prognosis in patients with multiple myeloma.	purposethe aim of this study is to determine the diagnostic and prognostic role of baseline spinal magnetic resonance imaging (mri) in patients with multiple myeloma.materials and methodswe enrolled patients newly diagnosed with multiple myeloma from 2004 - 2011 at a single center. abnormal mri findings that were not detected in radiographs have been analyzed and categorized as malignant compression fractures or extramedullary plasmacytoma. the bone marrow (bm) infiltration patterns on mri have been classified into five categories.resultsa total of 113 patients with a median age of 65 years (range, 40 to 89 years) were enrolled in the study. malignant compression fractures not detected in the bone survey were found in 26 patients (23.0%), including three patients (2.6%) with no related symptoms or signs. extramedullary plasmacytoma was detected in 22 patients (19.5%), including 15 (13.3%) with epidural extension of the tumor. of these 22 patients, 11 (50.0%) had no relevant symptoms or signs. the presence of malignant compression fractures did not influence overall survival ; whereas non - epidural extramedullary plasmacytoma was associated with poor overall survival in the multivariate analysis (hazard ratio, 3.205 ; 95% confidence interval [ci ], 1.430 to 9.845 ; p=0.042). during the follow - up for a median of 21 months (range, 1 to 91 months), overall survival with the mixed bm infiltrative pattern (median, 24.0 months ; 95% ci, 22.9 to 25.1 months) was shorter than those with other patterns (median 56 months ; 95% ci, 48.9 to 63.1 months ; p=0.030).conclusionthese results indicate that spine mri at the time of diagnosis is useful for detecting skeletal lesions and predicting the prognosis in patients with multiple myeloma.
a 30 year old male presented to the emergency department complaining of acute severe left loin pain with a visual analogue score of 10 radiating to his groin. his chest was clear and abdominal examination revealed left renal angle tenderness with voluntary guarding. laboratory investigations revealed white cells of 15 10/l (reference range 411 10/l) with a neutrophilia of 13 10/l (reference range 1.57 10/l). the patient s main concern was that he had a trapped nerve from a weights session the night before. he was initially diagnosed with renal colic and admitted for intravenous opioid analgesia and fluid resuscitation. ct kidney, ureter, bladder the next day was difficult to interpret due to paucity of intra - abdominal and retroperitoneal fat but revealed left sided calcifications adjacent to the expected position of the vesicoureteric junction. no proximal obstruction could be visualised and therefore excretory urogram was arranged to confirm the calcification position. laboratory tests were repeated including liver function tests revealing an ast of 378 u / l (reference range 1036 u / l). the following day ct urogram reported the pelvic calcifications to be phleboliths lying outside the urinary tract. the patient was reassessed and found to still be very tender in lumbar area, but also noted to have paravertebral paraesthesia overlying a 10 cm 15 cm area. flexion and extension of the spine significantly worsened his pain, as did left hip flexion. further specifics to the history were clarified ; he had participated in a deadlifting weight training session targeting the lumbar muscle groups. the ct urogram was then re - examined, and the possibility of enlargement and decreased density of the left paravertebral muscles was raised, with an mri suggested for further investigation (fig. 1). the mri confirmed a diagnosis of erector spinae rhabdomyolysis and compartment syndrome with increased uptake in the left paravertebral muscles suggestive of oedema (fig. the role of surgical fascia decompression was limited due to the risk of introducing infection. renal function was monitored closely with daily urea, electrolytes, base excess and serum ph being performed. over the following days he was discharged around two weeks post admission with significant improvement in back pain and function. at one week post discharge loin pain accounts for 2535% of emergency urological admissions, but only 64% due to renal calculi confirmed on ct. acute paravertebral lumbar compartment syndrome is a rare differential diagnosis of loin pain, but this case highlights the need for a systematic approach when assessing patients. compartment syndrome (cs) has various aetiologies and was first described by von volkmann in 1881. cs is caused by raised intra - compartmental pressure of the interstitium over its capillary perfusion pressure impairing capillary flow. interruption of the local microcirculation causes endothelial destruction, capillary leak, protein loss and accumulation of fluid in the interstitial and intracellular spaces resulting in lack of blood flow to tissues and ischaemia. untreated, ischaemic tissues can be irreversibly damaged, releasing high levels of muscle enzymes with resulting rhabdomyolysis and acute kidney injury. initial delay in diagnosis influenced the decision to treat our patient conservatively, as it has been previously demonstrated that in cases of cs with delayed diagnosis > 48 h the post - operative risk of infection out weights the benefits of surgery. on review of the literature, in all cases reported of pvcs the main presentation has been intractable lumbar / flank pain with or without radiation 612 h following exertion. on examination lumbar erector spinae muscles are tense and swollen with board like rigidity and an overlying area or paraesthsiae. in some cases patients have been found to have tenderness on deep abdominal palpation with reduced or absent bowel sounds. lumbar lordosis may be reduced and pain was often worse on flexion of the hip and extension and flexion of the spine. whilst exertional cs is well described in the hand, forearm, arm, leg and thigh,,,,, acute exertional paravertebral compartment syndrome (pvcs) has only been reported 15 times previously ; (table 1) on three occasions in downhill skiers,,, once in a surfer and 11 times in weight lifters participating in lumbar specific exercises such as dead lifts,,,,,,,,,. acute pvcs has also presented following direct trauma to the area and after surgical procedures such as abdominal aortic aneurysm repair and roux - en - y gastric bypass,,,. cs of the extremities is well known and presents classically with severe intractable pain, paraesthesia, pallor, paresis and pain on passive stretching. not all of these are seen in paravertebral lumbar cs and therefore a high index of suspicion is required. in two other case reports the patients were also originally mis - diagnosed with ureteric colic, causing delay in initiating appropriate management. on review of the literature, in all cases reported of pvcs the main presentation has been intractable lumbar / flank pain with or without radiation 612 h following exertion. on examination lumbar erector paravertebral lumbar paraesthesiae was frequently commented on and many patients complained of tenderness on deep abdominal palpation with reduced or absent bowel sounds. lumbar lordosis may be reduced and pain was often worse on flexion of the hip and extension and flexion of the spine. all patients with pvcs presented with features of rhabdomyolysis with raised creatine phosphokinase, ast, alt, and raised urinary and serum myoglobin. t2 weighted mri is the gold standard for diagnosis of pvcs with oedema of the affected muscle group. intra - compartmental pressures can be measured with slit catheter insertion to confirm the diagnosis, but given the clinical, biochemical and radiological findings in our case we did not feel that it was required. there is no consensus on the treatment of acute exertional pcvs and currently case reports provide the only standard for comparison. out of the 15 cases reported 7 underwent thoraco - dorsal fasciotomy with immediate improvement in physical symptoms and biochemical parameters. all patients treated surgically recovered well and all were able to return to their previous physical activities over the following 6 months. 8 of the 15 cases were treated conservatively and recovered well from the acute compartment syndrome, although several developed chronic exertional back pain. conservative measures involve aggressive fluid resuscitation and monitoring of urine output to optimise renal perfusion and prevent acute kidney injury. assessment of patients with loin pain requires a systematic approach and it is important for differential diagnoses to be considered. paravertebral lumbar compartment syndrome is a rare cause of lumbar back pain / loin pain but one that should be considered, particularly in active young males. early diagnosis is vital to prevent the potential complications of untreated rhabdomyolysis and to allow consideration of surgical decompression. pvcs should be managed with aggressive fluid resuscitation and monitoring of urine output to avoid acute kidney injury. early surgical decompression has good short and long term outcomes, but frequently initial mis - diagnosis causes delay necessitating conservative treatment. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request.	highlightswe present a rare case of exertional paravertebral lumbar compartment syndrome that was initially misdiagnosed as ureteric colic.exertional lumbar paravertebral compartment syndrome typically present with intractable lumbar back pain 612 h following lumbar specific exercise.there is currently no consensus on management with both conservative and surgical techniques used and case reports the only standard for comparison.long term back pain is more likely in patients managed conservatively.
its etiology is unknown, although cardiovascular disease, hemodynamic compromise, gastric outlet obstruction, alcohol ingestion, hypoxemia, hypercoagulable state, infection, and trauma have been suggested as possible causes. a 67-year - old female presented to an outside hospital with exertional chest pain associated with diaphoresis and dyspnea. she had no previous history of hypertension, diabetes, dyslipidemia, or cigarette smoking. cag findings showed visually a 80% to 90% diffuse stenosis in the distal left circumflex artery (lcx), a 70% to 80% tandem stenosis in the proximal and mid left anterior descending artery (lad) (fig. 1a), and a 50% to 60% diffuse stenosis in the mid right coronary artery (rca). intravascular ultrasound revealed that the minimal luminal area was 2.25 mm and external elastic membrane cross sectional area was 13.5 mm at the ostium. we planned percutaneous coronary intervention (pci) for the distal lcx and proximal / mid lad and medical treatment for the mid - rca. however, shortly after balloon inflation for the proximal lad, the patient complained of severe chest pain, and then her blood pressure abruptly fell to 70/40 mmhg. cineangiography showed major dissection in the proximal lad and thrombolysis in myocardial infarction flow grade i to ii (fig. we made an immediate stent deployment by the cross - over technique to the proximal lad from the left main coronary artery. nitroglycerin and adenosine were infused simultaneously via the intracoronary arterial route. after approximately 20 minutes, her vital signs returned to normal and her chest pain subsided. six hours after the index procedure, the patient experienced a large amount of hematemesis. emergency gastrofibroscopy (gfs) was performed to determine the cause of the sudden hematemesis. upper endoscopy showed black macerated mucosa in the mid third of the esophagus and circumferential mucosal necrosis with a huge adherent blood clot in the distal third of the esophagus (fig. she was diagnosed with acute esophageal necrosis, based on the gfs finding and her past history that did not include alcohol consumption, liver disease, varices, peptic ulcer disease, abdominal aortic surgery, nonsteroidal anti - inflammatory drug use, gastroparesis, or previous gastrointestinal bleeding. she was started on pantoprazole (40 mg, twice daily) and continued on aspirin, clopidogrel, and atorvastatin. 3d) months after the event, follow - up upper endoscopy revealed complete resolution of the esophageal lesion. black esophagus, also known as acute necrotizing esophagitis or acute esophageal necrosis, was first described by goldenberg. in 1990. six cases of black esophagus were seen at the mayo clinic, rochester from 1997 through 2003, and 46 cases were reported in the english - language literature from 1963 through 2003. identified 10 cases of acute esophageal necrosis in a review of more than 80,000 upper endoscopies, with an incidence of only 0.0125%. gurvits. demonstrated that the major risk factors include histories of cardiovascular disease, hemodynamic compromise including shock, and severe third spacing. reported a case of black esophagus associated with hypotension that developed during an acute coronary event. also, he reported that endoscopically, the lesion often appears circumferential and black, with friable or macerated mucosa, usually involving the distal two thirds of the esophagus. histopathology usually shows necrotic debris and mucosal and submucosal necrosis with a local inflammatory response. the esophagus receives an intricate segmental vascular supply that separates this organ into three parts : upper, middle, and distal esophagus. the arterial network of the upper esophagus is derived from the descending branches of the inferior thyroid arteries. the mid - esophageal blood supply is derived from the bronchial arteries, right third or fourth intercostal arteries, and numerous small esophageal arteries off the descending aorta. the distal esophagus receives its blood flow from branches off the left gastric artery or left inferior phrenic artery, but variations are common. therefore, since the esophagus has a rich intramural arteriovenous network complementing the segmental blood flow, spontaneous esophageal ischemia followed by necrosis is rare. the overall mortality of acute esophageal necrosis reported in the largest case review to date was high (32%). our patient had no severe comorbidities except for the underlying coronary three - vessel disease, and the esophageal lesion improved with only conservative treatment using an oral proton - pump inhibitor. coronary arterial dissection associated with therapeutic coronary intervention occurs in approximately 32% to 41% of cases. in our case, coronary arterial dissection during the pci led to sudden hypotension, which was suspected to cause acute esophageal ischemia and then necrosis. interventional cardiologists should be aware that not only acute coronary events or chronic cardiovascular diseases but also acute hemodynamic changes that can take place during coronary intervention may cause acute esophageal necrosis.	acute esophageal necrosis is uncommon in the literature. its etiology is unknown, although cardiovascular disease, hemodynamic compromise, gastric outlet obstruction, alcohol ingestion, hypoxemia, hypercoagulable state, infection, and trauma have all been suggested as possible causes. a 67-year - old female underwent a coronary angiography (cag) for evaluation of chest pain. cag findings showed coronary three - vessel disease. we planned percutaneous coronary intervention (pci). coronary arterial dissection during the pci led to sudden hypotension. six hours after the index procedure, the patient experienced a large amount of hematemesis. emergency gastrofibroscopy was performed and showed mucosal necrosis with a huge adherent blood clot in the esophagus. after conservative treatment for 3 months, the esophageal lesion was completely improved. she was diagnosed with acute esophageal necrosis. we report herein a case of acute esophageal necrosis occurring in a patient undergoing percutaneous coronary intervention.
abnormalities in retinal vasculature may reflect similar changes in the blood vessels in other parts of the body, suggesting systemic changes, and unlike the complicated invasive procedures that must undergo a patient to assess the cardiac, renal and other tissue 's microvasculature, a dilated fundus examination can be performed with a routine screening. these organs share pathophysiological mechanisms, such as endothelial dysfunction and the inflammatory process, leading to circulatory abnormalities and decreased vascular reactivity. in some cases, ophthalmic evaluation may lead to the diagnosis of a life - threatening systemic disorder. here, we describe the case of a patient in whom the findings of retinal vasculature led to the diagnosis and management of a hypertensive emergency secondary to an advanced chronic renal disease. a 23-year - old male without previous medical history presented to the emergency department of our institution for evaluation because of sudden and painless vision loss in both eyes of over 1 week without any other systemic complains like headache, dizziness, nausea or vomiting. at evaluation, the best - corrected visual acuity in the right eye was 20/800 and 20/400 in the left eye., both eyes showed mild papillary edema, rectified retinal vessels, with loss of the normal retinal - vein relation due to arteriolar narrowing, macular edema, cotton wool spots all over the arcades and involving the peripapillary area, flame - shaped hemorrhages and vascular tortuosity in the retinal periphery (fig. fluorescein angiography was evident for vascular tortuosity and hyperfluorescent areas with late leakage in the temporal and peripapillary areas in both eyes (fig. 2a, b), and the right eye showed capillary closure in the foveal avascular zone (fig. no optical coherence tomography was required at this moment of the evaluation. on systemic evaluation, the patient had an arterial blood pressure of 220/140 mm hg confirmed on several occasions. considering the diagnosis of secondary hypertension due to the age of the patient, he was referred for a multidisciplinary approach, not requiring any ophthalmic therapy at this moment. the laboratory tests are shown in table 1, while other examinations including liver function tests, coagulation tests and serum electrolytes were within normal limits. a renal ultrasound showed smaller than normal kidneys, the right kidney had dimensions of 65 35 50 mm (normal size 100 50 30 mm), with loss of corticomedullary differentiation and increased echogenicity. the left kidney had dimensions of 66 40 42 mm, with poorly defined edges, with loss of corticomedullary differentiation and increased echogenicity (fig. 3). a renal biopsy showed glomeruli with open capillary loops and normal basement membranes, a well - preserved interstitium without fibrosis nor tubular atrophy, preglomerular interstitial arteriolar vessels with nodular hyaline wall thickening, and mild reduction of lumen. the diagnosis of chronic renal failure was made, and the patient underwent kidney transplantation from a living related donor. the visual acuity of the patient gradually improved to a best - corrected visual acuity of 20/50 in both eyes after normalization of the blood pressure. hypertension is a disease that affects more than 1 billion of individuals throughout the world and is one of the leading causes of death. up to 1% of all patients present hypertensive crises, which can be divided into hypertensive urgency if the condition is characterized only by increase of tensional levels, or hypertensive emergency, the latter being a situation that requires immediate reduction of blood pressure because of acute or progressive end - organ damage. in adolescents and young adults with a hypertensive crisis, a secondary cause of hypertension must be suspected, and among them, renovascular disease is one of the most frequent causes, accounting from 0.2 to 32% of cases. other causes of secondary hypertension must also be considered such as antiphospholipid syndrome, aldosteronism or pheochromocytoma. it has been found that 32% of the patients with hypertension and nephropathy have hypertensive retinopathy because the altered regulatory mechanism appears as thinning of the retinal arterioles and sclerosis that can lead to occlusion and microinfarcts clinically observed as cotton wool spots. the generalized arteriolar narrowing reflects persistent arteriolar damage secondary to long - standing hypertension, and the hemorrhages, microaneurysms and cotton wool spots are related to acute blood pressure elevation. our patient presented with visual loss and bilateral findings on examination that made us suspect systemic diseases, and the blood pressure lead us to vascular pathologies. this case is similar to other previously reported cases in which retinal abnormalities in young patients suggested a systemic condition. hypertensive retinopathy can cause a spectrum of clinical manifestations ranging from arteriolar narrowing to papilledema. this patient was classified as grade 3 retinopathy according to the keith - wagener - barker classification system and as grade 3 according to the scheie classification. malignant hypertension presents with fibrinoid necrosis of afferent arterioles and focal glomeruli necrosis. in secondary malignant hypertension, choroidopathy, the most important markers of damage are micro- or macroalbuminuria, which were present in our patient. treatment of a hypertensive crisis consists in the immediate control of blood pressure to prevent further end - organ damage. renal atrophy secondary to chronic renal failure leads to the definitive treatment of renal replacement because the medical management alone does not provide long - term systemic control. the comprehensive management of patients with suggestive ophthalmic findings is important because many of them can represent initial manifestations of systemic diseases and besides causing loss of vision may threaten the patient 's life ; this case highlights the importance of a correlation of the ophthalmologic manifestations with systemic abnormalities as well as to make appropriate and multidisciplinary approaches.	the purpose of this paper was to report the case of a 23-year - old patient suffering from bilateral acute visual loss who received the diagnosis of hypertensive retinopathy. after systemic evaluation, he was diagnosed with bilateral renal disease and chronic renal failure, requiring a kidney transplantation to manage the systemic illness, followed by gradual improvement of his visual acuity.
mosquitoes were sampled in a rural area, on the santa rosa farm (24.78951s 047.78068w, south american datum 69), located at the serra azul neighbourhood, prefeito ivo zanella state highway, sp-222, km 87, iguape (fig. 1). fig. 1 : location of collection area in the municipality of iguape, state of so paulo, brazil, 2012. mosquito collections were carried out monthly over the course of three summer months and three autumn months, totaling six months of field collections (from january - june 2012). a latin square collection design was adopted to avoid potential biases caused by ecological and climatic peculiarities associated with the traps locations (kline 2002) that might influence their performance - either positively or negatively of the traps. three locations were randomly selected on the santa rosa farm, except for the distance among the traps that was somewhat fixed. accordingly, each trap was separated from each other by approximately 200 m. based on the published literature records, in order to avoid interference among the traps they should be kept separate by distances that normally vary from 30 - 50 m (cilek & hallmon 2005, hiwat. to ensure that there was no interference among the traps, we adopted a distance of approximately 200 m. all traps were installed and removed at the same time and period in day one and rotated in the consecutive two days. the cdc - lt and cdc - a were installed at approximately 1.5 m above ground level in a way that the light source, the lurex and co2 release point were at the same distance of the mmi inlet relative to the ground level. the traps were switched among the three locations over consecutive days and were left running for 12 h per day (from 06:00 pm-06.00 am), totaling 36 h per month for each trap. the cdc - lt trap was chosen because it is widely used in entomological surveys. another choice was the cdc trap without light source, but with attractive. according to forattini. (1990), the use of the co2 and lactic acid as attractive in traps increases mosquitoes capture. the mmi was chosen to compare its effectiveness with other traps used in most entomological studies. thus, in the present study, we evaluated a cdc - lt, a cdc - a and an mmi with co2 plus lurex3. the co2 used in the cdc was obtained from a compressed gas cylinder with a controlled flow rate of 450 ml co2 per minute. the release of the co2 in the trap was controlled by a low - pressure valve (swagelok, usa). the cartridge of the lurex3contained 4.88 g of lactic acid incorporated to 13.8 g of a gel matrix, thus releasing 230 mg / day of lactic acid (hoel. the mmi simulates the human presence by releasing co2, heat, humidity and lactic acid provided by the cartridge of the lurex3, identical to that used in the cdc - a. the data obtained with the cdc - lt were employed as the baseline for comparative analyses. this procedure was adopted because the cdc - lt has been widely employed for surveying mosquito fauna (hutchings. mosquitoes were individually identified employing the morphological keys proposed by lane (1953), galindo. (1954), correa and ramalho (1956), consoli and loureno - de - oliveira (1998) and forattini (2002). the efficiency of traps was measured using two parameters : the diversity of species sampled by each trap and the performance of each trap, i.e., how each trap undertook the mosquito sampling. for this, the time spent working on the basis of abundance and species selectivity at the level of subfamily and tribe was analysed. biodiversity (kindt & coe 2005) and venneuler (chen & boutros 2011) of the program r v.2.15.2. the homogeneity of variances was assessed by levene s test and the normality of abundance data was examined by the shapiro - wilk and kolmogorov - smirnov statistical tests. the diversity of species per trap was estimated by the diversity index obtained in the profile of the rnyi series (melo 2008) that generalises total richness (= 0), diversity (shannon - weiner = 1 ; simpson - yule = 2) and dominance (berger - parker = inf). the kruskal - wallis (kw) test (p > 0.05) was used to assess differences among the rnyi diversity index estimated for each trap. the bonferroni test was employed to perform multiple comparisons among the traps testing each pair of means. bootstrap values were used to estimate the total species richness expected for each trap. jaccard (cj) and sorensen (cn) indexes were used to address the similarity of the species captured by the traps. the selectivity of traps estimated as part of trap performance was evaluated by the pearson chi - squared test () using the spss v.17.0 program. correspondence analysis (spss program v.15.0) which seeks to synthesise the mass of data with ease of implementation and interpretation was employed as a complement to the test and can graphically display the association observed, because of similar categories are placed more closely to each other (hoffman & franke 1986, carvalho. this study showed the relationship among the selectivity of each trap relative to species grouped on the basis of the subfamilies and tribes they belong to and the trap employed. the venn diagram was constructed to graphically illustrate the species shared by each pair of traps, by all the traps and those captured exclusively by each individual trap. of these, 860 individuals (4.25%) were damaged and were not identified. consequently, 18,156 specimens were identified and grouped into 64 species and 11 taxonomic units (table i). the cdc - lt captured 18.82% (3,417) of all samples, with an average of 625 mosquitoes per month or 17 insects per hour. the cdc - a captured 26.10% (4,739) of the sample, with an average of 838 individuals per month, i.e., 23 mosquitoes / hour. the mmi trap captured 55.08% (10,000) of the insects representing an average of 1,706 specimens per month or 47 culicidae / hour. the statistical test to check the homoscedasticity of the variances of the observed data between the three traps showed a p value = 0.001 with a statistical significance of p > 0.05. regarding the tests for assessing the normality of the data distribution, it was seen that for each of the trap the values presented by the shapiro - wilk and kolmogorov - smirnov tests were p = 0.000 for both tests, with a 5% level of significance, indicating that the mosquito abundance observed in the traps did not have a normal distribution. thus the necessary conditions for the employment of the parametric tests were met ; accordingly non - parametric statistics were adopted for the analysis of the data. table ispecies and taxonomic units abundance per sampling trap in rural areas in the tropical atlantic rainforest, southeastern brazil traps n (%) species / taxonomic unitabctotal (n)anophelinae anopheles (anopheles) costai 3 (1.08)64 (23.02)211 (75.9)278 anopheles (anopheles) intermedius 1 (7.14)1 (7.14)12 (85.71)14 anopheles (kerteszia) bellator 0 (0)9 (13.85)56 (86.15)65 anopheles (kerteszia) cruzii 0 (0)54 (21.86)193 (78.14)247 anopheles (nyssorhynchus) evansae 1 (100)0 (0)0 (0)1 anopheles (nyssorhynchus) galvaoi 0 (0)0 (0)2 (100)2 anopheles (nyssorhynchus) oswaldoi 0 (0)0 (0)1 (100)1 anopheles (nyssorhynchus) triannulatus 0 (0)0 (0)4 (100)4 total5 (0.82)128 (20.91)479 (78.27)612aedini ochlerotatus (protomacleaya) argyrothorax 0 (0)0 (0)1 (100)1 ochlerotatus (chrysoconops) fulvus 0 (0)2 (100)0 (0)2 ochlerotatus (ochlerotatus) scapularis 4 (2.26)56 (31.64)117 (66.1)177 ochlerotatus (protoculex) oligopistus 0 (0)1 (100)0 (0)1 ochlerotatus (protoculex) serratus 11 (16.67)42 (63.63)13 (19.7)66 ochlerotatus (protoculex) serratus / nubilus 23 (11.79)112 (57.44)60 (30.77)195 psorophora (grabhamia) cingulata 1 (4.35)10 (43.48)12 (52.17)23 psorophora (janthinosoma) ferox 2 (15.38)3 (23.08)8 (61.54)13 psorophora (janthinosoma) lutzii 0 (0)7 (50)7 (50)14 sallumia hortator 1 (100)0 (0)0 (0)1 total42 (8.52)233 (47.26)218 (44.22)493culicini culex (aedes) amazonensis 10 (30.30)15 (45.45)8 (24.24)33 culex (culex) chidesteri 3 (100)0 (0)0 (0)3 culex (culex) eduardoi 1 (100)0 (0)0 (0)1 culex (culex) nigripalpus 228 (13.02)381 (21.76)1,142 (65.22)1,751 culex (culex) quinquefasciatus 2 (8.33)1 (4.17)21 (87.5)24 culex (culex) sp.30 (81.08)3 (8.11)4 (10.81)37 culex (culex) sp. coronator group6 (100)0 (0)0 (0)6 culex (microculex) imitator 1 (100)0 (0)0 (0)1 culex (microculex) neglectus 1 (100)0 (0)0 (0)1 culex (microculex) pleuristriatus 0 (0)0 (0)1 (100)1 culex (melanoconion) akritos 71 (55.9)5 (3.94)51 (40.16)127 culex (melanoconion) eastor 5 (100)0 (0)0 (0)5 culex (melanoconion) evansae 1 (100)0 (0)0 (0)1 culex (melanoconion) faurani 0 (0)1 (100)0 (0)1 culex (melanoconion) misionensis 1 (100)0 (0)0 (0)1 culex (melanoconion) oedipus 1 (100)0 (0)0 (0)1 culex (melanoconion) pedroi 39 (59.1)27 (40.9)0 (0)66 culex (melanoconion) putumayensis 2 (100)0 (0)0 (0)2 culex (melanoconion) rabelloi 6 (100)0 (0)0 (0)6 culex (melanoconion) ribeirensis 691 (43.51)628 (39.55)269 (16.94)1,588 culex (melanoconion) sacchettae 1,360 (32.04)1,862 (43.86)1,023 (24.1)4,245 culex (melanoconion) sp. atratus group1 (100)0 (0)0 (0)1 culex (melanoconion) sp. melanoconion section171 (72.15)46 (19.41)20 (8.44)237 culex (melanoconion) spissipes 4 (57.14)3 (42.86)0 (0)7 culex (melanoconion) vaxus 2 (100)0 (0)0 (0)2 culex (melanoconion) zeteki 1 (50)0 (0)1 (50)2 lutzia (lutzia) bigoti 1 (100)0 (0)0 (0)1 total2,639 (32.38)2,972 (36.46)2,540 (31.16)8,151mansoniini coquillettidia (rhynchotaenia) albicosta 4 (22.22)3 (16.67)11 (61.11)18 coquillettidia (rhynchotaenia) chrysonotum 227 (3.46)1,144 (17.45)5,186 (79.09)6,557 coquillettidia (rhynchotaenia) hermanoi 17 (13.39)34 (26.77)76 (59.84)127 coquillettidia (rhynchotaenia) juxtamansonia 0 (0)2 (50)2 (50)4 coquillettidia (rhynchotaenia) venezuelensis 352 (20.29)151 (8.7)1,232 (71.01)1,735 mansonia (mansonia) titillans 1 (20)1 (20)3 (60)5 total601 (7.11)1,335 (15.81)6,510 (77.08)8,446sabethinii limatus durhami 0 (0)6 (28.57)15 (71.73)21 limatus flavisetosus 1 (11.11)0 (0)8 (88.89)9 runchomyia (runchomyia) reversa 1 (5.26)6 (31.58)12 (63.16)19 wyeomyia (phoniomyia) davisi 0 (0)0 (0)4 (100)4 wyeomyia (phoniomyia) fuscipes 0 (0)0 (0)1 (100)1 wyeomyia (phoniomyia) galvaoi 0 (0)0 (0)18 (100)18 wyeomyia (phoniomyia) longirostris 0 (0)1 (20)4 (80)5 wyeomyia (phoniomyia) mulhensi 0 (0)0 (0)1 (100)1 wyeomyia (phoniomyia) pallidoventer 0 (0)0 (0)1 (100)1 wyeomyia (phoniomyia) palmata 0 (0)1 (50)1 (50)2 wyeomyia (phoniomyia) pilicauda / incauda 0 (0)0 (0)5 (100)5 wyeomyia (phoniomyia) near lassali 0 (0)0 (0)1 (100)1 wyeomyia (phoniomyia) near longilostris 0 (0)0 (0)3 (100)3 wyeomyia (phoniomyia) quasilongirostris 0 (0)2 (16.67)10 (83.33)12 wyeomyia (phoniomyia) theobaldi 0 (0)0 (0)88 wyeomyia confusa 2 (1.1)55 (30.22)125 (68.68)182 wyeomyia felicia / pampeithes 0 (0)0 (0)26 (100)26 wyeomyia mystes / finlayi 0 (0)0 (0)3 (100)3 wyeomyia airosai / howardi / luteoventralis 0 (0)0 (0)6 (100)6 total4 (1.22)71 (21.71)252 (77.06)327uranotaeniini uranotaenia (uranotaenia) geometrica 10 (100)0 (0)0 (0)10 uranotaenia (uranotaenia) incognita 1 (50)0 (0)1 (50)2 uranotaenia (uranotaenia) mathesoni 7 (100)0 (0)0 (0)7 uranotaenia (uranotaenia) natalie 5 (100)0 (0)0 (0)5 uranotaenia (uranotaenia) pulcherrima 103 (100)0 (0)0 (0)103 total126 (99.21)0 (0)1 (0.79)127total specimens3,417 (18.82)4,739 (26.1)10,000 (55.08)18,156total species / taxonomic unit47355075a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito magnet independence. a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito magnet independence. the results of the test showed the presence of a positive association between the cdc - lt and species of the culicini and uranotaeniini tribes. the species / taxonomic units belonging to the culicini tribe represented 77.23% of the total abundance sampled by the trap (= 4594.040 ; p < 0.000). as compared with the other traps, the cdc - lt was highly selective for species of the ura- notaeniini tribe, capturing 126 individuals, representing 99.21% of the total species of the genus uranotaenia collected by the traps. a, there was a positive association between the trap and representatives of the culicini and aedini tribes. thus 62.72% of the mosquitoes captured in the trap belonged to the culicini (2,972 insects), whereas 4.91% were of the aedini (233 individuals). the mmi showed a positive association with species of the mansoniini and sabethini tribes and the anophelinae subfamily. mosquitoes of the mansoniini represented 65.51% (6,510 individuals) of the specimens obtained in the mmi, 252 mosquitoes were of the sabethini and 479 belonged to the anophelinae subfamily. the mmi was capable of capturing a greater number of anopheles than the cdc - lt and the cdc - a (table ii), about 78% of anopheles collected. the results of the correspondence analysis are given in fig. 2, which shows the associations of the traps with the subfamilies and tribes. table iispecies and taxonomic units abundance grouped into tribes and subfamily per sampling trap in rural areas in the tropical atlantic rainforest, southeastern brazil a n (%) b n (%) c n (%) culicinae aedini42 (1.23)233 (4.91)218 (2.18) culicini2,639 (77.23)2,972 (62.72)2,540 (25.4) mansoniini601 (17.58)1,335 (28.17)6,510 (65.1) sabethini4 (0.12)71 (1.5)252 (2.52) uranotaeniini126 (3.69)0 (0)1 (0.01)anophelinae5 (0.15)128 (2.7)479 (4.79)total3,417 (100)4,739 (100)10,000 (100)a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito magnet independence ; = 4594.040 ; gl = 10 ; p = 0,000. a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito magnet independence ; = 4594.040 ; gl = 10 ; p = 0,000. 2 : correspondence analysis graphically represents the associations of the traps (cdc - a : cdc with co2 and lactic acid ; cdc - lt : cdc light trap ; mm : mosquito magnet) and the tribes and subfamily. to address the diversity of culicids per trap, only individuals identified to the species level the results of the kw test indicated that the observed differences between the rnyi diversity index (fig. 3) were statistically significant only as regards richness (kw = 19.338 ; p = 6.321 e-05). the results of the bonferroni test indicated that the mmi presented a significant difference as compared to the cdc - lt (p = 6e-05) and the cdc - a (p = 0.0012). however, the cdc - a showed no significant difference (p = 0.6059) compared with the cdc - lt. the other rnyi diversity index were not statistically significant, shannon - weiner (kw = 0.9298 ; p = 0.6282), simpson - yule (kw = 2.3813 ; p = 0.304) and berger - parker dominance (kw = 3.4419 ; p = 0.1789). the expected values obtained by the bootstrap analyses were closer to the observed richness in the mmi data because the species captured represented 88.7% of the expected species (sobserved = 44 ; bootstrap = 49.6). the cdc - lt was able to sample 83.5% (sobserved = 44, bootstrap = 52.7) and the cdc - a 76.7% (sobserved = 33, bootstrap = 43) of the expected species. the mmi and cdc - lt traps presented the same number of species, with some species common to both traps and other species that were captured by one specific trap. the cj (0.57) and the cn (0.72) values showed similarities between species captured by the cdc - a and mmi traps (supplementary data 1). the venn diagram (fig. 4) illustrated species distribution in each trap, as well as those that were shared by two or three traps. a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito magnet independence. 4 : venn diagram illustrating the similarity of the mosquito species captured and shared between traps in the municipality of iguape, state of so paulo, brazil. a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito magnet independence. results of the analyses conducted for the present study clearly demonstrated that the mmi is more efficient at capturing mosquitoes than the cdc - lt or the cdc - a. in this context, the mmi captured 2.0 and ~2.7 times more culicids than the cdc - a and the cdc - lt, respectively. (2010) when comparing the performance of different models of mm with the cdc trap. recently, de s and sallum (2013) obtained similar results by employing the set of traps in different locations and with lower sampling effort. the traps used in this study captured species of importance to public health. among them it is worth mentioning the coquillettidia venezuelensis that is competent to transmit the oropuche virus and the eastern equine encephalitis virus (eeev) (forattini 1965, rosa. anopheles cruzii and anopheles bellator are local vectors of plasmodium sp. (deane. 1984, forattini. 1996) that can cause disease in humans. ochlerotatus scapularis, captured in great abundance by mmi, is involved in the transmission of several viruses, as for instance, the melon virus, ilhus virus, rocio virus and venezuelan equine encephalitis virus (spence. 1962, arnell 1976, forattini. culex nigripalpus is a vector of the saint louis virus, west nile virus and eeev (forattini. the cdc - a captured a larger number of ochlerotatus serratus and oc. serratus / nubilus with vector competence for the yellow fever virus (yfv), ilhus, oropuche, aura, tocara and san luis viruses (forattini 1965, vasconcelos. culex ribeirensis with vector competence for the eeev (santos - neto & lozovei 2008) was captured with greater abundance by the cdc - lt. regarding selectivity, the cdc - lt showed a statistically significant positive association with species of the culicini and uranotaeniini tribes. as the uranotaeniini species feed on the blood of amphibians (cupp. 2004), the attractant factor was the light source of the cdc - lt that was absent from the cdc - a. the traps were linked to kairomones that attract insects that carry the blood obtained from mammals, including humans (dugassa. 2013), thus the absence of mosquitoes of the genus uranotaenia in the mmi was also observed by de s and sallum (2013). the cdc - a showed a positive association with species of the culicini and aedini tribes. it is noteworthy that the cdc - a estimated the greatest abundance of oc. the presence of these mosquitoes in the cdc - a indicates the possibility of employing it for monitoring mosquito vector species of medical importance. for instance, for surveillance of species that is potentially involved in the introduction, establishment and dispersion of the yfv in new areas. according to cardoso. (2010a), a new subtype of yfv had been isolated from specimens of oc.. results of a study conducted by laporta and sallum (2011) in a preserved area of the atlantic forest in southeastern sp, using cdcs traps associated with co2 and 1-octeno-3-ol, showed that oc. serratus was the second most abundant species sampled in the preserved forest area. considering that oc. serratus is present in both preserved forest areas as well as in rural areas surrounded by secondary forests we may be permitted to hypothesise that the species acts as a bridge vector for dispersing the yfv from forest to rural cycles. the mmi was positively associated with species of the mansoniini tribe and with coquillettidia chrysonotum as the most abundant species. chrysonotum has not yet been incriminated in the transmission of pathogens to humans ; however, its aggressive blood feeding behavior (forattini 2002) is a cause of discomfort and annoyance to humans and domestic animals. as has been seen, the mmi trap performed well in sampling species of mosquitoes that have the potential to become pests in urban and rural environments. a second model of trap, the mm professional (mmpro), has been employed for the control of culicoides spp in the peridomestic environment in coastal areas of florida, united states of america (cilek & hallmon 2005). the mmpro is capable of sampling several other groups of bloodsucking insects as well as mosquitoes. furthermore, the mmpro has been utilised for assessing biodiversity parameters and species distribution in urban, rural and natural environments in belgium (versteirt 2013). in relation to the subfamily anophelinae, the mmi trap sampled the largest number of representatives of the genus anopheles (479 individuals).. currently, almost 99.5% of human malaria cases reported in brazil are the amazon region. some cases do occur also outside the area of active transmission, in extra - amazonian areas, where malaria is manifested as isolated cases or in small outbreaks occurring mainly under the conditions associated with the atlantic forest biome (oliveira - ferreira. bellator are local vectors of species of the genus plasmodium sp. that cause malaria in humans. the transmission cycle of the pathogen may involve monkeys of the genus cebus and allouata (oliveira - ferreira. 2010, yamasaki. 2011). in the study area, the mmi collected a higher number of an. thus, in the light of the results of this study within the context of entomological surveillance, the mmi was more effective for monitoring mosquito species of public health importance. other models of mm trap have been tested for surveillance of those anopheles species that are vectors of plasmodium. (2012) examined the effectiveness of mm liberty plus (mmlp) regarding the method of capture by human attractant in an area where malaria is endemic, in the state of bolivar, venezuela. they argued that the mmlp can be employed for monitoring anopheles populations. in a study conducted in the state of mato grosso, brazil, 2011) compared the mm defender with human attraction for the sampling of anopheles. the results showed that mm defender was efficient because it captured the highest number of species and estimated the greater abundance of anopheles triannulatus, anopheles benarrochi, anopheles oswaldoi, anophles peryassui and anopheles rangeli. interestingly, this trap did not have the same positive effect in collecting anopheles darlingi. (2011a), in a study in suriname, found that the mm trap has potential for use as an alternative for collecting anopheles aquasalis. the various mm models have been developed to capture synanthropic mosquitoes in open domiciliary environments (cilek & hallmon 2005). however, multiple studies have been conducted to address the effectiveness of the trap in environments with distinct environmental, climatic and ecological conditions, including areas of tropical forest. despite the presence of variations, the results suggest that the trap has potential for use in monitoring activities of mosquito fauna in different regions of the world, since it is able to capture diverse species compositions. in this study, when the composition of species sampled by the mmi was compared with that observed in the cdc - a, greater similarity could be observed between them. this was proven by the similarity index, a result also observed by de s and sallum (2013). the similarity is probably related to attractants (co2 or lurex3) used in the traps, which permitted the collection of species that can carry blood from mammals and other vertebrates. traps which have specific odours attractive to anthropophilic species, particularly combined with co2, may provide improved results in the sampling of these species and have the potential to be effective as methods for capture, for example, in entomological monitoring and ecological studies of malaria vectors (jawara. 2009, 2011, hiwat. it is noteworthy that the flow rate of co2 might influence the mosquitoes collected. on the other hand, the species composition was different (supplementary data 2). the use of cdc - lts for capturing diurnal mosquitoes is not appropriate because it depends on the attractiveness of a point source of light. to improve the performance of the collections made with the cdcs, chemicals attractants (laporta & sallum 2011) or associating ultraviolet (uv) light with the cdc increases the effectiveness of the trap, both for the species sampled and for obtaining males (hutchings. however, other groups of insects may be also captured by a cdc - uv trap, what is not adequate in studies conducted in preserved or even rural environments. insects outside the focus group may be unnecessarily removed from the environment, sometimes in great number. it is noteworthy that neither the mmi nor the cdc - a captured specimens of any other group of insects besides culicidae - due to the use of specific attractants. it is necessary to adopt different, new methods of capture if one is to achieve better sampling in order to assist study of the epidemiological surveillance of vectors. the mmi, despite requiring greater care in its installation, use and transport, presented good performance both in its ability to sequester a large number of mosquitoes from the environment, as in the capture of culicidae of importance to public health.	traps are widely employed for sampling and monitoring mosquito populations for surveillance, ecological and fauna studies. considering the importance of assessing other technologies for sampling mosquitoes, we addressed the effectiveness of mosquito magnet independence (mmi) in comparison with those of the cdc trap with co2 and lurex3 (cdc - a) and the cdc light trap (cdc - lt). field collections were performed in a rural area within the atlantic forest biome, southeastern state of so paulo, brazil. the mmi sampled 53.84% of the total number of mosquitoes, the cdc - a (26.43%) and cdc - lt (19.73%). results of the pearson chi - squared test (2) showed a positive association between cdc - lt and species of culicini and uranotaeniini tribes. additionally, our results suggested a positive association between cdc - a and representatives of the culicini and aedini tribes, whereas the mmi was positively associated with the mansoniini and sabethini as well as with anophelinae species. the mmi sampled a greater proportion (78.27%) of individuals of anopheles than either the cdc - lt (0.82%) or the cdc - a traps (20.91%). results of the present study showed that mmi performed better than cdc - lt or cdc - a in sampling mosquitoes in large numbers, medically important species and assessing diversity parameters in rural southeastern atlantic forest.
with increased interest in esthetic dentistry, bleaching of discolored teeth, either vital or nonvital, has become popular. difficulties in color matching and achieving the natural appearance are possible drawbacks of full - coverage restorations. in contrast, nonvital bleaching is a noninvasive procedure ; it is less time consuming and economical (1, 2). tooth discoloration varies in etiology, appearance, location, and severity (3). it could be classified as intrinsic, extrinsic, or both according to its location and etiology (4). extrinsic discoloration is caused by chromogens derived from habitual intake of dietary sources such as wine, coffee, tea, carrots, oranges, chocolate, tobacco, mouth rinses, or plaque on the tooth surface (5), while intrinsic discoloration typically results from systemic or local causes. systemic causes include drug - related (tetracycline), metabolic, fluorosis, and genetic (hyperbilirubinemia, amelogenesis imperfecta, and dentinogenesis imperfecta). local causes include pulp necrosis, intrapulpal hemorrhage, pulp tissue remnants after endodontic therapy, endodontic materials, coronal filling materials, root resorption, and aging (6). over the years are oxalic acid, calcium hypochlorite, hydrogen peroxide, carbamide peroxide, and sodium perborate (2). the most commonly used agents for bleaching endodontically treated teeth are 30%35% hydrogen peroxide and sodium perborate either in combination or separately (7). two basic techniques have been used to bleach discolored nonvital teeth : thermocatalytic and walking bleach. both methods have lost favor due to the potential risk, which includes cervical resorption, under- or over - lightening, the possibility of color regression, and external root resorption. recently, a variety of products and techniques have been developed to resolve or minimize the side effects of the bleaching process. this development has been paralleled with rising interest among patients in correcting esthetic problems with their dentition (8, 9). therefore, in the current study, many modifications have been done for bleaching techniques in an attempt to minimize the risk and side effects of the bleaching process. a 13-year - old boy, who complained of discolored and unaesthetic appearance of his upper central incisor, was selected from the pedodontic clinic in the shibin elkom teaching hospital (egypt) in june 2013. the child was free from systemic disorders, and he was not under any medications that cause darkening or staining of the teeth. a diagnosis of nonvital maxillary right central incisor was made, based on the vitality test, which was performed by using an electric pulp tester ; thus, the shade guide of the discolored tooth was assessed under normal daylight with a vita porcelain shade guide (vita zahafabrik) ; also, a pre- and post - bleaching photograph was taken for the patient. conventional endodontic treatment was done for the patient, and, after successful endodontic treatment, the bleaching process was undertaken using 35% hydrogen peroxide gel. the gingiva was protected by water - soluble cream (vaseline) applied to soft tissues, and rubber dam isolation was achieved ; then, 12 mm of the gutta - percha was removed in an apical direction beyond the cemento - enamel junction. the tooth was then washed with 3% hydrogen peroxide solution, rinsed and dried. to assure a barrier between the sealed root canal and the bleaching gel (mechanical seal), usa, union broach co., long island city, ny) was then expressed into the opened pulp chamber and on the labial surface, and curing light was applied to activate the bleaching gel from the labial and palatal sides (10). then, the pulp chamber was rinsed and dried and obdurated with calcium hydroxide to be left in the pulp chamber for 1 week before the final or permanent filling material (light cure composite resin) (11, 12). clinical evaluation was recorded by comparing the tooth shade with its original one before treatment using the vita porcelain shade guide and photographs ; also, radiographic evaluation was done at 1, 3, 6, and 9 month intervals by taking periodical radiographs for the patient using the paralleling technique. the automatically developed radiographs were digitized using a full color flatbed umax scanner (umax data system, inc. usa) with a transparency adapter connected to a standard ibm compatible personal computer having a pentium iii intel processor and a 15 in. the scanned images were saved in a 28 gigabyte hard disk in tagged image format files (tiff) with adobe photoshop version 5.5 (adobe system incorporated, seattle, wa) (13). as shown in figure 1, periapical radiographs were taken prior to bleaching of teeth, immediately after bleaching, and at 1, 3, 6, and 9 months after bleaching. the pre - bleaching assessment for the tooth was diagnosed as having periapical pathosis ; during the follow - up period, there were signs of healing since the start of root canal treatment. the presence of resorption was also assessed radiographically using the digital subtraction technique (14), and there was no evidence of cervical or progressive apical resorption (figure 2). the present study was undertaken to evaluate the trials made to minimize the side effects of bleaching agents ; hence, teeth bleaching is a harmless process, but some conditions have to be respected : correct and complete soft tissue isolation (gums, lips, cheeks), in order to protect them from eventual burns caused by the peroxide. some modifications have been done in an attempt to minimize the risk of cervical or apical resorption ; thus, a base of 12 mm glass ionomer cement was placed over the root filling material to assure a mechanical barrier between the sealed root canal and the bleaching gel, which is in agreement with other studies (8, 9) but is in disagreement with friedman. (15), as they did not use an intermediate lining prior to the bleaching material. another modification added to the bleaching technique was that on reaching the desired shade guide ; thus, the pulp chamber was obturated by calcium hydroxide for seven days before the final filling material was placed. this was necessary to allow for elimination of residual oxygen, which interferes with the polymerization of the filling material and to neutralize and render the medium alkaline that reduces the risk of cervical resorption (16). radiographic follow - up using digital subtraction technique showed no evidence of any cervical resorption or any progressive alteration in the periapical area around the tooth, which may be attributed to the placement of the mechanical seal that prevents the leakage of hydrogen peroxide ; hence, a reduced potential risk of invasive resorption was noted (17). this case report demonstrates the successful management of a discolored nonvital tooth using 35% hydrogen peroxide gel (opalescence xtra) as bleaching material, effectively and safely, by following modifications and precautions to eliminate the side effects of peroxides. no evidence of cervical resorption of the tooth was observed during the follow - up period ; furthermore, there were signs of improvement related to the periapical region. more advanced studies still needed to gather more information about the stability of results and to detect any adverse effect that could appear.	the aim of this study is to report a case of a nonvital, discolored, maxillary central incisor bleached by 35% hydrogen peroxide gel with the use of glass ionomer cement as a mechanical barrier in an attempt to minimize the undesirable side effects of intracoronal bleaching. the patient was a 13-year - old boy complaining of a discolored nonvital upper - right central incisor and was selected for this study from the pedodontic clinic in the shibin elkom teaching hospital in june 2013. after successful endodontic treatment, the tooth was bleached by 35% hydrogen peroxide gel (opalescence xtra), activated by a standard curing light unit, and evaluated for any periapical changes by a periapical radiograph for a nine - months follow - up period. radiographically, there was no evidence of cervical or apical resorption during the study period.
meprins are zinc metalloproteases that are most abundantly expressed in the brush - border membranes (bbms) of proximal kidney tubules. several studies have demonstrated a role for meprins in the pathology of ischemia / reperfusion (ir) induced renal injury. pretreatment of mice with the meprin inhibitor, actinonin, and disruption of the meprin gene both protect mice against ir - induced renal injury [13 ], suggesting that presence of meprins enhances acute kidney injury. however, the mechanisms by which meprins modulate kidney injury in ir are not fully understood. meprins consist of two subunits, and, resulting in two protein isoforms. meprin a is a homooligomer of - subunits or a heterodimer of - subunits, while meprin b is a homooligomer of - subunits. while meprin a and meprin b are highly similar and have several shared substrates, there are unique substrates specific for each isoform. identifying meprin substrates in the kidney has provided insights on how meprins modulate kidney injury in ir. based on the currently known substrates, meprins could enhance kidney injury via proteolytic processing / degrading of cytoskeletal proteins (e.g., villin and actin) and tight junction proteins (e.g., occludin and e - cadherin) [5, 6 ]. in ir, meprins have been shown to be redistributed from the bbm to the cytosol and basolateral membranes. this redistribution brings meprins in close proximity to proteins in other cell compartments that could be proteolytically processed and thus impact the cellular response to ir. meprin targets in the cytosol and basement membrane include cell signaling molecules (e.g., the catalytic subunit of protein kinase a, pka c) [7, 8 ] and the extracellular matrix (ecm) proteins (e.g., laminin, fibronectin, nidogen-1, and collagen) [912 ]. the injury associated with ir is due, in large part, to reduced oxygen delivery to renal tissue. the normal response to hypoxia is mediated by hypoxia response genes, which include hypoxia inducible factors (hifs), for example, hif-1 and hif-2. hif-1 and hif-2 have cell - type specific effects on gene expression. in the kidney, hif-1 is the predominant form in epithelial cells, while hif-2 is predominant in interstitial fibroblast and endothelial cells. hif-1 target genes encode proteins that enable cells to survive oxygen deprivation by providing oxygen - independent means of atp production such as glucose transporters and glycolytic enzymes or by inhibiting hypoxia - induced apoptosis for example, through survival factors like insulin - like growth factor 2 (igf2). studies with a yeast two - hybrid system showed that os-9 interacts with the carboxyl - terminal tail of meprin. this interaction is significant because os-9 has also been shown to interact with hif-1 and prolyl hydroxylase, proteins which mediate the cell 's response to changes in oxygen concentration. although os-9 is localized in the endoplasmic reticulum, it is present in both nuclear and cytoplasmic protein extracts, suggesting that it could traffic hif proteins between the nucleus and the er during the hypoxic response. the current study was conducted to determine if os-9 is a meprin substrate and whether there is a correlation between meprin expression and proteolytic processing of os-9 in vivo and in vitro. the findings will provide new insights on how meprins modulate kidney injury under hypoxic conditions such as ir - induced acute kidney injury. twelve - week - old wild - type and meprin double knockout (ko) mice on a c57bl/6 background were used for these studies. the mice were housed at the north carolina a&t state university laboratory animal resource unit (laru), greensboro, north carolina. all the mice were kept under a 12 : 12 light : dark cycle and provided rodent chow and fresh water ad libitum. the animal procedures used were approved by the north carolina a&t state university institutional animal care and use committee (iacuc). ischemia reperfusion was induced in wt and meprin ko mice (n = 6 for each genotype) by bilateral clamping of the renal pedicle for 26 minutes followed by reperfusion for 6 h or 24 h as previously described [1, 4 ]. control mice (n = 4 for each genotype) were sham - operated without clamping the renal pedicle. the mice were sacrificed at 6 h or 24 h after ir by using isoflurane asphyxiation followed by cervical dislocation. sections of the kidney were fixed in carnoy 's fixative (60% ethanol, 30% formalin, and 10% acetic acid) overnight and then transferred to 70% ethanol. the kidney tissues were subsequently paraffin embedded at the penn state hershey histology core laboratories. the remaining kidney tissues were wrapped in aluminum foil, snap - frozen in liquid nitrogen, and stored at 80c until used for protein extraction. to confirm kidney injury, the levels of blood urea nitrogen (bun) were determined using bun slides (cat. # 1532332, ortho - clinical diagnostics, rochester ny) read on a vitro dt60 ii analyzer (ortho - clinical diagnostics). the kidney tissues were thawed on ice and homogenized in ice - cold edta - free lysis buffer with protease inhibitors. proteins were fractionated by differential centrifugation as previously described [4, 7, 17 ] to obtain a bbm- and cytosolic - enriched protein fractions. some kidney tissues were extracted in ripa buffer without any fractionation (herein referred to as total protein). extracted kidney proteins were aliquoted and stored at 80c until used for western blot analysis. we first determined the kidney protein fraction that had the most abundant os-9 levels by loading 60 g of total (nonfractionated kidney proteins), cytosolic-, and bbm - enriched kidney proteins onto 10% sds - page gels. the proteins were transferred to nitrocellulose membranes and western blot analysis was used to evaluate levels of os-9. our data showed that os-9 proteins were most abundant in the cytosolic - enriched protein fraction (figure 1(a)). for this reason, subsequent analysis utilized cytosolic - enriched protein fractions. to determine whether meprins are capable of proteolytically processing os-9 present in kidney tissue, activated purified forms of recombinant homomeric meprin a (-) and meprin b (-) were incubated with 60 g of cytosolic - enriched kidney proteins from meprin double ko mice (which lack endogenous meprins) in tris buffer (20 mm tris and 150 mm nacl, ph 7.5) at 37c for 4 h. the recombinant homomeric meprin a and meprin b used were purified from stably transfected human embryonic kidney (hek293) cells and activated using limited trypsin proteolysis as previously described [4, 18 ]. briefly, meprins were incubated with trypsin (1 : 20 ratio) in 20 mm tris, 150 mm nacl, ph 7.5, at 37c for 30 min (meprin a) or 1 h (meprin b). the trypsin and sti were removed by running the reaction mixtures through a sephadex g25 column. meprin activity was measured by using the substrate azocasein (sigma - aldrich, mi). product fluorescence was measured with a hitachi f2000 fluorescence spectrophotometer using an excitation wavelength of 320 nm and an emission wavelength of 417 nm. to confirm that the cleavage was meprin b - specific, tris buffer and latent forms of meprin a and meprin b that were not trypsin - activated were used as negative controls. additional negative controls used meprin b which was preincubated with 30 m of the meprin inhibitor, actinonin for 1 h. the products obtained from coincubating meprins with protein lysates were electrophoretically separated and western blot analysis with anti - os-9 specific antibodies (cat. # ta301503, origene, rockville md) was used to evaluate for degradation / fragmentation of os-9. our preliminary data showed that meprins cleave os-9 present in kidney proteins. to confirm that degradation of os-9 was meprin - specific, we incubated purified human os-9 (cat. # tp316820, origene, rockville, md) with activated forms of meprin a and meprin b and used western blot analysis to evaluate protein fragmentation / degradation. to this end, 4 nm activated homomeric meprin a (-) and 4 nm meprin b (-) were incubated with 92 nm purified os-9 in a total reaction volume of 5065 l. equal volumes of reactants (7 l) were taken out at 0, 0.25, 0.5, 1, 2, 3, and 4 h. additional incubations were done for shorter time points totaling 1 h, with samples being taken out at 5 minute intervals. proteolysis was stopped by addition of sds sample buffer followed by boiling for 5 minutes. the proteins were electrophoretically separated on 10% sds gels, and western blot analysis with anti - os-9 specific antibodies (origene, rockville, md) was used to evaluate changes in os-9 levels and fragmentation over time. because os-9 has been shown to interact with the hypoxia response factor, hif-1, we chose to work with an in vivo hypoxia model (ischemia / reperfusion) which causes acute kidney injury. to evaluate meprin degradation of os-9 in vivo, cytosolic - enriched kidney protein fractions from wt and meprin ko mice subjected to ir the levels of os-9 in individual protein samples from sham - operated control kidneys and kidneys subjected to 6 h ir were quantified by optic densitometry using bio - rad 's gs800 calibrated densitometer. to determine whether meprin b interacts with os-9 in vitro, mdck cells were transfected with a meprin cdna construct using the lipofectamine method as previously described [1921 ]. the cdna construct sequence was analyzed at the penn state hershey genomic core laboratories and the sequence confirmed using blast alignment before transfecting the cells. stably transfected cell lines were established using hygromycin b supplemented dulbecco 's modified essential medium (dmem). western blot analysis and immunofluorescence staining were used to confirm expression of meprin b proteins by the transfected mdck cells. meprin transfected and mock transfected control mdck cells were seeded at 1 10 in 100 mm dishes and cultured in dmem supplemented with 10% fbs and antibiotics / antimycotics at 37c and 5% co2 until they were 8090% confluent. judith bond, penn state college of medicine, hershey, pa) were also used. the hek293 cells were similarly cultured in mem media supplemented with 10% fbs and antibiotics / antimycotics. to induce hypoxia, the cells were serum - starved by overnight incubation in serum - free media supplemented with 0.1% bsa. the cells were then exposed to 125 m cobalt chloride (cocl2), a hypoxia mimic for 03 h. cells for immunofluorescence staining were cultured in 8-well slide chambers and similarly exposed to 125 m cocl2. cell protein lysates were fractionated into cytosolic-, nuclear-, and membrane - enriched fractions using previously described protocols [5, 20 ]. all cell lysis buffers used were supplemented with protease inhibitors with edta (roche diagnostics, indianapolis, in). bio - rad 's protein reagent (cat. # 500 - 0006) was used to determine the protein concentrations. western blot analysis was used to confirm meprin b protein expression by the transfected mdck cells, to evaluate the levels of hif-1 and os-9 in kidney proteins and mdck protein lysates, and for evaluating fragmentation and/or degradation of os-9. the protocols used were as previously described [4, 7 ]. to confirm that cocl2 treatment induced hypoxia, nuclear - enriched proteins were probed for hif-1 by incubation in anti - hif-1 antibody (mouse monoclonal, cat. # ab16066, abcam, cambridge, uk), diluted 1 : 1000 in tbs - t. to probe for os-9, cytosolic- and nuclear - enriched protein fractions were incubated in rabbit polyclonal anti - os-9 antibodies (cat. # ta301503, origene, rockville, md), diluted 1 : 1000 in tbs - t. the intensities of the protein bands on the x - ray film were quantified by optic densitometry using bio - rad 's gs800 calibrated densitometer and quantity one software. the levels of tubulin were used as a loading control for determining the relative ods for os-9 and hif-1. immunofluorescence staining was used to evaluate protein expression for hif-1 and os-9 in cultured cells exposed to cocl2. the mdck cells were cultured in slide chambers until 8090% confluent and exposed to cocl2 as described above. the cells were fixed by heating at 70c for 10 minutes in freshly made 1% paraformaldehyde. the cells were then permeabilized in 0.2% triton - x 100 in pbs for 10 min at room temperature (rt). nonspecific binding sites were blocked in 5% normal goat serum in pbs with 0.1% triton - x-100 for 1 h at room temperature. this was followed by incubation in primary antibodies at rt for 1 h or overnight at 4c in a humidified chamber. the antibodies used were rabbit polyclonal anti - meprin b (hmc77, a gift from dr. judith bond, pennsylvania state university, hershey, pa) diluted 1 : 400, mouse monoclonal anti - hif1 (abcam, cambridge, uk) diluted 1 : 200, and rabbit polyclonal anti - os-9 (origene, rockville, md) diluted 1 : 200. boston, ma) diluted 1 : 1000 or anti - mouse alexa fluor555 (cat. the slide sections were imaged using a confocal microscope (carl zeiss microscopy, llc, thornwood, ny) with axiovision software. two - way anova with pairwise comparisons was employed to evaluate protein levels following quantitation of the protein bands by optic densitometry. western blot analysis of mouse kidney proteins detected os-9 (~88 kda) predominantly in the cytosolic - enriched kidney protein fractions (figure 1(a)). incubation of cytosolic - enriched kidney proteins from meprin ko mice with activated meprin b resulted in degradation of os-9 present in the kidney proteins. this degradation was not observed when proteins were incubated with activated meprin a (figure 1(b)). to confirm that the degradation of os-9 was meprin - specific, activated forms of recombinant homomeric mouse meprin a (-) and rat meprin b (-) were incubated with purified human os-9 for 04 h at 37c. a time - dependent degradation of the purified os-9 was observed in reactions with activated meprin b, but not in proteins incubated with activated meprin a or tris buffer without meprins (figures 2 and 3). degradation of os-9 was not observed in control reactions incubated with latent meprin b or reactions in which activated meprin b was preincubated with the meprin inhibitor, actinonin (figure 3(b)). however, we did not observe accumulation of os-9 intermediate fragments when purified os-9 was incubated with meprin b. to determine whether meprins proteolytically process os-9 present in kidney tissue in vivo, western blot analysis was used to evaluate fragmentation of os-9 in cytosolic - enriched kidney proteins from wt and meprin ko mice subjected to ir - induced acute kidney injury. kidney injury was first confirmed by assays for blood urea nitrogen (bun) (figure 4(b)). the bun levels were significantly higher in mice subjected to ir injury when compared to sham - operated controls at 6 and 24 h after ir (p 0.05 and p 0.0001, resp.). bun levels at 24 for wt were also significantly higher than for the meprin ko at the same time point (p 0.05). the pattern for markers of kidney injury was consistent with data from previous studies where the plasma creatinine levels were significantly higher in wt mice when compared to meprin knockout counterparts at 6 and 24 h after ir. the western blot data showed fragmentation of the os-9 present in samples from wt mouse kidneys (which express normal levels of meprin a and meprin b) at 6 h after ir. the ir - associated fragmentation produced a unique ~60 kda os-9 fragment in proteins from wt kidneys at 6 h after ir. this fragmentation was not observed in proteins from meprin ko kidneys (which are deficient in meprin a and meprin b) subjected to ir or wt control kidneys not subjected to ir (figure 4(a)). the data suggest that the presence of both meprins and hypoxia / atp - depletion associated with ir plays a role in the cleavage of os-9 to produce the 60 kda fragment. a second fragment (~37 kda) however, its presence did not correlate to ir - induced kidney injury. to evaluate in vitro interactions between meprin b and os-9, we used meprin transfected madin - darby canine kidney (mdck) cells and human embryonic kidney (hek293) cells. we first used western blot analysis to determine localization of os-9 in mdck cells exposed to cocl2. our data showed that os-9 is predominantly present in the nuclear - enriched protein fraction (figure 5(a)). western blot analysis also confirmed expression of meprin b in the meprin transfected mdck (figure 5(b)) and hek293 cells (data not shown). exposure of the cells to the hypoxia mimic, cocl2, resulted in a time - depended stabilization of nuclear hif-1 leading to increased levels of hif-1 in both mdck (figure 6) and hek293 (figure 7) cells. we then determined changes in the nuclear levels of os-9 in mdck and hek293 following exposure to cocl2. in mock transfected cells this increase was observed at 0.5 h after cocl2 exposure and was sustained through the 3 h time course. in meprin transfected cells on the other hand, the levels of os-9 significantly decreased (5075% decrease ; p 0.001) following exposure to cocl2 (figures 6 and 7). immunofluorescence staining and confocal microscopy also showed that both the nuclear and cytosolic levels of os-9 increased following exposure to cocl2 in nontransfected mdck cells (figure 8(a)). however, this increase was not observed in meprin transfected cells, where in contrast the os-9 levels decreased in a time - dependent manner following exposure to cocl2 (figure 8(b)). an equally important observation was the correlation between meprin b degradation of os-9 and the relative levels for nuclear hif-1 (figures 6 and 7). in mock transfected mdck cells, we observed a more modest increase in hif-1 (~2-fold at 2 h). however for meprin transfected mdck cells, the fold - change in hif-1 was significantly higher (~3 fold at 2 h) (p 0.0001). mdck cells also had higher basal levels of hif-1 when compared to hek293 cells, for which hif-1 levels were below detectable levels. the relative quantities for hif-1 were significantly higher in meprin transfected hek293 cells when compared to the mock transfected control cells. acute kidney injury caused by ischemia / reperfusion (ir) occurs in nearly 5% of hospitalized patients. the reperfusion phase of ir is associated with oxidative stress, cellular dysfunction, and altered signal transduction. in ir - induced renal injury, disruption in the cytoskeleton leads to loss of the brush - border, a breakdown of cell junctions, and the incorrect relocalization of sodium - potassium atpases from the basal surface to the apical surface. this reperfusion event has been shown to be the causative agent of oxidative injury to tubular cells. however, specific proteases that are activated in ir and their substrates are not fully understood. meprins are metalloprotease that are abundantly expressed in the brush - border membranes (bbms) of proximal kidney tubules. changes in the level of expression and localization of meprins have been associated with the pathology of ir - induced kidney injury in mice and rats [1, 10, 24, 25 ]. while meprins are normally localized in the bbm of proximal tubules, redistribution of meprins to the cytosol and basolateral compartments this redistribution brings meprins in close proximity with protein targets in these compartments, which include mediators of the hypoxia response. the cells response to hypoxia is primarily mediated by hypoxia inducible factors (hifs), which consist of two subunits, an oxygen - regulated subunit and a constitutively expressed subunit. under normoxia conditions, hif - prolyl - hydroxylase promotes degradation of hif. however, hypoxia inhibits hif - prolyl - hydroxylases, resulting in rapid accumulation of hif, which then binds to hif to form a heterodimer. the hif dimers translocate into the nucleus where they activate genes that promote adaptation to hypoxia by counteracting oxidative stress and improving cell survival. in the kidney, hif-1 is expressed in tubular cells while hif-2 is predominantly in peritubular cells, renal interstitial fibroblast cells, and endothelia cells. while hifs are central in mediating the response to hypoxia, multiple mechanisms are involved in the pathophysiology of hypoxia - induced renal injury. osteosarcoma amplified 9 (os-9), an endoplasmic reticulum (er) lectin, is a protein coding gene located on 12q13 chromosome of humans. os-9 is part of the endoplasmic reticulum - associated degradation (erad) machinery and erad substrates and aids in the transfer of misfolded proteins. using rnai, os-9 was shown to be required for efficient ubiquitination of glycosylated erad substrates. there is increasing evidence that os-9 is a critical component of the oxygen - signaling that upregulates hif-1. os-9 negatively regulates hif-1 by binding to the hif-1 and prolyl - hydroxylases (phds) promoting degradation of one of its subunits. the presence of os-9 promotes interaction of hif-1 with prolyl - hydroxylase domain proteins phd2 or phd3, leading to hydroxylation and von hippel - lindau (vhl) binding. by promoting degradation of hif-1, os-9 could potentially modulate the hypoxia response and is a key player in the pathophysiology of renal ir. studies using a yeast two - hybrid system showed that meprin interacts with os-9. however, it was not known whether meprins are capable of cleaving os-9 and what the significance of such cleavage would be. the present study evaluated the interaction between meprin metalloproteases and os-9 in vivo and in vitro. our data shows that meprin metalloproteases are capable of proteolytically processing os-9 in vitro and in vivo. coincubation of meprins with kidney proteins from meprin ko mice (which lack endogenous meprins) and purified os-9 confirmed that the os-9 cleavage was meprin b - specific and was inhibited by the meprin inhibitor, actinonin, at molar concentrations previously demonstrated to inhibit meprin activity. to the best of our knowledge previous studies have demonstrated differences in substrate and cleavage site preferences for the meprin protein isoforms, with meprin a selecting a variety of small and hydrophilic amino acids (e.g., proline residues) while meprin b prefers acidic residues (e.g., asp / glu - n peptidase). the present data further shows that hypoxia could induce meprin b degradation of os-9 in vitro and in vivo. a distinct 60 kda os-9 fragment was observed in the cytosolic kidney proteins from wild - type mice subjected to ir - induced renal injury, but not in samples from meprin ko mice counterparts (which lack endogenous meprins). interestingly, we did not observe accumulation of the 60 kda os-9 fragment when meprin b was incubated with purified os-9. this could suggest that while meprin b cleaves os-9, other factors present in the kidney tissue could play a role in stabilizing the cleavage products. degradation of os-9 was similarly observed in meprin transfected mdck and hek293 cells exposed to the hypoxia mimic, cocl2, suggesting that hypoxia enhances meprin degradation of os-9. studies that identify the meprin cleavage sites on os-9 are thus needed to determine the functional implications of the meprin - os-9 cleavage. because os-9 is believed to shuttle the hypoxia inducible factor (hif) between the nucleus and er using immunofluorescence staining and confocal microscopy, we further showed that, in nontransected kidney cell lines, there was a time - dependent redistribution of os-9 from the nucleus to the cytosol over a 3 h period following exposure to cocl2. however, in meprin transfected cells, the hypoxia - induced cytosolic accumulation of os-9 did not occur. taken together, the data suggest that meprin b is partly responsible for the in vivo os-9 fragmentation and that meprin b degradation of os-9 blocks the cytosolic accumulation of os-9 in the mdck and hek293 cells. the current data thus demonstrates a link between the previously reported interaction between os-9 and meprin to the pathology of acute kidney injury induced by ir. indeed, the fold - change in nuclear hif-1 levels was significantly higher in meprin expressing cells under hypoxic conditions than in the nontransfected cells. previous work from our lab has shown that meprins are capable of proteolytically processing / degrading cytoskeletal kidney proteins, for example, actin and villin, the catalytic subunit of protein kinase a (pka c), and protein kinase c (pkc), all in an isoform - specific manner. furthermore, ir - induced acute kidney injury resulted in meprin - associated changes in pka c profiles in mouse kidney tubules, leading us to conclude that the pka signaling pathway is involved in the hypoxia response. other known kidney meprin substrates include extracellular matrix (ecm) proteins [912 ], tight junction proteins, for example, e - cadherin and occludin [5, 31 ], and proinflammatory cytokines, for example, il-1, il-6, and il-18 [21, 32, 33 ]. of all the meprin substrates identified to date, os-9 provides the most direct link to the hypoxia response and provides new insights on how meprins modulate tubular injury in ir. the hif-1/os-9 interaction also links the hypoxia response to mitochondrial energy expenditure and reactive oxygen species (ros) formation. this is supported by previous studies which showed upregulation of os-9 in response to er stress. the present study and previous work from our group have provided evidence that meprins modulate the pathophysiology of ir - induced kidney injury via multiple mechanisms. further investigations are needed in elucidating these mechanisms because the knowledge is important in development of therapeutic targets for acute kidney injury.	meprin metalloproteases play a role in the pathology of ischemia / reperfusion- (ir-) induced renal injury. the endoplasmic reticulum - associated protein, osteosarcoma-9 (os-9), has been shown to interact with the carboxyl - terminal tail of meprin. more importantly, os-9 interacts with the hypoxia inducible factor-1 (hif-1) and the prolyl - hydroxylase, proteins which mediate the cell 's response to hypoxia. to determine if os-9 is a meprin substrate, kidney proteins from meprin knockout mice (ko) (which lack endogenous meprins) and purified human os-9 were incubated with activated forms of meprin a and meprin b, and western blot analysis was used to evaluate proteolytic processing of os-9. fragmentation of os-9 was observed in reactions with meprin b, but not meprin a. to determine whether meprin b cleaves os-9 in vivo, wild - type (wt) and meprin ko mice were subjected to ir - induced renal injury. fragmentation of os-9 was observed in kidney proteins from wt mice subjected to ir, but not in meprin ko counterparts. transfection of kidney cells (mdck and hek293) with meprin cdna prevented accumulation of os-9 following exposure to the hypoxia mimic, cocl2. these data suggest that meprin interaction with os-9 plays a role in the hypoxia response associated with ir - induced renal injury.
weight loss in obese asthma patients improved morbidity and lung function ; however, the mechanisms underlying the relationship between asthma and obesity are unclear. it is suggested that low - grade systemic inflammation associated with obesity plays a role. the metabolic syndrome is a common metabolic disorder that may result from the increasing prevalence of obesity. metabolic syndrome is a cluster of cardiometabolic risk factors characterized by abdominal obesity, insulin resistance, and chronic systemic inflammation. positive associations with lung function impairment have been reported for components of the metabolic syndrome, such as hypertension, type diabetes mellitus [6, 7 ], low - density lipoprotein cholesterol, and overall obesity. in recent large cohort studies, it has been shown that there is also a relationship between lung function impairment and the metabolic syndrome [1012 ]. however, all the aforementioned studies included overweight as well as normal weight subjects and were therefore not specifically targeted to examine these issues in the morbidly obese. data on the association between lung function impairment and the metabolic syndrome in the morbidly obese are limited. the mechanisms underlying the relationship between impaired lung function and the metabolic syndrome are unclear. the chronic low - grade systemic inflammation that is associated with obesity might explain this relationship. our hypothesis is that this low - grade systemic inflammation causes inflammation in the lungs and, hence, lung function impairment. we therefore performed a study to investigate (1) the association between lung function and the metabolic syndrome in morbidly obese subjects and (2) to determine the effect of systemic inflammation on the relationship between lung function impairment and the metabolic syndrome. the subjects included in this study were consecutive patients who underwent preoperative screening for bariatric surgery in the sint franciscus gasthuis in rotterdam, the netherlands, between october 2009 and may 2011. eligibility criteria for bariatric surgery were age between 18 and 60 years and body mass index (bmi) either 40 kg / m or 35 kg / m combined with the presence of comorbidity, for example, diabetes mellitus, hypertension, or proven obstructive sleep apnea syndrome (osas). subjects underwent baseline physical examinations that included routine assessment of anthropometry, blood pressure, pulmonary function, and blood samples. body mass index was calculated as weight (in kg) divided by height (in m) squared. abdominal circumference was measured directly to the body surface midway between the lower rib margin and the ileac crest. the epworth sleepiness scale questionnaire was used to assess osas and the gerd questionnaire for gastro - esophageal reflux disease (gerd). all subjects gave informed consent, and the local ethics committee (toetsingscommissie wetenschappelijk onderzoek rotterdam e.o, trial no. metabolic syndrome was diagnosed according to the national cholesterol education program 's adult treatment panel iii report (ncep atp - iii) criteria when 3 of the following 5 risk factors were present : abdominal obesity, an elevated level of serum triglycerides, low serum level of high - density lipoprotein cholesterol (hdl - c), elevated blood pressure, and high serum glucose level or treatment for any of these disorders. spirometry was performed with vmax spirometer (vmax sensormedics viasys, type encore 20/22/229/62 encore, cardinal health, usa) before and after 400 g of inhaled salbutamol, with subjects in a sitting position and nose clips in place according to the american thoracic society / european respiratory society statement. all values obtained were related to height, age, and gender and expressed as percentage of their predicted value (reference ers 1993). the pulmonary function results are prebronchodilator values unless specifically noted. exhaled nitric oxide (feno) was measured with niox mino (aerocrine, sweden) and expressed in parts per billion (ppb). plasma - cholesterol, hdl - cholesterol, glucose, triglycerides, and crp were measured using lx 20 and dxc analyzers (beckman coulter, miami, fl, usa). total ige and specific plasma ige were determined with a solid - phase two - step chemiluminescent immunoassay on the immulite 2000 (siemens, los angeles, ca). a positive inhalation screen was defined as at least one increased amount of specific ige against one of the following allergens : aspergillus fumigatus, house - dust mite, cat, dog, grass, birch, or herbs. blood cell counts and 5-part leukocyte differentiation were determined automatically using lh750 analyzers (beckman coulter). hba1c was determined using a tosoh g8 hlc-723 analyzer (tosoh bioscience, tokyo, japan). insulin was measured using radio immunoassay (ria) dsl1600 (diagnostic systems laboratories, webster, tx, usa). vitamin d was determined by ria or chemiluminescence (lia) on liason analyzers (diasorin, stillwater, mn, usa). unadjusted between - group comparisons were performed using student 's t test or the chi - square test, where appropriate, and mann - whitney u test for nonparametric comparisons (eosinophils). crp, ige, insulin, lipoprotein - a, and vitamin b6 were not normally distributed (standard error of kurtosis and skewness below 3 or above 3) and were therefore log transformed. backward linear regression analysis was used to investigate which component of the metabolic syndrome is related to the fev1/fvc ratio. variables associated with fev1/fvc ratio in univariate analysis at a p - value of < 0.1 were examined in the multiple linear regression analysis. since the fev1/fvc ratio as percentage predicted is already corrected for height, age, and gender, we did not add these variables to the model. as eosinophils and abdominal circumference were correlated, we selected only eosinophils to prevent collinearity. results were evaluated at 95% confidence interval at a two - sided p < 0.05. a total of 452 subjects were included in the study (97 males and 355 females). also 293 subjects (64.8%) fulfilled the criteria for metabolic syndrome. patients with the metabolic syndrome were significantly older (mean 43 years), were less often female (73.7%), and had a larger abdominal circumference in centimeters (mean 134.3 cm) compared to patients without the metabolic syndrome (mean 37 years, 87.4%, and mean 126.8 cm, resp.). patients with the metabolic syndrome did not have a higher total leukocyte count or neutrophil percentage in the peripheral blood (p = 0.253), but they did have a significantly higher percentage of eosinophils (p = 0.002) and monocytes (p = 0.044) compared to patients without the metabolic syndrome (table 2). other parameters of systemic inflammation such as crp, complement c3, and complement c4 did not show significant differences between the two groups (table 2). we did find a low, but significant, correlation between eosinophils and abdominal circumference (spearman correlation coefficient 0.270, p < 0.001) and crp and abdominal circumference (spearman correlation coefficient 0.146, p = 0.010). the subjects with the metabolic syndrome showed a significantly lower fev1/fvc ratio a measure for bronchial obstruction compared to the group without the metabolic syndrome (76.7% and 78.2%, resp., p = 0.032), while no difference was observed regarding fev1 or fvc (table 3). although there was no significant difference in fef2575 between subjects with and without the metabolic syndrome, the fef75 was significantly lower in the metabolic syndrome group (p = 0.036). log transformed feno a measure for bronchial inflammation showed no difference between the two groups. there was no difference in the prevalence of self - reported asthma between subjects with or without the metabolic syndrome (21.0% versus 19.6%, resp., p = 0.723). in a subgroup analysis of subjects with self - reported asthma, we found no significant difference in the use of inhaled corticosteroids, body mass index, pack years, fev1 (% predicted), fev1/fvc (% predicted), feno, blood eosinophils, or crp between subjects with or without the metabolic syndrome (data not shown). in a subgroup analysis of subjects with and without reversibility (fev1 12%), we found that only 50% of subjects with reversibility (n = 30) had self - reported asthma. although subjects with reversibility did use more often inhaled corticosteroids (13.3% versus 4.4%, p = 0.032), there was no significant difference in feno, blood eosinophils, or crp between subjects with and without the metabolic syndrome. since the fev1/fvc ratio was the only variable of spirometric function tests which was different between subjects with and without the metabolic syndrome and our hypothesis was that the metabolic syndrome causes a lower fev1/fvc ratio not the other way around there was an association between the peripheral blood eosinophils percentage and the fev1/fvc ratio (univariate linear regression coefficient = 0.806, p = 0.006). abdominal circumference and osas (epworth sleepiness scale (ess)) were significantly related to the fev1/fvc ratio (univariate linear regression analysis), whereas bmi, gerd, crp and monocytes were not significantly related to the fev1/fvc ratio (table 4). missing values of ess were not correlated to other variables. since the metabolic syndrome was associated with the fev1/fvc ratio, we investigated which of the components of the metabolic syndrome contributed to this relationship. only hypertension was significantly related to fev1/fvc ratio (regression coefficient = 1.612, p = 0.038 in backward linear regression analysis) (table 5). after correction for the use of inhaled corticosteroids and the number of pack years, we found an association between blood eosinophils and the fev1/fvc ratio (adj. this study shows that obese patients with the metabolic syndrome have a higher proportion of blood monocytes and eosinophils and a lower fev1/fvc ratio, indicating airway obstruction, than obese patients without the metabolic syndrome. blood eosinophils (%) in morbidly obese subjects were related to fev1/fvc, whereas monocytes were not. after adjustment for multiple variables, the relationship between fev1/fvc ratio and eosinophils remained intact. although the differences are small, it strengthens our hypothesis that the presence of the metabolic syndrome could play a role in lung function impairment, through the induction of systemic inflammation, in particular mediated by blood eosinophils. whether this also leads to asthma on the long term still remains to be elucidated. to our knowledge, this is the first study concerning lung function and the metabolic syndrome in a cohort of only morbidly obese subjects. secondly, comorbid conditions associated with obesity such as osas and gerd were taken into account in the current study. thirdly, adiposity was not only assessed with bmi, but also with abdominal circumference. although one would expect that all our subjects have the metabolic syndrome, we found a 65% prevalence of the metabolic syndrome in our group, which corresponds with 60% of the morbidly obese in the nhanes iii cohort. various studies have shown that obesity causes a modest reduction in total lung capacity (tlc) and a larger reduction in functional residual capacity (frc). however, we were unable to perform a tlc measurement in all subjects to rule out a restrictive pattern. this could explain the high mean fev1/fvc ratio in our subjects, and therefore we have used the fev1/fvc ratio as a continuous variable and did not use a cutoff value of 70% predicted. furthermore, in contrast to the fev1/fvc, the fev1 is influenced by bmi ; hence, our focus is on the fev1/fvc ratio. we did not perform methacholine - provocation tests ; so, a definitive diagnosis of asthma was often not possible. since misdiagnosis of asthma is a relevant issue, also in the obese, we felt more comfortable using objective parameters instead of a presumed diagnosis. since the metabolic syndrome was associated with the fev1/fvc ratio in univariate analysis, we investigated which of the components of the metabolic syndrome contributed to this relationship. hypertension was the only component that was significantly associated with a reduced fev1/fvc ratio after multiple backward regression analysis. hypertension may have the strongest association with systemic inflammation, as proposed by irace.. showing that there is indeed a relationship between abdominal circumference (in cm) and the fev1/fvc ratio. interestingly, we found a correlation between blood eosinophil percentage and abdominal circumference, where we found no correlation between blood eosinophil percentage and bmi, indicating that it is mainly the place of the fat that matters. this suggests that the increased abdominal circumference of subjects with the metabolic syndrome causes the relative eosinophilia. several epidemiological studies have already noted a relationship between some components of metabolic syndrome and leukocytes [20, 21 ]. several studies showed an increased eosinophil percentage in obesity or in the metabolic syndrome [20, 21 ]. c - reactive protein (crp) is a traditional marker of inflammation and is well correlated with bmi. even though our study found no difference in crp between the two groups we did not use high - sensitivity crp measurements monocyte and eosinophils percentage in the blood were higher in those with the metabolic syndrome. our study suggests that increased numbers or circulating eosinophils could be a specific manifestation of the systemic inflammation associated with the metabolic syndrome. the fat tissue is a source of adipokines, which are considered to play a role in the low - grade systemic inflammation in obesity. leptin mainly produced by adipose tissue is a proinflammatory agent. serum leptin is markedly increased in obese humans, correlating to bmi ; it activates eosinophils and increases their survival. the leptin / adiponectin ratio is a marker of insulin resistance and the metabolic syndrome. thus, both leptin and possibly eotaxin could contribute to relative eosinophilia in the obese. unfortunately, we do not have data on leptin, adiponectin, and eotaxin to further support their role in the observed eosinophilia. another question is whether this relative eosinophilia in the peripheral blood also has local effects on the bronchial tissue and causes or enhances the bronchial inflammation as seen in asthma. asthma - like symptoms are common in patients with the metabolic syndrome, and their pulmonary function is impaired [34, 35 ]. although we did find differences in pulmonary function between subjects with and without the metabolic syndrome, there was no difference in prevalence of self - reported asthma between the two groups. the characteristics of airway inflammation in asthma (exhaled nitric oxide (eno) and inflammatory cells in induced sputum) have been investigated in obesity and are still subject to debate. de winter - de groot. showed that a higher bmi was associated with a higher eno in healthy patients. several studies have found no or even an inverse relationship between the number of sputum eosinophils and bmi. induced sputum or bronchial biopsies could have helped to solve this question but were unfeasible in this study. since there was no difference in the use of inhaled corticosteroids, this could not have influenced our results of eno. we did not measure tlc values ; so, we were not able to firmly assess a restrictive lung function pattern. also, we did not perform methacholine - provocation tests ; so, a definite diagnosis of asthma was often not possible. the fev1/fvc ratio, however, is easy to measure and widely used in clinical practice. although the differences in fev1/fvc and eosinophils between subjects with and without the metabolic syndrome were small, the difference was significant despite the fact that our study groups consisted of unselected subjects. there probably is a selection bias since we only included patients who were willing to undergo bariatric surgery. it is widely known that most of the subjects who undergo bariatric surgery are females. we did not include a non - obese control group as comparison for low - grade inflammation, and we can not fully exclude that smoking might have contributed to a state of low - grade inflammation. however, we corrected for smoking in the multiple regression analysis. finally, we realize that a cross - sectional association between metabolic syndrome and lung function can not establish causality. have recently shown that the metabolic syndrome predicts a steeper fev1 decline over time, suggesting that the systemic inflammation produced by metabolic syndrome may impact the progression to abnormal lung function in a longitudinally followed cohort. in summary, our study shows that there is a small, but statistically significant, difference in eosinophils and fev1/fvc between subjects with and without the metabolic syndrome. although the differences we have found were relatively small, it might support our hypothesis that the presence of the metabolic syndrome may influence lung function impairment, through the induction of systemic inflammation, in particular, mediated by blood eosinophils. further research with a firm diagnosis of asthma and assessment of peripheral airway inflammation is necessary. moreover, in order to establish causality between the metabolic syndrome and lung function impairment, longitudinal studies in morbidly obese patients before and after bariatric surgery are needed.	background. obesity and asthma are associated. there is a relationship between lung function impairment and the metabolic syndrome. whether this relationship also exists in the morbidly obese patients is still unknown. hypothesis. low - grade systemic inflammation associated with the metabolic syndrome causes inflammation in the lungs and, hence, lung function impairment. methods. this is cross - sectional study of morbidly obese patients undergoing preoperative screening for bariatric surgery. metabolic syndrome was assessed according to the revised ncep - atp iii criteria. results. a total of 452 patients were included. patients with the metabolic syndrome (n = 293) had significantly higher blood monocyte (mean 5.3 versus 4.9, p = 0.044) and eosinophil percentages (median 1.0 versus 0.8, p = 0.002), while the total leukocyte count did not differ between the groups. the fev1/fvc ratio was significantly lower in patients with the metabolic syndrome (76.7% versus 78.2%, p = 0.032). blood eosinophils were associated with fev1/fvc ratio (adj. b 0.113, p = 0.018). conclusion. although the difference in fev1/fvc ratio between the groups is relatively small, in this cross - sectional study, and its clinical relevance may be limited, these data indicate that the presence of the metabolic syndrome may influence lung function impairment, through the induction of relative eosinophilia.
low back pain (lbp) is the most common problem in the world and is a major cause of disability, and work absenteeism, and has a high cost of treatment. a recent study reported the one year prevalence of lbp is more than 80%, and 1020% of cases develop into chronic lbp1. a cause of non - specific lbp is tension, soreness and/or stiffness in the lower back region for which it is not easy to identify a specific cause of pain. several structures in the back, including the joints, discs and connective tissues, may be causes of lbp symptoms2. an abnormal curvature of spine is associated with chronic non - specific lbp symptoms3. thus, an important way of relieving and preventing lbp symptoms is suggested to be maintenance of the appropriate spine curvature of the lower back4. we previously reported that physical therapy including exercise and massage is an effective treatment for the release of muscle stiffness, amelioration of pain, and improvement of physical functions5,6,7. physical therapy can provide beneficial effects for muscle relaxation and the spinal alignment in lbp. in addition to the treatment of physical therapy, instrument can be provided to support the lumbar spine and to reduce and prevent lbp symptoms, and lumbar support is frequently used to treat patients with lbp8. appropriate lumbar lordosis decreases disc pressure by changing the distribution of the load between the disc and apophyseal joints and also reduces the tension in the intervertebral ligaments that can cause disc degeneration9, 10. wearing a lumbar support may increase intra - abdominal pressure that can decrease disc pressure, limit disc compression, and reduce disc pain11. to create a better back supporter, the lumbar spinal curvature of an individual was designed to support the lumbar spine following the recommendation of a previous study. measurements of lumbar spinal curvature in the normal population have shown that lumbar spinal curvature differs among age groups and genders12. however, there is lack of evidence to support the effect of a bcp in the treatment of chronic non - specific lbp patients. the aim of this study was to investigate the effects of a bcp on pain, rom and the functional disability of patients with chronic non - specific lbp. a randomized control trial was conducted with chronic non - specific lbp patients, who were assigned to receive a standard physical therapy treatment plus bcp or only physical therapy treatment. this study was approved by the ethics committee for human research of khon kaen university (protocol no. he552347) and it was prospectively registered with the united states clinical trials registry (nct01911806). patients were diagnosed as having chronic nonspecific lbp by medical doctors at the department of physical therapy, phiboonmungsaharn hospital, thailand. each patient was given a personal interview by a research assistant who had one year s experience in orthopedic physical therapy and a physical examination by a medical doctor. participants aged between 2069 years old who presented with lbp for more than three months and had moderate to severe pain on a numerical rating scale (score 4) were eligible. participants who had underlying systematic or visceral diseases, specific conditions such as neoplasm, ankylosing spondylitis, previous low back surgery, cauda equina syndrome, nerve root symptoms, or pregnancy were excluded. the estimated sample size was calculated, based on the result of a pilot study. the standard deviation of the pain score (numeric rating scale : nrs) after treatment of both groups was used to calculate the sample size for a power of 80% at 5% significance and a drop - out rate of 20% was allowed for the estimate on of the final sample size. the patients who met the above inclusion criteria were randomly allocated to receive either the treatment with physical therapy alone (control group, cg), or the treatment with physical therapy and bcp (bcp group) using stratified random allocation by age group (group 1 = 2029 years old, group 2 = 3039 years old and group 3 = 4069 years old) with block sizes of 2, 4 and 6. 1.procedural flow and follow - up chart shows the design of the procedural flow and follow - up. a total of fifty - two participants with chronic non - specific lbp were eligible for the study. details of the demographic data and health status are presented in table 1table 1.baseline characteristics of the participantscharacteristicsbcpcontotalnumber of patients262652age (years) ; meansd38.511.039.712.239.111.52029 years ; n (%) 7 (27.0)7 (27.0)14 (27.0)3039 years ; n (%) 7 (27.0)7 (27.0)14 (27.0)male 4069 years ; n (%) 6 (23.0)6 (23.0)12 (23.0)female 4069 years ; n (%) 6 (23.0)6 (23.0)12 (23.0)gender ; n of female11 (42.3)13 (50.0)24 (46.2)weight (kg) ; meansd60.0 10.262.38.161.19.2height (cm) ; meansd160.57.3164.47.8162.47.8bmi ; meansd23.23.023.02.223.12.6bcp : back care pillow group ; con : control group ; sd : standard deviation ; bmi : body mass index. procedural flow and follow - up chart bcp : back care pillow group ; con : control group ; sd : standard deviation ; bmi : body mass index participants in each group received six sessions of 30 minutes treatment for two weeks conducted by the same licensed physical therapist who was blinded to the intervention. the participants in the bcp group additionally received a bcp of a specific size for an age group s standard lumbar spinal curvature determined in a previous study12. each subject was asked to wear the bcp during the daytime in the treatment period and to continue using it during the 12-week follow up. all outcomes were measured before and after the 2-week treatment, and at the end of the 12-week follow up. the scale ranged from 0 (no pain) to 10 (severe pain)13. lumbar range of motion (lrom) was measured using the modified - modified schober test. the examiner put his thumbs on the inferior margin of the posterior superior iliac spine. an ink mark was drawn along the midline of the lumbar spine horizontal to the posterior superior iliac spine (lower landmark). while the examiner held the tape firmly against the skin, he marked a second line 15 cm above the original one (higher landmark). then, the subject was asked to do an active anterior flexion of the trunk without increasing the pain. the difference in the initial distance between the skin markings in the neutral position and the measurement in the flexion position was used to indicate the amount of lumbar flexion. after each measurement, the skin marks were removed by rubbing with alcohol to ensure blinding of the next examiner to the landmarks14. functional disability was measured using the thai version of the roland - morris disability questionnaire (rmdq). the scale ranges from 0 (no disability) to 24 (severe disability)15. one - way repeated measures anova was employed to compare continuous outcome variables between baseline and after the treatment in the intervention group and cg. analysis of covariance (ancova) was performed to compare differences in outcome measures between the two treatment groups and to estimate the adjusted mean differences and the 95% confidence intervals of each outcome measure in each group. for statistical significance, the baseline characteristics of the participants are shown in table 1 ; there were no significant differences between the two groups. the intra - tester reliability of the modified - modified schober method showed correlation coefficients ranging from 0.99 to 1.00 for the objective measurement of lumbar flexion and extension rom in healthy volunteers. after two weeks of treatment and at the 12-week follow up, the pain level and lrom in the bcp group and cg showed significant improvement from the baseline (table 2table 2.outcome measures after 2 weeks of treatment and the follow up of 12 weeksoutcomesgroupsbaselineshort - term effectiveness(after 2 weeks of treatment)long - term effectiveness(12 weeks follow - up)nrs (scores)bcp6.81.51.81.2 1.11.2con6.41.54.11.1 4.41.5lrom - flexion (mm)bcp17.50.619.50.5 20.20.5con17.61.118.31.0 18.81.1lrom - extension (mm)bcp14.40.313.10.6 12.70.4con14.40.414.00.4 14.00.5rmdq (scores)bcp16.25.35.93.7 2.32.7con14.46.311.75.9 9.95.2bcp : back care pillow group ; con : control group ; sd : standard deviation ; nrs : numerical rating scale ; lrom : lumbar range of motion ; rmdq : roland morris disability questionnaire. values are presented as meansd. significantly different (p<0.01) from the baseline.). when the results after two weeks of treatment and at the end of the 12-week follow up were compared against the baseline, the pain level and lrom in the extension position showed significant improvements in the bcp group, while the cg did not show any significant differences. there were significant differences in the changes in pain intensity and lrom between the bcp group and cg (table 3table 3.comparison of the adjusted mean and 95% ci of outcome measures (adjusted for baseline using ancova) at each assessment timeoutcomesshort - term effectiveness(after 2 weeks of treatment)long - term effectiveness(12 weeks follow - up)bcpcondifference(95% ci)bcpcondifference(95% ci)nrs (scores)1.84.1 2.3 (1.6 to 2.9)1.14.4 3.4 (2.6 to 4.1)lrom - flexion (mm)19.518.31.3 (1.5 to 1.0)20.318.71.5 (1.9 to 1.1)lrom - extension (mm)13.114.0 0.9 (0.7 to 1.2)12.714.0 1.4 (1.1 to 1.6)rmdq (scores)5.312.3 7.0 (5.3 to 8.7)1.810.3 8.5 (6.9 to 10.2)bcp : back care pillow group ; con : control group ; sd : standard deviation ; nrs : numerical rating scale ; lrom : lumbar range of motion ; rmdq : roland morris disability questionnaire. significant difference between bcp and con (p<0.001).). bcp : back care pillow group ; con : control group ; sd : standard deviation ; nrs : numerical rating scale ; lrom : lumbar range of motion ; rmdq : roland morris disability questionnaire. bcp : back care pillow group ; con : control group ; sd : standard deviation ; nrs : numerical rating scale ; lrom : lumbar range of motion ; rmdq : roland morris disability questionnaire. there were significant decreases in the rmdq score at each assessment time in both groups (table 2). in the comparison of the groups, the bcp group showed more improvement in functional disability than the cg (table 3). this study demonstrated that bcp plus physical therapy for chronic non - specific lbp decreased pain intensity, improved lrom and functional disability better than standard physical therapy after 2 weeks of treatment and at the end of a 12-week follow up. bcp maintained the decrease in pain intensity and the improvement in lrom in the extension position for 12 weeks after the 2-week treatment. in the comparison of the bcp group and cg, a mean difference in the nrs of greater than 2 is generally considered the minimum acceptable when looking for a significant clinical difference between groups16, 17. the clinical findings of the present study are similar to those of calmels.8 who used a lumbar belt for patients with lbp. they found a decrease in pain intensity and rmdq at 30 and 90 days after treatment in the intervention group. other studies have also shown that the pain intensity and the functional status improved in patients with lbp who used a lumbar support for over 12 months ; however, there were no significant clinical changes. the benefits of bcp are possibly explained by mechanisms through which bcp maintain an appropriate lumbar curvature, preserving normal lumbar curve stability, and adaptation of muscle activity to the appropriate lumbar curve. in addition, the appropriate lordotic curve may decrease intra - disc pressure by changing the distribution of loads between discs18. decreased pressure on the apophyseal joints may decrease disc degeneration decreasing pain in the apophyseal joints19. the effect on spinal structure may also improve pain intensity ; however, the lumbar range of motion provided no evidence to support our hypothesis of decreased pressure on the apophyseal joints. limitations of the present study were the small sample size, even though the sample size was calculated based on a pilot study. further studies will need to investigate different populations, e.g. office workers, for longer periods. finally, the mechanisms underlying the beneficial effects of bcp also need to be determined.	[purpose ] the aim of this study was to investigate the effect of a back care pillow (bcp) on pain, lumbar range of motion (lrom) and functional disability of patients with chronic non - specific low back pain (lbp). [subjects and methods ] fifty - two subjects who were aged between 2069 years old, who presented with lbp of more than 3 months duration with a numerical rating scale (nrs) value of at least 4 were randomly assigned to treatment (bcp) and control (con) groups. participants in each group received six sessions of the 30 minutes treatment for two weeks. the bcp group was asked to wear the bcp during the daytime during the study period. pain, lumbar rom and functional disability were assessed before and after the 2-week treatment, and at the end of a 12-week follow up. [results ] after the 2-week treatment and 12-week follow up, all outcomes had improved in both groups ; the bcp group had maintained the decrease in pain intensity and improved lumbar rom in the extension position after the 12-week follow up, and showed better improvements in all outcomes at 2 weeks and after the 12-week follow up. [conclusion ] bcp combined with physical therapy had better pain, lumbar rom and functional disability outcomes than physical therapy alone.
this waste is disposed by different methods i.e. recycling, composting, waste treatment, incineration and landfill (waste disposal on land). the job process of waste disposal areas involves i.e. waste collectors, waste sorters and workers at landfill. in india the amount of hw generated is of the order of 7.243 million tons per annum (tpa) and out of this 1.4 million tons are recycled, 0.4 million tons are incinerated and the rest is disposed by landfill. there are 454 waste generating units in bangalore at karnataka, india which generated 1.023 million tons per annum and out of the 0.47 million tons are recycled, 0.33 million tons incinerated and the rest is disposed by landfill. the processes of waste disposal areas are covered under section of 6, 8 and 25 of environment (protection) act of hazardous waste management 1989. according to a report published during 2000 by the ministry of environment and forests, govt. of india, the composition of waste stream at columbia landfill in missouri have reported 41% paper, 21% organic, 16% plastic, 6% metal, 3% glass and 13% other waste. the handling and disposal of waste cause environmental pollution, which create breeding grounds for pathogenic organisms and spreading of infectious diseases. occupational health hazards associated with waste disposal area are infections to the skin, blood, eyes and intestines. the workers involved in waste disposal areas have reported higher level of respiratory, dermatological and musculo - skeletal effects.[612 ] other health hazards reported as increased chromosomal aberrations occurrence of stillbirths and congenital malformations in general population residing near the landfill. the waste disposal areas (collection and disposal) have reported higher levels of respirable dust, bioaerosols like endotoxins, (13) - -d - glucan, viable fungi and it spores and fungal extra - cellular polysaccharides.[1519 ] a significant association was noticed between airways inflammation and microbial components of endotoxin and (13) - -d - glucan in waste disposal areas.[2022 ] the occurrence of occupational respiratory symptoms was resulted from airways inflammation which is caused due to exposure of toxins, pro - inflammatory and allergens. the airways inflammatory mediatory mechanism involves in two types viz, allergic and non - allergic respiratory symptoms. the allergic respiratory symptoms reflect immune specific inflammation in which antibodies like ige and igg plays major role. the higher levels of serum total ige were reported in art conservators and museum workers exposed to fungi, tetrachloroethylene exposure from dry cleaning process, strawberry green house workers, laboratory animal workers chlorohexadine used by health care workers wheat flour exposed workers from bakery industry grain storage godowns workers. no reports are available in the literature regarding the relationship between occupational health hazards and serum total ige among workers involved in waste disposal area. the present study was undertaken to evaluate the relationship between occupational health hazards and serum total ige among workers involved in hazardous waste disposal area. the survey was performed in 180 study subjects working in hazardous waste areas of bangalore at karnataka, india during the year 2009. the first group consisted of 60 subjects occupied in landfill disposal area, the second group consisted of 60 subjects engaged in compost process and the third group consisted of 60 office workers with no exposure of biological hazards from waste disposal area as control group. the study subjects were interviewed with the american thoracic society (ats) standard questionnaire for respiratory symptoms. the other work related symptoms were collected through a questionnaire developed by ray. 2 ml of whole blood was collected from each subject in test tubes and centrifuged at 3000 rpm for 10 min at 4c and serum was separated used for the determination of immunoglobin e concentration. the concentrations of serum total immunoglobin - e were measured in workers involved in hazardous waste management area by using with enzyme - linked immunosorbent assay (elisa) kits (drg international inc, usa). the absorbance of standards and samples were measured by using thermo scientific multiskan ex reader (usa) at 450 nm. taking the mean of the duplicate readings and subtracting the absorbance of the average zero standards measured the absorbance of each standards and samples. plot the standard curve with standard concentration on the x - axis and absorbance on the y - axis. the levels of serum total immunoglobin - e expressed as iu / ml. the levels of serum total immunoglobin - e were expressed as geometric mean (gm). the proportion of morbidity conditions among waste disposal workers and controls were compared by using f - test. the differences of serum total immunoglobin - e between groups were computed by using non - parametric mann whitney - u test. the study subjects were interviewed with the american thoracic society (ats) standard questionnaire for respiratory symptoms. the other work related symptoms were collected through a questionnaire developed by ray. 2 ml of whole blood was collected from each subject in test tubes and centrifuged at 3000 rpm for 10 min at 4c and serum was separated used for the determination of immunoglobin e concentration. the concentrations of serum total immunoglobin - e were measured in workers involved in hazardous waste management area by using with enzyme - linked immunosorbent assay (elisa) kits (drg international inc, usa). the absorbance of standards and samples were measured by using thermo scientific multiskan ex reader (usa) at 450 nm. taking the mean of the duplicate readings and subtracting the absorbance of the average zero standards measured the absorbance of each standards and samples. plot the standard curve with standard concentration on the x - axis and absorbance on the y - axis. the levels of serum total immunoglobin - e were expressed as geometric mean (gm). the proportion of morbidity conditions among waste disposal workers and controls were compared by using f - test. the differences of serum total immunoglobin - e between groups were computed by using non - parametric mann whitney - u test. the demographic details of mean age, experience, body mass index (bmi) and frequency distribution of life style confounding factors (smoking, alcohol consumption and chewing of tobacco products) of study subjects is shown in table 1. demographic detail of waste disposal area workers the morbidity conditions of solid waste workers occupied in landfill area, compost and administrative sections were reported in figure 1. the respiratory complaints were significantly increased in land fill area (p=0.006) and compose plant workers (p=0.0006) as compared to controls. the musculoskeletal complaints were significantly increased in compose plant workers (p=0.0008) as compared to controls. the other health hazards were not shown any significant difference among waste disposal workers when compared to controls. shows the morbidity pattern of landfill, compose plant workers and controls the geometric mean levels of serum total immunoglobin - e in subjects according to job category are presented in figure 2 the levels of serum total ige level was significantly increased in landfill area workers (p=0.027) compose plant workers (p=0.020) as compared to controls. the levels of serum total ige was increased by 28.3% in landfill area workers and 30.8% in compose plant workers as compared to controls. highest increase was noticed in compose plant workers as compared to land fill area workers. shows the geometric mean levels of serum total ige in different category of workers the levels of serum total immunoglobin - e in subjects according to occurrence of morbidly conditions in hazardous waste management area the serum total ige levels were increased in the subjects having health related symptoms as compared to no symptoms. the results of study found no significant relationship was found between the levels of serum total ige and occurrence of health related symptoms or past respiratory disease. the present study assessed the relationship between occupational health hazards and serum total immunoglobin - e in workers engaged in hazardous waste area. the occupational health hazards reported as respiratory followed by musculo - skeletal, dermatological, gastrointestinal, injuries and nose and eye problems. bunger, reported 24% respiratory, 13.8% skin and 7% of gastrointestinal complaints in hazardous waste workers (hww) who had 3 years of mean duration of exposure. the present study has reported higher health complaints in hww because they had 5 years of mean duration of exposure. vrijheld, have reported health complaints such as headaches, sleepiness, respiratory, psychological, gastrointestinal, cancers, adverse pregnancy outcomes, abnormalities in liver and renal function of workers exposed from landfill area. goorah, have reported respiratory symptoms such as bronchitis 26.1%, copd 15.1% and asthma 13%, 26.9% of skin irritation, 19.9% nasal irritation, 22.9% eye irritation and gastrointestinal problems (5.25% diarrhea, 11.1% constipation, 10.3% abdominal pain, 14.9% vomiting and nausea) in landfill area workers from mare chicose in mauritius with 30 years of exposure. most of the studies indicated that the workers involved in these hazardous waste industries are exposed to high levels of microorganisms causes higher prevalence of respiratory symptoms and airways inflammation. the present study also indicated higher level of respiratory symptoms as compared to other health hazards. the waste disposal areas (collection and disposal) have reported higher levels of respirable dust, microbial component like endotoxins, (13) - -d - glucan, viable fungi and its spores and fungal extra - cellular polysaccharides.[1519 ] higher respiratory symptoms and airways inflammation are associated with higher levels of neutrophil counts and production of pro - inflammatory cytokines. coenen, have reported significant association between high endotoxin exposure (72 eu / m) and serum concentration of igg in waste collectors. wouters, have reported 27% increased levels of serum total ige in waste collectors involved in municipal waste collecting facility at dutch cities. marth, also reported higher levels of serum total ige in employs engaged in waste sorting facility. in the present study we undertaken to find out the relationship between occurrence of health hazards and serum total ige in workers involved in waste disposal sites. we found that the levels of serum total ige were significantly increased in land fill area and compose plant workers as compared to controls. skorska, have reported significantly increased levels of serum total ige in workers occupationally exposed to organic dust from hatchery process and found non- significant relationship between the levels of serum total ige and occurrence of health related symptoms or past respiratory disease. in the present we also found increased levels of serum total ige in land fill area and compose plant workers and no significant relationship was found between the levels of serum total ige and occurrence of health related symptoms or past respiratory disease. the levels of serum total in land fill area and compose plant workers but no significant relationship was found between the levels of serum total ige and occurrence of health related symptoms or past respiratory disease.	background : the exposures of bio - aerosols have reported higher occupational health hazards, the association between serum total ige levels and job categories and occupational health hazards of waste disposal area was limited. the present study was undertaken to assess the relationship between occupational health hazards and serum total ige in waste disposal area.materials and methods : one hundred eighty subjects working in waste disposal areas in different parts of bangalore at karnataka, india were enrolled into the study in 2009. using questionnaire the respiratory morbidity and other work related problems in hww was carried. the levels of serum total ige in study subjects were determined by using enzyme linked immunosorbent assay kits (drg international inc, usa). the differences of serum total ige levels between the groups were computed by using non - parametric mann - whitney u test. spss 10.0 for windows version of statistical software was used in the analysis.results:the levels of serum total ige was significantly increased in landfill area (p=0.027) compose plant workers (p=0.020). the morbidity conditions such as respiratory and musculoskeletal found significantly higher in waste disposal workers as compared to controls.conclusion:the levels of serum total ige was significantly increased in land fill area and compose plant workers but no significant relationship was found between the levels of serum total ige and occurrence of health related symptoms or past respiratory disease.
technical evolution in posterior lumbar interbody fusion (plif) devices has witnessed the application of cage implants fabricated from various metallic and non metallic materials with a varying range of geometric design. cages fabricated from poly - ether - ether - ketone (peek), when impacted with autologous bone graft, have been shown to promote spinal interbody fusion with high fusion rates and good to excellent clinical outcome reported in the literature [6, 1113 ]. more recently a resorbable implant fabricated from poly - l - lactide - co - d, l - lactide (pldlla) was introduced into clinical practice as a structural support and bone graft containment device intended to aid lumbar spinal interbody fusion [2, 4, 10, 16 ]. although early papers were overwhelmingly promising, appraisal of the current literature shows that little subsequent clinical data on resorbable cages have emerged and clinical application of these implants seems to be diminishing. whereas several authors have reported fusion rates as high as 92100% after plif with the aid of a pldlla cage [25, 810 ], virtually none of these studies have prospectively assessed patient - centred clinical outcome measures in their entire study population. the objective of this study was to compare the clinical outcome of lumbar interbody fusion using telamon peek with that of telamon pldlla hydrosorb (both medtronic sofamor danek, memphis, tn, usa) fusion devices in a prospective randomized clinical trial, at a minimum of 2-year follow - up. twenty - six patients with chronic back pain and irradiating lower extremity symptoms were randomly assigned to undergo instrumented posterior lumbar interbody fusion whereby either a non resorbable telamon peek cage or a resorbable telamon pldlla cage was implanted to aid fusion. all patients had degenerative spondylolisthesis and in addition either a canal stenosis, foramen stenosis or both. the diagnosis was supported by pre - operative radiographs (antero - posterior and lateral views as well as flexion and extension views) and mri - scans in all patients. the study protocol was approved by the institutional review board and ethical committee, whilst an informed consent was obtained from each patient. follow - up visits were at 3, 6, 12 and 24 months after surgery. a 10-point vas score, a vas score of zero indicates no pain whilst a vas score of 10 indicates the worst imaginable pain. for the sf-36 questionnaires a physical component score (pcs), mental component score (mcs) and eight summary scores (physical functioning, role physical, bodily pain, mental health, social function, vitality, role emotional, and general health) were calculated for each patient. scores were normalized to the general dutch population so that a score of 50 represents the score of the general population. preoperative, intra - operative, and follow - up examination data were entered into a study - specific database. repeated measures analyses of variance were performed on all dependent variables to test for differences between pre - operative values and post - operative values. in addition, correlation between outcome variables was assessed using the pearson correlation (r) for continuous, and the point - biserial correlation coefficient (rpb) for correlation between dichotomous and continuous variables. for categorical and ordinal variables a chi - square analysis was performed, and a fisher s exact two - sided p value was computed. to test for difference in performance between cages, the relative post - operative scores (difference between pre- and post - operative values) peek cages were implanted in 14 patients and resorbable pldlla cages were implanted in 12 patients. one patient (peek cage) was lost to follow - up at 1 year after surgery but was subsequently traced for a 2-year post surgery assessment. considering the whole study population, the average vas score for back pain and leg pain, respectively, improved from 6.1 [standard error of mean (sem) 0.4 ] prior to surgery to 4.0 (sem 0.6, p = 0.012) and from 5.0 (sem 0.5) to 2.7 (sem 0.6, p = 0.07) at 2 years after surgery. the average preoperative odi of 20.5 (sem 1.3) improved to 13.9 (sem 2.2, p = 0.014) at 2 years after surgery, whilst average values for the sf-36 physical component score improved from 30.4 (sem 1.8) prior to surgery, to 38.8 (sem 2.9, p = 0.004) at 2 years of follow - up. the vas, odi and sf-36 pcs all showed significant correlations varying from r = 0.42 (vas legs and vas back, p = 0.029) to r = 0.83 (sf-36 pcs and odi, p = 0.000). when compared with preoperative values, all clinical parameters significantly improved in the peek group at 2 years after surgery compared with preoperative values with the exception of sf-36 general health, sf-36 mental health and sf-36 role emotional scores (table 2). no single clinical parameter showed significant improvement at 2 years after surgery compared with preoperative values in the pldlla patient group. only six patients (50%) in the pldlla group showed more than 10% improvement in the vas cores for leg and back pain as well as the odi, as opposed to 10 patients (71%) in the peek group. one - third of the patients in the pldlla group actually reported more than 10% worsening of their pain scores and odi. figure 1 shows the degree of decline in pain scores for each treatment group at various time intervals of follow - up. both treatment groups demonstrate a greater improvement at 612 months which is not sustained at 2 years of follow - up. based on ct scan assessment, 13 out of 14 patients demonstrated solid fusion in the peek group (92% fusion rate) whereas 6 out of 12 patients in the pldlla group (50% fusion rate) showed solid fusion. fusion rate was significantly related to vas legs (rpb = 0.49, p = 0.01), vas back (rpb = 0.40, p = 0.04) and sf-36 pcs (rpb = 0.40, p = 0.04). there was a significantly higher rate of subsidence in the pldlla group compared with the peek group (p = 0.0414).table 1baseline characteristicspeekpldllap valuemean age years (range)44 (2069)53 (3967)0.085female sex no. (%) 54380.452radiating pain left leg no.780.632 right leg no.1090.261lumbar segment affected l4l5250.130 l5s11170.130 l6s11vas scores for back pain6.5 (sd 2.0)5.6 (sd 2.2)0.210 for leg pain4.9 (sd 2.5)4.5 (sd 2.5)0.833oswestry disability index23 (sd 6)18 (sd 7)0.542sf-36 scores pcs27 (sd 10)35 (sd 7)0.029 mcs45 (sd 11)47 (sd 9)0.683table 2preoperative and 2-year follow - up clinical data in both treatment groupspeek n = 14p valuepldlla n = 12p valuepreoperative (sem)postoperative (sem) 2 yearspreoperative (sem)postoperative (sem) 2 yearsvas leg pain4.9 (0.7)2.3 (0.7)0.0084.5 (0.7)3.3 (8.7)0.311vas back pain6.5 (0.5)4.1 (0.7)0.0285.6 (0.6)3.9 (7.4)0.117odi23 (2)14 (3)0.01518.1 (2.1)14.0 (3.9)0.300sf-36 pcs26.8 (2.6)41.5 (3.0)0.00534.7 (2.1)37.5 (4.7)0.368sf-36 mcs45.0 (2.9)49.0 (2.4)0.23746.7 (2.6)46.5 (3.0)0.953sem standard error of meanpcs physical component scoremcs mental component scorefig. 1a and b average change in vas scores (+ sem) at 3, 6, 12 and 24 months after surgery. greater improvement is seen in the peek group baseline characteristics preoperative and 2-year follow - up clinical data in both treatment groups sem standard error of mean pcs physical component score mcs mental component score a and b average change in vas scores (+ sem) at 3, 6, 12 and 24 months after surgery. greater improvement is seen in the peek group three patients (all with non fusion) demonstrated signs of osteolysis in the pldlla treatment group. two patients out of the six radiological failures (pldlla group) reported in the preliminary paper have been revised. one case with symptomatic non fusion and screw breakage in s1 underwent an anterior interbody fusion using a peek cage whilst a postero - lateral fusion was performed in a second patient. despite the small patient population, this study demonstrates that plif significantly improves pain (vas scores) and disability (odi) (fig. plif with the aid of peek cages have been more extensively studied, with clinical outcome as high as 86% improvement based on patient - centred outcome measures [6, 11 ]. our result of 71% improvement in the peek group is in concordance with the findings in the literature. most published data on resorbable cages to date, however, have focused on relatively short - term surgical outcome rather than patient - centred outcome measures of pain, disability and capacity for work. coe and vaccarro published a series of 31 patients who underwent plif with the aid of pldlla implants in which only 16 patients were assessed using sf-36 questionnaires. they reported statistically significant improvement in the 12- and 24-month mean pain scores and 24-month mean role physical scores compared with the preoperative scores on the same scales. these findings contrast with our current results in the pldlla patient group. at a minimum of 2-year follow - up, only 50% of the patients in the pldlla group demonstrated improvement in the vas scores for leg and back pain as well as the odi, as opposed to 71% in the peek patient group. the poorer clinical outcome (based on vas scores, odi and sf-36) with the pldlla implant is consistent with the poorer fusion rate reported in the pldlla patient group, and casts some serious doubts on the efficacy of pldlla cage in relieving symptoms and enhancing posterior lumbar spinal interbody fusion. we belive that the low fusion rate observed with pldlla cages is a result of early mechanical failure leading to loss of structural support prior to the establishment of a fusion mass. preclinical in vivo studies have demonstrated this fact, with loss of mechanical integrity of pldlla cage implants as early as 3 months after implantation, a feature not observed in a plla cage another resorbable counterpart [14, 15 ]. this preclinical finding suggests that the composition of the two pla isomers in the pldlla cage may confer inherent time - dependent mechanical weakness as compared with the mono isomer plla cage. perhaps an even more important factor that could have lead to early mechanical failure and non fusion in the pldlla cage is the method of sterilization. preclinical in vitro studies have demonstrated the adverse effects of irradiation sterilization techniques on the time - dependent mechanical properties of pla - based polymers. therefore, e - beam irradiation may have contributed to a preliminary breakdown of the cage and consequently the higher rate of non union in our study (fig. 2a and b average change in scores for sf-36 physical component score and oswestry (+ sem) at various time intervals of follow - up. indicates a significant difference between peek and hydrosorb groupsfig. 3radiographs of a 48-year - old patient at 1 day (a) and 1 year (b) after surgery showing subsidence and non fusion a and b average change in scores for sf-36 physical component score and oswestry (+ sem) at various time intervals of follow - up. indicates a significant difference between peek and hydrosorb groups radiographs of a 48-year - old patient at 1 day (a) and 1 year (b) after surgery showing subsidence and non fusion worsening of symptoms seen in 33% of the patients in the pldlla group remain worrisome, and may be a result of the lower fusion rate proffered by the resorbable cage. this is supported by significant correlations between poor fusion rates on the one hand and worsening of pain scores on the other hand. the occurrence of osteolysis and a relatively high rate of subsidence associated with the pldlla cage may also be important contributing factors in the poorer clinical outcome as observed in this patient group. the higher rate of subsidence is most likely related to early mechanical failure which in itself can be an important contributory factor in the occurrence of osteolysis. it is, however, important to note that a potential underlying low - grade infection has not been ruled out in these cases of osteolysis. in both treatment groups, initial improvement at 612 months after surgery is apparently not sustained at 2 years of follow - up. we have found no underlying reason for worsening of symptoms at 2 years in our study population. following up on our preliminary report, the 2-year results confirm the superiority of the peek implant to the resorbable pldlla implant in aiding spinal fusion and alleviating symptoms following plif in patients with degenerative spondylolisthesis associated with either canal stenosis or foramen stenosis or both and emanating from a single lumbar segment.	previous papers on resorbable poly - l - lactide - co - d, l - lactide (pldlla) cages in spinal fusion have failed to report adequately on patient - centred clinical outcome measures. also comparison of pldlla cage with a traditionally applicable counterpart has not been previously reported. this is the first randomized prospective study that assesses clinical outcome of pldlla cage compared with a poly - ether - ether - ketone (peek) implant. twenty - six patients were randomly assigned to undergo instrumented posterior lumbar interbody fusion (plif) whereby either a peek cage or a pldlla cage was implanted. clinical outcome based on visual analogue scale scores for leg pain and back pain, as well as oswestry disability index (odi) and sf-36 questionnaires were documented and analysed. when compared with preoperative values, all clinical parameters have significantly improved in the peek group at 2 years after surgery with the exception of sf-36 general health, sf-36 mental health and sf-36 role emotional scores. no clinical parameter showed significant improvement at 2 years after surgery compared with preoperative values in the pldlla patient group. only six patients (50%) in the pldlla group showed improvement in the vas scores for leg and back pain as well as the odi, as opposed to 10 patients (71%) in the peek group. one - third of the patients in the pldlla group actually reported worsening of their pain scores and odi. three cases of mild to moderate osteolysis were seen in the pldlla group. following up on our preliminary report, these 2-year results confirm the superiority of the peek implant to the resorbable pldlla implant in aiding spinal fusion and alleviating symptoms following plif in patients with degenerative spondylolisthesis associated with either canal stenosis or foramen stenosis or both and emanating from a single lumbar segment.
overview. ovarian cancer (oc) is a common condition in women scenario and it represents the principal cause of death from gynaecologic cancer in united states. it is estimated that 21.290 cases will have been diagnosed in 2015 and that 14.180 women will have died due to this malignancy. about 90% of oc are epithelial carcinomas and 70% of those have a serous histology. death rate from oc declined from 1970s to 1990s but it has since then remained stable. in light of these discouraging data, the development of novel therapies for oc has become a priority. recent better molecular characterization and immune system identification are the starting point of future research in immunotherapy. undoubtedly, the next decade will see immunotherapy coming to the clinic use alongside standard regimes and it is possible that it could replace cytotoxic chemotherapy in combination strategies. therefore, in addition to possessing expertise with immunotherapy, oncologists will be expected to conduct trials of novel agents in combination with standard treatment. thus, it is paramount to focus the attention on maximising the knowledge of the more important component of the immune system. loss in this challenge could run the risk that oncologists will take a passive role in the development of new strategies. this paper reviews the rationale for immunotherapy and the main approaches under investigation in oc, with a special focus on the role of checkpoint inhibitors. we will briefly describe the human immune system in an attempt to provide a means of understanding how it relates specifically to the clinical practice. the role of immunotherapy in cancer treatment has been identified decades ago, because of beneficial effect of severe induced infection on tumour regression. at that time, coley showed that inoculation with streptococcal organisms resulted in the shrinkage of inoperable bone and soft - tissue sarcomas. however, severe criticisms due to the inconsistency of the method and results emerged in the scientific community, across the years. one explanation was that other physicians, who tested his treatment, did not report the same excellent effect. these results, as well as the concurrent development of radiotherapy and chemotherapy, determined immunotherapy to slowly disappear from treatment cancer scenario. since coley 's death, immunology has represented an active research field and, nowadays, immunotherapy is considered again a valid treatment option in different types of cancer. the human immune system can be divided in two components : the innate and the adaptive immunity. the innate immune system consists of natural killer (nk) cells, dendritic cells, and macrophages and neutrophils, whereas b cells and t cells, including cytotoxic (cd8 + t or ctl) cells, helper (cd4 + t) cells, and nk t cells, are specific of the adaptive immunity. the innate immunity provides a first line response against pathogens in a nonspecific manner ; it has no immunologic memory and it is not able to recognize antigen. thus, in terms of tumor immunology, its contribution is marginal and limited to secreted cytokines that recruit immune cells. on the other hand, the adaptive immune system plays a central role in the antitumor immune response, due to its capability in processing nonself cells. firstly, the presentation of an antigen to a t cell receptor (tcr) by a major histocompatibility complex (mhc) molecule on antigen presenting cells (apcs). secondly, the interaction of the cd28 receptor on t cells to b7 costimulatory molecules (b7 - 1 and b7 - 2) on apcs. the distinction between self and nonself is provided by cancer - specific antigens that are expressed by tumor cells. tumor antigens are traditionally divided in two classes : tumor specific antigens (tsas) and tumor associated antigens (taas). for instance, oncospermatogonal antigens are expressed by tumor cells as well as normal spermatocytes ; carcinoembryonic antigen (cea) is expressed on fetal tissues and in several cancer types. theoretically, the immune system recognizes nascent transformed cells, in order to prevent progression to clinical tumor it is an extrinsic tumor suppressor mechanism that consists of 3 sequential phases : elimination, equilibrium, and escape. transformed cells are recognized and eradicated by the innate and adaptive immune system. cd8 + t cells, cd4 + t cells, natural killer (nk) cells, and nk t cells secrete interferon- (inf-) to inhibit tumor cell proliferation and angiogenesis, whereas macrophages and dendritic cells are processed to phagocytise and remove tumor cells killed. the cells that are not eliminated in this phase may then enter the equilibrium phase, in which their development is prevented by adaptive immunologic mechanism. cd8 + t cells and dendritic cells secrete inf- and interleukin- (il-) 12, respectively, and preserve tumor cells in a steady state. this is a functional state in which latent tumor cells are specifically controlled by the adaptive immunity. this dynamic balance can persist for long period, sometimes exceeding 20 years. in response to immune system, tumor cells can change their characteristics in immune resistant cells and therefore escape from immune system suppression. in this final phase, tumor cells emerge and become clinically apparent, because they are no longer blocked by immunity. the generation of immune resistant tumor cells can occur in several ways : through loss of tumor antigens expression ; through downregulation of mhc ; through the overactivation of the prooncogenic transcription factor stat3 ; through the overexpression of antiapoptotic effector bcl-2 ; through the expression of inhibitory cell surface molecules, such as programmed cell death 1 ligand 1 (pd - l1), cytotoxic t - lymphocyte associated protein-4 (ctla-4), and fas ligand (fasl), which directly kill cytotoxic cd8 + t cells. otherwise tumor cell escape can be a consequence of an immunosuppressive state established in the tumor microenvironment. this condition may result from the secretion of immunosuppressive cytokines, like il-4, il-1, vascular endothelial growth factor (vegf), and prostaglandin - e2 (pge2), which recruit regulatory cells. particularly, the secretion of il-4 recruits macrophages that inhibit cd4 + t cells, by expressing transforming growth factor- (tgf-), il-10, and platelet - derived growth factor (pdgf), whereas the secretion of il-1, vegf, and pge2 determines the accumulation of myeloid - derived suppressor cells that blocks t cell function. over the last decade we need to integrate the potential understanding of the immunoediting process from the 3es and the tumor characteristics to conduct the optimal treatment. it is difficult to define a clear role of immunotherapy ; nonetheless it is reasonable to hypothesise that any immune molecule capable of activating this process might have a useful role in eradication of nascent tumor cells. at this time it is paramount that oncologists are familiar with the immunoediting process so that they can have a role in the rational development of innovative clinical trials. the stabilization of equilibrium state, as well as the inhibition of tumor escape mechanisms, should be clinical endpoints. current immunotherapies for cancer treatment include therapeutic vaccines, cytokines, immune modulators, immune checkpoint inhibitors, and adoptive t cell transfer. therapeutic vaccines are designed to treat established cancers and may be used in the induction of the tumor - directed immune response of the patients through the introduction of tumor antigens. the other approaches such as immune checkpoint inhibitors and adoptive t cell transfer are designed to augment anticancer immunity against cancer. nowadays, one of the most promising strategies seems to be the takeover of immune cell - intrinsic checkpoints that are induced on t cells activation. the blockade of one of these checkpoints, such as ctla-4 or the programmed death 1 (pd-1) receptor, has recently been found to be active to achieve an immune - modulation approach in the treatment of solid tumors [15, 16 ]. the immune checkpoint blockade targeted agents might represent breakthrough drugs in the treatment of solid tumors and have generated greater expectations in the field of cancer immunotherapy, even in oc [17, 18 ]. t cells activity is regulated by a great number of different molecules, as well as immune - modulatory signals, both costimulatory and coinhibitory. to avoid inappropriate t cell activation, resulting in autoimmunity, negative regulators of t cell immunity, including ctla-4 and pd-1, are needed. in fact both ctla-4 and pd-1 are key immune checkpoint proteins and represent a further promising immunotherapeutic target. ctla-4 is a member of the cd28:b7 immunoglobulin superfamily, typically low - expressed on the surface of naive effector t cells and regulatory t cells (tregs). when naive t cells are stimulated through the tcr, ctla-4 is upregulated and competes with cd28 for b7 and, finally, determines the suppression of t cell activity. it was found that the antitumor effect of ctla-4 blockade might be obtained also by depletion of treg, as revealed in a model of mouse melanoma, in which both the augmentation of t effector cell function and inhibition of treg activity through the blockade of ctla-4 manage to obtain a strong antitumor response. pd-1 is expressed on chronically stimulated t cells, as well as tregs, activated b cells, and nk cells. differently from ctla-4, which regulates t - lymphocytes at the level of initial activation, pd-1 regulates immunity at multiple phases of the immune response, including its effect on effector t - lymphocyte activity in the peripheral tissues. experimental models showed that pd-1 deficient mice present enhanced immunity with phenotypes characterized by autoimmune cardiomyopathy and a lupus - like syndrome [24, 25 ]. the activity of pd-1 is related to its interaction with its ligands, pd - l1 (b7-h1) and pd - l2 (b7-dc). both ligands, especially pd-1, are expressed on many hematologic and nonhematologic human tumors. generally, in human cancer, when pd-1 binds with cells bearing one of its ligands, t cell activity is attenuated (phenomena known as peripheral tolerance), which prevents these t cells from rejecting the tumor at the tissue level, and tumors can thereby employ the pd-1 inhibitory pathway to silence the immune system. based upon the findings of preclinical studies, suggesting the involvement of these molecules in immune control, various agents blocking ctla-4, pd-1, or pd - l1 or other immune molecules are currently investigated in ovarian cancer (oc) treatment. the ctla-4 is currently being investigated as a single or combinatorial therapy in clinical trials involving several cancer types. ipilimumab and tremelimumab are fully human igg1 or igg2 antibodies, respectively, that antagonize the ctla-4 immune checkpoint. the majority of clinical data derived from studies in patients with melanoma. in these studies ctla-4 blockade has yielded objective responses to such an extent that ipilimumab was fda approved to treat metastatic or unresectable melanoma in 2011 [29, 30 ]. experience in oc is actually based on small population studies but results seem to be interesting. firstly showed [31, 32 ] antitumor effects in patients with stage iv oc patients. initially, they reported that a single infusion of ipilimumab (3 mg / kg) in two - stage iv oc patients previously vaccinated with granulocyte - macrophage colony - stimulating factor modified irradiated autologous tumor cells (gvax), was well tolerated, and triggered a decrease or stabilization of ca-125 levels of several months ' duration. in order to clarify the toxicity and antitumor efficacy, they treated additional 9-stage iv oc subjects by using the same antibody dose and schedule (with the exception of one patient). in one patient, an objective radiographic response was noted and multiple infusions of anti - ctla-4 antibody every 3 to 5 months have maintained disease control over 4 years ; furthermore, 3 out of 9 patients had stable disease of 6 (ongoing at the moment of paper 's publication), 4, and 2 months ' duration, as measured by ca-125 levels and radiographic criteria, in the absence of serious toxicities. tumor regression correlated with the cd8+/treg ratio, suggesting that other forms of therapy that target treg depletion might have a synergistic effect when combined with the tumor vaccine and ctla-4 antibody molecules. these findings prompted a phase ii clinical trial to evaluate ipilimumab as monotherapy in platinum - resistant oc patients (nct01611558). ipilimumab can cause significant immune - related adverse events (aes), and the more common observed side effects include diarrhea, colitis, and dermatitis. less common severe immune - related adverse events include hypophysitis, thyroiditis, and hepatitis. tremelimumab (previously known as ticilimumab) is a fully human igg2 monoclonal antibody to ctla-4. in contrast to ipilimumab, a large phase iii trial in melanoma did not demonstrate improved pfs or os compared with cytotoxic chemotherapy although durable responses were observed in some patients. much speculation have been done about the potential reasons for this clinical result, because both phase iii clinical trials [14, 29 ] testing ipilimumab succeeded in showing improved os. it has been proposed that human igg1 (the ipilimumab subclass) binds with a higher affinity to fcrs than human igg2 (the subclass of tremelimumab) does, therefore suggesting that tremelimumab might determine a ctla-4 antibody mediated treg - cell depletion to a lesser extent [36, 37 ]. the combination of tremelimumab and a pd-1 inhibitor (see below) is currently ongoing, in a phase i study including ovarian and cervical cancer patients. the therapeutic benefit obtained with ctla-4 inhibition led to the effort in identifying other potential immune checkpoint inhibitors that should have been more specific and equally efficacious and have less immune toxicity. pd-1 and pd - l1 inhibitors were identified as potentially accomplishing those requirements. differently from ctla-4, which regulates t - lymphocytes at the level of initial activation, pd-1 regulates immunity at multiple steps, including exerting its effect on effector t - lymphocyte activity in the peripheral tissues. several monoclonal antibodies have been developed that block the pd-1 system, either by interactions with the pd-1 receptor or with its specific ligands. nivolumab (also known as bms-936558 or mdx1106) is a fully human igg4 monoclonal antibody that targets pd-1. a phase i / ii clinical trial tested the safety and efficacy of nivolumab at doses of 0.1 to 10.0 mg / kg of body weight intravenously every 2 weeks for up to 12 cycles until disease progression or a complete response occurred. patients with advanced melanoma, non - small - cell lung cancer, prostate cancer, renal cancer, and colorectal cancer were enrolled. among the 296 patients, those with metastatic melanoma achieved the higher rates of objective responses (27.6%), with a median os of 16.8 months ; conversely responses were not observed in colon and prostate cancer patients. responses were seen in both pd - l1 positive and negative patients, even if with lower extent. common treatment - related adverse events included fatigue, diarrhea, pruritus, rash, nausea, and decreased appetite. grade 3 or 4 treatment - related adverse events were seen in 14% of patients. treatment - related serious adverse events were noted in 11% of patients and included pneumonitis (3%, and grade 3 or 4 in 1%), colitis, hepatitis, thyroiditis, and hypophysitis. recently, at the 2014 asco meeting, the first clinical trial of nivolumab treatment against platinum - resistant oc has been presented. a total of 18 evaluable patients were treated with nivolumab : 10 patients were administered 1 mg / kg and 8 patients were administered 3 mg / kg, each every 2 weeks for 1 year. starting at week 8, patients were assessed every 8 weeks and patients with disease progression were taken off study. there were two serious treatment - related aes : one patient in the 1 mg / kg group experienced grade 3 fever, disorientation, and gait disorder and one patient in the 3 mg / kg category experienced grade 3 fever and deep - vein thrombosis. other grade 3/4 treatment - related aes included hypothyroidism (two patients, both in the 1 mg / kg group) ; heart arrhythmia (one patient, in the 3 mg / kg group) ; and lymphocytopenia (one patient, in the 1 mg / kg group). the 3 mg / kg dose may be more favourable (25%) than 1 mg / kg (10%). two patients in the 3 mg / kg group experienced complete response (cr ; response rate 25%). among those receiving 1 mg / kg nivolumab, one experienced a partial response (10% response rate) and two patients experienced stable disease (sd). further researches are investigating biomarkers predicting response. a further molecule investigated in oc is pembrolizumab (mk-3475, formerly known as lambrolizumab), a humanized igg4 monoclonal antibody against pd-1. it was found to be active in treating both melanoma and nsclc [4143 ], similar to nivolumab. actually, no randomized trial has compared the two agents, which are surely different in binding affinities, nivolumab being a fully human igg4, and pembrolizumab is humanized recently, an interim analysis with pembrolizumab showed preliminary signal for clinical efficacy in recurrent oc. in addition to antibodies targeting pd-1, several different anti - pd1-l1 monoclonal antibodies, such as bms-936559, mpdl3280a, medi4736, and msb0010718c, have been developed which might enhance immune function. it was found that the ligand / receptor interaction inhibits the t - lymphocyte response by inhibiting the kinases involved in t - lymphocyte activation via phosphatase activity and other signaling pathways. bms-936559 is a high - affinity, fully human igg4 monoclonal antibody that binds pd - l1 and that blocks pd - l1 from binding its two known receptors pd-1 and cd8. it was safe in a phase i trial that included 17 oc patients in escalating doses of 0.310 mg / kg iv every 14 days in 6-week cycles for up to 16 cycles or until the patient had a complete response or confirmed disease progression and observed durable tumor regression and prolonged stabilization of disease. common side effects included fatigue, infusion reactions, diarrhea, arthralgia, pruritis, rash, nausea, and headache. in the trial, only oc patients at the 10 mg / kg dose achieved objective responses : 1 (6%) with a partial response and 3 (18%) with stable disease lasting more than 24 weeks. msb0010718c is a fully human igg1 monoclonal antibody targeting pd - l1. unlike other pd - l1 targeting agents, it is a native fc receptor, allowing for antibody dependent cell mediated cytotoxicity. in a phase i trial 27 patients with refractory malignancies were treated with msb0010718c at 1, 3, 10, and 20 mg / kg twice weekly. eleven patients in the study had received prior treatment with an immunotherapy. at the 3 and 10 mg / kg doses, the drug was found to inhibit 93.8% and 93.2% of the pd - l1 receptor on peripheral leukocytes. additionally, a linear pk profile was found, with a maximum concentration of the drug achieved at 1.52 hours following infusion. at the 20 mg / kg dose, a dose - limiting immune - related adverse event was noted. in this trial also oc patients were included and, interestingly, a larger subsequent meta - analysis of the company developing the drug (merck), including 23 patients ' oc cohort, showed 48% of patients reaching stable disease and 17% getting a partial response within 30 weeks of treatment start, though 13 had been taken off the drug. noteworthily, the responses came despite 77% of patients having already failed at least three lines of therapy. more recently, efficacy data from the 23 patients followed up for more than 2 months (range 28 months) were presented. four patients (17.4%) achieved an unconfirmed partial response, 11 (47.8%) patients had stable disease, and 2 patients had > 30% tumor shrinkage after progression. toxicity was manageable and only 2 patients (8.7%) experienced grade 3 drug - related aes. other molecules, such as mpdl3280a and medi4736, are currently investigated in phase i trials including oc patients. tumor tissue can be considered a darwinian microenvironment that selects the better strategy to elude the immune system. expression of specific ligands, such as pd - l1 and ctla-4, in the stroma or in the tumor cells associated, is paramount to improve growth and resistance to immune attack. it depends on both tumor type and histology, and therefore it also represents the major limitation of immunotherapy. maybe the identification and characterization of similar patients population, as well as tumor histology, could provide data to facilitate the development of novel treatment strategies. immune check point inhibitors may have a synergic mechanism in multimodality treatment and thus a positive effect on overall survival, with a tolerable toxicity profile. in the last years, immunotherapy has achieved an important role in the fight against cancer and also, in oc immunological phenomena, has been demonstrated to play a central role. immune checkpoint inhibitors have shown clinical activity in several cancers, especially melanoma, and they represent a major step forward in the fight against cancer. these novel therapies will likely play a role also in oc given the potential for rapid, durable responses and their favourable toxicity profiles. their function in the treatment of patients with oc remains to be defined but initial results seem to be promising. next challenges should be the clinical development of combinatorial approaches and further defining patients who benefit from immune checkpoint monotherapy and patients who require potentially more active albeit more toxic combination regimens. finally, the definition of potential biomarkers that can determine which immune checkpoint pathway or pathways dominate in a particular tumor will be crucial to guide the choice of inhibitor. the possibility of using immunotherapy in oc is still restricted to clinical trials but it is reasonable to expect that over the next years important advances in oc immunotherapy will be made, running further phase ii and iii trials development.	ovarian cancer is the most important cause of gynecological cancer - related mortality, with the majority of women presenting with advanced disease. although surgery and chemotherapy can improve survival rates, it is necessary to integrate alternative strategies to improve the outcomes. advances in understanding the role of immune system in the pathogenesis of cancer have led to the rapid evolvement of immunotherapy, which might establish a sustained immune system response against recurring cancer cells. recently, it has emerged that powerful immunologic effector cells may be blocked by inhibitory regulatory pathways controlled by specific molecules often called immune checkpoints, which turn off the immune system. similarly, cancer cells are able to use these checkpoints to avoid immune control and rejection. inhibition of these inhibitory pathways represents a potent strategy in the fight against cancer and is currently under investigation with encouraging results in some cancers, such as melanoma. in ovarian cancer researches are still in an early phase, but with promising results. in this review we will explore the rationale of immunotherapy in ovarian cancer with a special focus on these emerging molecules.
tocopherols have a saturated phytyl tail, whereas tocotrienols have an unsaturated isoprenoid side chain, which contains three double bonds. on the chromanol ring, the 4 variants (-, -, -, and -) are determined by the number and position of methyl groups. the predominant form of vitamin e in tissues is -t, and its deficiency leads to ataxia in humans. the effects of vitamin e on the pathogenesis of human cancers have been investigated in many studies, and the results are generally inconsistent (reviewed in refs (3 and 4)). studies on the correlation between tocopherols and cancer risk have shown that lower levels of tocopherols are associated with increased risk of lung, breast, and some other types of cancers. in an earlier clinical study, intake of -t and -carotene however, results from other studies including a large - scale human clinical trial do not support a protective role of -t in cancer prevention. this discrepancy may be explained by the fact that other forms of vitamin e or a mixture of different tocopherols may be superior to -t alone in preventing cancer development. since epidemiological studies indicate a protective role of vitamin e in cancer prevention, while clinical trials using -t was not effective, it is important to determine the effect of the different tocopherols in the vitamin e family in cancer prevention. previous studies have investigated the effects of -t and -t in different types of cancers (reviewed in refs (3 and 4)), and recent studies from several laboratories demonstrated that a mixture of tocopherols that contains 13% -t, 1.5% -t, 57% -t, and 24% -t had potent preventive effects in a number of carcinogenesis models. however, studies on the anticancer effect and mechanisms of action for -t on prostate cancer are still lacking. the present study was therefore designed to explore the anticancer activity and mechanisms of action for -t in prostate cancer. in the present study, we determined the effects of -t on growth and apoptosis in different prostate cancer cells cultured in vitro. we also determined the in vivo effects of -t using a mouse subcutaneous xenograft tumor model. our study provides the first evidence for the stronger activities of -t on growth inhibition and apoptosis stimulation in human prostate cancer cells as compared to -t, -t, and a mixture of tocopherols. our study also demonstrated that -t had a more potent inhibitory effect than -t on the formation and growth of prostate lncap tumors in scid mice. human prostate cancer lncap, vcap, and cwr22rv1cells were obtained from the atcc (rockville, md, u.s.a.). the extracellular matrix matrigel was obtained from corning co. (tewksbury, ma, u.s.a.). the various human prostate cancer cells were maintained in rpmi-1640 culture medium as described in our earlier study. cell viability was determined by the trypan blue exclusion assay. at the end of the experiments trypan blue solution (0.4%) was mixed with cell suspension in a ratio of 1:4, and the mixtures were kept at room temperature for 2 min for the cells to be stained with the dye. the number of viable cells was counted under a microscope (nikon optiphot, tokyo, japan). apoptotic cells were identified in propidium iodide (pi) stained cells by morphology. at the end of the experiment, cells were spined to slides using a shandon cytospin centrifuge (thermo scientific, waltham, ma, u.s.a.) and fixed in a solution containing 50% acetone and 50% methanol. the cells were then stained with pi at a concentration of 1 g / ml. a microscope (nikon optiphot, tokyo, japan) was used to identified apoptotic cells according to the morphological changes characteristic of apoptosis as described earlier. after treatment, proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) and transferred to nitrocellulose membrane. after blocking nonspecific binding sites with blocking buffer, the membrane was incubated overnight at 4 c with psa primary antibody (cbl-252, millipore co, billerica, ma, u.s.a.). the -actin was used as a loading control. following removal of the primary antibody, the membrane was washed three times with tbs (pbs containing 0.05% tween 20) buffer at room temperature and then incubated with fluorochrome - conjugated secondary antibody (santa cruz biotechnology inc. final detection was done with an odyseey infrared imaging system (li - cor biotechnology, lincoln, ne, u.s.a.). the cwr-22rv1/ar cell line that stably expresses the ar - luciferase reporter gene cwr-22rv1/ar cells were treated with dihydrotestosterne (dht), -t and -t for 24 h. after treatment, the cells were harvested in a reporter lysis buffer and the luciferase activities were measured using luciferase assay kits from promega (madison, wi, u.s.a.), as described in our recent study. scid mice originally obtained from taconic farms (germantown, ny, u.s.a.) were bred in the animal facility in the susan lehman cullman laboratory for cancer research. human prostate cancer lncap cells (2.0 10 cells / mouse) were suspended in culture medium and matrigel (1:1) and injected subcutaneously into the back of the animals. the mice were randomly assigned to 3 experimental groups in the same day of the tumor cell injection. group 1 was the control and the mice in this group were fed ain 93 m diet. when the subcutaneous tumor formed, tumor length and tumor width were measured once every third day and calculated as tumor size (length width, cm). the animal experiment was carried out under the rutgers university institutional animal care and use committee (iacuc)-approved protocol (ru02 - 001). proliferation of lncap tumor cell was measured by determining the number of mitotic cells, and apoptosis of the tumor cells was assessed by immunohistochemical staining for activated caspase-3. at the end of the experiment, tumor tissues were fixed in buffered formalin for 24 h and then with ethanol for 48 h. paraffin blocks of tumor tissues were prepared and paraffin sections of tumor tissues were processed for h&e staining. a microscope (nikon optiphot, tokyo, japan) activated caspase-3 was determined immunohistochemically using an anti activated caspase-3 antibody (# af835, r&d systems ltd., minneapolis, mn). the number of cells with positive staining of activated caspase-3 was counted and expressed as % caspase-3 cells. we used anova (analysis of variance) and multiple comparison (tukey - kramer test) to perform statistical analyses in the present study. differences in cell viability, apoptosis and ar activity in cells from different treatment groups were analyzed. differences in tumor size and body weight between different treatment group in the in vivo study were also analyzed. in initial studies, the effects of -t, -t, -t, and -tmt on growth and apoptosis in human prostate cancer lncap cells were determined. as shown in figure 1a, the different tocopherols dose - dependently inhibited the growth of cultured lncap cells. -t had a stronger inhibitory effect on the growth of lncap cells than the other tocopherols (figure 1a). additional experiments showed that treatment of lncap cells with different tocopherols resulted in a concentration - dependent increase in apoptosis. as shown in figure 1b, -t had a more potent effect on inducing apoptosis in lncap cells than other tocopherols. inhibition of growth and induction of apoptosis in lncap cells by -t, -t, -t, and -tmt. human prostate cancer lncap cells (0.5 10 cells / ml) were seeded in rpmi medium with 10% fbs and incubated 24 h to allow the cells attach to the dish. then the cells were cultured in medium containing 1% fbs and treated with various concentrations of -t, -t, -t, or -tmt. the treatment time was 96 h. the trypan blue exclusion assay was used to determine cell viability. morphological assessment of pi - stained cells was used to determine apoptosis and the number of apoptotic cells is shown in (b). different superscripts (a, b, and c) indicate statistical differences in the number of viable cells or percent apoptotic cells among different tocopherol isoforms (p 0.05). in vivo effects of -t and -t. the mice received ain93 m diet (10 mice), ain93 m diet containing 0.3% -t (10 mice), or ain93 m diet containing 0.3% -t (10 mice) for 48 days. (a) tumor growth curve (measured as tumor size ; cm) in the control, 0.3% -t - treated, and 0.3% -t - treated groups. human prostate cancer lncap cells were injected subcutaneously into scid mice as indicated in the materials and methods. tumor size (cm) and tumor weight (g) were measured at the end of the experiment. tumor cell proliferation was measured by determining the number of mitotic cells in the xenograft tumors. we found that treatment of the mice with -t in diet resulted in a strong inhibition of tumor cell mitosis (table 2). the differences in % mitotic cells between the -t - treated group and the control group, and between the -t - treated group and the -t - treated group were statistically significant (p 0.05) in % mitotic cells between the control group and the -t - treated group (table 2). as shown in table 2 and figure 6, treatment with -t also resulted in a strong increase in the number of caspase-3 cells. the differences in the percentage of caspase-3 between the -t - treated group and the control group, and between the -t - treated group and the -t - treated group were statistically significant (p 0.05). effects of -t and -t on expression of activated caspase-3 in prostate lncap xenograft tumors. immunohistochemistry with an activated caspase-3 antibody was performed in paraffin sections of lncap tumors collected from the experiment described in figure 5. representative micrographes of caspase-3 immunostaining in lncap tumors from the control (a), -t - treated (b), and -t - treated (c) mice are shown. the tumors from mice at the end of the experiment were analyzed for proliferation and apoptosis. < 0.001 compared to the control or to the 0.3% -t - treated group. in the present study, we demonstrated for the first time that -t had a potent inhibitory effect on the growth of prostate cancer cells cultured in vitro and grown as subcutaneous xenograft tumors in scid mice. in initial studies, we determined the effects of -t, -t, -t, and a mixture of tocopherols on cultured prostate cancer cells and found that -t had stronger effects on growth inhibition and apoptosis stimulation than -t, -t, and the tocopherol mixture. in subsequent studies, we found that -t more potently inhibited the growth of lncap tumors in scid mice than -t. it has recently been shown by a number of laboratories that -t or a mixture of different tocopherols, -tmt, are more active than -t for inhibiting prostate cancer. accumulating evidence from experimental studies suggests that the -t used in clinical trials may not be the correct form of vitamin e for cancer prevention. the strong inhibitory effect of -t on prostate cancer shown in our present studies indicates that -t may be a good candidate for future clinical trial of prostate cancer prevention. the mechanisms by which different tocopherols inhibit the growth and induce apoptosis in prostate cancer cells are still largely unknown. although many studies investigated the mechanisms of action for -t and -t in cancer cells, there are very few studies for the mechanisms of anticancer activities of -t. a previous study showed that combination of -t with -t induced apoptosis in lncap cells by induction of cytochrome c release, activation of caspase-9 and caspase-3, and cleavage of poly - adp - ribose polymerase. recent studies by our collaboration team demonstrated that suppression of lung tumorigenesis by -t was associated with its ability to inhibit the formation of 8-ohdg, -h2ax, and nitrotyrosine, as well as to induce cell apoptosis. in another study, -t was found to induce ppar- and pten, and reduce pakt levels in breast cancer cells. it is known that the ar signaling pathway is important for growth and survival of prostate cancer cells. earlier studies have shown that analogues of -t induced transcriptional repression of ar, inhibited ar activity and decreased the level of psa. however, the effect of -t on ar signaling was not reported. in the present study, we demonstrated that -t had a more potent inhibitory effect than -t on ar activation in prostate cancer cells as determined by the luciferase reporter gene expression assay. in addition, -t more potently decreased the level of psa than -t. suppression of the ar signaling may be one of the mechanisms by which -t inhibits the growth and induces apoptosis in androgen sensitive prostate cancer cells. preliminary results from our recent studies showed inhibitory effect of -t on the growth of pc-3 and du145 cells (data not shown). this result indicates that -t also inhibited androgen - independent prostate cancer cells and that mechanisms other than ar signaling may be involved. further studies are needed to explore the mechanisms of action for -t in androgen - independent prostate cancer cells. since -t showed strong effects on growth inhibition and apoptosis in cultured prostate cancer cells, we used the xenograft prostate tumor model to determine the in vivo effect of this tocopherol. we found in our earlier studies that 0.3% of different tocopherols in diet were effective. therefore, 0.3% of -t and -t in diet were used in our present in vivo study. our study showed that treatment with 0.3% -t in diet decreased the number of mice that formed lncap tumors. administration of 0.3% -t in the diet strongly inhibited the formation and growth of subcutaneous lncap tumors. in a recent pilot study, we found low plasma levels of -t (0.160.47 m) in prostate cancer patients received 2 capsules of mix - tocopherol (containing 128 mg -t, 200 mg -t, and 71 mg -t per capsule) for 7 or 14 days (data presented in the 2011 aacr annual meeting). the concentrations of -t and -t to inhibit cultured prostate cancer cells are higher than the plasma levels of these compounds. in addition, -t may interact with endogenous factors such as cytokines within tumor tissues leading to stronger anticancer effects. mechanistic studies showed that treatment of mice with -t inhibited proliferation as reflected by decreased mitosis, and stimulated apoptosis as reflected by increased caspase-3 in lncap tumors. this result indicates that the strong inhibitory effect of -t on the growth of lncap tumors in scid mice may be mediated by inhibition of proliferation and stimulation of apoptosis in the tumors. to our knowledge, this is the first report for an inhibitory effect of -t on the formation and growth of human prostate tumors in a xenograft model. in summary, the present study showed that -t had more potent effects on inhibiting proliferation and inducing apoptosis in prostate cancer cells than -t. the strong activities of -t were associated with suppression of ar activity and decreased level of psa. results from the in vivo study demonstrated a strong inhibitory effect of -t on the formation and growth of lncap tumors in scid mice without apparent toxicity. clinical studies for determining the potential preventive efficacy of -t on prostate cancer patients are warranted.	in the present study, the effects of -tocopherol (-t) on growth and apoptosis of human prostate cancer cells were determined and compared with that of -tocopherol (-t), a commonly used form of vitamin e. treatment of human prostate cancer cells with -t resulted in strong growth inhibition and apoptosis stimulation, while the effects of -t were modest. the strong effects of -t on the cells were associated with suppression of androgen receptor (ar) activity and decreased level of prostate specific antigen (psa) that is a downstream target of the ar signaling. in the in vivo study, we found that -t had a more potent inhibitory effect on the formation and growth of prostate xenograft tumors than that of -t. moreover, -t inhibited proliferation and stimulated apoptosis in the tumors. the present study identified -t as a better form of vitamin e than -t for future clinical studies of prostate cancer prevention.
dialysis requires more self - management than any other medical treatment to control risk factors associated with increased mortality. a patient starting dialysis, aged 60 years, may be informed that they have a 50% chance of surviving five years, but observational studies suggest this can be improved by controlling intake of fluids, salt, fat, phosphate, and potassium, attending regularly for dialysis, monitoring blood pressure, cholesterol, phosphate, and haemoglobin, and taking prescribed medication to control these factors, plus weekly injections to improve haemoglobin. few people are able to understand and manage all aspects simultaneously, and their relative importance is unknown. meaningful discussion between clinician and patient about these issues, and phosphate control in particular, is currently not possible and unsurprisingly adherence to prescribed regimens is poor [3, 4 ]. we will address the significance of one risk factor for mortality in dialysis patients serum phosphate to facilitate and inform future patient - clinician discussions and enhance the shared decision making process. serum phosphate increases in chronic kidney disease and by the time the patients are on dialysis, high serum phosphate is found in more than 40% of dialysis patients and is linked with a 40100% increased mortality risk in retrospective, observational studies [5, 6 ]. 27 observational studies were included in a meta - analysis which examined the relationship between dysregulated mineral metabolism and all - cause or cardiovascular mortality or cardiovascular events in patients with chronic kidney disease (ckd) or end - stage renal disease (esrd, which is when they need to start dialysis treatment). though there were limitations in the analysis noted by the authors due to the low number of studies included and the quality of the data obtained from them, a greater risk of all - cause and cardiovascular mortality was seen with elevated phosphate concentrations. the dialysis outcomes and practice patterns study (dopps), a prospective cohort study in 25,588 patients with esrd receiving haemodialysis, showed an increased risk of cardiovascular mortality with serum phosphate concentrations of 5.15.5 mg / dl (1.61.78 mmol / l) and an increase in all - cause mortality at serum concentrations over 6.0 mg / dl (1.94 mmol / l). it is recognised that such studies are useful for generating research hypotheses but can not definitively establish cause / effect relationships. consequently it is believed important to control phosphate, but whether this improves patient outcomes remains unknown, since no randomised interventional trials have been undertaken. dietary control and dialysis are insufficient to normalise phosphate, and tablets are required to bind phosphate in the gut. the available phosphate binders can be broadly classified into calcium containing phosphate binders like calcium carbonate and calcium acetate and non - calcium containing binders like lanthanum (fosrenol trade name) and sevelamer (renvela and renagel trade names for sevelamer carbonate and sevelamer hydrochloride, resp.) and aluminium containing and iron containing phosphate binders. this classification is important because several small interventional randomised controlled trials have looked at difference in clinical outcomes between calcium containing phosphate binders and the common non - calcium containing ones like lanthanum and sevelamer. jamal. published a meta - analysis of 11 such studies, 9 of them in dialysis patients, and compared outcomes between patients with chronic kidney disease taking calcium - based phosphate binders and those taking non - calcium - based binders. they concluded that non - calcium - based phosphate binders were associated with a decreased risk of all - cause mortality compared with calcium - based phosphate binders in patients with chronic kidney disease. multiple clinical trials have further shown an increase in coronary artery calcification with calcium - based binders compared to non - calcium - based binders [1118 ]. there is an increasing acceptance among clinicians that calcium containing phosphate binder is not optimum therapy and has resulted in greater use of non - calcium containing binders which are ten times more expensive than the calcium containing binders. a study of patients ' perspectives of phosphate binding medication identified gaps in understanding of the concept of phosphate control and the role of medication [3, 19 ]. even when binders are taken correctly, achieving normal phosphate levels is difficult, and more than 25% of patients remain above the current opinion - based target range of 1.11.7 mmol / l, and more than 40% are above the true normal limit of 1.4, there is a significant gap between the recommendations and the serum phosphate concentrations achieved by patients in clinical practice. the important contributing causes to the lack of effective phosphate control are nonadherence to a low phosphate diet and to phosphate binder medication. despite the investment in expensive binder medication (up to 3,000 per patient / per annum, 30% of the 27000 prevalent dialysis patients in uk, works out to more than 24 m per annum) and large pill burden (up to 15 pills with meals daily in some cases) with significant gastrointestinal side - effects, we do not know if lowering serum phosphate is of benefit. a review of all available evidence by the international kidney disease improving global outcomes (kdigo) expert group concluded, the extensive review exposed significant gaps in our knowledge robust studies of a large sample size addressing the following issues should be given priority : does lowering phosphate our trial will examine the feasibility of conducting such a study of large sample size, which we hope will ultimately answer the questions posed by kdigo. the primary endpoint is the percentage of study participants achieving, and being maintained within, the higher and lower target ranges for phosphate, over the duration of the maintenance phase of the study. the secondary endpoints are the following : percentage of greater manchester kidney physicians agreeing to enter patients into a study which includes a higher range group.percentage of eligible invited participants willing to be randomised into a study which includes a higher range group.percentage of participants achieving consistent control of serum phosphate in each group over a 10-month maintenance period.drop-out rate from the study due to adverse events, kidney transplantation, intercurrent illness, and death. these numbers will inform the power calculation for the larger national study.pill burden per participant required to control serum phosphate.adherence to therapy.number of participants willing to participate in communicare patient support programme.mean symptom score assessed by pittsburgh dialysis symptoms index.incidence of major vascular events, defined as nonfatal myocardial infarction or any cardiac death, any stroke, or any arterial revascularisation excluding dialysis access procedures (expected mortality of around 14% per annum in patients on dialysis). percentage of greater manchester kidney physicians agreeing to enter patients into a study which includes a higher range group. percentage of eligible invited participants willing to be randomised into a study which includes a higher range group. percentage of participants achieving consistent control of serum phosphate in each group over a 10-month maintenance period. drop - out rate from the study due to adverse events, kidney transplantation, intercurrent illness, and death. incidence of major vascular events, defined as nonfatal myocardial infarction or any cardiac death, any stroke, or any arterial revascularisation excluding dialysis access procedures (expected mortality of around 14% per annum in patients on dialysis). the design is a dual - centre, pan - manchester prospective randomised parallel group study, with titration to target (2 months) and maintenance phase (10 months). the total number of participants planned is 100 at randomisation (up to 300 at consent). the study will be conducted across two large renal units in greater manchester which cover a population of about 3.2 million. prevalent dialysis patients will be recruited from the renal units and their associated satellite dialysis units which give a target dialysis population of about 1100 patients with 900 patients on haemodialysis. documented approval has been obtained from appropriate ethics committee and the cmft research and development department. the study conforms to international conference on harmonization of good clinical practice guidelines and with the declaration of helsinki. written informed consent will be obtained from each patient before any study - specific procedure takes place. participation in the study and date of informed consent of patients will be documented appropriately in each patient 's files. safety monitoring committee is in place to review saes as is trial steering committee to review progress and trial management committee to oversee the conduct of the trial. the medical records of all the dialysis patients in the greater manchester area will be accessed by the study personnel that are also a part of the direct care team (some study personnel will be members of the direct care team). a retrospective screening of their previous serum phosphate levels (which would have been done as part of their routine monthly blood tests) will be completed. patients with a mean level of > 1.4 mmol / l over the past 3 months, and taking an oral phosphate binder, will be identified and flagged up. inclusion criteria are as follows : male and female patients aged 30 years or above, on dialysis for at least 6 months (to ensure no recovery of renal function), and under the supervision across pan - manchester sites. patients less than 30 years of age have a low rate of vascular events and will not be recruited.serum phosphate level of 1.7 mmol / l or greater after wash - out (discontinuation) of previous phosphate binding medication.able to achieve renal association standards for quality of dialysis on the most recent test of dialysis efficacy. this would be a urea reduction ratio of 65%.able to communicate in english because communicare package (the package is explained in treatments) is currently available only in english.able to consent. male and female patients aged 30 years or above, on dialysis for at least 6 months (to ensure no recovery of renal function), and under the supervision across pan - manchester sites. patients less than 30 years of age have a low rate of vascular events and will not be recruited. serum phosphate level of 1.7 mmol / l or greater after wash - out (discontinuation) of previous phosphate binding medication. able to achieve renal association standards for quality of dialysis on the most recent test of dialysis efficacy. able to communicate in english because communicare package (the package is explained in treatments) is currently available only in english. exclusion criteria are as follows : living donor renal transplant planned in the next 12 months.serum parathyroid hormone greater than 800 pg / ml (85 pmol / l) on 2 consecutive 2- or 3-monthly blood tests. such patients probably have uncontrolled hyperparathyroidism which adversely influences serum phosphate levels and needs treatment in its own right.known intolerance of both oral sevelamer and lanthanum carbonate.medical history that might limit the individual 's ability to take the trial treatments for the duration of the study (e.g., history of cancer other than nonmelanoma skin cancer or recent history of alcohol or substance misuse). living donor renal transplant planned in the next 12 months. serum parathyroid hormone greater than 800 pg / ml (85 pmol / l) on 2 consecutive 2- or 3-monthly blood tests. such patients probably have uncontrolled hyperparathyroidism which adversely influences serum phosphate levels and needs treatment in its own right. medical history that might limit the individual 's ability to take the trial treatments for the duration of the study (e.g., history of cancer other than nonmelanoma skin cancer or recent history of alcohol or substance misuse). prior to randomisation, the number of potentially eligible participants will be identified (medical records) and each will be sent the participant information sheet with an invitation to attend a screening clinic appointment. written informed consent will be sought from those who attend the appointment and are willing to participate (3 weeks). they will undergo an assessment of adherence at baseline using modified basel assessment of adherence scale for immunosuppressives (baasis). patients whose average serum phosphate is more than or equal to 1.7 mmol / l despite ongoing therapy with phosphate binders will be randomised. patients whose average serum phosphate is less than 1.7 mmol / l, and who are taking phosphate binders, will enter a 3- to 5-week wash - out period from their previous phosphate binder and receive standard dietary advice from a renal dietician. serum lipids will be measured and treated according to uk renal association guidelines with a statin and/or ezetimibe. equivalent lipid levels would be required in the larger definitive trial to exclude the possibility of this influencing mortality. this is because two non - calcium containing phosphate binders, sevelamer and colestilan (bindren trade name), also reduce serum cholesterol. sevelamer has been observed to reduce absorption of advanced glycated end - products, bacterial toxins, and bile acids, suggesting that it may reduce inflammatory, oxidative, and atherogenic stimuli in addition to its lowering of serum phosphate. provided that serum phosphate level rises to greater than or equal to 1.7 mmol / l after wash - out, each participant will be randomised to either the lower range (lrg) or higher range (hrg) phosphate group. this minimises the possibility of an individual being randomised to hrg but whose phosphate level will not reach 1.7 mmol / l. those randomised to lrg will undergo a stepped approach to aid achievement of the lower range phosphate target. the treatment for each study visit is summarised in table 1. during each of their dialysis sessions in the first week after randomisation, participants will be given access to, and encouraged to utilise, the communicare online patient adherence support programme. this comprises a patient questionnaire, developed by the london school of behavioural science, which highlights individual patient information needs and concerns related to taking phosphate binding medication. questionnaire results give participants access to online tailored, personalised (but reproducible and standardised) information or info bytes to help address the concerns they have about taking phosphate binding medication. the package also provides training for the study staff to enable them to discuss phosphate control knowledgeably with participants. there is a paper version of the communicare package which can also be used. the lrg participants will be able to access the communicare online support programme at any time but will be specifically encouraged to do so again during dialysis session in month 4 and month 8. they will recommence oral phosphate binding medication with either lanthanum carbonate or sevelamer (either carbonate or hydrochloride), titrated on a weekly basis with meals to achieve serum phosphate of 0.8 to 1.4 since sevelamer reduces serum lipid levels, those individuals taking a statin for cholesterol reduction may require dose adjustment. the dose adjustment of statin will be completed by the study clinicians according to their clinical judgement, with a view to maintaining the serum cholesterol according to the standards set by the renal association. this is a validated questionnaire which we have modified to reflect phosphate binders instead of immunosuppressants. this questionnaire has been validated in kidney transplant patients on immunosuppressive medications and in hiv patients who are on antiretroviral medications. both of these groups need to take their medications on a regular timely basis. this criterion applies to the administration of phosphate binders which need to be taken regularly and with each meal to be effective. all patients will have their adherence assessed at baseline ; only the patients randomised to the lrg will continue to have their adherence assessed once every 4 weeks. those randomised to hrg will recommence oral phosphate binding medication in the weeks following randomisation, with either lanthanum carbonate or sevelamer, titrated on a weekly basis to achieve a serum phosphate level of 1.82.4 mmol / l. the participants will have a range of licensed phosphate binding medication to choose from chewable tablets (lanthanum fosrenol), tablets to be swallowed (sevelamer renvela, renagel), and granules that can be mixed with water and consumed (fosrenol, renvela)as first - line therapy. changes to the phosphate binders and the cholesterol medications during the study will be documented in a drug dosing diary which will be carried by the patient. phosphate in all participants will be monitored on a monthly basis from week 8 onwards, with medication adjustments as necessary to maintain results within range. all haemodialysis patients have blood taken routinely for biochemical and haematological measurements on a monthly basis ; attempts will be made to ensure that the study blood tests coincide with the routine monthly blood tests ; this ensures a reduction in the number of additional blood tests required by this study during the maintenance phase. a blood sample will be collected at the consent visit, at randomisation, and every 12 weeks thereafter to measure the serum level of parathyroid hormone (pth). all dialysis patients have this blood test once every three months as part of routine clinical care. efforts will be made to ensure that the study blood test coincides with their routine test to minimise the number of extra blood tests. participants will be asked to gift an extra 5 ml of blood with every blood sample collected for parathyroid hormone. this will be stored in the biobank at the renal research laboratories, manchester royal infirmary, for future biomarker analysis. all participants will complete the pittsburgh dialysis symptoms index again at month 6 and month 12 (study end). oral vitamin d dosage will be altered if necessary to ensure good control of serum calcium pth. they will be commenced on one of the two non - calcium containing phosphate binders determined by their preference (chewable, swallowed, and granules). the dosage of the medication will be increased once a week during the titration phase. the changes to drugs and dosages done as part of the study will be recorded in a drug dosing diary which the patient will carry. phosphate binders are prescribed in daily doses divided according to the estimated phosphate content of the meals. for some patients, this is three equal doses, whilst for others it might be two different daily doses. standard practice is for this advice to be given by the prescribing physician or by a renal dietician. the dosing schedule shown in table 3 is only a guide and the phosphate binders can be dosed according to clinician judgement. it is expected that many patients in the hrg will not require a phosphate binder. however, at any stage during the study, oral non - calcium containing phosphate binder will be introduced if the serum phosphate level exceeds the upper limit of 2.4 mmol / l. the intention would be to reduce and stabilise the phosphate level within the specified range of 1.72.4 mmol / l. table 4 outlines the titration regime, but if a patient 's serum phosphate exceeds 2.4 mmol / l for the first time during, for example, week 4, then they should commence titration at that point. therefore this table describes titration steps rather than study weeks. the participants in both of the groups are allowed to switch their phosphate binding medication at any stage in the study to one of the other non - calcium containing formulations. they will then be changed to their trial phosphate binding medication of choice at a dose determined by the trial physicians. the target is to achieve the desired range of serum phosphate with a dose and combination that is convenient to the patient in order to encourage adherence. the dose of the study medications can be altered to maintain the serum phosphates in the desired range in the maintenance period on a monthly basis, according to the discretion of the study clinicians. no formal sample size calculation has been conducted given the exploratory and evaluative nature of the study. we will randomise 100 patients in total to the lower range group and higher range group. assuming an overall attrition rate of 25% at 12 months, 75 patients will complete the data - monitoring period. this will be sufficient to allow the monitoring of logistical aspects of this study (such as recruitment, randomisation, and attrition). detailed statistical evaluation will not be undertaken, and therefore the sample size is chosen to be representative of the manchester dialysis population (1100 in total). it will be large enough to address the outcome measures but small enough to facilitate timely recruitment and follow - up. we will be able toestimate a confidence interval for the proportion of patients achieving consistent control of serum phosphate in each group, estimate the major cardiovascular event (including death) rate and a confidence interval for this parameter, observe and record time - to - event data. estimate a confidence interval for the proportion of patients achieving consistent control of serum phosphate in each group, estimate the major cardiovascular event (including death) rate and a confidence interval for this parameter, observe and record time - to - event data. these calculations will help to inform the sample size calculation for a multicentre randomised controlled trial, for which mortality and cardiovascular event rate are expected to be the primary outcome measures but only if sufficient events are observed. we will also monitor and summarise recruitment and attrition rates and collect data on reasons for study withdrawal. table 5 gives a list of assessment measures used for the different endpoints in the study. this study will inform design of a large definitive trial of the effect of phosphate on mortality and cardiovascular events in dialysis patients, starting 2016/17. if reduction of phosphate improves life expectancy, then both clinicians and patients will be better informed and will be able to address this issue more certainly and appropriately, despite current drawbacks of phosphate binding medication. the time, inconvenience, and expense associated with phosphate control would be justified. if there is no benefit to reducing phosphate to a prespecified range, then patients may be relieved of the burden of excessive binding medication and side - effects. savings from reduced prescriptions could be redirected to develop other methods believed to improve patients ' quality and quantity of life, for example, increased provision of home dialysis therapies, with benefits to nhs capital / revenue expenditure. a study which could definitively show a cause - effect relationship between serum phosphate levels and clinical outcomes like mortality and cardiovascular events will be a game - changer in the management of dialysis patients.	background. retrospective, observational studies link high phosphate with mortality in dialysis patients. this generates research hypotheses but does not establish cause - and - effect. a large randomised controlled trial (rct) of about 3000 patients randomised 50 : 50 to lower or higher phosphate ranges is required to answer the key question : does reducing phosphate levels improve clinical outcomes ? whether such a trial is technically possible is unknown ; therefore, a study is necessary to inform the design and conduct of a future, definitive trial. methodology. dual centre prospective parallel group study : 100 dialysis patients randomized to lower (phosphate target 0.8 to 1.4 mmol / l) or higher range group (1.8 to 2.4 mmol / l). non - calcium - containing phosphate binders and questionnaires will be used to achieve target phosphate. primary endpoint : percentage successfully titrated to required range and percentage maintained in these groups over the maintenance period. secondary endpoints : consent rate, drop - out rates, and cardiovascular events. discussion. this study will inform design of a large definitive trial of the effect of phosphate on mortality and cardiovascular events in dialysis patients. if phosphate lowering improves outcomes, we would be reassured of the validity of this clinical practice. if, on the other hand, there is no improvement, a reassessment of resource allocation to therapies proven to improve outcomes will result. trial registration number. this trial is registered with isrctn registration number isrctn24741445.
implant rupture has been reported as an uncommon reason for failure of a revision total hip arthroplasty (tha). this rare type of stem failure poses a surgical challenge since the distal portion of the stem is usually well fixed due to significant bony ingrowth and removal can create femoral perforations, femoral fractures or could require the creation of a cortical window. the use of hollow trephine reamers in order to over - drill the well - fixed distal femoral component has been reported as a satisfactory technique in recent reports. we hereby present two cases in which this complication occurred and present how they were managed, the clinical result obtained, and discuss the possible causes leading to this rare complication. a 70-year - old male had undergone uncemented tha for the treatment of primary osteoarthritis in 1996. comorbilites included cardiovascular disease without angor since 2003, diabetes mellitus type 2 and hypertension. the primary implant was revised to a 190-mm - long, 11-mm - diameter extensively porous coated straight stem (echelon, smith & nephew) and to a 56- mm - diameter acetabular component (reflection, smith & nephew). through surgery an anterior metaphyseal window was performed in order to facilitate removal of the primary implant and it was closed by using three 1.6 mm dall - miles cable system (stryker). an intra - operative greater trochanter fracture was noted right after extraction. the proximalfemur fracture was managed with the use of another 1.6 mm dall miles cable system. the postoperative period was uneventful, and the patient was discharged. during follow - up the patient remained asymptomatic, and the radiological studies depicted a progressive healing of the trochanteric fracture. 25 months after hip revision the patient consulted again, explaining an increase in pain intensity, focusing mainly on the thigh while walking. this time the radiographic study revealed a transverse femoral stem fracture at the junction of the proximal and the middle third, at the level of the third cable (fig. radiological evolution after revision surgery ; x - ray ap views : i.a.) 1 year after primary hip arthroplasty revision ; 1.b.) 25 months after hip revision ; the femoral stem fracture can be observed at the junction of the proximal and the middle third ; 1.c.) 1 year after revision of the fatigued femoral stem femoral stem at revision surgery, proximal release of the femoral stem was easy because it was loose. the distal portion of the femoral component was removed by using a series of hollow trephines (depuy, warsaw, ind) since the distal portion of the implant was well fixed. the new femoral component implanted was a non - cemented, 200-mm - long 16mm - diameter modular stem with a 60 mm modular metaphysis (lima corporate) (fig2). radiological evolution after revision surgery ; x - ray ap views : 2.) 37 months after revision surgery ; the femoral stem fractured at the junction between de proximal loose part and distally well - fixed zone and 2.) 1 year after revision of the fatigued femoral stem intra - operative cultures were negative ; and the patient was discharged after a satisfactory postoperative evolution, with a total hospital stay of 13 days. at one - year follow - up, the patient walks with the aid of one stick and refers no pain, with a harris hip score 79.19. the present patient is a 73-year - old female with a pathological history of hypertension, dyslipidaemia and hiatal hernia. she underwent cementless tha for the treatment of primary osteoarthritis 12 years before, and in 2006 the patient was diagnosed of aseptic loosening. during the revision surgery, a trochanteric extended osteotomy was performed to remove the femoral stem ; it was revised to a 190-mm - long, 12-mm - diameter extensively porous coated straight stem (echelon, smith & nephew) and to a 52-mm - diameter acetabular component (reflection, smith & nephew). the trochanteric osteotomy was closed using one 1.6 mm dall - miles system cable. in october 2009 the radiographic study revealed a femoral stem fatigue fracture just proximal to the diaphyseal isthmus [fig.2 ]. she underwent revision surgery : as in the previous case, the proximal part of the broken femoral stem was released easily due to its loosening ; on the other hand, the distal part of the femoral stem was firmly attached to the bone and again a series of hollow trephines (depuy, warsaw, ind) were used. in order to expose the distal femur and to guide the trephines, a transverse osteotomy was performed one centimetre distal to the rupture site. due to the increase of the diameter of the medullar canal after using the trephines, the failed femoral stem was revised to a non - cemented, bowed 16mm - diameter, 200-mm - long, modular stem with a 70 mm modular metaphysis (lima corporate). the patient was discharged after an uneventful postoperative period of 15 days. at 18 month harris hip score is 72.7 and the follow - up radiographies show a correct integration of the implant [fig.2 ] a 70-year - old male had undergone uncemented tha for the treatment of primary osteoarthritis in 1996. comorbilites included cardiovascular disease without angor since 2003, diabetes mellitus type 2 and hypertension. the primary implant was revised to a 190-mm - long, 11-mm - diameter extensively porous coated straight stem (echelon, smith & nephew) and to a 56- mm - diameter acetabular component (reflection, smith & nephew). through surgery an anterior metaphyseal window was performed in order to facilitate removal of the primary implant and it was closed by using three 1.6 mm dall - miles cable system (stryker). an intra - operative greater trochanter fracture was noted right after extraction. the proximalfemur fracture was managed with the use of another 1.6 mm dall miles cable system. the postoperative period was uneventful, and the patient was discharged. during follow - up the patient remained asymptomatic, and the radiological studies depicted a progressive healing of the trochanteric fracture. 25 months after hip revision the patient consulted again, explaining an increase in pain intensity, focusing mainly on the thigh while walking. this time the radiographic study revealed a transverse femoral stem fracture at the junction of the proximal and the middle third, at the level of the third cable (fig. radiological evolution after revision surgery ; x - ray ap views : i.a.) 1 year after primary hip arthroplasty revision ; 1.b.) 25 months after hip revision ; the femoral stem fracture can be observed at the junction of the proximal and the middle third ; 1.c.) 1 year after revision of the fatigued femoral stem femoral stem at revision surgery, proximal release of the femoral stem was easy because it was loose. the distal portion of the femoral component was removed by using a series of hollow trephines (depuy, warsaw, ind) since the distal portion of the implant was well fixed. the new femoral component implanted was a non - cemented, 200-mm - long 16mm - diameter modular stem with a 60 mm modular metaphysis (lima corporate) (fig2). radiological evolution after revision surgery ; x - ray ap views : 2.) 37 months after revision surgery ; the femoral stem fractured at the junction between de proximal loose part and distally well - fixed zone and 2.) 1 year after revision of the fatigued femoral stem intra - operative cultures were negative ; and the patient was discharged after a satisfactory postoperative evolution, with a total hospital stay of 13 days. at one - year follow - up, the patient walks with the aid of one stick and refers no pain, with a harris hip score 79.19. the present patient is a 73-year - old female with a pathological history of hypertension, dyslipidaemia and hiatal hernia. she underwent cementless tha for the treatment of primary osteoarthritis 12 years before, and in 2006 the patient was diagnosed of aseptic loosening. during the revision surgery, a trochanteric extended osteotomy was performed to remove the femoral stem ; it was revised to a 190-mm - long, 12-mm - diameter extensively porous coated straight stem (echelon, smith & nephew) and to a 52-mm - diameter acetabular component (reflection, smith & nephew). the trochanteric osteotomy was closed using one 1.6 mm dall - miles system cable. in october 2009 the radiographic study revealed a femoral stem fatigue fracture just proximal to the diaphyseal isthmus [fig.2 ]. she underwent revision surgery : as in the previous case, the proximal part of the broken femoral stem was released easily due to its loosening ; on the other hand, the distal part of the femoral stem was firmly attached to the bone and again a series of hollow trephines (depuy, warsaw, ind) were used. in order to expose the distal femur and to guide the trephines, a transverse osteotomy was performed one centimetre distal to the rupture site. due to the increase of the diameter of the medullar canal after using the trephines, the failed femoral stem was revised to a non - cemented, bowed 16mm - diameter, 200-mm - long, modular stem with a 70 mm modular metaphysis (lima corporate). the patient was discharged after an uneventful postoperative period of 15 days. at 18 month harris hip score is 72.7 and the follow - up radiographies show a correct integration of the implant [fig.2 ] extensively porous coated stems are commonly used in revision hip arthroplasty, and rupture of the implant is rare. several factors can be related to this breakage : those related to the implant itself, and those that depend on the patient. implant related risk factors involve : the stem diameter, the material composing the implant and the use of cables. stems with smaller radii are considered substantially more susceptible to fatigue fracture, and the use of metals with a high young modulus (such as cobalt - chrome) is preferable. each of the two patients in the present series presented a mechanical failure of an extensively porous coated stem (echelon, smith & nephew), which is a cobalt - chrome, non - modular, fully porous - coated, distally slotted, and fluted implant. this stem is available in two lengths : 190 mm (straight stem) and 260 mm (bowed stem) ; and diameters from 11 to 22 mm are available. it is important to outline that some manufacturers do not produce implants with a diameter lesser than 14 mm in order to avoid implant fracture. in a previous series by landa. only one patient had an implant diameter lesser that 13 mm ; in our cases both patient&s implants had a small diameter, 11 mm and 12 mm respectively. although the use of cables has not been proven to be a risk factor for fracturing a prosthetic revision stem, it is possible that they serve as a fulcrum for the cantilever bending forces and may predispose to fractures in particular when they erode into the cortical bone. patient related risk factors involve : age, body mass index (bmi), and poor proximal bone stock. younger aged patients probably have an increased activity, which may lead to more cycles of cantilever loading. some works describe cohorts, who usually are under 65 years old [1, 8 ] ; nevertheless this is not the case in our study where the mean age was actually 72 years old. it is / unknown to what degree age might be a factor in fatigue failure of revision femoral stems. charnley reported an inordinately high rate of stem fractures in patients who weighed more than 88 kg, however, the bmi of presented cases were not elevated (their bmi was 27,67kg / m2 and 24,75 kg / m2 respectively). one of them had a trochanteric fracture in the primary implant extraction process and the other one had to undergo a trochanteric osteotomy in order to retrieve the primary implant. these two processes healed with no complication in the postoperative period but while doing so, and coupled with distal bony ingrowth, it would have created a cantilever force that was the most likely cause of metal fatigue and ultimate failure of the femoral component with cyclic bending stress. the use of trephines for extraction of the distal portion of a cylindrical broken femoral stem has previously been described. a transverse osteotomy one centimetre distal to the distal ruptured part of the implant was conducted. this allowed the trephines to be self - guided in the reaming process making the final extraction easier and preserving the bone stock as much as possible. the teeth on the trephines are quickly worn out during reaming of the well - fixed prosthesis ; multiple trephines must be available, particularly if the prosthesis is long. these drill - powered reamers build up considerable heat, and continuous irrigation is necessary [12 - 13 ]. it is also noticeable that in both cases thigh pain was a common reason of symptomatic complaining previously to the occurrence of the stem fracture.. however, thigh pain is a relatively common finding after total hip arthroplasty revision using long stems, and nowadays there are not imaging techniques to detect an impending fracture of the stem. few ruptures of extensively porous coated stems have been previously reported in the literature. many of the characteristics that were previously described, were present in our two patients ; mainly, poor proximal bone support and extended trochanteric osteotomy. in addition, the use of a small diameter stem (defined as less than 14 mm) was used in both cases. other factors such as young age or elevated bmi were not present in our patients. although there is not a consensus about the possible role of the previous use of cables in the development of the stem rupture, we consider that they could also play a role. finally, in order to ease the revision procedure, the use of hollow trephines is recommended and, as presented in one of the cases, a transverse osteotomy of the femur can be considered in order to aid in the removal of the broken hardware. rupture of the stem in revision total hip arthroplasty is a rare condition, however risk factors can be identified in order to prevent them. stem rupture is a challenging situation that should be assessed by specialized hip units with a very detailed preoperative planificaction.	introduction : mechanical failure of femoral stems of revision hip arthroplasty has been rarely reported. in the current study, the cause of two stem fractures, which occurred in vivo, was analysed with use of clinical and radiological data, and the functional result after revision is presented.case report : two patients, a 70-year - old male and a 73-year - old female, both of mediterranean ethnic, and both patients underwent a revision total hip replacement to an uncemmented extensively porous coated stem. both stems suffered an implant fatigue in vivo at three years and at two years follow - up respectively.conclusion:revision total hip arthroplasty is a procedure that will be performed more often the following years due to aging of population. any orthopaedic surgeon performing hip surgery should be aware of the risk factors that can lead to total hip arthroplasty failure. in the analysed cases we can learn that the main factors related to this failure included the use of a small size stem (inferior to 14 mm), an inadequate proximal osseous support because of trochanteric osteotomy, and a reduced preoperative bone stock. although the use of cables has not been stated as a predisposing factor, we consider that they could also play a role in the development of this rare complication.
experience has shown that routine examination and diagnostic procedures is not enough to evaluate pelvic pathology of infertile women. the ability to observe and treatment the uterus, fallopian tubes, and ovaries during laparoscopy has made it a gold standard to evaluate pelvic pathology (2). similarly, visualizing the uterine cavity and identifying the possible pathology has made hysteroscopy an essential part of infertility evaluation. the abnormalities of pelvic and uterus can resolved in combined hysterolaparoscopy, such as the lesion of tubal morphology and patency, ovarian morphology, and uterine cavity abnormalities at the same time (3). therefore, the aim of this study was to assess the effects and safeness of combined hysterolaparoscopy on evaluation the causes of infertility. this retrospective study was conducted at the department of gynecology (the third affiliated hospital of sun yat - sen university, guangzhou, china) from january 2011 to april 2014. patients between 21 and 43 years of age with infertility were included in this study. infertility is defined as the failure of a couple to conceive pregnancy following 12 months of unprotected intercourse. primary infertility patients were those who had never conceived before, while secondary infertile patients had at least one prior conception, irrespective of the outcome. patients with abnormal ovulation who were treatment six cycles but failed to get a pregnancy, patients who were suspected with fallopian tube abnormalities or endometriosis or patients with unexplained infertility were suggested to do this operation. out of 132 patients, 71 (53.8%) women had primary infertility and the rest 61 (46.2%) had secondary infertility. the patients in secondary infertility group were elder compared to primary infertility group (30.154.54 vs. 32.84.5.25 years, p = 0.002). abnormalites detected in laparoscopy were more common than those in hysteroscopy both in primary infertility group and in secondary infertility group. endometriosis and pelvic inflammatory disease were the most common abnormalities detected in laparoscopy in two groups (table 1). prevalence of lesions detected in laparoscopy the most common intrauterine pathology in both the groups was uterine polyps (47.89% in primary infertility group ; 29.51% in secondary infertility group). out of 12 patients having malformation uterus, only one was double uterus and double cervical with double vagina (table 2). prevalence of lesions detected in hysteroscopy there was no major surgical or anesthetic complication in any of our patients, other than mild abdominal pain. hysterolaproscopy may appear to be invasive, but it may become more beneficial, as diagnosis and therapeutic interventions can be done at the same sitting. the decisions for artificial reproductive technique can be taken in time after the evaluation of hysterolaparoscopy (4). other than mild abdominal pain, there were no major surgical or anesthetic complications in any of our patients. pelvic inflammatory disease and endometriosis is still the two major lesions found among infertility women in more than 90% reports (6, 7). the patients already be treated by special doctors for some cycles and have no baby before operation. this could explain why the incidence of pelvic inflammatory disease and endometriosis was higher than other studies. the gold standard technique for finding these disorders is laparoscopy, which is a good predictor of future spontaneous pregnancy in infertile couples with unexplained infertility (9). snowden reported that the false negative rate of hysterosalpingography (hsg) was 13% and the false positive rate was 16% (10). hsg and laparoscopy are the two classic methods for evaluation of tubal pathology and are complementary to each other. although pelvic sonography and hsg are good enough to exclude gross intrauterine pathology, subtle changes need be found and treated with hysteroscopy. hysteroscopy is good at treatment of proximal obstruction of fallopian tube and laparoscopy is good at treatment of peritubal adhesions and hydrosalpinx. hysteroscopy and laparoscopy are the two methods for evaluation and treatment of tubal pathology and are complementary to each other (4). uterine pathologies were the cause of infertility in about 15% of infertile couples and were diagnosed in about 50% of infertile women (11, 12). among all congenital uterine abnormalities, septate uterus is the most common cause associated with highest reproductive failure rate (13, 14). the incidence of asymptomatic endometrial polyps in infertile women has been reported to range from 10% to 32% (15, 16). developmental uterine anomalies have long been associated with pregnancy loss and obstetric complications, but the ability to conceive is generally not affected. this can explained why the incidence of uterine anomalies in primary infertility group was similar to those in secondary infertility group in this study. it is a very useful tool that can detect and treat various structural abnormalities in multiple sites like pelvis, tubes, and uterus in the same time, especially in couples with normal ovulation and sperm quality. hysterolaparoscopy may be recommended as the first and final procedure for evaluation of female infertility.	objectives : to evaluate the effects and safeness of combined hysterolaparoscopy on evaluation the causes of infertility.methods:this retrospective study was conducted at the department of gynecology (the third affiliated hospital of sun yat - sen university, guangzhou, guangdong, china) from january 2011 to april 2014. patients aged 2143 years with infertility were included in this study. the prevalence of different lesions was collected to analyze.results:132 infertile patients were included, 71 (53.8%) women had primary infertility and the rest 61 (46.2%) had secondary infertility. laparoscopic abnormalites were more common than hysteroscopy abnormalites both in primary infertility group and secondary infertility group. pelvic inflammatory disease (59.09 %) and endometriosis (29.55%) were the most common abnormalities in two groups. the most common intrauterine pathology was uterine polyps and the most common uterine malformation was uterine septum in two groups. out of 12 patients having malformation uterus, only one was double uterus and double cervical with double vagina. there was no major surgical or anesthetic complication in any of our patients, other than mild abdominal pain.conclusion:hysterolaparoscopy is an effective and safe tool in comprehensive evaluation of infertility to diagnosis and treat the lesions of pelvic and uterus in the same time. hysterolaparoscopy may be recommended as the first and final procedure for evaluation of female infertility.
the wide availability and technical improvement of magnetic resonance imaging (mri) has led to an increase in detection of vestibular schwannoma (vs) at an early stage. the natural development of vs remains uncertain as growth percentages between 30% and 90% have been reported, depending at least in part on the length of the observation period [2, 4 ]. so far, no clinical parameters have been identified to correlate with vs growth [57 ], and therefore, vs growth is objectified by performing consecutive mri [2, 5 ]. if growth is found on mri, an intervention may be chosen, such as surgical resection or radiation therapy [6, 8 ]. most patients therefore enter the so - called wait and scan policy for a certain period, in which the audiovestibular symptoms are monitored regularly and vs growth is measured on consecutive mri. radiologists generally use two - dimensional measurements to assess vs growth, although volume measurements seem to provide more accurate growth assessment [1, 9, 10 ] since vs shows asymmetric growth in all directions. little information is published about the diameter increase or volume increment between subsequent images that constitute to vs growth beyond measurement error [912 ]. usually, when an increase in tumor diameter or volume is found, it is considered to be growing, but validation of this observation is lacking. especially when invasive treatment decisions are based on these observations, it is of great importance to find the most suitable method to assess growth of vs on mri and to provide a definition of growth beyond measurement error. this study focuses on the accuracy and reproducibility of vs volume measurements compared to two - dimensional measurements to determine vs growth on mri. the hypothesis is that measurement of tumor volume with specific area tracing software is a more accurate tool compared to two - dimensional measurements for determining tumor growth. all patients who received an mri scan of the cerebellopontine angle (cpa) between january 2003 and march 2008 in our tertiary referral center were analyzed retrospectively. patients were included in this study if a radiological diagnosis of a vs was made, resulting in 102 patients. thirty patients were excluded who had been treated by surgery or radiotherapy, resulting in 72 patients. mri images of 68 patients, 32 (47%) men and 36 (53%) women, age range of 3684 years, median age of 63.5 years, were available ; one scan was available in 21 patients, two scans were available in 22 patients, three scans in ten patients, four scans in eight patients, five scans in four patients, and six scans in three patients, resulting in a total of 165 scans suitable for analysis. in patients with more than one scan, all examinations were performed at 1.5 t (gyroscan, powertrack 6000, philips, best, the netherlands) using a head neck coil (philips, best, the netherlands). the mr protocol consisted of axial 2d se t1-weighted images (tr / te, 550/15 ms ; slice thickness, 3 mm ; inter slice gap, 0.3 mm ; number of slices, 12;fov, 180 mm (rfov 80%) ; and matrix 256 256), axial 3d tse t2-weighted images (tr / te, 3,000/250 ms ; slice thickness, 0.35 mm ; number of overcontiguous slices, 30 ; fov, 130 mm (rfov 80%), and matrix 256 256) covering the skull base, and contrast - enhanced (gadolinium 0.2 ml / kg body weight) axial 3d iso t1-weighted images (tr / te, 8.9/4.6 ms ; slice thickness, 1 mm ; fov, 256 (rfov 80%) ; and matrix 256 256) covering the entire skull base and cranium. all patients underwent the same mri protocol with similar parameters and planes of acquisition to ascertain an optimal correlation in serial scans. two readers, experienced in head and neck imaging, independently performed the measurements on contrast - enhanced t1-weighted images (ce t1-wi) and on the corresponding t2-weighted images (t2-wi). both observers were blinded to each other s mr assessments and clinical information. for two - dimensional assessment of vs, the maximum diameter was measured in three diameters : anteroposterior (ap), mediolateral (ml) [including the portion in the internal auditory canal (iac) ], and craniocaudal (cc) (fig. volume assessment was done on a stereotactic radiotherapy treatment planning station, fitted with iplan rt image version 3advanced contouring workstation (brainlab oncology solutions, feldkirchen, germany). mr images were uploaded in this system, and area tracing software was used to outline the vs on each mr image that contained tumor tissue (fig. if there was a sharp contrast with surrounding tissue, the auto brush function (surrounding the vs automatically) was used. manual segmentation was necessary in cases in which differentiation with surrounding tissue was difficult because of the high sensitivity of this autotracer. each segmentation result was checked visually. by tracing the vs surface on all slices, fig 1a, b contrast - enhanced t1-weighted image with a vestibular schwannoma in the cerebellopontine angle (cpa) on the right side. a measurements in the axial plane : x is the maximum mediolateral, and y the maximum anteroposterior dimension ; b in the coronal plane axial contrast - enhanced t1-weighted image shows a right - sided vestibular schwannoma (asterisk) with a large cerebellopontine angle component. b three dimensional representation of a vestibular schwannoma (vs), integrating the surface of all slice intervals. the small intracanalicular (a) and large extracanalicular (b) portion of the vs can easily be identified a, b contrast - enhanced t1-weighted image with a vestibular schwannoma in the cerebellopontine angle (cpa) on the right side. a measurements in the axial plane : x is the maximum mediolateral, and y the maximum anteroposterior dimension ; b in the coronal plane, the z demonstrates the maximum craniocaudal dimension a : example of area tracing with volume software. axial contrast - enhanced t1-weighted image shows a right - sided vestibular schwannoma (asterisk) with a large cerebellopontine angle component. b three dimensional representation of a vestibular schwannoma (vs), integrating the surface of all slice intervals. the small intracanalicular (a) and large extracanalicular (b) portion of the vs can easily be identified to compare reproducibility of the measurements in different size categories, vs were classified into four stages, as defined by hasegawa. : stage a, intracanalicular vs ; stage b, vs extending into the cpa ; stage c, vs compressing the brain stem ; and stage d, vs deviating the fourth ventricle. these parameters were evaluated for measurements by two different readers at one point in time. spss 15.0 statistical software (spss, chicago, il, usa) was used to perform the statistical calculations. for evaluation of interobserver reproducibility, we used baseline measurements of the mri scans from the 68 patients for both readers, which can be considered as independent observations, whereas consecutive measurements within patients are correlated. agreement parameters measure the ability to achieve the same value in two measurements and give an indication of the size of measurement errors. the bland and altman plot is the most robust method to quantify agreement in clinical measurements. here, the differences between measurements (on the y - axis) are plotted against the mean of two measurements (on the x - axis). the visual representation of agreement illustrates the magnitude and range of the differences, bias or outliers, and the relation between the magnitude of the differences and the magnitude of the mean values. these limits of agreement are used to define the smallest detectable difference (sdd) as 1.96 the standard deviation (sd) of the mean difference in measurements between the two readers. we can define a tumor to have grown when the difference between two measurements falls outside this interval. in this way, it will be possible to discriminate between stable tumors and growing tumors, according to our measurements. for both two - dimensional and volume measurements, the sdd for absolute differences and for differences relative to baseline [sdd (%) ] was calculated. differences relative to baseline were calculated using the following formula : (ab)/[(a + b)/2 ] 100, in which a is the result of reader a and b the result of reader b. the sdd and sdd (%) were presented for four vs stages : a, b, c, and d. use of the sdd (%) enables comparison of the different unities involved in the two different measurement techniques (millimeter and cubic centimeter). because, in clinical practice, all three diameter measurements (cc, ap, and ml) are equally essential in assessing vs progression, we considered the diameter with lowest agreement as the limiting factor in diameter measurements. we compared the sdd (%) of this diameter with the sdd (%) of volume measurements. reliability parameters assess whether measurements can be used to distinguish patients from each other despite measurement error. the iccs are defined as the ratio of the variance among patients (patients variability) over the total variance (among patients, among readers, plus the error variance), which is expressed as a dimensionless number, being one (perfect reliability) in the most ideal case. icc was calculated for interobserver diameter and volume vs measurement and was also presented for four vs stages : a, b, c, and d. these parameters were evaluated for measurements by two different readers at one point in time. spss 15.0 statistical software (spss, chicago, il, usa) was used to perform the statistical calculations. for evaluation of interobserver reproducibility, we used baseline measurements of the mri scans from the 68 patients for both readers, which can be considered as independent observations, whereas consecutive measurements within patients are correlated. agreement parameters measure the ability to achieve the same value in two measurements and give an indication of the size of measurement errors. the bland and altman plot is the most robust method to quantify agreement in clinical measurements. here, the differences between measurements (on the y - axis) are plotted against the mean of two measurements (on the x - axis). the visual representation of agreement illustrates the magnitude and range of the differences, bias or outliers, and the relation between the magnitude of the differences and the magnitude of the mean values. these limits of agreement are used to define the smallest detectable difference (sdd) as 1.96 the standard deviation (sd) of the mean difference in measurements between the two readers. we can define a tumor to have grown when the difference between two measurements falls outside this interval. in this way, it will be possible to discriminate between stable tumors and growing tumors, according to our measurements. for both two - dimensional and volume measurements, the sdd for absolute differences and for differences relative to baseline [sdd (%) ] was calculated. differences relative to baseline were calculated using the following formula : (ab)/[(a + b)/2 ] 100, in which a is the result of reader a and b the result of reader b. the sdd and sdd (%) were presented for four vs stages : a, b, c, and d. use of the sdd (%) enables comparison of the different unities involved in the two different measurement techniques (millimeter and cubic centimeter). because, in clinical practice, all three diameter measurements (cc, ap, and ml) are equally essential in assessing vs progression, we considered the diameter with lowest agreement as the limiting factor in diameter measurements. we compared the sdd (%) of this diameter with the sdd (%) of volume measurements. reliability parameters assess whether measurements can be used to distinguish patients from each other despite measurement error. the iccs are defined as the ratio of the variance among patients (patients variability) over the total variance (among patients, among readers, plus the error variance), which is expressed as a dimensionless number, being one (perfect reliability) in the most ideal case. icc was calculated for interobserver diameter and volume vs measurement and was also presented for four vs stages : a, b, c, and d. bland and altman plots were constructed using data of the baseline mr images of the 165 scans from 68 patients (figs. 3 and 4). the sd for each reader (a and b) and the sd of the mean difference between readers are presented in table 1. the sdd and sdd (%) for absolute differences and differences relative to baseline mr images, respectively, are presented in table 2. in table 3 3bland and altman plot of baseline two - dimensional maximum craniocaudal (cc) dimension measurements on contrast - enhanced t1-weighted images (ce t1-wi). the values on the y - axis represent the measurement differences between the two readers and their mean difference (thin line). 4bland and altman plot of baseline volume measurements on contrast - enhanced t1-weighted images (ce t1-wi). the values on the y - axis represent the measurement differences between the two readers and their mean difference (thin line). interobserver differences are larger in smaller vestibular schwannomastable 1interobserver agreement parameters based on baseline t1-weighted, contrast - enhanced images (ce t1-wi) and t2-weighted images in 68 patients.baselinetwo-dimensional measurements on ce t1-wi (mm)volume measurements on ce t1-wi (cm)ccapmlvolreader a, mean (sd)8.40 (4.60)8.70 (4.90)12.70 (5.70)0.85 (1.74) a (n = 29)5.005.068.320.13 b (n = 27)9.059.7114.280.63 c (n = 6)12.5812.2318.121.29 d (n = 6)17.5018.3321.624.71reader b, mean (sd)8.20 (4.80)8.60 (5.10)13.20 (5.70)0.81 (1.69) a (n = 29)4.595.038.580.13 b (n = 27)9.279.8615.060.63 c (n = 6)11.5313.1018.281.06 d (n = 6)18.3318.9322.284.57meandiff (sddiff)0.12 (1.53)-0.18 (1.14)-0.48 (1.08)0.01 (0.055)baselinetwo - dimensional measurements on t2-wi (mm)volume measurements on t2-wi (cm3)ccapmlvolreader a, mean (sd) (n = 68)7.60 (4.5)8.20 (5.00)11.90 (5.50)0.72 (1.49)reader b, mean (sd) (n = 68)7.20 (4.00)8.40 (4.90)12.10 (5.00)0.72 (1.50)meandiff (sddiff)0.35 (1.41)0.18 (1.37)0.21 (1.69)0.07 (0.069)the rows of reader a and b of each imaging modality present the means and standard deviations of the measurements assessed by these readers. the means and standard deviations are also subdivided according to vestibular schwannoma stage for ce t1-wi. the mean difference row presents the mean differences between the measurements of reader a and b and the sd of these differencesa intracanalicular, b extracanalicular, c compressing brain stem, d deviating fourth ventricle, sd standard deviation, cc craniocaudal dimension, ap anteroposterior dimension, ml mediolateral dimension, vol volume, meandiff mean of the observer difference, sddiff standard deviation of the differences between observer a and b, ce t1-wi contrast enhanced t1-weighted images, t2-wi t2-weighted imagestable 2sdd and sdd (%) for measurements performed on t1-weighted, contrast - enhanced (ce t1-wi) images and t2-weighted images.ccccapapmlmlvolvolsdd (mm)sdd (%) sdd (mm)sdd (%) sdd (mm)sdd (%) sdd (cm)sdd (%) t1 a d (n = 68)2.9840.32.2328.32.1220.90.1119.7t2 a d (n = 68)2.7640.12.6934.32.6930.520.1430.1t1 a (n = 29)2.4152.81.4928.01.9627.10.0528.9t1 b (n = 27)3.2334.52.5326.52.3317.80.0610.4t1 c (n = 6)3.6032.73.0528.72.4914.90.078.70t1 d (n = 6)2.9618.82.9023.71.085.000.265.70the sdd and sdd(%) for ce t1-wi are subdivided according to vestibular schwannoma (vs) stage : a intracanalicular vs, b vs extending into the cerebellopontine angle, c vs compressing brain stem, d vs deviating the fourth ventriclesdd smallest detectable difference, sdd (%) smallest detectable difference relative to baseline measurements, cc craniocaudal dimension, ap anteroposterior dimension, ml mediolateral dimension, vol volumetable 3intraclass correlation coefficients (icc) for two - dimensional and volume measurements on t1-weighted, contrast - enhanced images (ce t1-wi) and t2-weighted images with their 95% confidence intervals.ccapmlvolt1 a d0.947 (0.9150.967)0.974 (0.9570.984)0.978 (0.9650.987)0.999 (0.9991.000)t2 a d0.943 (0.9090.965)0.961 (0.9380.976)0.948 (0.9170.968)0.999 (0.9980.999)t1a (n = 29)0.564 (0.2600.768)0.862 (0.7290.932)0.947 (0.8920.975)0.987 (0.9730.994)t1b (n = 27)0.674 (0.3930.841)0.802 (0.6040.907)0.889 (0.7670.949)0.988 (0.9730.994)t1c (n = 6)0.840 (0.3150.975)0.917 (0.5880.988)0.968 (0.8220.995)0.999 (0.9931.000)t1d (n = 6)0.966 (0.8100.995)0.980 (0.8830.997)0.990 (0.9420.999)0.999 (0.9921.000)icc values for ce t1-wi are subdivided per vs stage : a intracanalicular vs, b vs extending into the cerebellopontine angle, c vs compressing brain stem, d vs deviating the fourth ventriclecc craniocaudal dimension, ap anteroposterior dimension, ml mediolateral dimension, vol volume bland and altman plot of baseline two - dimensional maximum craniocaudal (cc) dimension measurements on contrast - enhanced t1-weighted images (ce t1-wi). the values on the y - axis represent the measurement differences between the two readers and their mean difference (thin line). interobserver differences are larger in smaller vestibular schwannomas bland and altman plot of baseline volume measurements on contrast - enhanced t1-weighted images (ce t1-wi). the values on the y - axis represent the measurement differences between the two readers and their mean difference (thin line). interobserver differences are larger in smaller vestibular schwannomas interobserver agreement parameters based on baseline t1-weighted, contrast - enhanced images (ce t1-wi) and t2-weighted images in 68 patients. the rows of reader a and b of each imaging modality present the means and standard deviations of the measurements assessed by these readers. the means and standard deviations the mean difference row presents the mean differences between the measurements of reader a and b and the sd of these differences a intracanalicular, b extracanalicular, c compressing brain stem, d deviating fourth ventricle, sd standard deviation, cc craniocaudal dimension, ap anteroposterior dimension, ml mediolateral dimension, vol volume, meandiff mean of the observer difference, sddiff standard deviation of the differences between observer a and b, ce t1-wi contrast enhanced t1-weighted images, t2-wi t2-weighted images sdd and sdd (%) for measurements performed on t1-weighted, contrast - enhanced (ce t1-wi) images and t2-weighted images. the sdd and sdd(%) for ce t1-wi are subdivided according to vestibular schwannoma (vs) stage : a intracanalicular vs, b vs extending into the cerebellopontine angle, c vs compressing brain stem, d vs deviating the fourth ventricle sdd smallest detectable difference, sdd (%) smallest detectable difference relative to baseline measurements, cc craniocaudal dimension, ap anteroposterior dimension, ml mediolateral dimension, vol volume intraclass correlation coefficients (icc) for two - dimensional and volume measurements on t1-weighted, contrast - enhanced images (ce t1-wi) and t2-weighted images with their 95% confidence intervals. icc values for ce t1-wi are subdivided per vs stage : a intracanalicular vs, b vs extending into the cerebellopontine angle, c vs compressing brain stem, d vs deviating the fourth ventricle cc craniocaudal dimension, ap anteroposterior dimension, ml mediolateral dimension, vol volume two - dimensional and volume assessments of vs were performed on both ce t1-wi (cc dimension, t1cc ; ap dimension, t1ap ; ml dimension, t1ml ; volume, t1vol) and t2-wi (cc dimension, t2cc ; ap dimension, t2ap ; ml dimension, t2ml ; volume, t2vol). the dimension with highest sdd (%) was taken as a limiting factor, when comparing both imaging modalities. with two dimensional measurements, the sdd (%) for t1cc appeared to be equal to t2cc (40.3 and 40.1) however, the t2ap dimension showed higher sdd (%) compared to t1ap : 34.3 versus 28.3, respectively, the icc was consistently higher in t1cc, t1ap, and t1ml directions, compared to t2cc, t2ap, and t2ml directions (0.947, 0.974, and 0.978 versus 0.943, 0.961, and 0.948), reflecting higher reliability for ce t1-wi in two - dimensional measurements. for volume measurements, similar results were obtained : sdd (%) for t1vol was 19.7 compared to 30.1 in t2vol. t1ml were 40.3, 28.3, and 20.9, respectively (table 2). all three dimensions are equally essential in estimating vs growth. because the t1cc dimension is the limiting factor in these two - dimensional measurements, with its lowest agreement, it was used to compare the two - dimensional measurements with volume measurements. the sdd (%) for t1cc was considerably higher than the sdd (%) for volume measurements (t1vol) : 40.3% versus 19.7%. the sdd (%) decreased with increasing tumor size from stage a to type d vs, in both two - dimensional and in volume measurements (table 2). in all tumor stages, volume measurements were associated with smaller sdd (%) compared to cc dimensional measurements. the icc revealed that both two - dimensional and volume measurements showed high interobserver reliability. however, volume measurements revealed a higher reliability compared to the three diameter measurements (0.999 versus 0.947, 0.974 and 0.978). reliability increased with tumor size, and volume measurements were more reliable in all vs stages (table 3). sdd for two dimensional measurements on ce t1-wi varied from 2.12 to 2.98 mm. for volume measurements on ce t1-wi, two - dimensional and volume assessments of vs were performed on both ce t1-wi (cc dimension, t1cc ; ap dimension, t1ap ; ml dimension, t1ml ; volume, t1vol) and t2-wi (cc dimension, t2cc ; ap dimension, t2ap ; ml dimension, t2ml ; volume, t2vol). the dimension with highest sdd (%) with two dimensional measurements, the sdd (%) for t1cc appeared to be equal to t2cc (40.3 and 40.1) however, the t2ap dimension showed higher sdd (%) compared to t1ap : 34.3 versus 28.3, respectively. therefore, ce t1-wi showed highest agreement in two dimensional measurements. in addition, the icc was consistently higher in t1cc, t1ap, and t1ml directions, compared to t2cc, t2ap, and t2ml directions (0.947, 0.974, and 0.978 versus 0.943, 0.961, and 0.948), reflecting higher reliability for ce t1-wi in two - dimensional measurements. for volume measurements, similar results were obtained : sdd (%) for t1vol was 19.7 compared to 30.1 in t2vol. the sdd (%) values for t1cc, t1ap, and t1ml were 40.3, 28.3, and 20.9, respectively (table 2). all three dimensions are equally essential in estimating vs growth. because the t1cc dimension is the limiting factor in these two - dimensional measurements, with its lowest agreement, it was used to compare the two - dimensional measurements with volume measurements. the sdd (%) for t1cc was considerably higher than the sdd (%) for volume measurements (t1vol) : 40.3% versus 19.7%. the sdd (%) decreased with increasing tumor size from stage a to type d vs, in both two - dimensional and in volume measurements (table 2). in all tumor stages, volume measurements were associated with smaller sdd (%) compared to cc dimensional measurements. the icc revealed that both two - dimensional and volume measurements showed high interobserver reliability. however, volume measurements revealed a higher reliability compared to the three diameter measurements (0.999 versus 0.947, 0.974 and 0.978). reliability increased with tumor size, and volume measurements were more reliable in all vs stages (table 3). sdd for two dimensional measurements on ce t1-wi varied from 2.12 to 2.98 mm. for volume measurements on ce t1-wi, vs are benign neoplasms, originating from the neurolemnal sheath of the eighth cranial nerve and are predominantly found in the cpa and in the internal auditory canal (iac). the incidence of vs varies from 1 to 2/100,000 [2, 12, 16, 17 ], although postmortem histopathological examinations show a higher incidence of about 2.7%. this discrepancy indicates that the vast majority of vs never become symptomatic, reflecting very slow or arrested growth. therefore, the wait and scan policy has gained popularity as an alternative or prelude to surgery and radiation therapy. this can be justified, as growth is known to be extremely variable with most vs remaining stable or showing minimal growth for many years [16, 19 ]. the goal of this regimen is to minimize therapeutic risks and complications and to preserve an optimal quality of life in selected patients. because no single reliable clinical feature exists that predicts tumor growth [57 ], mri is the mainstay in the conservative management of vs [2, 5 ]. it is essential to use a measuring method that provides reliable measurements with a high interobserver agreement, as change in size is besides its clinical presentation the most relevant parameter. some authors use the largest ap and/or ml dimension, sometimes combined with the cc dimension [5, 8, 1924 ]. others use the guidelines for measuring vs described by the american academy of otolaryngology, head and neck surgery (aao - hns) [6, 7, 25 ]. firstly, two - dimensional measurements assesses vs growth in maximal three directions, while a vs shows asymmetric growth in all possible directions ; therefore, a two - dimensional assessment does not approach real tumor growth. secondly, in vs with large diameters, a small increase in diameter corresponds to a much larger increase in vs volume than a similar increase in diameter in a small vs. volume measurements can be performed in several ways ; some authors consider vs to be ellipsoid and calculate the volume using a mathematical formula [11, 21, 26, 27 ]. however, this method has shown to produce a large overestimation of vs volume [1, 11 ]. others have performed true volumetric measurements by using (semi)automatic software to calculate vs volume [1, 9, 11, 12, 21, 28, 29 ]. according to the few studies comparing two - dimensional versus non - formula - based volume measurements, the vs volume measurements are more accurate compared to two - dimensional measurements [1, 9, 10 ]. other authors disagree with this [16, 21, 27 ], and also in clinical practice, most clinicians keep relying on two - dimensional measurements. however, the results of this study indicate that vs volume measurements, especially on the ce t1-wi, produce a better interobserver agreement and reliability compared to the two - dimensional measurements. this study therefore indicates that ce t1-wi volume measurements should replace two - dimensional measurements in evaluating vs growth. the difference in interobserver agreement and reliability between the two measurement methods is of clinical significance because invasive treatment decisions are based on these observations. therefore, the measurement method with highest agreement on reliability is necessary in assessing vs growth. an exception may be made concerning small (stage a) vs. in these small intracanalicular vs, the cc dimension does not play an important role, since the diameter of the iac is usually quite constant. when one uses the aao - hns guidelines, the cc direction is not taken into account at all. then, the ap direction is the limiting factor of the remaining ap and ml directions (table 2). in stage a vs, under these conditions, we found that measurement error [sdd(%) ] of both two - dimensional and volume measurement techniques are comparable (ap 28.0, ml 27.1, and vol 28.9). therefore, both measurement techniques can be used to evaluate stage a vs. in larger vs (stages b and c), both ap and ml dimensions show higher measurement error [sdd (%) ] compared to volume measurements. in stage d vs, the sdd (%) of the ml dimension equals the sdd (%) of volume measurements : 5 versus 5.7, respectively. this can be explained by the fact that the ml dimension is the longest distance in two - dimensional vs measurements, and measurement error will decrease when measurement distances increase. this occurs both in two - dimensional and in volume measurements and should be taken into account when evaluating growth in different vs stages (table 2 and figs. 3 and 4). however, apart from this low sdd (%) of the ml dimension in stage d vs, the far worse sdd (%) of the ap dimension should also be taken into account in evaluating vs growth with two - dimensional measurements, thus pleading for the use of volume measurements in the assessment of stage b, c, and d vs. overall, contrast - enhanced images are necessary, both in volume and two - dimensional measurements, to maintain a high reproducibility, since it facilitates differentiation of vs from surrounding tissue. the calculated agreement [sdd and sdd (%) ] and reliability (icc) values were compared with findings reported in the literature. studies addressing the change in length and volume exceeding measurement error (sdd) are reported inconclusively. in two - dimensional vs measurements, many authors simply state a 1- or 2-mm increase in subsequent scans as evidence of growth without any statistical justification [2, 68, 20, 22, 31 ]. according to this present study, sdd varies between 2.12 mm for the ml dimension and 2.98 mm for the cc dimension on ce t1-wi (table 2). this indicates that there is a measurement error in two - dimensional measurements, which is not recognized when a measured increase in size of only 1 or 2 mm between follow - up scans is considered as tumor growth, and treatment decisions should be used with caution when using these arbitrary criteria. in volume measurements, an absolute increase above which one can consider a vs to have grown was not found in the literature. this study revealed sdd ranging from 0.05 to 0.26 cm (types a d) on ce t1-wi (table 2). volume increase expressed as sdd (%) varied from 15 to 89% in the literature [912 ]. however, the numbers of patients in these studies were small [9, 11, 12 ], and generally, not only baseline measurements of the vs were taken into account [9, 10, 12 ]. therefore, the percentages reported in the literature could be questioned. in this study, their contour method, similar to our volume method, had an interobserver reliability of 0.96, which is comparable to the icc of 0.99 in this study. firstly, the assessments of reproducibility parameters were based on interobserver differences and not on intraobserver differences. it was assumed that this approach better reflects clinical practice, where it is usual that different clinicians assess subsequent scans. secondly, brainlab volume software is not widely available in radiology departments thirdly, performing these volume measurements takes a little more time compared to the conventional two - dimensional measurements. in our experience, vs contouring and volume calculation typically took a few minutes. this could be a limiting factor in introducing this method for vs in daily clinical practice. in the literature, 1025 min for manual segmentation has been described, although these calculations were performed with different software and on older systems [10, 11 ]. however, there is still a human component that is responsible for an interobserver difference. it is desirable to develop software able to perform even better automated volume measurements in order to further diminish reader - related measurement error. ce t1-wi volume measurements show better interobserver agreement and reliability compared to two - dimensional measurements for the assessment of growth of vs. small intracanalicular vs form an exception. when evaluating vs growth, one has to take vs baseline characteristics into account because sdd (%) strongly depends on vs size. the 1- or 2-mm difference commonly used to define growth of vs in consecutive scans in two - dimensional measurements lies within measurement error and should not direct clinical practice.	introductiona conservative treatment strategy is often proposed as a primary treatment option in the management of vestibular schwannomas (vs). in this wait and scan policy, audiovestibular symptoms are monitored regularly, and vs growth is measured on consecutive magnetic resonance images (mri). the aim of this study is validation of two - dimensional versus volume mri assessment in the longitudinal follow - up of vs and to define tumor growth beyond measurement error.methodsmri scans of 68 consecutive patients with vs were analyzed retrospectively. two - dimensional and volume measurements on contrast enhanced (ce) t1- and t2-weighted images were performed independently by two readers. smallest detectable differences (sdd) were calculated, and intraclass correlation coefficients (iccs) were determined for both assessment methods.resultstwo-dimensional and volume measurements both showed best reproducibility on ce t1-weighted images. sdd for differences relative to baseline mri [sdd (%) ] for two - dimensional measurements had a higher interobserver error compared to volume measurements (40% versus 19.7%), which decreases when tumor size increases. the icc for two - dimensional measurements in three directions was 0.947, 0.974, and 0.978 and for volume measurements 0.999.conclusionvolume measurements are more accurate compared to two - dimensional measurements for the evaluation of vs growth. these measurements are assessed preferably on ce t1-weighted images. sdd (%) strongly depends on vs size. sdd between consecutive scans exceeds the common clinical applied criterion of 1 or 2 mm growth to define growth.
the current treatment for patients with liver metastases from colorectal cancer is multimodal, including liver resection, chemotherapy, targeted therapies (monoclonal antibodies), interventional radiology, and radiotherapy. the complete resection of liver metastases results in 5-year overall survival rates that range from 21% to 58% [13 ], which are significantly higher than those rates that are achieved by nonsurgical therapies (5-year survival rates less than 5%). thus, the only potentially curative therapy in patients with colorectal liver metastases (crlm) includes complete resection of the liver metastases. at present, crlms are considered resectable when the following criteria are met [5, 6]:the complete resection of all known disease can be achieved, at least two contiguous liver segments can be preserved, with adequate vascular inflow and outflow, with biliary drainage, the remnant liver volume is adequate to avoid postoperative liver failure. the complete resection of all known disease can be achieved, at least two contiguous liver segments can be preserved, with adequate vascular inflow and outflow, with biliary drainage, the remnant liver volume is adequate to avoid postoperative liver failure. in patients with a healthy liver, the volume of the future liver remnant (flr) should represent more than 25% of the total liver volume (tlv) to avoid postoperative liver failure [79 ]. however, in patients with chronic liver disease or chemotherapy - induced liver injury, a minimum of 40% of the tlv should be preserved [912 ]. therefore, although the frontiers of liver resection have broadened over the previous two decades, approximately three quarters of patients with crlm are not eligible for an initially curative liver resection (r0) after a preoperative evaluation. a single, very large liver metastasis, the resection of which would not spare a sufficient volume of liver parenchyma to avoid postoperative liver failure. multiple bilobar liver metastases, the complete resection of which would not preserve a sufficient volume of functional liver parenchyma. crlm involving or located in close proximity to either the bifurcation of the portal vein or the confluence of the three hepatic veins with the inferior vena cava (ivc). in this case, the resection of the liver metastasis would not allow for the preservation of a minimum of two adjacent liver segments with adequate vascular inflow and outflow. a single, very large liver metastasis, the resection of which would not spare a sufficient volume of liver parenchyma to avoid postoperative liver failure. multiple bilobar liver metastases, the complete resection of which would not preserve a sufficient volume of functional liver parenchyma. crlm involving or located in close proximity to either the bifurcation of the portal vein or the confluence of the three hepatic veins with the inferior vena cava (ivc). in this case, the resection of the liver metastasis would not allow for the preservation of a minimum of two adjacent liver segments with adequate vascular inflow and outflow. until 20 years ago, the only available treatment for these patients was palliative chemotherapy, the goals of which were to increase progression - free and overall survival ; however, there was no prospect of a cure. although survival rates increased with the advent of new chemotherapeutics (such as oxaliplatin and irinotecan) and targeted therapies (e.g., bevacizumab, cetuximab, and panitumumab), the current survival rates for these cases are still modest compared to those that can be achieved by liver resection. therefore, several therapeutic strategies were introduced to achieve a complete resection in these patients. in figure 1, we schematically present those situations in which metastases are considered unresectable and the therapeutic options that are available for conversion to resectability. in certain instances, although a minimum of two adjacent segments with appropriate vascular inflow and outflow, and biliary drainage can be preserved following the complete resection of crlm, the volume of the remaining liver parenchyma may be insufficient to avoid postoperative liver failure. such situations are generally encountered in patients who (1) require a right trisectionectomy, or (2) when a right hemihepatectomy must be performed, but the volume of the left hemiliver is prohibitively small (figures 1(a) and 1(b)). to avoid postoperative liver failure in these patients, it is advisable to attempt to increase the volume of the flr prior to the liver resection. this goal may be achieved by initially performing portal vein embolization (pve) or ligation (pvl). if the volume of the flr following a pve / pvl increases sufficiently to prevent the risk of postoperative liver failure, liver resection should be performed 48 weeks later. this therapeutic strategy is based on the observation that increasing the volume of the flr improves the function of the residual liver parenchyma following the hepatectomy [16, 17 ]. the reports of kinoshita and makuuchi revealed that the ligation or embolization of the right portal vein induces a process of atrophy - hypertrophy of the liver (increasing the safety of liver resection) in patients with hepatocellular carcinoma or hilar cholangiocarcinoma [18, 19 ]. therefore, other authors applied the same procedure in patients with crlm whose flr was insufficient to avoid postoperative liver failure [10, 2022 ]. the rationale for such an approach is that the embolization or ligation of the right portal branch abolishes the portal inflow into the right hemiliver, leading to its atrophy ; alternatively, the portal inflow into the left hemiliver increases, causing hypertrophy of the flr. this approach permits the performance of the scheduled hepatectomy (while concomitantly reducing the risk of fatal liver failure) in more than 50% of patients who were otherwise unresectable due to a small flr. concerns regarding the comparative effectiveness of pve versus pvl have been raised by certain authors. in an animal model, furrer. revealed that the hypertrophy of the left hemiliver significantly increased following pvl versus pve. these authors hypothesized that the entrapment of a greater number of macrophages in the embolized liver (due to the foreign - body reaction that is induced by the material used for embolization) explains this result. their conclusion that pvl is superior to pve in inducing a regenerative response of the remnant liver is in contrast to that of wilms., who stated that although pvl and pve both induce liver hypertrophy, pve is the most effective technique to increase the flr. these authors stated that pve - induced vascular occlusion is more durable than that induced by pvl. furthermore, the cause of the inferior regeneration in the ligation group was reported to be the formation of collaterals between the occluded and nonoccluded portions of the liver. to avoid this undesirable situation, lastly, in addition to these experimental studies, a retrospective study of 35 patients revealed that pvl and pve are similar in terms of both increasing the flr and the conversion to resectability rate. pve- or pvl - induced liver hypertrophy involves both segments 2 - 3 and segment 4. most patients that are subjected to pvl / pve require a right trisectionectomy. in such patients, the principal objective of this maneuver is to increase the volume of segments 2 - 3 and not the volume of segment 4 (which is resected). to primarily increase the volume of the left lateral section, certain authors state that the optimum approach is to concomitantly occlude the right portal vein and the portal branches to segment 4 (right trisection portal vein embolization - r3pe). in 2000, nagino. presented results that support this hypothesis, demonstrating that the volume gain of the left lateral section was higher in patients with r3pe relative to patients who received right portal vein embolization. to date, we have performed r3pl in one patient, and the results were outstanding : the percent of flr gain was 16.22%, whereas this metric was 10.8% in the patients who received a right portal branch ligation. however, another study that was published in 2005 failed to confirm these results, revealing that the mean volume of segments 2 - 3 following embolization and the rate of the segments 2 - 3 volume increase were similar between the patients who received a r3pe and those that received a standard right portal vein embolization. therefore, definitive conclusions can not be drawn regarding the usefulness of the embolization of the portal branches to segment 4, and further studies are required to clarify this subject. most centers now prefer to routinely perform only right portal branch embolization / ligation in such patients. to achieve a more marked and rapid hypertrophy of the flr following portal vein ligation, schnitzbauer.. recently recommended the association of right portal vein ligation with in situ liver transection / splitting. using this approach, the authors achieved a significant and more rapid hypertrophy of the flr, enabling subsequent curative liver resection during the same hospitalization. the authors concluded that this technique induces the rapid and more robust growth of the flr than is reported with portal vein occlusion alone. moreover, this approach allows for the performance of a staged liver resection during a single hospital stay. two formulas can be used to calculate the percent of the flr gain following pve / pvl:(volume of the flr following pve volume of the flr prior to pve) 100/volume of the flr prior to pve, % flr following pve % flr prior to pve. (volume of the flr following pve volume of the flr prior to pve) 100/volume of the flr prior to pve, % flr following pve % flr prior to pve. in a series of 30 patients, the percent of the flr gain (calculated using the first formula) was 42%, whereas the flr gain ranged from 9.7% to 13% in different series using the second formula [21, 27, 30 ]. in most patients, these percentages are generally sufficient to allow for a safe resection of liver metastases. when pve is planned, bevacizumab should be used with caution given that aussilhou. revealed the detrimental effect of this medication on the flr gain ; this effect is especially strong in patients who are older than 60 years and received more than six cycles. however, other authors have demonstrated that this monoclonal antibody does not impair liver regeneration following pve. the most severe complications of right pve are liver hematoma, liver abscess, thrombosis of the left portal vein, portal hypertension, and cholangitis. in a meta - analysis that was performed by aboulkhir., the morbidity rate following pve was 2.2%, and the mortality was zero. the resectability rate following pve / pvl in different centers ranges from 60% to 88% [10, 21, 22 ]. the primary reason of failure to perform the curative hepatectomy is not insufficient hypertrophy of the flr but rather the progression of the disease. in our series, 5 of 13 patients (38%) exhibited disease progression following pvl, precluding a curative liver resection. the morbidity and mortality rates that were observed following curative hepatectomy were less than 35% and 4%, respectively, in most series [10, 22 ]. the 5-year survival rate of these patients was approximately 38% [10, 21 ]. it must be noted that in patients with liver metastases in the flr, this approach is not recommended, due to the risk of rapid growth of these metastases. such patients should undergo a two - stage liver resection (see below) to clear the remnant liver prior to the pve. the term two - stage liver resection has been used by a small number of authors to define a strategy that consists of a single liver resection that is performed following pvl and which does not include a sequential liver resection herein, we shall use the nomenclature of two - stage liver resection for those procedures that consist of two consecutive hepatectomies. this therapeutic strategy is used in patients with multiple bilobar crlm, whose resection will not spare a sufficient amount of liver parenchyma to avoid postoperative liver failure. these patients usually require a right hepatectomy or a right trisectionectomy along with wedge resections of the metastases that are located in the left hemiliver or in the left lateral section (figures 1(c) and 1(d)). to avoid such extensive resections, which are accompanied by a high risk of postoperative fatal liver failure, it is recommended that a complete resection of the liver metastases be achieved in a two - stage surgical procedure. in the first stage, a limited resection of the metastases from the left hemiliver or the left lateral section (future liver remnant) is performed. in stage two (following the regeneration of the flr), the bulk of the metastatic burden is resected by a right hepatectomy or trisectionectomy. flr regeneration is essential to minimize the risks of hepatic failure following the second operation. thus, pve / pvl may be suitable in patients with small flr to increase the safety of the second hepatectomy. to facilitate the second operation and to avoid disease progression between the first and the second intervention, it may be useful to deliver systemic or locoregional chemotherapy to shrink the metastatic bulk. to minimize the inhibitory effects of the chemotherapeutic drugs on liver regeneration, the chemotherapy should be begun three weeks following the first hepatectomy. this sequence is necessary, as liver regeneration is essential to the feasibility of the second resection. such a therapeutic approach is especially useful in patients with synchronous bilobar crlm given that (1) it avoids the cumulative risks of a simultaneous primary tumor resection and major hepatectomy, and (2) it allows for the evaluation of the chemosensitivity of the liver metastases and the guiding of the adjuvant therapy following the second operation. the resection of the primary tumor is performed in the first stage, along with a limited resection of the metastases from the future liver remnant (generally from the left hemiliver or the left lateral section). short - course chemotherapy (systemic or loco - regional) should begin three weeks later. if the residual lesions will be stable or responsive to chemotherapy, the second liver resection should be performed. the results of such an approach were first published by the paul brousse group in 2000. in other series, the resectability rate ranged from 66% to 75% [27, 33 ]. among the published series, the morbidity rates following the first resection were less than 31%, and the mortality rates were zero [33, 34 ]. the morbidity rate following the second liver resection ranges from 45% to 56% [27, 33, 34 ]. despite these relative high morbidity rates, the mortality was zero in most series [27, 33 ] nonetheless, a mortality rate of 15% following the second operation was reported by adam.. the authors explained that this result was a consequence of the combination of (1) the diminished tolerance of such patients to perioperative complications due to their advanced neoplastic disease and (2) the effects of the adjuvant procedures that were used to facilitate liver resection (chemotherapy, pve). the 3-year survival rates of these patients ranged from 35% to 54% [27, 33, 34 ], with a median survival of 44 months from the diagnosis of liver metastases. in selected patients with multiple bilobar colorectal liver metastases, a " two - stage " liver resection could be avoided, by performing ultrasonically - guided hepatectomy. the implementation of ultrasonography in liver surgery dramatically alters the approach to liver metastases, permitting a more accurate diagnosis and challenging the traditional paradigms of liver resection. intraoperative ultrasound (ious) allows for the detection of additional crlm that were not revealed by preoperative imaging methods and is the most accurate technique for detecting liver tumors [36, 37 ]. however, standard ious may miss lesions that are smaller than 1 cm, especially in patients who are undergoing preoperative chemotherapy, whose crlm exhibit a similar echo - pattern to that of the surrounding liver parenchyma. the use of contrast - enhanced ious (ce - ious) was demonstrated to improve the detection of crlm and is the most sensitive and specific method for the diagnosis of crlm. in the mid 1980s, ious was first used to guide the puncture and balloon occlusion of the portal branch that feeds the portion of the liver to be resected, allowing for limited anatomical liver resections instead of major hepatectomies [3941 ]. this technique decreased the risk of postoperative liver failure and was recommended principally in patients who have hccs on liver cirrhosis. in addition, ious offers a better estimation of the spatial relationships between the liver tumors and the intrahepatic vessels, permitting the resection of liver masses with the preservation of intrahepatic vascular structures even when the tumors are located in close proximity to major intrahepatic vessels. furthermore, even when major hepatic vein(s) must be resected, color doppler ious findings provide reliable information that may lead to the preservation of a portion of the liver parenchyma that is drained by those vein(s), avoiding major hepatectomies [4245 ]. thus, a novel liver resection technique was developed in recent years that is referred to as ultrasonically guided hepatectomy. this technique opened the door to new procedures that allow for radical but conservative liver resections, reducing the requirement for major hepatic resections [4648 ]. because many patients exhibit colorectal liver metastases that are considered unresectable due to the insufficient remnant liver parenchyma following major hepatectomies, the use of this surgical technique (which spares a significant amount of functional liver parenchyma) allows for the complete resection of the metastases, reducing the risk of developing postoperative liver failure. ultrasonically guided liver resection decreases the requirement for major hepatectomies, obviating the requirement for portal vein occlusion prior to the liver resection and/or the necessity of a two - stage liver resection in selected patients. in patients with crlm that are located in close proximity to major hepatic veins or near the first - degree portal branches, a major hepatectomy is still the main surgical option at most centers. if the remnant liver volume following a major hepatectomy is critically small, a liver resection following portal vein occlusion, either by pve or pvl, is generally recommended. in such instances, the patient is exposed to an interventional radiology procedure or a laparotomy prior to the curative liver resection. the development of the ultrasonically guided hepatectomy in recent years permits a more limited liver resection of poorly located crlm, avoiding the necessity of a prehepatectomy pve / pvl. in a series of 22 patients who presented poorly located liver tumors and who were scheduled for initial ultrasonically guided liver resection, a limited resection with or without hepatic vein preservation was achieved in 91% cases, providing lower morbidity rates than major resections following pve and no mortality. the rate of local recurrence (at the transection surface) was zero at a mean follow - up period of 23 months. because this approach avoids portal vein occlusion (and its associated morbidity), moreover, the resectability rate following portal vein occlusion does not exceed 6088%, due to either insufficient hypertrophy of the remnant liver or disease progression in the interval between the portal vein occlusion and the liver regeneration [21, 22, 49 ]. when an initial ultrasonically guided hepatectomy is performed, the risk of disease progression is avoided, and the hypertrophy of the flr is no longer necessary. thus, the resectability rate that is achieved by the ultrasonically guided approach appears to be higher than those that are achieved by pve / pvl, broadening the indications for curative surgery in cases of crlm. in patients with multiple bilobar crlm, the ultrasonically guided technique may also represent an effective alternative to the two - stage the advantages of this approach over the two - stage liver resection are the comfort of the patient, a lower morbidity rate, and an increased possibility of repeat resections if the patient develops recurrent metastases [5052 ]. furthermore, the recurrence rate following one - stage ultrasonically guided liver resection was similar to that reported after two - stage liver resection. due to the aforementioned benefits, the one - stage ultrasonically guided liver resection should be part of the armamentarium of the liver surgeon, especially in the context of patients with complex tumoral presentations. this therapeutic strategy was first presented by the paul brousse group in 1996 and is recommended in patients with a small number of large crlm, the resection of which would not spare a sufficient amount of functional liver to prevent postoperative liver failure (figure 1(e)). the goal of this approach is to downsize the liver metastases to an extent that allows for their complete resection. therefore, a chance of a potentially curative liver resection is available to patients who otherwise may have only benefited from palliative treatment. until 20 years ago, the only efficient chemotherapeutic regimen that was used in patients with unresectable crlm consisted of 5-fluorouracil (5-fu) and folinic acid. although this chemotherapy increases the overall survival rates and the progression - free survival rates of these patients, the response rates were less than 23%, and only anecdotal cases of liver metastases that shrink sufficiently to allow for a subsequent curative hepatectomy were reported [5456 ]. the advent of new chemotherapeutic agents such as oxaliplatin and irinotecan led to significantly better results. the response rates that have been achieved by folfox and folfiri regimens range from 40% to 56% [5759 ]. a strong correlation was observed between the response rates and the resection rates of patients with initially unresectable clrm. thus concluded that resectability should be considered a new endpoint for preoperative chemotherapy, focusing on the curative potential of this oncosurgical treatment. the paul - brousse group published their updated results in 2004, reporting a 12.5% rate of conversion to resectability in 1104 patients with initially unresectable crlm (following an average of 10 courses of chemotherapy). apart from these very large series of patients, other centers have subsequently reported similar results in smaller numbers of selected patients who presented with initially unresectable crlm that were rendered resectable by different chemotherapy regimens [6163 ]. in many reports, the morbidity rates following hepatectomy in patients with initially unresectable crlm [14, 62, 64 ] ranges from 23% to 28%, which are similar to those rates that are observed in patients with initially resectable crlm. however, certain authors have reported significantly higher incidences of postoperative complications in patients who receive resections following downsizing chemotherapy, raising concerns regarding the deleterious effects of the preoperative chemotherapy on the liver parenchyma (see below) [65, 66 ]. the postoperative mortality rates are reported to be less than 2% in most centers, which are similar to those rates that have been achieved in patients who did not receive preoperative chemotherapy [14, 53, 62, 66 ]. the 5-year survival rate of patients who were rendered resectable by chemotherapy was 33% in the paul brousse group, a rate that was higher than those that were achieved by new palliative chemotherapeutic regimens in similar patients. although this survival rate is significantly lower than what can be achieved in patients with initially resectable crlm (p value = 0.01), the 5-year disease - free survival rate of 22% that was reported in initially unresectable patients appears to fully justify the efforts to render to resectability, these patients with otherwise dismal prognosis. the addition of targeted therapies (e.g., bevacizumab, cetuximab, and panitumumab) to chemotherapy regimens may be useful in further increasing the rate of conversion to resectability in initially unresectable lesions. this hypothesis was confirmed in a series of patients whose liver metastases were refractory to previous rounds of conventional chemotherapy. the advent of cetuximab to the next - line chemotherapy rendered 7% of these patients resectable, with morbidity and mortality rates of 50% and 3.7%, respectively, and a median survival of 20 months., demonstrated that monoclonal antibodies in combination with conventional chemotherapy have no detrimental effects on the safety of liver resection. furthermore, gruenberger. revealed that bevacizumab has little detrimental impact on liver regeneration following hepatectomy. however, it should be noted that the use of vascular endothelial growth factor inhibitors (e.g., bevacizumab) prior to major surgery increases the risks of bleeding and wound healing complications. this therapy should be discontinued 58 weeks prior to the surgical intervention [68, 70 ]. if the chemotherapy is continued, the metastases may regrow and again become unresectable, closing the window of opportunity for a potentially curative hepatectomy. therefore, if the systemic disease is controlled, the liver resection should be scheduled as soon as the metastases become resectable. (ii) if the chemotherapy is continued beyond the point when the metastases become resectable, it is possible the liver metastases will become smaller and will no longer be visible on imaging (ct / mr / pet scans). such metastases are referred to as vanishing metastases. unfortunately, this radiological complete response or clinical complete response is achieved in fewer than 20% of cases [73, 74 ]. in one - third of patients with radiological complete response, a laparotomy may reveal small metastases that were missed by the imaging methods or residual scars, the resection of which would reveal viable tumor cells. alternatively, in patients without macroscopic residual tissue (on laparotomy) and negative (contrast - enhanced) intraoperative ultrasound, pathologic examination of the resected specimens, including liver segments where the metastases where initially located, revealed viable metastatic cells in 75% of cases [74, 75 ]. an indirect confirmation of the presence of viable tumor cells at the sites of the former metastases (which were invisible on laparotomy) is given by certain reports. in a series of patients with 31 crlm that disappeared following chemotherapy and which were not observed on laparotomy after a one - year follow - up, 23 (74%) metastases recurred in situ. the survival benefit that is achieved by performing liver resection in patients with clinical complete response following chemotherapy was revealed by another study. fourteen patients with radiological complete response following chemotherapy were not subjected to a liver resection, achieving a 5-year overall survival of 14% and a median survival of 30 months. in 25 patients who suffered from initially unresectable crlm that were rendered to resectability by a folfoxiri regimen and further resected, the 5-year survival rate was 43%, and the median survival was significantly higher (61 months, p value = 0.006). for these reasons, laparotomy is mandatory in patients with vanishing metastases, with the aim of resecting the macroscopic residual metastatic tissue or the sites of the initial crlm (blind resection). the resection of the metastases sites is a very demanding operation, especially in patients with (initially) multiple metastases located deep in the liver. in such cases, computer - based virtual surgery planning is very useful, merging pre- and postchemotherapy computed tomography data. information that is processed using a computer is then intraoperatively transferred to the liver surface using an image - guided stereotactically navigated ultrasound dissector, enabling the surgeon to perform the resection. in patients with initially multiple bilobar crlm that become invisible following chemotherapy and that can not be identified intraoperatively, it is frequently impossible to assume a complete resection of the metastatic sites. thus, a small number of these missing metastases will remain. in such situations, elias. recommended the placement of a chemotherapy catheter in the hepatic artery, allowing for a hepatic arterial infusion (hai) with oxaliplatin, along with systemic 5-fu and folinic acid. using this approach, following a median follow - up period of 51 months the recurrence rate following hai with oxaliplatin was significantly lower than those that were noted in patients who were treated by systemic chemotherapy alone (p value = 0.01). alternatively, it should be noted that a small number (415%) of crlm that were treated by systemic chemotherapy prior to the liver resection achieved a complete pathologic response [72, 73, 77 ]. the complete pathologic response was observed both in patients with or without a radiologic complete response. the predictive factors for a complete pathologic response were age less than 60 years, maximum metastasis diameter of less than 3 cm, cea levels at diagnosis below 30 ng / ml, an objective response following chemotherapy and the use of hepatic arterial infusion chemotherapy. the addition of bevacizumab to the oxaliplatin - based chemotherapeutic regimens did not appear to increase the incidence of the complete pathologic response (11.3% versus 11.6% ; p value = 0.59). patients with complete pathologic response achieved uncommonly high survival rates (76% at 5 years). (iii) treatment with new chemotherapeutic drugs induces alterations of the nontumoral liver parenchyma, potentially impacting the results of the liver resection. the initial belief was that use of irinotecan may cause nonalcoholic fatty liver disease (nafld), which represents a spectrum of diseases. the mildest form of nafld is macrovesicular steatosis, and the most severe form is non - alcoholic steatohepatitis (nash). although a study that was published in 2003 revealed a correlation between prior treatment with chemotherapy and steatosis and highlighted the fact that morbidity rates following liver resections were significantly higher in patients with marked steatosis, more recent studies have reported differing results. in 2006, moreover, this latter study noted that the mortality rate was significantly higher in patients with steatohepatitis (14.7%) than in patients without steatohepatitis (1.6%, p value = 0.001). the first study to reveal a correlation between oxaliplatin and non - tumoral liver parenchyma injury was published in 2004. the results indicated that 78% of the patients who were preoperatively treated with oxaliplatin exhibited sinusoidal alterations. these results were subsequently confirmed by other reports [8385 ], which revealed that oxaliplatin - based preoperative chemotherapy was associated with sinusoidal dilatation and congestion, peliosis, and venoocclusive disease. one of these studies reported that only long - course oxaliplatin - based chemotherapy (6 or more cycles) is significantly associated with sinusoidal injury. however, in a series that was presented by vauthey., the risk of sinusoidal dilatation did not appear to increase with the duration of chemotherapy (although their patients received relatively short - course treatments). interestingly, the addition of bevacizumab to oxaliplatin - based regimens appears to reduce the incidence and severity of hepatic injury. the impact of these liver injuries on the clinical outcome of the patients who received resections following oxaliplatin - based chemotherapy was assessed in several reports. none of these studies reported increased mortality rates following liver resection in patients with oxaliplatin - related sinusoidal injury. two trials revealed that a limited course (fewer than 6 cycles) of oxaliplatin - based therapy was not associated with increased morbidity rates following liver resection [81, 86 ]. however, karoui. observed a statistically higher incidence of postoperative complications in patients who underwent a major hepatectomy following preoperative chemotherapy when compared with the patients who were subjected to a similar liver resection without preoperative chemotherapy. similarly, nakano. observed that sinusoidal injury was significantly associated with increased morbidity and longer hospital stays in patients who underwent a major hepatectomy. the above - mentioned pitfalls that are associated with preoperative chemotherapy justify the scheduling of the liver resection as soon as the metastases become resectable. this type of approach is especially recommended in patients with multiple bilobar crlm who present with fewer than 3 liver metastases in the flr, with each of these metastases being less than 3 cm in maximum diameter (figure 1(f)). another indication for this approach is when one or a small number of metastases are anatomically poorly located (e.g., in close proximity to the confluence of the three hepatic veins and the inferior vena cava or at the bifurcation of the portal vein). the operation consists of the resection of the main tumor bulk (generally by a right trisectionectomy) and thermal ablation of the unresected metastases from the remnant liver (frequently the left lateral section). this approach could be performed in a two - stage manner in patients with synchronous unresectable crlm, as described by lygidakis.. in the first stage, the following procedures are performed : (1) the resection of the primary colorectal tumor, (2) the ligation and transection of the relevant (right or left) primary portal branch, (3) the ablation of the metastatic nodules in the contralateral hemiliver, and (4) the insertion of an arterial catheter into the hepatic artery for locoregional chemo(immuno)therapy. the second stage of the operation consists of the resection of the tumoral liver (usually by right hemihepatectomy or trisectionectomy). thermal ablation can be achieved using radiofrequencies, microwaves, lasers, or cryotherapy. to increase the chances of a complete hyperthermic ablation, the pringle maneuver can be performed during the ablation. [27, 90 ] of patients undergoing combined liver resection and thermal ablation of unresectable crlm appear to be similar to those of patients with initially unresectable crlm that are rendered resectable by other therapeutic strategies. a retrospective study that was published in 2004 reported a significantly higher local recurrence rate following resection combined with rfa (5%) than was observed following complete resection of the crlm (2%). however, the patients were not stratified according to the maximum diameter of the ablated crlm in this study. it was recently demonstrated that the best results achieved by thermoablation are observed in patients whose crlm were less than 3 cm in maximum diameter. thus, a significantly higher rate (p value = 0.0001) of sustained complete ablation was achieved in patients whose lesions were less than 3 cm (66.7%) than for the patients with metastases that were larger than 3 cm (33.3%). one retrospective study (including resectable and unresectable crlm) revealed that the local recurrence rate following the radiofrequency ablation (rfa) of metastases that were less than 3 cm in maximum diameter was 1.3% after a follow - up period of 33 months. moreover, another retrospective study (including patients with single crlm treated by rfa or liver resection) determined that the 5-year overall and local recurrence - free survival rates were similar for patients with crlm that were smaller than 3 cm who were treated either with rfa or liver resection. however, in patients with multiple bilobar crlm who are treated with a combination of rfa and liver resection, the overall and disease - free survival rates were significantly lower than for patients who underwent a complete resection of crlm but significantly higher than in the patients who were treated by chemotherapy alone. revealed in a study of 57 patients with initially unresectable crlm that the overall survival rates were similar for patients who underwent a complete liver resection following conversion chemotherapy and those who received liver resection combined with cryotherapy. these results justify liver resection combined with thermal ablation of initially unresectable crlm in patients who fulfill the above - mentioned criteria. however, due to the lower recurrence rates achieved by the ultrasonically guided liver resection technique, this approach may be more suitable than liver resection combined with thermal ablation. unfortunately, there are still many patients with multiple bilobar colorectal cancer liver metastases who are not amenable to complete resection by any of the above - mentioned therapeutic strategies. in such patients, the only chance of complete removal of the liver metastases is total hepatectomy followed by liver transplantation (figure 1(g)). this approach was used in the early period of liver transplantation, achieving 1- and 5-year overall survival rates of 62% and 18%, respectively. due to organ shortages, it was considered that the allocation of an organ to a patient with such a short life expectancy following the transplantation was not ethically acceptable. however, the above - mentioned survival rates appear to be higher than those that can be achieved by palliative treatment, suggesting that even using the therapeutic options that were available thirty years ago, liver transplantation offered a higher survival benefit than the best palliative treatment that is currently available. furthermore, due to recent progress in the fields of posttransplant immunosuppression and medical oncology and due to the more refined methods that are used in selecting patients to receive tailored therapies (based on reliable pathologic and biologic markers), improved survival rates could be achieved for selected patients who undergo liver transplantation. moreover, the ethical issues could be challenged by the use of a living donor liver transplantation given that, in such instances, the willing donation is directed toward a certain patient and not to the community. meanwhile, it is also considered unethical to offer a marginal graft to a patient with a good chance of long - term survival following liver transplantation. because the number of available marginal grafts has increased in recent years, it may be acceptable to allocate such organs in selected patients with crlm, at least in the setting of controlled trials. although the available data do not support liver transplantation as a routine procedure in patients with crlm, we believe that a discussion of the current advances in this field and of the recently published results is worthwhile and should encourage debate on this issue. by reviewing the largest series of patients undergoing liver transplantation for unresectable crlm, it was revealed that 66% of patients with histologically negative lymph nodes were genetically positive for micrometastases when mutant allele - specific amplification (masa) method was used to search for micrometastases in dna from the regional lymph nodes of the primary colorectal cancer. those patients who were both genetically and histologically negative exhibited a significantly longer overall survival (p value = 0.011) than the other patients. thus, kappel. concluded that the genetic detection of micrometastases by masa may be a powerful prognostic indicator for selecting patients with colorectal liver metastases who could benefit from liver transplantation. over the previous two decades, certain further developments have emerged that may improve patient selection and the results of liver transplantation. for example, the advent of mdct, gadolinium - enhanced mri, and pet / ct scans has improved the detection of extrahepatic metastases, permitting the better selection of patients with colorectal cancer that had metastasized only to the liver. recent studies have identified several biological parameters (such as the expression of p53, thymidylate synthase, ki-67, k - ras, and human telomerase reverse transcriptase, as well as the type, density, and location of immune cells within the tumor) that may be more sensitive predictors of outcome in patients with crlm than are the current histopathological methods that are used to stage colorectal cancer [98, 99 ]. using a panel that incorporates these parameters, it may be possible to identify a highly selected group of patients who could greatly benefit from liver transplantation. moreover, it has been hypothesized that the progress in posttransplant immunosuppressive therapy may decrease recurrence rates and improve the survival of patients who undergo liver transplantation for malignant disease, primarily due to the use of m - tor inhibitors. such immunosuppressive agents (sirolimus, temsirolimus) inhibit tumor growth and proliferation and exhibit antiangiogenic effects. these effects are in contrast to traditional immunosuppressive drugs, which appear to promote malignant cell proliferation [100, 101 ]. over the past several years, taking into account the better expertise of transplant surgeons and the above - mentioned progress in both the selection of patients with crlm and in the efficacy of posttransplant immunosuppressive regimens, certain authors have argued that the outcome of selected patients who undergo liver transplantation for unresectable liver metastases from colorectal cancer may be significantly improved. for these reasons, certain authors have proposed a rational revisitation of the concept of liver transplantation in such patients. thus, a pilot study (seca - study) that aims to assess the survival and quality of life in patients receiving pretransplant chemotherapy, liver transplantation for unresectable crlm, and posttransplant sirolimus - based immunosuppressive regimen began in norway in november 2006. the preliminary data of this study reveal a 94% survival rate after a median 25 months of postoperative follow - up and an excellent quality of life. however, only 40% of these patients are disease - free after a median follow - up period of 25 months. favorable results were also recently reported in two patients with crlm who were treated with liver resection followed by hepatic artery infusion chemotherapy and who underwent a liver transplantation for intraarterial chemotherapy - induced sclerosing cholangitis. the two patients were disease - free at 2 and 5 years following transplantation, respectively. based on these disparate results, definitive conclusions can not be drawn ; however, due to ethical considerations (i.e., organ shortage), liver transplantation with grafts from brain - dead donors can not be accepted until 5-year survival rates exceed 50%. however, if the results that are achieved by liver transplantation will become significantly higher than those can be achieved by nonsurgical therapies, it may eventually be difficult, in the future, to defend the prohibition of living - donor liver transplantation or liver transplantation with marginal grafts in highly selected patients with unresectable crlm. such a position would be difficult given that ethical considerations would no longer be valid in such situations. selected patients with initially unresectable crlm may be rendered resectable following portal vein embolization or ligation, resulting in an important survival benefit or even a cure. two - stage hepatectomies (with / without pve / pvl) may be performed safely, achieving complete resection of liver metastases and long - term survival. the use of ultrasonographically guided hepatectomies decreases the requirement for major hepatectomies, portal vein occlusion and two - stage liver resections in patients with crlm that are close to the hepatocaval confluence or in cases of multiple bilobar disease. this approach provides (1) an improved comfort and safety profile over two - stage liver resections and major hepatectomies following pve / pvl and (2) a similar oncological benefit to other strategies. liver resection following conversion chemotherapy in previously unresectable patients may offer a considerable survival benefit. rfa could be combined with liver resection to increase the number of patients who are eligible for complete removal and ablation of crlm. in the future, highly selected patients with unresectable crlm and favorable prognostic factors who receive liver transplantations with grafts from marginal donors or from living donors could achieve better survival rates than would be possible with palliative treatment. however, further studies and perioperative treatment improvements are required before this procedure achieves social acceptance.	although the frontiers of liver resection for colorectal liver metastases have broadened in recent decades, approximately 75% of these patients present with unresectable metastases at the time of their diagnosis. in the past, these patients underwent only palliative treatment, without the chance of a cure. in the previous two decades, several therapeutic strategies have been developed that render resectable those metastases that were initially unresectable, thus offering the chance of long - term survival and even a cure to these patients. the oncosurgical modalities that are available include liver resection following portal vein ligation / embolization, two - stage liver resection, one - stage ultrasonically guided liver resection, hepatectomy following conversion chemotherapy, and liver resection combined with thermal ablation. moreover, in recent years, certain authors have recommended the revisiting of the concept of liver transplantation in highly selected patients with unresectable colorectal liver metastases and favorable prognostic factors. by employing such therapies, the number of patients with colorectal liver metastases who undergo a potentially curative treatment could increase to 40%. the safety profile of these approaches is acceptable (morbidity rates as high as 45%, mortality rates of less than 5%). furthermore, the 5-year survival rates (approximately 30%) are significantly increased over those that were achieved with palliative treatment.
periodontal diseases are among the most prevalent dental diseases affecting people worldwide as well as in the indian community. periodontal diseases cause loss of teeth and also affect systemic health with the oro - hematogenous spread of bacteria. the related systemic diseases include cardiovascular diseases, preterm delivery of low birth weight, osteoporosis, diabetes mellitus (dm) and respiratory infections. periodontal diseases are infectious in nature, and bacteria play a major role in its initiation and progression. it occurs as a result of complex interactions between periodontopathic microorganisms and the host tissues. this process is modified by the status of the host immune system, genetic factors and a complex array of environmental exposures. chronic periodontitis is associated with accumulation of plaque and calculus and has a slow to moderate rate of disease progression ; however, periods of rapid destruction are also observed. the increase in the rate of disease progression is caused by the impact of local, systemic or environmental factors that influence the normal host - bacterial interaction. as periodontitis is an infectious disease, it is mainly associated with a group of gram - negative bacteria such as actinobacillus actinomycetemcomitans (aa), porphyromonas gingivalis and bacteroides forsythus. of these, p. gingivalis and b. forsythus are found in chronic periodontitis, whereas aa is found in cases of aggressive periodontitis. p. gingivalis is an anaerobic, gram - negative, rod - shaped and highly virulent organism implicated as a major pathogen in destructive periodontal disease with its ability to adhere and invade oral epithelium. it is also implicated to be involved in the development of systemic diseases due to systemic inflammation with increased circulating cytokines and mediators, direct infection and cross - reactivity / molecular mimicry between bacterial antigens and self - antigens. it has been detected in heart valve lesions and atheromatous plaque, amniotic fluid of pregnant women with threatened premature labor and placentas from cases of preterm delivery, dm, respiratory diseases and osteoporosis subjects. dm is a metabolic disorder characterized by hyperglycemia due to defective secretion or activity of insulin. diabetes increases the glucose concentration in the gingival crevicular fluid and decreases the salivary levels of epidermal growth factor which plays an important role in wound healing. these modifications in gcf affect plaque composition which is supported by an increased amount of plaque and increased numbers of gram - negative anaerobes. the consequences include impaired cellular functions, impaired host defense, vascular alterations, prolonged inflammation, impaired bone formation or repair ultimately resulting in tooth mobility and premature loss of teeth. there is a two - way relationship between dm and periodontitis, where local periodontal infection can exacerbate and help in the progression of systemic complications. the systemic exposure to periodontal pathogens in the periodontium worsens the low - grade systemic inflammation present in diabetes. the consequences are alterations in glucose metabolism and regulation resulting in difficulties in maintaining optimal glycemic control, which in turn increase the development and progression of diabetic - related complications such as periodontitis. p. gingivalis is frequently detected in various distant organs such as the liver, cardiovascular tissue, cerebrospinal fluid and tubal - ovarian locations. p. gingivalis is also able to invade intracellularly without the signs of apoptosis and necrosis. it has virulence factors such as collagenase, aminopeptidase and trypsin - like enzyme activity. the structural components of p. gingivalis include lipopolysaccharide and fimbriae that trigger intracellular signaling events. p. gingivalis in the oral cavity moves to the liver and is responsible for glycogen synthesis through akt / gsk-3 signaling in the liver cells. hence, p. gingivalis in the oral cavity contributes to the pathogenesis of dm by affecting hepatic glycogenesis through akt / gsk-3 signaling. local factors such as plaque with a complex array of environmental exposures also play an important role in the periodontal destruction. this involves cigarette smokers who develop severe periodontitis up to five times more than nonsmokers. smokers have been associated with deeper pockets and greater attachment loss, more pronounced furcation involvement and increased alveolar bone loss. as both dm and smoking are important and significant risk factors for periodontitis, it is very important to know the role of p. gingivalis and its implications. hence, from our study, we intend to quantify the p. gingivalis count using real - time polymerase chain reaction (pcr). hence, we used real - time pcr which provides a sensitive, efficient and reliable method for quantification of p. gingivalis. our aim is to observe the relative involvement of p. gingivalis in dm patients having periodontitis with tobacco smoking habit and to compare them with group ia (no periodontal diseases) and group ib (periodontal disease with no systemic pathology). the study was conducted on the subgingival plaque of patients with chronic periodontitis, with and without type ii dm associated with smoking habit from our institution. group i (control) : (a) fifteen healthy individuals without periodontal and systemic pathologies. group ii (study) : (a) twenty newly diagnosed cases of dm having chronic periodontitis without tobacco smoking habit. (b) twenty newly diagnosed cases of dm having chronic periodontitis with tobacco smoking habit. the sample was placed in a vial containing phosphate buffer at 4c and transported to the laboratory for further procedures. dna was isolated from samples by modified cetyltrimethylammonium bromide method by centrifuging at 10,000 rpm for 10 min to pellet down the bacterial cells. the quantity of the isolated dna was checked in ultraviolet - visible spectrophotomer (vivaspec biophotometer, germany). from the stock, 1 l dna was mixed with 49-l sterile distilled water to get fifty times dilution. the melting temperature was calculated, and the synthesized primers were purified by high - performance liquid chromatography. the quantified dna was used to detect the presence of p. gingivalis using the specific primers, and primer optimization was done in a gradient pcr at the annealing temperature of 55c [tables 1 and 2 ]. amplified products were resolved on 2% ethidium bromide - stained agarose gel along with a 100 bp dna ladder polymerase chain reaction design and synthesis polymerase chain reaction temperature profile electrophoresis results were analyzed in gel documentation system and photographed. of 70 samples, 46 were male (65.71%) and 24 were female (34.28%) [table 3 ]. distribution of study subjects by gender the study subjects were found to be in the age group between 2 to 5 decades [table 4 ]. distribution of study subjects in each group by age the maximum chronic generalized periodontitis were seen in group iia and localized periodontitis in group iib [table 5 ]. distribution of study subjects based on their periodontal status the maximum p. gingivalis count were observed in group iia and minimum count in group ia [table 6 and graph 1 ]. it was also observed that the p. gingivalis count was significantly reduced in group iib compared to nonsmokers. comparison of porphyromonas gingivalis scores in all the study groups comparison of porphyromonas gingivalis scores in all the study groups (groups ia, ib, iia and iib) pair - wise comparison of all the groups with respect to p. gingivalis scores as given by tukey 's multiple post hoc procedures is presented in table 7. pair - wise comparison of four groups with respect to log porphyromonas gingivalis scores by tukey 's multiple post hoc procedures periodontitis is a disease that affects the periodontium which is characterized by the loss of periodontal attachment including the alveolar bone. the bacterial etiology of periodontal disease is complex, with a variety of organisms responsible for the initiation and progression of the disease. the microbe which causes periodontitis contains primarily gram - negative rods and cocci, filaments, flagellated rods and spirochetes. many of these organisms are also present in periodontally healthy individuals and can exist in commensal harmony with the host. the mere presence of pathogen alone is not sufficient to cause disease but should be above a determined threshold to cause periodontal disease. p. gingivalis has been implicated to be associated with many systemic diseases, where multiple fold increase is seen. these pathogens gain entry into circulation through the ulcerated epithelium and exposed capillaries during periodontal inflammation and may induce systemic symptoms. the interrelationship between periodontitis and dm provides an example of systemic disease predisposing to oral infection, and once that infection is established, the oral infection exacerbates systemic disease. the effects of smoking in oral cavity include bad breath, tooth discoloration, inflammation of the salivary gland openings, increased plaque and tartar on the teeth, risk of leukoplakia and gum diseases leading to tooth loss. nicotine, a major smoke component, induces periodontal collagen degradation by increasing expression of the collagenase matrix metalloproteases which results in detachment of the periodontal ligaments. p. gingivalis persistence in the oral cavity of smokers is attributed to a compromised immune response and/or increased bacterial virulence. despite increased p. gingivalis, smokers consistently display reduced clinical inflammation due to lower levels of proinflammatory cytokines at diseased sites. as periodontal diseases are polymicrobial infections with various etiologic factors, accurate quantitation of the number of cells of individual bacterial species in dental plaque samples is needed for understanding the bacterial etiology of periodontitis. the real - time pcr provides precise counts through direct monitoring of the increasing amount of pcr product throughout the enzymatic assay and is the most sensitive method with detection limits of 10 genome copies. hence, we used real - time pcr method for quantification of p. gingivalis. in group ia, p. gingivalis counts ranged from 0 to 1561 with a mean of 525. it implies that p. gingivalis exists in commensal harmony with other organisms present in the periodontally healthy individuals. the study is supported by other studies, where lyons. and van winkelhoff. showed the presence of p. gingivalis in healthy subjects by pcr assay and concluded that this organism may also be a normal inhabitant of a periodontally healthy dentition. in group ib, p. gingivalis counts ranged from 288 to 21697 with a mean of 4178. it implies that p. gingivalis is one of the major pathogens implicated in the development and progression of the periodontal disease. hence, we can observe the substantial increase in the p. gingivalis count when compared to healthy individuals from our results. our study results are supported by kirakodu. and socransky., who conducted a study on quantification of p. gingivalis from subgingival plaque by quantitative pcr assay which showed an increased prevalence of p. gingivalis organisms ranging from 6 10 8.6 10 in chronic periodontitis patients. in group iia, p. gingivalis count ranged from 204 to 81691 with a mean of 19140. it shows that high glucose levels in type ii dm patients lead to the development of periodontogenic flora due to reduced oxygen production and defense cells. hence, diabetic patients have an increased susceptibility for more severe periodontal disease due to increased prevalence of p. gingivalis. in our study, group iia had statistically significant increase in the p. gingivalis counts by 40 folds than group ia. our study results are supported by campus. and ebersole., who conducted a study using pcr assay and concluded that these patients have an increased susceptibility for severe periodontal disease and increased prevalence of p. gingivalis. in group iib, the p. gingivalis counts ranged from 0 to 8684 with a mean of 1238. the counts of p. gingivalis have significantly decreased by 16-fold (p = 0.0002) when compared to group iia. this is because the organism can not thrive in the altered environment that is attributed to heat and tobacco smoke extracts despite increased blood glucose levels. the studies done by bagaitkar and zeller. showed increased prevalence for p. gingivalis as compared to nonsmokers ; whereas studies done by zambon and kinane and chestnutt gave inconclusive results about the prevalence of p. gingivalis in smokers, where they concluded that no statistically significant difference was found between smokers and nonsmokers. although the previous studies have shown increased prevalence of this organism in smokers compared to nonsmokers, our study showed conflicting results where the count of this organism was significantly reduced. however, none of the previous studies have included both the parameters of diabetes and smoking together for assessing p. gingivalis count in periodontitis subjects. we chose quantitative real - time pcr as it is more efficient and sensitive compared to other methods and provides precise counts through direct monitoring of the increasing amount of pcr product throughout the enzymatic assay. subgingival plaque samples were chosen over other samples such as saliva and gingival crevicular fluid for better yield and to understand bacterial etiology of periodontitis. p. gingivalis is one of the major etiological agents in periodontal destruction and in recent years, it is gaining importance because of its implications in the systemic pathologies such as dm. thus, by assessing its quantity, we can analyze its role as a pathogen and the risk associated with this organism as studies have shown that it exacerbates the systemic pathology. a quantitative real - time pcr is an efficient and sensitive method which allows us to study specific organisms and their role in various pathologies. further studies with larger sample size should be conducted to confirm the findings of our study.	introduction : periodontal diseases, if left untreated, can lead to tooth loss and affect at least one tooth in 80% of adults worldwide, with the main cause being a bacterial plaque. among subgingival plaque bacterial species, porphyromonas gingivalis has been implicated as a major etiological agent causing tooth loss. diabetics and smokers are two patient groups at high risk for periodontal disease. the increase in the number of this organism with the coexistence of other pathogenic microbes leads to rapid destruction of the periodontium, premature loss of teeth and also because of its virulence has implications in systemic pathology. our aim was to observe the involvement of p. gingivalis in diabetes mellitus (dm) patients associated with periodontitis with and without tobacco - associated habits and to compare them with periodontitis patients having no other systemic pathologies.materials and methods : subgingival plaque samples from a total of seventy subjects were included in the study. dna was isolated from the collected sample and was quantified using spectrophotometer for standardizing the polymerase chain reaction. the quantity of the isolated dna was checked in a ultraviolet - visible spectrophotomer.statistics:one-way anova and tukey 's multiple post hoc procedures were carried out.results:the maximum score of p. gingivalis was seen in periodontitis patients having dm, whereas the least score was seen in periodontitis patients having dm with tobacco smoking habit compared to the other groups.conclusion:p. gingivalis count is significantly reduced in periodontitis patients having dm with smoking habit ; it is concluded that p. gingivalis might not be a key causative organism responsible for the periodontal destruction in case of smokers despite the dm condition. the decrease in counts may be attributed to change in the local environment like chemical (tobacco nitrosamines) and physical changes preventing the growth of p. gingivalis.
dental amalgams have been widely used in dentistry for over 160 years and are still being used due to their good clinical performance, strength, durability and reasonable price. in low - copper amalgams, different electrolytic potentials in various phases make it susceptible to galvanic corrosion in its most electropositive phase (2) especially in the interface region.[2 - 5 ] corrosion products formed by the interaction of metallic ions from amalgam with chlorine and oxygen in the oral environment fill this gap. they consequently create a potential to seal the tooth / amalgam restoration interface.[6 - 8 ] on the other hand, there are less electrolytic potentials and galvanic corrosion in high - copper amalgams. marginal microleakage due to setting contraction of high - copper amalgams has been a cause for dentists ' concern about patients ' post - operative sensitivity and secondary caries.[9 - 10 ] however, it takes some time for the microleakage of high - copper amalgams to be sealed by corrosion products. it would be advantageous if these interfacial gaps could be sealed as quickly as possible by accelerated corrosion products.[11 - 12 ] in addition to the galvanic corrosion, other types of corrosions are expected to take place in amalgam restorations, as it would be at the vicinity of some factors such as tension,[13 - 14 ] oral bacterial flora,[15 - 16 ] and ph changes. acidic environment accelerate corrosion phenomenon.[17 - 18 ] applying adhesives as an intermediary layer between enamel / dentin and amalgam reduces secondary caries and microleakage significantly.[20 - 22 ] it also increases bonding23 and diminishes postoperative sensitivity.[24 - 26 ] corrosion resistance of alloys is reduced in a lower ph environment. due to the acidic functional monomers of self - etched adhesives (sea), the resultant interfacial structure becomes more hydrophilic and may create an interfacial acidic environment that would be a more efficient condition for interfacial corrosion for amalgam restorations. electrochemical tests (ects) can be considered as the testing techniques for evaluation of potential corrosion and its behavior. the aim of this study was to evaluate the effect of seas with different ph levels on corrosion behavior of high - copper amalgams and its induction potential for self - sealing ability in the early post setting hours using ect techniques. thirty intact second mandibular bicuspid teeth extracted for the orthodontic treatment purposes were used in this study. initially, the coronal segments of teeth were sectioned just short of the cementoenamel junction (cej) with a diamond disk (drendel & zweiling 942fdiamant gmbh ; kalletal, germany) and using a low speed hand piece and cooling water spray. the occlusal part at the cuspal base level was removed just inside the dentinoenamel junction (dej) using the same instruments and technique. to standardize the samples of 5.0 mm occlusogingival thickness and make identical cylindrical cavity preparations of 4.5 mm in diameter and 4.7 mm in depth with a 0.3 mm remaining dentin base at the occlusal side of the samples, attempts were made to prepare the occlusal and cervical sections parallel together within the above - mentioned dimensions. (figures1a and 1b) schematic model of the sample preparation for the electrochemical tests. a : tooth sample with a 5.0 mm thickness, b : cavity with 4.5 mm diameter and 4.7 mm depth, c : coating the external and internal walls of the cavity with hydrophobic resin, d : amalgam filling without ar / liner, e : prepared d - sample embedded in epoxy resin, f : amalgam filling with ar / liner g : prepared f - sample embedded in epoxy resin. the cavities were prepared from the pulpal toward the occlusal part (figure 1b) with a # 57-fissure bur (brasseler usa dental ; savannah, georgia, usa). samples were then placed for two minutes in an ultrasonic device (micro 10+sonic ; unindent s.a. anios international dental group, genve, switzerland) with distilled water to clean any remaining cut debris. these samples were then kept in a 37c incubator (thermo ; hatfield, pa 19440, usa) for 12 hours. to leave the opposing side of the cavity floor intact for the testing purposes, a polyethylene cylinder with 4.5 mm of internal diameter and 3.0 mm length was then carefully placed and secured with cured hydrophobic resin (margin bond ; coltne / whaledent ag, altstatten, switzerland). to seal the none - testing surfaces, all sample surfaces except for the cavity floor and its opposing external surface were etched with 37% phosphoric acid gel (ultra - etch ; ultradent products inc. one layer of mentioned hydrophobic resin was carefully applied on these surfaces and initially cured by scanning the curing light (blue phase c8 ; ivoclar/ vivadent, liechtenstein) for 20 seconds at the intensity of 600 mw / cm. the samples were post - cured using a laboratory light cure unit (triad 2000 ; dentsply international, york, pa, usa) for 80 seconds. a second layer of hydrophobic resin was applied and cured in the same manner (figure 1c). a 33 1/2 inverted cone diamond bur (dia ; f gso10014, italy) was used to refresh the interfacial surfaces (floor of the cavities) to guarantee the resin removal. the samples were then washed, cleaned and dried using the same procedures previously described. the samples were randomly divided into five main groups. the first main group was left with no ar/ liner (no) as a positive control group. in other main groups, each containing of six samples was assigned to a certain ar / liner, which was applied on the cavity floor according to manufacturer s instructions : i - bond (ib), clearfil s (s), single bond (sb) and varnish (v) (table 1). each main group was divided into two subgroups according to the types of the used amalgams (table 1). considering various electrochemical behaviors, one high - copper spherical alloy (tytin ; kerr, usa) and one admixed alloy (ana 2000 ; the mixed amalgam was condensed into the assigned cavity, while a piece of copper wire of 0.7 mm diameter was inserted about 3.0 mm deep into the cavity and submerged in the condensed amalgam. each prepared sample was then mounted in epoxy resin (araldit cy219 ; hardener hy5160, ciba - geigy, switzerland) in such a way to leave the opposing surface exposed (figure 1d-1 g). details of the defined groups used in this study the electrochemical measurements were performed using gill ac laboratory potentiostat (acm instrument, uk). in order to check the steady state, the prepared sample (working electrode) as the third electrode was held for 20 minutes in fusayama - meyer artificial saliva solution. the open circuit potential (ocp) of each sample was measured in a period of 1200 seconds. the linear polarization resistance (lpr) test was performed at a constant sweep rate of 10 mv / min and in the potential range of -15 to + 15 mv around the final monitored ocp value. the ocp and lpr measurements were carried out after the initial set up (20 minutes), 18 and 44 hours of immersion. in potentio- dynamic polarization (pdp) of the samples, the potential was applied by a constant sweep rate of 30 mv / min ranging from - 250 to + 250 mv (with respect to the sample ocp value). a water bath was utilized to control the electrolyte temperature at 371c during the entire exposure times. mean ocp values of the samples with ana 2000 and tytin amalgams showed (in all samples except ana - no and tytin - v) that the mean ocp values reached a steady state after 18 hours of immersion (figure 3a). mean ocp values of the ana - v and ana - ib were the highest (least corrosion potential) and lowest (most corrosion potential) respectively (figure 3b). mean ocp values of the tytin - no and tytin - ib samples were the highest (least corrosion potential) and lowest (most corrosion potential) respectively (figure 3). ana 2000 (a) and tytin (b) groups with varying cavity coatings (ar / liner or nothing). in lpr tests, the effect of different ar s on polarization resistance (rp) values (electrical resistance) of ana 2000 and tytin amalgams has been measured and all values are represented in table 2. the rp values of almost all samples have reached nearly a steady state and have diminished slightly after the 44-hour immersion time. the rp values of ana - v and tyt - v were the highest (lowest corrosion rates). contrarily, the ana - ib and tyt - ib samples, with the lowest ph values, represent the lowest rp values (i.e. highest corrosion rates) among the other groups (table 2). lpr values (rp values) of ana 2000 and tytin amalgams with different ar / liner in all tested groups figure 4 shows the pdp curves of all tested groups in artificial saliva media. although the values of ecorr and icorr were different, the diagrams in each group were roughly the same. table 3 represents the extracted parameters from these curves. according to this table, tyt - ib had the lowest ecorr and the highest icorr among the groups (i.e. highest corrosion rate). the highest value of ecorr was witnessed in tyt - no and lowest value of icorr was that of tyt - v (i.e. lowest corrosion rate). one of the influential parameters that can affect the corrosion reactions on the amalgam surface is the ph of the aqueous environment.[17 - 18 ] the ar / lining materials used in this study had various ph levels. the ph of the ar could influence the ph of the diffused electrolyte in ar / amalgam interface. as it was expected for ar, consequently, the lowest ocp value of the ana - ib sample could be attributed to its lowest ph value (table 1 and figure 3). on the other hand, in all samples except ana - no, the mean ocp values reached steady state after 18 hours of immersion (figure 3). considering this and ocp values of ana - no and ana - v samples, ocp values of the tyt - no and tyt - ib samples were the highest and lowest respectively (figure 3b). notwithstanding the lower ph of clearfil s bond (table 1) in comparison to single bond, the ana - s group presented a higher ocp value. considering the ocp values of ana - no and tyt - no samples, it could be stated that the ana - no sample (with approximate ocp value of -230 mv / sec) showed a more active corrosion behavior than tyt - no (figure 3). like ocp, lpr method is a non - destructive method for evaluation of corrosion resistance. in lpr technique, a potential is applied to a freely corroding sensor element (here amalgam as a working electrode) and the resulting linear current response is measured. in samples with ar layers, the decrease in rp values can be ascribed to the water - uptake phenomenon in such polymeric layers with microleakage.[28 - 30 ] however, in the groups without ar layers, the significant rp drop after the initial immersion could be attributed to the occurrence of both the anodic and cathodic reactions at an active state leading to higher dissolution rates. as the results showed, the rp values of ana - v and tyt - v were the highest (i.e. lowest corrosion rates) in comparison to the other samples in other groups. contrarily, the ana - ib and tyt - ib samples, with the lowest i - bond ph values, represent the lowest rp values that mean higher corrosion rates. in other words, it can be inferred that the acidic nature of the i - bond results in formation of higher amounts of corrosion products that can fill the gap at amalgam/ dentin interface and it consequently diminishes the microleakage. a microleakage study is recommended for further substantiating the result. by comparing the rp values of the ana - no and tyt - no samples, it can be deduced that the corrosion rate of the ana sample is slightly higher than the tytin sample at longer immersion times. this superior corrosion resistance behavior of the tytin sample can be attributed to its more homogenous microstructure.[31 - 32 ] concerning the ph values of clearfil s bond and single bond, it is expected that the samples with single bond reveal lower rp values while a converse behavior is observed in lpr results. it should be noted that the above - mentioned samples show the same unpredicted behavior in ocp results. these results can be related to the occurrence of other rate - controlling reactions but the mechanism is not completely understood. in addition, it may be concluded that other factors such as the chemical composition, the molecular structure, degradability or the microleakage may also outweighed the acidity of the ar s. in samples without an ar layer, the ana - no sample had a lower ecorr value in comparison with the tyt - no sample (figure 4a and table 3). concerning their anodic and cathodic tafel slopes, the ana - no sample revealed a more active corrosion behavior (with a slightly higher icorr value) as compared to tyt - no sample (table 3) which could lead to lower microleakage in ana - no sample. this can be ascribed to the ana 2000 heterogeneous microstructure (admixed) in comparison with more homogenous spherical microstructure in tytin. [31 - 32 ] pdp curves of tested groups a : tytin / ana - no, b : tytin / ana - ib, c : tytin / ana - sb, d : tytin / ana - s3 and e : tytin / ana - v. on the other hand, it should be considered that the corrosion rate of different groups is not only influenced by the type of amalgam and ph value [17,31 - 32,34 ] but also by the liner properties such as their degradability and water absorption affinity.[27 - 30 ] these factors may also be correlated with the liner porosity, the differences in the molecular structures and the densities of the polymeric chains in self - etch dentin bonding agents as well. besides, as it has already been shown, the degree of polymerization could affect the amount of water absorption, therefore, the authors suggest evaluating the effect of ar degree of polymerization on interfacial corrosion potential and behavior of the amalgams. the application of either chlorhexidine on the dentin surface separately or a new adhesive layer with added chlorhexidine to its formula to control degradability of ar may influence the corrosion production pattern / rate at the amalgam/ dentin interface. comparing the ana - ib and tyt - ib samples, both samples had almost equal ecorr values (figure 4b and table 3). besides, the tyt - ib sample had a higher icorr value compared to the ana - ib sample (table 3), which means lower corrosion resistance or higher corrosion rate. therefore, it can be deduced that in presence of the i - bond (ph= 1.6), the surface activity of the tytin amalgam was more affected as compared to ana 2000 amalgam. on the other hand, the formation of a corrosion product layer was facilitated more, and thus, the gaps at amalgam / dentin interface have been more rapidly filled (table 3). comparing samples with i - bond liners and the control groups (without any liner), a reduction in ph value resulted at higher corrosion rates. the ana - s sample had a lower ecorr value compared with tyt- s sample while both of them showed almost the same corrosion resistance (figure 4c and table 3). by comparing the icorr values of clearfil s samples with the control groups, it can be observed that there might have been a competition between the ph and stability of this ar layer, which affected the overall surface activity. as a result, it can be observed that the created microleakage in ana - s3 sample (with an acidic ph) could be restored even at a lower rate as compared to the ana - no samples. extracted parameters from pdp curves in figure four of tested groups both ana - sb and tyt - sb samples again revealed an approximately similar icorr values although the ana 2000 sample value was slightly lower. upon comparing the higher corrosion rates of sb groups with control groups, it can be deduced that the microleakage has been reduced faster. in the varnish groups, furthermore, in the presence of a more stable layer in the varnish groups, the corrosion rates were considerably lower and hence, the sealing process would be delayed. in addition, it should be noticed that the pdp results were consistent with other previous electrochemical measurements. therefore, the authors believe that supplementary experiments and investigations should be performed for further confirmation of the suggested method. considering the limitations of this study, it can be concluded that applying some self - etch adhesives as a liner beneath the high - copper amalgam restorations may increase interfacial corrosion potential and self - sealing ability of high - copper amalgams by increasing corrosion phenomenon potential. the lowest ph level of self - etch adhesive showed the highest whilst, the highest ph level of liners revealed the lowest susceptibility to corrosion and self - sealing ability of high - copper amalgams.more specifically, i - bond with the lowest ph value showed the highest corrosion rate whilst the varnish with the highest ph value revealed the lowest susceptibility to corrosion. clearfil s and varnish demonstrated lower corrosion rates in comparison to the control group ; therefore, these agents are not suggested for the reduction of microleakage in high copper amalgam restorations. the admixed amalgam had a higher corrosion rate in comparison with the spherical one.	statement of the problem : similar to conventional amalgam, high - copper amalgam alloy may also undergo corrosion, but it takes longer time for the resulting products to reduce microleakage by sealing the micro - gap at the tooth / amalgam interface. purpose : the aim of this study was to evaluate the effect of self - etch adhesives with different ph levels on the interfacial corrosion behavior of high - copper amalgam restoration and its induction potential for self - sealing ability of the micro - gap in the early hours after setting by means of electro - chemical tests (ects). materials and method : thirty cylindrical cavities of 4.5 mm x 4.7 mm were prepared on intact bicuspids. the samples were divided into five main groups of application of adhesive resin (ar)/ liner/ none (no), on the cavity floor. the first main group was left without an ar/ liner (no). in the other main groups, the types of ar/ liner used were i - bond (ib), clearfil s3 (s3), single bond (sb) and varnish (v). each main group (n=6) was divided into two subgroups (n=3) according to the types of the amalgams used, either admixed ana 2000 (ana) or spherical tytin (tyt). the ects, open circuit potential (ocp), and the linear polarization resistance (lpr) for each sample were performed and measured 48 hours after the completion of the samples. results : the tyt - no and tyt - ib samples showed the highest and lowest ocp values respectively. in lpr tests, the rp values of ana - v and tyt - v were the highest (lowest corrosion rate) and contrarily, the ana - ib and tyt - ib samples, with the lowest ph levels, represented the lowest rp values (highest corrosion rates). conclusion : some self - etch adhesives may increase interfacial corrosion potential and self - sealing ability of high - copper amalgams.
from the mid-1970s south africa grew increasingly isolated within the international community, including the medical community, and in 1976 the medical association of south africa (masa) decided to withdraw from the world medical association (wma) because diplomatic pressure had prevented it from attending two of the international organisation s world medical assemblies. yet despite an international academic boycott, even as late as the 1980s, some south african civil society organisations with cordial relations with the country s government continued to enjoy membership of certain prestigious international organisations, such as the wma. indeed, in august 1980 the american medical association (ama) launched a campaign for their south african counterparts in the masa to be readmitted to the wma. the transnational anti - apartheid activists who opposed the masa s readmission to the world body argued that, in their exoneration of the doctors who had failed to treat the fatally injured steve biko whilst he was in police detention in september 1977, the masa had failed to uphold the principles enshrined in the wma s tokyo declaration against torture and the geneva declaration (an updated version of the hippocratic oath). the wma readmitted the south africans at its meeting in lisbon in september 1981. anti - apartheid health activists within south africa and their international allies, continued to campaign for the masa s expulsion, however, and framed its readmission to the wma as a matter of conscience. by contrast, their opponents in the wma derided them as individuals concerned with anti - apartheid activists efforts in this regard were unsuccessful as the masa was not subsequently expelled from the wma. its defiance of the international academic boycott against south africa did, however, cost the wma its relationship with the world health organisation (who) and meant that many national medical associations disaffiliated from the former. south africa s membership of the wma only ceased to be a serious politically difficult issue for the international medical association when the country became democratic in 1994. this article explores the paradox of south africa s readmission to the wma in the wake of the biko doctors scandal, despite an international academic boycott. it describes the ways in which anti - apartheid activism led to internal rifts within the wma. the activists alleged that the wma had engaged in unprincipled, racist and pro - free - market behaviour. this activism came to diminish the moral reputation and the number of national member organisations of the wma, which was an international health organisation (iho). the activists characterised the wma as having conferred moral authority on the masa, which was closely aligned to the national party government, which followed a racially discriminatory policy of apartheid. they also held that in a cold war context, the renewal of the masa s wma membership was aimed at bolstering the ama s influence in the international organisation. these activists believed that this placed the ama in an excellent position to promote free market - focused approaches to health care delivery, internationally. the topic of steve biko s death in detention has received considerable attention and it has served as an important case study in discussions of medical ethics and the nature of racial discrimination in apartheid era medicine. similarly, the diverse array of anti - apartheid health - related non - governmental organisations has also been an area of interest to historians of medicine. biko s death in detention and the contours of health politics within south africa in the period examined in this article are very important topics which continue to merit attention in their own rights. this paper, however, takes a different, transnational historical angle on these events and, instead, focuses on the impacts of racism in south african medicine upon the internal politics of an iho. while there is a small literature dealing with the history of the wma, scholars have tended not to discuss its controversial stance during the 197694 apartheid era in detail. instead, studies have predominantly focused on how the organisation s ethical projects were shaped by post - war revelations that some european physicians had perpetrated nazi atrocities, as well as on its declarations and role in formalising ethical standards for research involving human subjects. a critical exception to this is laurel baldwin - ragaven.s book an ambulance of the wrong colour, which is based upon testimony given at the health sector hearings of south africa s truth and reconciliation commission (trc) in 1997. briefly mention the controversy around the masa s readmission to the wma in 1981, they do not provide a detailed analysis of the development of domestic and transnational advocacy against it. such multi - country advocacy can be understood within a wider framework of transnational activism, which margaret keck and kathryn sikkink have termed activism beyond borders. according to keck and sikkink such transnational activism occurs when activists based in different countries communicate, share resources and work together to press for changes to policies of which they are critical. there is a rich literature on transnational anti - apartheid activism, which points to the importance of shared, morally resonant framings of apartheid in generating support for the anti - apartheid movement in diverse countries. for example, hkan thrn has argued that exchanges of information, knowledge and symbolic practices between activists in different countries were key activities in the transnational anti - apartheid movement. similarly, audie klotz has argued that the enforcement of an international norm of racial equality promoted by anti - apartheid activists focusing on the influence of transnational anti - apartheid health activism on the internal politics of the wma, this article develops the literature on transnational anti - apartheid activism in general by describing activists roles in opposing racism in international medicine. the transnational anti - apartheid health activism discussed here is relevant to our understanding of ihos in the enforcement of medical ethics. in particular, it points to the deficiencies of an international medical professional association such as the wma as an adjudicator of last resort in a case where serious human rights violations had been perpetrated by physicians. the masa was a founder member association and attended the wma s first general assembly in 1947. this iho had a range of objectives including upholding the reputation and interests of the medical profession and assisting the world s people in attaining an improved state of health. the fledgling association quickly developed codes of medical ethics and established a relationship with the who. from its earliest days, the wma was dependent upon funding from the american medical association (ama) for its financial survival and a substantial proportion of this came from the leaders of us pharmaceutical companies. among the principles of social security the wma adopted in 1947 were the ideas that all medical services should be controlled by physicians, and that doctors should not be full - time salaried servants of the government or social security bodies a position which was very similar to that taken by the ama in the 1950s and 1960s. the ama was also plagued by racial conflict in this period : its 1968 conference was interrupted by civil rights activists who expressed their opposition to the exclusion of black physicians from membership of some southern chapters. it voted to end such discrimination at the same gathering, however, perceptions that racism lingered in the organisation persisted among many black physicians. in 1972 the ama left the international medical body because of what tessa richards has framed as disagreements over funding and voting strengths. the withdrawal of the americans from the organisation caused a crisis of legitimacy the soviet union had never been a member and the people s republic of china had no presence in the organisation and so with the ama s exit, it did not include doctors from three of the world s great powers. the departure of the americans also influenced the canadians in their decision to leave. in 1974 the organisation moved its secretariat from new york to ferney - voltaire in france. from the mid-1970s there had been increasing efforts by certain governments to isolate south african professional organisations, including its medical association because of its government s policy of apartheid. in 1975 the japanese government refused south african delegates visas to visit the country to attend the world medical assembly which was held in tokyo that year. the masa expressed its fury at its representatives being declined visas by the japanese government by arguing that as a founder member of the wma, in good standing it had an absolute right to be allowed to attend all world medical assemblies. the following year, the ghanaian government followed suit when ghana hosted the world medical assembly. that same year, 1976, the masa resigned from the wma in disgust at this diplomatic pressure. in 1979, after negotiations with the belgian surgeon dr andr wynen, who was then the part - time secretary - general of the wma, the americans re - joined, on condition that the international medical body changed its bylaws so that the number of votes a national body had within the iho depended upon the number of members it had declared i.e. paid for. this meant that a wealthier nation such as the us with more doctors who were potential members of its medical association (in this case, the ama) had far more votes at the wma than a less affluent country such as nigeria, with a medical association which was poorer by virtue of having fewer members. by 1983 the usa had thirty five votes, west germany fourteen, japan fourteen and the rest of the forty - seven member countries had only one or two votes each. voting strengths within the wma was controversial in the case of the vote to readmit the masa to the international association. the dispute over the role of physicians in relation to biko s maltreatment and death in detention was central to the development of a rift within the wma over apartheid. the soweto uprising of 1976 was inspired by biko s black consciousness (bc) writings. biko had studied at natal university s medical school, whose student - body was all black. this legislation broadly criminalised extra - parliamentary opposition as it defined terrorism as consisting of any act aimed at changing the economic or social system or fostering animosity between the races. biko died from brain injuries at a pretoria hospital twenty - six days into his detention. the depth of the international outrage over biko s death in detention and the wider crackdown on anti - apartheid opposition which had occurred post - soweto was also reflected in the united nations security council s unanimous vote in favour of resolution 418 which instructed states to stop supplying armaments to south africa. in november 1977 an inquest was held into biko s death in detention in which no one was found responsible for the loss of his life. the presiding magistrate referred part of the evidence which had been presented to the south african medical and dental council (samdc, hereafter referred to as the medical and dental council). in terms of section 45 of the medical, 56 of 1974, courts were to send the council evidence which appeared to implicate doctors in having engaged in improper or disgraceful professional conduct. a group of physicians also lodged a complaint with the medical and dental council about the behaviour of the two doctors, who had treated biko in detention. this group consisted of members of the black transvaal medical society and physicians with relevant specialist expertise who supported the organisation, including the head of a renal unit, a general medical lecturer and a neurosurgical registrar. the transvaal medical society was a voluntary organisation of black medical doctors, dentists, pharmacists, nurses and paramedics. their complaint was largely based upon evidence given at the inquest by the doctors who had treated biko. the black medical society and their physician supporters argued that a reading of the inquest record clearly shows a prima facie case of improper and/or disgraceful conduct on the part of lang and tucker, the doctors who had treated biko in detention. three years after biko s death, the medical and dental council reached a decision on the anti - apartheid doctors complaint. on 24 april 1980, the committee of preliminary inquiry of the medical and dental council found that there was no prima facie evidence of disgraceful conduct by the doctors involved in biko s care. jonathan gluckman, a private pathologist based in johannesburg, had performed a post - mortem examination on biko s body at the request of his family. gluckman was also a member of the federal council of the masa, to which he sent a letter signed by thirty - eight of its members calling for it to conduct an inquiry into whether tucker was fit to remain a member of the organisation. a critical component of the dispute which evolved over the medical association s presence in the wma was the decision the south africans subsequently took on this issue. the cape midlands branch of the masa discussed the issue and found that a charge of unethical conduct against dr tucker could not be sustained and ordered that the case be closed. following the cape midlands branch s decision on tucker, the federal council of the masa decided that as far as it was concerned the case was also constitutionally and legally closed. in august 1980, a month after the masa closed the case against tucker, the ama extended an invitation to their south african counterparts to attend their annual meeting in chicago. dr marais viljoen, the secretary - general of the masa was quoted in a south african newspaper as having said that the friendly invitation of the a.m.a. to attend their meeting in chicago later this year is an indication of the acceptance of south africa in medical circles. the same report stated that there were also indications that the ama would be sending a delegation to south africa in the near future to examine the system of medical services there. according to an article in one of the masa s publications, dr wynen, the head of the wma, also apparently offered his support for the masa. masa representatives who attended the chicago meeting found that the only false note sounded during the meeting, as far as sa [south africa ] is concerned, took place during a meeting of the american medical association s board of trustees which had been requested by the secretary of the nigerian medical association, dr beko ransome - kuti, during which he criticised sa [south africa ] for its alleged policies of discrimination against blacks in general and black doctors in particular. the criticism was, however, short - lived when the ama trustees pointed out to him that many of them had been to sa and that his facts were incorrect. ransome - kuti of the nigerian medical association was the brother of the famous musician fela kuti, and publicly shared his anti - apartheid views. like his musician brother, the doctor was involved in broader activism for human rights and democracy in nigeria. both came from a family tradition of vocal civil society advocacy : their mother campaigned against unfair colonial taxation of women and their father had been an anglican priest and founder of the nigerian union of teachers. in february 1981 dr jack sammons, the executive vice - president of the ama said that the world s best medical services were to be found in the us, south africa, canada and australia, with west germany following closely. an article translated from the largely pro - government afrikaans - language die burger by anti - apartheid health activists provided a similar picture of dr sammons s impressions of south africa during his visit and quoted him as having said in south africa we can learn a lot about various aspects of medical care, such as financing, manpower utilisation and the organisation of a complex such as groote schuur [a large teaching - hospital in cape town ], which has many services. such a position bore critical similarities to the concept of constructive engagement which was an approach to us foreign policy towards south africa developed by dr chester crocker, the assistant secretary of state for african affairs in the reagan administration. at the core of constructive engagement was crocker s optimism that the administration of president p. w. botha was meaningfully and incrementally reforming the apartheid system, developments which were thought to be deserving of encouragement by means of maintaining a friendly relationship with leaders of the south african government. this policy was criticised by its opponents as overly circumspect about the possibility of offending pretoria and insufficiently informed of the demands of the black opposition. it was doubtless also shaped by the fact that south africa was deemed by hawks in washington dc to be a strategically important bulwark against the infiltration of foreign soviet - aligned forces into the region of southern africa. an article in a south african journal by and for anti - apartheid health activists also published in february 1981 was much more critical of the ama s trip to the country. it noted that the visit by the ama delegates was in spite of an academic boycott on links with south africa, a boycott which had been called by various international organisations including the united nations general assembly, the commonwealth conference and the organisation of african unity. the aim of such boycotts was to isolate south africa financially, militarily, academically and in the arena of sports, and to thereby exert peaceful pressure on south africa to end apartheid. anti - apartheid health activists who contributed to and edited the journal feared that the ama delegation s visit could have been a prelude to m.a.s.as attempt to gain readmission to the world medical association and part of south africa s policy to seek credibility and acceptance in the international community. the ama s visit would thus give the impression that south africa and its health care are not so bad and that international contact will help promote change in this country. the south african anti - apartheid health activists feared that subsequent to the visit, the ama would probably claim to have conducted its own unprejudiced examination of medical care in south africa and would offer to exert pressure on the south african doctors to make positive changes to the health system. they correctly thought that the visit would focus on the adequacy of south africa s training of physicians and the quality of care provided in hospitals and private practice and not on whether the country s health system met the needs of all its people. the anti - apartheid health workers suspected that the ama delegation would not come into contact with the migrant labour system, forced population relocation, the bantustan policy and the oppression and unemployment that are the background to health problems in south africa. any report which would have resulted from the visit would have been inaccurate in the activists view and, therefore, they saw meaningful change resulting from the americans visit which they condemned as a breach of the academic boycott. in the light of the biko controversy, the anti - apartheid health workers were dismayed that the [ama ] group notably declined to comment on south african hospital overcrowding or the treatment of detainees, because of their lack of knowledge [my emphases ]. this was the type of response we expected from this group and it is obvious they ignored (or were not shown) the desperate lack of health facilities in rural areas and black urban areas. in august 1981, with the september meeting of world medical assembly in lisbon a mere month away, anti - apartheid activists from various countries swung into action to counter - act what they saw as the ama s campaign for the readmission of the south africans to the wma. the anti - apartheid american committee on africa sent a memorandum to the british anti - apartheid movement s health committee on the issue in august 1981, in which they outlined their opposition to the south africans proposed readmission to the wma. they pointed out that the country s medical and dental council had taken two and a half years to reach a ruling on the biko doctors and had seen no reason for disciplinary action against them. then they asked for their british counterparts to join them in opposing the ama s expression of support for the south africans readmission and argued that to allow them back into the world body would be to condone both racism and the operation of a vicious double standard in the application of the hippocratic oath. similarly, in august 1981, the nigerian medical association had lodged a complaint with the australian embassy in london about the latter s national medical association s support for their south african counterparts. the australian government joined the anti - apartheid groups in opposing the ama s support for south africa s readmission to the wma. the australian medical association s president dr lionel wilson was quoted as having said of their support for the south africans bid for readmission to the wma that it was a difficult decision to make but the australian body had decided to support colleagues in south africa because they believed that solutions to sa s [south africa s ] racial problems were most likely to come through the efforts of compassionate, educated people who have been exposed to world opinion. indeed, wilson argued that we [the australian medical association ] believe masa is one of the few liberal organisations in sa [south africa]. in september 1981 it was reported that the british anti - apartheid movement had sent to the portuguese embassy in london a memorandum written by fifteen anti - apartheid organisations and addressed to all the wma s members, which had had to be smuggled out of south africa. according to the report, when the wma considered the proposal to admit the transkei association and readmit the south africans, the medical associations of nigeria, ghana and liberia threatened to leave the world medical body. the strength of opposition to the south africans readmission by many africans from other nations on the continent was demonstrated by a position paper on the issue which was written by two nigerian physicians, dr o. o. adekunle and dr beko ransome - kuti and sent to the british anti - apartheid movement. adekunle and ransome - kuti contrasted the opposition of the nigerian, liberian and ghanaian medical associations with the visit to south africa of the ama delegation who, they thought, were perfectly happy with the conditions there [in south africa]. they then moved on to note that in their view there was a critical disjuncture between the wma s declarations and the masa s behaviour. by contrast, the masa had been silent on human rights violations in the country, and, the nigerian doctors went on to state that there is a saying that silence means consent all evidence points to its acquiescence with discriminatory health policies of the apartheid government. the m.a.s.a. is obviously towing the line of its sister organisation the south african medical and dental council which itself has only two coloured members in a country of over 80% non - white [people ]. one of the memoranda sent by south african opponents of the masa s readmission to the wma was penned by the natal health workers association. it outlined the role of masa in directly and indirectly implementing the state s policy of apartheid and thereby perpetuating this form of oppression against the majority of its citizens ; the same policy of apartheid which the international community had committed itself to eradicate. apartheid medicine on the majority of our people [black south africans] and thereby violating all codes of medical ethics and negating all considerations of human rights. its memo went on to describe key facets of apartheid medicine : systematic racial discrimination in medical training (fewer black doctors were trained) ; racial disparities in rates of mortality and disease and an unduly high death rate among black patients due to substandard provision of medical care for them, partly due to a shortage of health professionals who were employed to cater for their needs. uphold the highest traditions and ethics of the medical profession and to ensure racial non - discrimination in health care. therefore, it called on the wma and all its progressive and democratic members to reject the application by masa for membership. masa office - bearers hit back against their black critics who opposed the association s readmission to the wma by attacking their credibility. in the days leading up to the vote on south africa s readmission to the world body, dr marais viljoen, the secretary - general of the masa was quoted as having asked of the black health groups who are they ? what are their objectives? he apparently claimed that his organisation did not condone the findings of the medical and dental council regarding the conduct of the doctors treating mr biko, but noted the council s findings. dr viljoen was also said to have rejected allegations that the medical association had not maintained its objectives or upheld the highest traditions and the ethics of the medical profession. this transnational campaign against the south africans readmission to the wma proved unsuccessful and the breakdown of votes on 25 september 1981 was reported as follows : australia, belgium, brazil, cuba, taiwan, west germany, italy, japan, portugal and the united states voted in favour of south africa s readmission.france, korea and spain abstained.ten votes recorded against the masa which were mostly african and asian countries, including india. australia, belgium, brazil, cuba, taiwan, west germany, italy, japan, portugal and the united states voted in favour of south africa s readmission. ten votes recorded against the masa which were mostly african and asian countries, including india. professor guy de klerk, speaking on behalf of the masa, said that it welcomed its readmission to the wma as a recognition of the high standards of medical ethics and care in the country. anti - apartheid activists in britain swung into action to denounce the outcome of the wma s vote. this was yet another example of transnational anti - apartheid activists challenging the moral authority of the nationalist government and civil society groups perceived as being aligned to it. in late september 1981, dr johnny fluxman of the british anti - apartheid movement s health committee argued that the wma voting system enables a handful of western countries to dominate the organisation and stifle any criticism by third world countries. he also pointed out that it did not represent any african, scandinavian or socialist countries, and that countries such as taiwan and transkei, not recognised by the un were members. fluxman added that fifteen anti - apartheid organisations within south africa had written to the wma asking it not to readmit the country s medical association but, instead approval has been given to masa and its cover - up of the murder of steve biko, and to south africa s bantustan policy the transkei bantustan has been admitted as a member alongside masa. he concluded his letter by calling upon the british medical association (bma) to resign from the wma, noting that they had already stated their opposition to the south africans readmission. meanwhile, within south africa, there were reports in early october that groups such as the black transvaal medical society feared that the acceptance of masa into the wma would lead to other south african organisations clamouring for international recognition putting up a relentless fight. for black medical bodies such as the transvaal medical society, apartheid and oppression violated all codes of medical ethics and the masa, being a predominantly white body, had directly and indirectly condoned this state of affairs. the society also held that the wma s re - acceptance of the masa would forever be regarded a breakthrough for apartheid and oppression of the black majority of the country and a damning and adverse blow to resistance against the status quo and domination of man by man. the newly readmitted masa covered the issue in an editorial in its journal, the south african medical journal, published on 10 october 1981. the journal reported that the breakdown of votes was seventy - seven in favour of its readmission, with ten against, and eight abstentions were registered. an assembly of doctors concerned with the practice of medicine and not infrequently in conflict with politicians (as papers read at the scientific sessions later in the week amply demonstrated). we are sure that the masa has a contribution to make to this body and that the wma will in no way be weakened by its presence. as the controversy over the south africans involvement in the wma deepened, the idea that a cordon sanitaire could be imposed between medical ethics and politics, would continue to be asserted by the masa and their international supporters. four months later, in january 1982, the issue was considered by the executive board of the world health organisation (who). the who was one of many un agencies which had restricted south africa s membership since 1960 after agitation by the newly independent african states. on 20 january 1982, h.e. alhaji yusuff maitama - sule, nigeria s representative to the un who also chaired its special committee on apartheid (established in 1964) sent a telegram to the executive board of the who on the matter of the masa s readmission to the wma. maitama - sule accused the wma of violating article ii of the international convention on the suppression and punishment of the crime of apartheid of 1973 : this convention defined apartheid as being a crime against humanity. the nigerian diplomat held that apartheid was an evil system which the non - white world had played a leading role in opposing because it represented an affront to their recently won freedom, independence, and i dare say, human dignity. he went on to add, let me take advantage of this appearance before who and eminent doctors to state that we in the special committee, and i might add in the organisation of african unity, consider the role being played by the masa and wma as not being too different from the role played by many nazi doctors during the second world war. maitama - sule then said that when the time of retribution came, the example of nuremberg would not be lost on the united nations and the african people. he ended by calling on the who executive board to terminate its relationship with the masa and the so - called transkei. an african diplomat to the un was publicly equating the actions of the wma, an organisation which was set up in the wake of the nuremberg trials and which had the promotion of medical ethics as one of its core aims, with those of nazi doctors. the wma s credibility as the keeper of the medical ethical creed was under serious attack at a critical international health institution. the who s executive board voted to discontinue official relations with the wma on 27 january 1982 by twenty - seven votes in favour, one vote against (the us) and one abstention. this decision was reversible provided the wma expelled the south africans. in subsequent years, this decision would be cited repeatedly by anti - apartheid activists to show that the wma had suffered a loss of prestige and was out of step with established international norms of racial equality in relation to how to deal with its south african member organisation. by this period, there was an increasing enforcement of such norms, internationally. on 5 february 1982, dr neil aggett a young physician and trade union organiser died in johannesburg after having spent seventy days in detention without trial. meanwhile, shortly after their readmission to the wma, the south africans were soon nominated and elected for some of its key positions. (marais) viljoen the masa s secretary - general was elected to the council of the wma and nominated to its medical ethics committee : a nomination he described as of major importance to the south african medical profession, whose medical ethics have frequently been questioned at an international level. this was a position to which he was appointed in 1983. viljoen was alleged by anti - apartheid activists to be both a national party supporter and a long - time member of its inner conclave, the secretive broederbond. with an unsuccessful campaign against the south africans readmission behind them, the health workers association (formerly the transvaal medical society) re - strategised and also began to campaign against the wma itself. at its national meeting in may 1982, an anonymous activist gave a speech on the wma s history, a summary of which was kept by the organisation. the full, written version of the speech is worth discussing at some length as it reveals what some anti - apartheid health workers based within the country thought the socio - economic and political forces were behind the ama s campaign for the readmission of their south african counterparts. the activist cited an article published in the south african medical journal in 1951 to claim that the best financial support for the wma had come from the great pharmaceutical firms of the united states many of which, as we have seen, had close ties with the ama. usa through the ama, attempted to impose its hegemony on the wma since its inception. the global advantages for the multinational pharmaceutical industry of an american dominated wma are obvious. their close association with ama would enable them to come into contact with the medical profession from many countries to whom they could promote their drugs. furthermore, health programmes and policies that were compatible with the financial interests of american capitalism would be promoted at a global level in the wma. the anti - apartheid health activist went on to argue that part of the impetus behind forming national medical associations was to block or slow the formation of national health services. the author also argued that exiled cuban doctors in miami had represented cuba in the wma since 1959. in the document s conclusions the anti - apartheid health activist claimed that the wma was an organisation which was not truly representative of the world medical profession because less than a third of the world s countries had medical associations affiliated to it. the activist went on to argue that the wma only allowed associations which were independent of governments as its members and that this was intended to exclude socialist governments from joining it. the article went on to add that this concept of independent is farcical as in most countries the medical associations are closely aligned with the state. this author again compared the wma to the who, by stating that while the who has made a significant contribution at an international level to the promotion of health care, wma s contribution to this sphere has been negligible. without significant reform to the wma s structure it was unlikely to make a significant contribution, in the author s view. reactionary organisation which could not be changed from within as the british medical association had apparently realised by this stage. the health workers association s new strategy on this issue had to be to oppose both the masa and the wma at national and international levels. by july 1982 the british medical association (bma) had officially terminated its relationship with the masa. in a letter to the editor of the south african medical journal, dr jonathan gluckman outlined his version of what had caused the termination of the relationship. as we have seen gluckman had performed a post - mortem examination on biko s body and was also a member of the executive committee of the masa and he met with the bma council s executive committee to discuss its decision to terminate its relationship with the masa. gluckman was a fierce opponent of the termination of the bma s relationship with the masa and following his meeting with representatives of its council he claimed that the british association which [he said ] was part of the trades union congress was dominated by the politics of the extreme left in britain, and that the effect of this is to erode the structures of the bma to the same extent as it has eroded the structure of great britain. he claimed that so vicious had been the attitude of the council of the bma, that its representatives for the then upcoming meeting of the wma had been instructed to vote against the readmission of the south africans and not to be influenced by any debate or arguments to the contrary which might have been forthcoming [his emphasis]. in july 1983 dr antonio gentil martins, the portuguese president of the wma was one of the guests of honour at the fifty - fourth meeting of the masa in cape town. also in attendance were representatives from the american medical association (the ama) and the west german medical association. the presence of these representatives of the wma reflected the fact that two years earlier the south africans had been readmitted to the international medical association. following his visit to south africa, dr martins spoke highly of the south african medical profession, and, according to an anti - apartheid activist newsletter, he had said, we found that the quality of health care available to all races was completely equal. he then apparently went on to argue that providing medical infrastructure was a political issue and not the responsibility of the profession. this was dr martins s second visit to south africa. on his first visit to the country, the portuguese surgeon had been quoted as having defended the masa s controversial approach to the scandal surrounding biko s death in detention discussed above. the only member of masa involved in the affair had been exonerated of all blame. masa therefore could not be held responsible for the treatment of mr biko. he claimed to be familiar with the problem of overcrowding in south african facilities and apparently said that providing the infrastructure for adequate medical services was a political issue and not the responsibility of masa. martins had seen the same technology available in hospitals serving black people and those serving whites and he apparently claimed that i saw no difference as far as the quality of care was concerned. he was also quoted as having argued that the low standard of education and lack of family planning among black people were among the worst problems facing south african medicine. martins apparently went on to say that no country can afford to provide hospital beds for a population growing as rapidly as that of south africa s blacks. such a racist, neo - malthusian argument that the poverty experienced by africans was their own fault due to their supposed uncontrolled fertility was a key element of apartheid thinking, as evident in the south african government s disproportionate spending on the promotion and provision of family planning when compared to their spending on the other health services for black people, including maternal and child health services. the masa had invited representatives to attend the same 1983 congress, and the president of the ama, dr frank j. jirka said that health services in this country [south africa ] compared favourably with those in the united states of america. one of the delegates was dr james h sammons (the executive vice president of the ama, as mentioned above) who was named the leading medical personality in the usa for 1983 by the times us news and world report, inter alia, referred to the masa s report on the medical treatment of prisoners and detainees. superb job and he hoped that the recommendations would be favourably considered by the authorities. dr sammons argued that the maltreatment of prisoners also occurred in america and he said unfortunately it happens far too often, and although there are problems, it is not excused by the problems. there was yet another call by the masa s supporters to separate politics. dr horst bourmer of the west german medical association apparently held that doctors, as doctors, should never become involved in politics, but should concern themselves with the improvement of medical care only. bourmer called for governments to grant medical associations autonomy on professional matters and then was quoted as having said that on the other hand i believe that [the ] exclusion of medical associations from international medical politics because of the policies of their governments is discrimination at its worst. humanity and fraternity should be the motto of all who belong to the medical profession. at its october 1983 meeting in vienna, the wma decided to hold its 1985 assembly in cape town on a clear day robben island a potent symbol of racial discrimination robben island contained a prison where several opponents of apartheid remained detained and whose most famous resident just over a year before had been nelson mandela, who had recently been moved to pollsmoor prison on the mainland, just outside cape town. in this context, at its first annual national conference in durban from 5 to 6 december 1983, the new anti - apartheid national medical and dental association (namda) resolved to oppose cape town s hosting of the wma. the new association aimed to unite all south african doctors and dentists opposed to apartheid in one nationwide organisation. unlike the masa, namda was affiliated with the united democratic front (udf), a national anti - apartheid civil society coalition. on 5 january 1984 eroded by a series of events which had cast doubt on the ability and willingness of wma to provide an international, representative forum for the resolution of important medical, professional and ethical issues. the press release mentioned that an undemocratic voting block system had resulted in the south africans having been readmitted to the international medical association against the wishes of the majority of countries belonging to wma, even though certain important issues affecting its application for re - admission had not at that time been resolved. these issues included the failure of the masa to adequately investigate the conduct of certain doctors who had examined steve biko before his death in police custody. the statement noted that the british association had tried to reform the wma constitution from within in the years subsequent to south africa s readmission. it also noted that the who had withdrawn consultative status from the wma over the issue and questioned the representative legitimacy of members such as the transkei association and the cubans who were represented by the free doctors of cuba based in miami. but british doctors were far from unanimous in supporting their national medical association s stance on this issue. the lancet s brief coverage of the withdrawal of the british medical association from the wma stated that its undemocratic voting system was at the heart of its decision on the issue. it also reported that a group of british doctors had formed who continued to support the wma. such dissenters within bma on south africa issue held views which were in keeping with a substantial slice of british public opinion. the conservative party government led by margaret thatcher had a policy which was in many respects similar to the constructive engagement of their american counterparts. moreover, in the early 1980s, cultural and economic ties between britain and south africa remained strong, despite calls for sanctions. back in south africa, dr r.d. le roex, chairman of the federal council of the country s medical association mentioned in his 1984 report for that body that it had hosted dr lionel l. wilson, the wma s council s former chairman and past president of the australian medical association. le roex expressed his appreciation for the role played by the masa in supporting of improvement of medical care for south african political detainees. he stated how much he valued the south africans ongoing membership of the wma, as such international contact was essential if the lofty ideals of the wma, viz. to achieve the highest international standards of medical education, medical science, medical art and medical ethics, and health care for all people of the world, are to be attained. le roex said that at the wma s meeting in singapore the south africans had had the chance to meet both those who were well informed and well disposed towards south africa and its health services and those with the opposite opinion. in his view their opponents were frankly hostile to the system of government in south africa and consequently also to our health care system and to the masa, a position which he viewed as being based on ignorance or misinformation, much of which is deliberately disseminated from this country by misguided colleagues [namda]. he saw namda s call for a boycott of the cape town assembly as based upon a malicious misrepresentation as the invitation was not a political statement of any kind. by contrast, the masa s chairman said that the south african government s only involvement would be the granting of visas to bona fide delegates. in late 1984 there were dramatic broader developments in the country s popular politics, which had also important effects on anti - apartheid activism abroad. there was a new wave of popular protests in several townships across the country which involved the civic associations and activists aligned to the udf and in many countries in the west images of police brutality towards the demonstrators were broadcast on television news, which broadened opposition to apartheid. in november 1984 the anglican archbishop desmond tutu tutu made a strong impression in the united states and his nobel win was associated with more radical and popular american anti - apartheid activism as represented by the civil disobedience actions of groups such as transafrica led by randall robinson. on 5 february 1985, three hundred physicians and other health professionals marched on the south african embassy in washington dc five of whom were arrested and later released. some of the thinking behind this growing us opposition to apartheid in south african medicine is suggested by a guest editorial published in the journal of the national medical association in july 1985. the national medical association was an organisation which represented african - american physicians and members of the communities that they served. charles h. wright began his article stating that dr philip m. smith, the president of the national medical association, had asked members to join him in protesting the proposed cape town meeting of the wma. he then moved on to discuss the scandal around biko s physicians collusion in his torture in detention and noted that while the national medical association s journal had covered the issue and while many of the world s medical societies reacted with revulsion towards south africa s efforts to ignore and cover up this event, the american medical association s officials reacted as if it was a non - event. the journal of the american medical association did not mention the controversy. wright pointed to the ama representatives two visits to south africa and also mentioned that in 1981 the american association had cast all its votes in favour of the country s readmission. he then stated his view that while a campaign for a cancellation of the world medical assembly in cape town was laudatory, a letter - writing campaign to the ama would not have intrude[d ] on its loyalty to its colleagues in south africa. he then called for national medical association members to stop paying for ama membership because a more certain way to make sure that biko, a former medical student did not die in vain is to divest in ama. he ended by stating that his only regret was that he could not join in such a protest as he had already divested from the ama in the late 1950s when the ama showed more concern for hungarian and cuban exiles than for black doctors. the wma s membership dwindled significantly because of its position on south africa. in 1985 doubtless at least partially due to such civil society pressure on the issue, the thirty - seventh (1985) world medical assembly was moved to brussels, belgium at short notice. meanwhile, a breakaway group was formed which consisted of the medical associations of denmark, finland, iceland, ireland, the netherlands, new zealand, norway and sweden, later joined by the british, canadians and jamaicans. according to an article published in the british medical journal in 1994, the group met annually and campaigned around demands such as : member associations of the wma should be truly representative of the medical profession in their country.member associations should be politically independent of their government.the wma needed to have a more democratic voting system. member associations of the wma should be member associations should be politically independent of their government. the wma needed to have a more democratic voting system. after a 1987 meeting of this group in canada, the group became known as the toronto group. in johannesburg, in november 1985, litigation launched by a group of medical academics at the university of the witwatersrand resulted in a supreme court decision which forced the south african medical and dental council to reconsider the case of drs tucker and lang in relation to their treatment of biko. lang continued to practise, however, and was promoted to the position of chief district surgeon in port elizabeth. for the rest of the 1980s the namda and the masa remained bitter foes because apartheid itself remained at the very heart of their dispute. however, the international medical association apparently responded to some of the toronto group s criticisms by implementing certain reforms such as agreeing with the principle that member associations should be truly representative of doctors in their own countries. it also switched to granting each member country one vote per 10 000 members instead of the previous one vote per five thousand members and resolutions on ethical issues now required a two - thirds majority of delegates present to pass. the nordic countries and britain had only just rejoined as late as 1995 when a dr anders milton a swedish nephrologist became new chairman of the wma and stated his relief that with the country s democratic transition it no longer had the political problem of south africa. milton acknowledged that this had been the main reason why the british and swedish associations had left and pledged to reach out to more countries and ensure not only that ethical declarations are taught at medical schools and discussed by practising doctors all over the world but that they are in daily use. transnational anti - apartheid activists efforts to isolate south africa and morally delegitimise the wma for failing to do so, therefore, had had a real impact on physicians support for an iho in a number of countries. in post - apartheid south africa, the issue of the biko doctors was re - examined in the truth and reconciliation commission s health sector hearings in 1997. in the spirit of nation reconciliation the namda and the masa merged in 1998 to become the south african medical association (sama). also in 1997, the south african medical and dental council (samdc) became the health professions council of south africa (hpcsa) as part of a broader package of reforms to the bring the regulation of doctors activities into alignment with the human rights values espoused in the country s new constitution (1996). today, the wma is an iho best known for its ethical declarations, including the declaration of tokyo on physicians and torture. yet, one of the clearest examples of the violation of this declaration occurred almost immediately after it was passed the maltreatment of south african activist steve biko by his doctors, physicians whom the country s medical association and medical and dental council were extremely hesitant to discipline until the end of the main period discussed in this article. less documented have been the circumstances around the readmission of the masa and the subsequent exodus of a substantial group of the wma s members because of perceptions that it had exonerated apartheid medicine, including physician collusion in torture a lacuna in existing literature that this article has aspired to make modest progress in addressing. the moral authority of the wma around the world also dwindled as physicians who were anti - apartheid activists in britain, the united states and south africa repeatedly denounced the organisation as racist and campaigned against it in the same breath as they opposed apartheid medicine. these transnational activists or activists beyond borders shared information with each other, including information which helped to generate morally influential framings of racial discrimination in south african medicine. yet they were ultimately unable to successfully press for the wma to reject the masa as a member organisation. what they were, however, able to accomplish was a diminution in the moral authority of the iho both among many physicians in different countries and several international health officials. they successfully drew attention to the fact that in the late twentieth century, senior office - bearers of an organisation fundamentally shaped to combat the horrors of nazi medicine had condoned medical aspects of a system of racial discrimination which had been cast in international law as being yet another crime against humanity. these activists, thereby, managed to reduce the number of member associations and press the who to end its relationship with the wma. the wma and its supporters on this issue derided their opponents as politicians concerned with ethics. anti - apartheid activists rejected such a division by pointing to the racism in south africa s health system and a lack of political freedom to highlight it. indeed, they drew attention to the fact that a former medical student (steve biko) and a doctor (neil aggett) were among the many activists tortured - to - death in detention. for such activists, these cases demonstrated that even health workers could not freely highlight issues of terrorists with all the dire consequences that entailed. the full reasons why the wma not only bucked international trends to isolate south africa by welcoming its medical association back into its fold but also promptly thereafter invited one the south africans to sit on its ethics committee have yet to be explored in further research. what is hard to dispute is that the wma and the ama (its member with the largest number of votes) generated perceptions among opponents of apartheid that it had welcomed the south africans back as part of a wider effort to pack the body with members who would uncritically support the ama an organisation with stances very different to those of the who on the ideal roles of physicians and the private sector in health systems. some african - american physicians opposed to apartheid saw the controversy as the latest chapter in a long history of institutionalised anti - black racism within the american member organisation. the most charitable interpretation of why some national associations chose to support south africa s readmission is that they might have hoped to have won them over to incremental liberal reforms to end apartheid in the manner of crocker s constructive engagement. apartheid s demise occurred almost two decades ago, but the notions that medical ethics should be depoliticised remain current. this article has discussed an important, under - examined, late twentieth - century example of racism in international medicine. medical ethicists have observed that the wma s role as an international adjudicator is limited by the voluntary nature of its international professional guidelines. the history offered in this article, therefore, suggests that transnational civil society actors must remain permanently vigilant to ensure that justice prevails in cases where physicians are accused of human rights violations.	this article describes the role of transnational anti - apartheid activism in south africa, britain and the united states in generating international moral outrage over the readmission of the medical association of south africa (masa) to the world medical association (wma), which had taken place in 1981 after it had withdrawn from that body in 1976. it discusses an example of a controversy where an international health organisation (iho) lost moral authority as a result of being accused of white supremacy and a pro - american engagement in cold war politics. at the time of its readmission to the wma, the masa was controversial because of its failure to strike off its membership roll one of the doctors implicated the death in detention of black consciousness leader steve biko in 1977. it details how these activists viewed the american medical association as having campaigned for the masa s readmission. the wma s readmission of the masa cost the former its relationships with the world health organisation (who) and the british medical association a dispute which continued until south africa s democratic transition of 1994. with its focus on transnational activism in relation to the wma and the effects of activists allegations of racism on its internal politics, this article contributes to the literature on the history of ihos. ultimately, this controversy shows the deficiency of international medical professional associations as ethical arbitrators of last resort.
the animals were 24 one- to two - year - old thoroughbreds (twelve males, twelve females) clinically free of any orthopaedic disorders and history. prior to a breaking and training period that begins in september and ends in april every year, twelve horses (six males, six females) were selected as subjects in the first study from december 2013 to april 2014. the other twelve horses were used in the second study from december 2014 to april 2015. the running (that is at a canter and a gallop) distances for 1 month before the dates of the ultrasonographic examination are presented in table 1table 1.changes of running distance for 1 month before the dates of the ultrasonographic examination in december, february, and april2013 (km / month)2014 (km / month)december9.6 6.39.8 5.2february47.7 1.834.0 9.6april54.0 18.145.6 7.6running was performed at a canter and a gallop.. running was performed at a canter and a gallop. the horses sdfts were ultrasonographically scanned on the same dates in december, february, and april. before the examination, the horses had been kept in pasture for at least 3 hr following their daily exercise. the horses were retained in a treatment stall, and then sedated with medetomidine (2 mg / head intravenous injection). after completely clipping the palmar aspect from the proximal to the distal metacarpus, a linear array transducer (50 mm of effective aperture length) with a broadband frequency between 5 and 12 mhz was directly placed onto the skin with abundant gel. using a mobile ultrasound system (iu22, philips medical systems corporation, tokyo, japan), three longitudinal (three equal lengths of the labelled 1 to 3 in order from proximal to distal metacarpus) and six transversal (separated by equal distance along the labelled 1a, 1b, 2a, 2b, 3a and 3b in order from proximal to distal, fig. after completely clipping the palmar aspect of the metacarpus, a linear array transducer was directly placed on the skin with abundant gel. the rounded sdft was clearly outlined in the 1a, 1b and 2a images ; however, the lateral and medial margins could not be visualized in the flattened sdft of the 2b, 3a and 3b images.) images from the proximal end (bottom of the accessory carpal bone) to the distal end (top of the proximal sesamoid bone) of the sdft were scanned. the maximum depth of the image was 3.5 cm, and the depth of focus was adjusted to the sdft. both gs and cd images were recorded for 15 and 30 sec at frame rates of 45 hz and 6 to 11 hz, respectively. after completely clipping the palmar aspect of the metacarpus, a linear array transducer was directly placed on the skin with abundant gel. the rounded sdft was clearly outlined in the 1a, 1b and 2a images ; however, the lateral and medial margins could not be visualized in the flattened sdft of the 2b, 3a and 3b images. blood flows in the tendon bundles were visualized in the longitudinal and transversal cd images, in which the region of interest was set and the gain setting was similar for all tendons, just below the noise level (7785% of maximal gain). the positive signals of cd flow are grade 1 (indicated by white arrows), small color activities that were fixed and rhythmically blinking.) was recorded and graded, as previously reported : grade 1, tiny to small color activities that were rhythmically blinking at a site, but for which flow rates were not measurable by pulsed doppler ; grade 2, unequivocal color activities that were pulsatile dots and for which flow rates were measured by pulsed doppler ; and grade 3, unequivocal linear color activities appearing as blood streams in a regular direction due to periodic changes in color. the longitudinal cd scan was inferior to the transversal one in detecting and recording the small color activities of grade 1 and 2, therefore we used only the transversal images for the cd data analysis. a transversal cd image of the sdft. the positive signals of cd flow are grade 1 (indicated by white arrows), small color activities that were fixed and rhythmically blinking. the horses sdfts were ultrasonographically scanned on the same dates in december, february, and april. before the examination, the horses had been kept in pasture for at least 3 hr following their daily exercise. the horses were retained in a treatment stall, and then sedated with medetomidine (2 mg / head intravenous injection). after completely clipping the palmar aspect from the proximal to the distal metacarpus, a linear array transducer (50 mm of effective aperture length) with a broadband frequency between 5 and 12 mhz was directly placed onto the skin with abundant gel. using a mobile ultrasound system (iu22, philips medical systems corporation, tokyo, japan), three longitudinal (three equal lengths of the labelled 1 to 3 in order from proximal to distal metacarpus) and six transversal (separated by equal distance along the labelled 1a, 1b, 2a, 2b, 3a and 3b in order from proximal to distal, fig. after completely clipping the palmar aspect of the metacarpus, a linear array transducer was directly placed on the skin with abundant gel. the rounded sdft was clearly outlined in the 1a, 1b and 2a images ; however, the lateral and medial margins could not be visualized in the flattened sdft of the 2b, 3a and 3b images.) images from the proximal end (bottom of the accessory carpal bone) to the distal end (top of the proximal sesamoid bone) of the sdft were scanned. the maximum depth of the image was 3.5 cm, and the depth of focus was adjusted to the sdft. both gs and cd images were recorded for 15 and 30 sec at frame rates of 45 hz and 6 to 11 hz, respectively. after completely clipping the palmar aspect of the metacarpus, a linear array transducer was directly placed on the skin with abundant gel. the rounded sdft was clearly outlined in the 1a, 1b and 2a images ; however, the lateral and medial margins could not be visualized in the flattened sdft of the 2b, 3a and 3b images. blood flows in the tendon bundles were visualized in the longitudinal and transversal cd images, in which the region of interest was set and the gain setting was similar for all tendons, just below the noise level (7785% of maximal gain). the positive signals of cd flow are grade 1 (indicated by white arrows), small color activities that were fixed and rhythmically blinking.) was recorded and graded, as previously reported : grade 1, tiny to small color activities that were rhythmically blinking at a site, but for which flow rates were not measurable by pulsed doppler ; grade 2, unequivocal color activities that were pulsatile dots and for which flow rates were measured by pulsed doppler ; and grade 3, unequivocal linear color activities appearing as blood streams in a regular direction due to periodic changes in color. the longitudinal cd scan was inferior to the transversal one in detecting and recording the small color activities of grade 1 and 2, therefore we used only the transversal images for the cd data analysis. a transversal cd image of the sdft. the positive signals of cd flow are grade 1 (indicated by white arrows), small color activities that were fixed and rhythmically blinking. the running distance for 1 month before the date of the ultrasonographic examination was increased in the order of december, february, and april in both of the two training periods. as the transversal gs images were obtained using a linear array transducer that was directly placed onto the skin with abundant gels and not attached to a coupler, the rounded sdft was clearly outlined in 1a, 1b, and 2a images, however the lateral and medial margins could not be visualized in the flattened sdft of 2b, 3a and 3b (fig. 1). there were no longitudinal or transversal gs images indicating injury in the sdfts in either of the two training periods. of 864 (6 images 2 forelimbs 24 horses 3 examinations) transversal cd images, 56 images (6.4%) showed the cd flows in sdft bundles (table 2table 2.among 864 transversal cd images, 56 images (6.4%) showed positive cd flows in sdft bundles), which were categorized as rhythmically blinking (grade 1, 32 images, as presented in fig. the cd flows were unevenly distributed at 1a, 1b, 2a and 2b, and were more frequently detected in april than in either december or february (table 3table 3.the distribution of transversal cd images showing positive cd flows in sdft bundles1a1b2a2b3a3btotaldecember 4210007february104000014april1461230035total28121330056). there were 7, 8 and 15 horses showing cd flows in december, february and april, respectively. a normal tendon is hypovascular, and angiogenesis or increased vascularity are associated with an injured tendon for much of the healing process. within the first 24 hr after injury (inflammatory phase), inflammatory cells are recruited to the injured site, and vasoactive and chemotactic factors are associated with an increase in vascular permeability and the initiation of angiogenesis [17, 22 ]. within a few days, tenocytes gradually migrate to the injury site, proliferate, and synthesize type iii collagen (proliferative phase) [19, 22 ]. after approximately six weeks, the repair tissue changes from cellular to fibrous (remodelling phase). the fibrous tissue gradually changes to scar - like tendon tissue over the course of one year (maturation stage) [10, 22 ], and the vascularity finally declines in the latter half of this stage [1, 22 ]. therefore, we hypothesised that the blood flows would also increase in equine sdft from a few days to six weeks after the injury, and that they might be indicative of tendon injury and the subsequent healing process. however, in this study of one- to two - year - old horses, no macroscopic injury was found in the 56 transversal gs images in which positive signals of blood flow were seen within sdft bundles. some of the positive flow signals disappeared and/or then reappeared during the study period. therefore, we speculate that the positive signals of blood flow observed in this study might be due to transient increases of blood flows in the inherent vessels in response to hypoxia and/or hyperthermia [11, 20, 25 ] of tendon tissue which were related to the increased exercise (the increased mechanical behaviour of the tendon), rather than increased vascularization associated with macroscopic injury of tendon bundles. based on the observance of increased doppler flow in achilles tendons that was associated with weekly badminton hours and badminton years, it was suggested that doppler flow may be a response to mechanical load on the tendon. since doppler activity increased in human achilles tendons after running exercise performed by both non - symptomatic subjects and symptomatic subjects in a rehabilitation program, it was proposed that all tendons of both healthy and diseased subjects, have a certain amount of vascularization, which may be manifested on demand up to a certain level. considering our result in reference to these human studies, it is possible that the blood flows observed in the sdft of the one- to two - year - old thoroughbreds were response to the increases in the intensity and distance of the running exercise during the training period. the main sources of blood supply are : the intrinsic systems at the myotendinous and osteotendinous junctions, and the extrinsic system through the paratenon or the synovial sheath. the ratio of the blood supply from the intrinsic systems to that from the extrinsic system varies in the different parts of the tendon. for example, the central third, which could be a zone of hypovascularity, receives 35% of its blood supply from the extrinsic system in the rabitt achilles tendon [14, 18 ]. an equine study reported that the blood supply of the normal sdft is primarily intratendinous rather than paratendinous as previously thought. in this study, the cd flows mainly increased in 1a (28 images), 1b (12 images), and 2a (13 images), which are proximal parts of the sdft. this result could be suggestive of increased blood supply from the intrinsic systems at the myotendinous junction. at the myotendinous junction, perimysial vessels from the muscle continue between the fascicles of the tendon, and extend to the proximal third of the human achilles tendon [6, 22 ]. although we have no data regarding the blood vessel distribution from the superficial digital flexor myotendinous junction to the proximal third of metacarpal sdft in horses, this result might be explained as the perimysial blood supply to the proximal third physiologically increasing in response to the increases in the running distance of the clinically healthy horses. in conclusion, the increase of intratendinous blood flows in the proximal parts of the sdft could be responses to the increase in exercise intensity as the training advanced. because no longitudinal or transversal gs image indicating macroscopic injury of the sdft were found in either of the two training periods, increase of cd flows in the proximal parts of the sdft are not necessarily predictive of tendon injury in the near future in the training period of young thoroughbreds.	abstractaim of this study was to evaluate the relationships of exercise and tendon injury with doppler flows appearing in the superficial digital flexor tendon (sdft) of young thoroughbreds during training periods. the forelimb sdfts of 24 one- to two - year - old thoroughbreds clinically free of any orthopaedic disorders were evaluated using grey - scale (gs) and color doppler (cd) images during two training periods between december 2013 to april 2015. twelve horses per year were examined in december, february, and april in training periods that began in september and ended in april. the sdft was evaluated in 3 longitudinal images of equal lengths (labelled 1, 2, 3 in order from proximal to distal), and 6 transversal images separated by equal lengths (labelled 1a, 1b, 2a, 2b, 3a and 3b in order from proximal to distal) of the metacarpus using both gs and cd. the running (canter and gallop) distance for 1 month before the date of the ultrasonographic examinations was increased in december, february, and april in both of the two training periods. cd flows defined as rhythmically blinking or pulsatory colored signals were found in 56 of 864 (6.4%) transversal cd images, in 28, 12, 13, and 3 images of 1a, 1b, 2a and 2b, respectively, and in 7, 14, and 35 images captured in december, february, and april, respectively. there were no longitudinal or transversal gs images indicating injury in the sdfts in either of the two training periods. the increase of cd flows in the proximal regions of the sdft are possibly related to the increase of the running distance during the training periods of the one- to two - year - old thoroughbreds. because no injury was diagnosed in the sdfts by gs images during the training periods, the increase of cd flows in the proximal parts of sdft is not necessarily predictive of tendon injury in the near future during the training period of young thoroughbreds.
it is widely accepted that the fundamental physics of high - tc superconductors (htsc) takes place in the two - dimensional cuo2-layers. on the other hand, several classes of htsc exist with a different number of cuo2-layers per unit cell, their transition temperature being strongly related to this number., one could mention interlayer interactions, charge imbalance, or quantum tunneling of cooper pairs [24 ]. experimental measurements, supported by theoretical investigations, show that the interlayer coupling and the third dimension more generally have a strong impact on angle - resolved photoemission spectroscopy (arpes) results [68 ]. depending on photon energy and polarization, different features are accentuated in the measured spectra [9, 10 ], while the real underlying quasiparticle spectrum remains hidden. in the last decade, the bisrcuo compounds bsco-2212 and bsco-2201 have been studied thoroughly, and several conclusions have been drawn from the results : high resolution arpes on bsco-2212 with suppressed superstructure reveals the presence of two fermi surface pieces : one hole - like, the other changing from electron to hole - like. heavily overdoped bsco-2212 shows a difference in bilayer band splitting for the normal and superconducting case. in the normal state, this is about 88 mev and gets renormalized to about 20 mev in the superconducting state. in the superconducting state, each one of the two split band develops its own peak - dip - hump structure (pdh). this is most probably due to the strong renormalization at about 60 mev produced by the interactions with spin fluctuations. bilayer splitting in the normal state only weakly depends on doping. in optimally doped bsco-2212 (bilayer), the quasiparticle in the (,0) region should look similar to that of bsco-2201 (monolayer) ; the enhanced linewidth in the bilayer material is attributed to correlation effects, more specifically (,) scattering due to antiferromagnetic fluctuations. in order to unravel the underlying mechanisms producing these effects, lda calculation done for ybco show that the interlayer hopping comes from copper s electrons. different models were used to describe the system of coupled 2d cuo planes, e.g., the bilayer hubbard model [14, 15 ], coupled two - leg spin ladders, tight binding extended hubbard model [17, 18 ], bilayer t - j model. from these calculations, the following conclusions can be drawn. the pdh structure can be explained by a coupling of the electronic excitations to magnetic resonances or spin fluctuations [20, 21 ]. at low doping, the coupling between the layers should be antiferromagnetic, and there might be contributions to superconductivity by interlayer cooper pairs, being formed by holes belonging to different layers. the reduction of the bilayer splitting with respect to the noninteracting tight binding model is attributed to the formation of spin bags in the layers, which increases the quasiparticle weight or / and antiferromagnetic interlayer order. in this paper, we address these issues by an alternative approach in which correlations are evaluated exactly at a short - range level of a cluster, and thus is expected to capture the interplay between short - range antiferromagnetic coupling and quasiparticle excitations. specifically, we use the variational cluster approach (vca) [22, 23 ] to solve the bilayer hubbard model. vca is an extension of cluster perturbation theory (cpt) [24, 25 ]. due to its variational nature, it allows for a treatment of symmetry breaking phases, in our case antiferromagnetism and/or superconductivity. the method has already been successfully been applied to a wide range of problems [23, 2629 ] and is based on the self - energy functional theory (sft) [30, 31 ]. we will illustrate the effects of bilayer splitting by displaying the spectral functions for the two bands. finally, we will discuss the reduction of the splitting due to correlation in both the normal as well as in the superconducting state. a single cuo2 layer on the x y plane is commonly described by the two - dimensional hubbard hamiltonian 1 in standard notation. as usual, we include a next - nearest hopping in order to reproduce the band structure observed in arpes experiments. as it is well known, for example, from lda calculations, the interlayer hopping displays a characteristic k structure.1 downfolding an 8-band hamiltonian for the bilayer compound yba2cu3o7 (ybco) gives a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vec{k}$\end{document}-dependent interlayer hopping, originating mainly from copper s and oxygen d - orbitals in the form : 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_\perp(k) \approx\tilde{t } \frac{v^2}{(1 - 2ru)^2}$$\end{document } with 3 and u another form factor, which we do not need to specify. a is the lattice constant on the layer, which for simplicity we take to be equal in the x and y direction. however, since rt/t=0.31, this term can be neglected and one obtains 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_\perp(k) \approx\frac{\tilde{t}}{4 } (\cos k_y-\cos k_x)^2. $ $ \end{document } in our approach, we need the hopping term in real space. fourier transformation yields three types of interlayer hopping terms, a vertical one t(x=0,\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta y=0)=\tilde{t}/4$\end{document }, a diagonal hopping (x=1, y1), and one along the x or y axis (x=0,2,y=0,2). the method used for approximating the ground - state properties of the system is vca. in a first step, the lattice is split up into identical clusters, which constitute the so - called reference system. the model then solved exactly on each cluster, and the single particle green s function g(z) of a cluster is calculated numerically, in our case by lanczos exact diagonalization. the disconnected clusters are then coupled within strong - coupling perturbation theory at leading order in the hoppings, yielding an approximation for the green s function of the whole lattice in the form : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g^{cpt}(z)=\bigl(g^{cl}(z)^{-1}-t\bigr)^{-1},$$\end{document } where t is a matrix describing intercluster hoppings (see, e.g., [23, 29 ] for details). a variational principle based on the self - energy functional approach has been formulated by potthoff. by introducing additional variational fields and optimizing the grand potential with respect to these fields, one can study broken - symmetry phases, such as magnetism or superconductivity [23, 26, 27 ]. details of vca can be found, e.g., in [26, 29 ]. in the present paper, we introduce the following variational fields, which within vca are just used for the determination of the self - energy and then subtracted perturbatively : staggered magnetic field 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_m = h_m\sum_{i\sigma}(-1)^\sigma e^{i\vec{q } \vec{r } } c_{i\sigma}^+c_{i\sigma},$$\end{document } with q=(,).superconducting field 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{sc}=h_{sc}\sum_{i, j}\frac{\eta_{i, j}}{2}(c_{i\uparrow}c_{j\downarrow } + c_{j\uparrow}c_{i\downarrow}),$$\end{document } where is the form factor which determines the symmetry of the superconducting order parameter, in our case d-wave.on-site energy 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{n}=\epsilon\sum_{i\sigma } n_{i\sigma}$$\end{document } which is needed for thermodynamic consistency. staggered magnetic field 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_m = h_m\sum_{i\sigma}(-1)^\sigma e^{i\vec{q } \vec{r } } c_{i\sigma}^+c_{i\sigma},$$\end{document } with q=(,). superconducting field 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{sc}=h_{sc}\sum_{i, j}\frac{\eta_{i, j}}{2}(c_{i\uparrow}c_{j\downarrow } + c_{j\uparrow}c_{i\downarrow}),$$\end{document } where is the form factor which determines the symmetry of the superconducting order parameter, in our case d - wave. on - site energy 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{n}=\epsilon\sum_{i\sigma } n_{i\sigma}$$\end{document } which is needed for thermodynamic consistency. the nearest - neighbor hopping t=1 sets the energy scale, and we take typical values u=8 and t=0.3 t (see, e.g.,). the interlayer hopping is chosen to be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde{t}\approx0.2$\end{document } close to the value estimated for bsco-2212 in. the spectral function a(k,) at half filling is plotted in fig. 1 along the path [(0,0),(0,),(,),(0,0) ] in the two - dimensional brillouin zone. the spectrum shows the asymmetric behavior of electron and hole filling induced by t : electrons are expected to first enter the brillouin zone around (,0), while holes first enter at (/2,/2). the interlayer hopping introduces a splitting between the kz=0 and kz= spectra, which for simplicity of language we will refer to as the bonding and antibonding bands. without correlations, we would expect the splitting of the bands to be given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$2t_{\perp}(k)\frac{(\cos(k_{x } a)-\cos(k_{y } a))^{2}}{2}$\end{document}. looking at the brillouin zone this means that along the diagonal kx = ky the two fermi points for the bonding and antibonding bands are exactly one over the other. when going away from this diagonal, the splitting grows until reaching a maximum near the (0,) and (,0) points. in fig. 2, we plot the spectral function of the bonding and antibonding bands at (0,), which clearly shows the interlayer splitting. the splitting is approximately u=0.32 t, which is reduced with respect to the value 0=0.4 t in the noninteracting case. 1spectral function a(k,) as a gray plot for the half filled bilayer hubbard model. results are shown for the bonding (k z=0) and antibonding (k z=) bandfig. 2spectral function at the (0,) point (maximum bilayer splitting) for the bonding (solid line) and antibonding (dashed) bands spectral function a(k,) as a gray plot for the half filled bilayer hubbard model. results are shown for the bonding (k z=0) and antibonding (k z=) band spectral function at the (0,) point (maximum bilayer splitting) for the bonding (solid line) and antibonding (dashed) bands at optimal doping, no bilayer splitting could be resolved in arpes measurements of bsco-2212. in order to analyze this effect, the spectral functions for the bonding and antibonding bands at (,0) in the superconducting case are displayed in fig. our calculations indeed suggest that the antibonding and bonding spectrum lie almost exactly over each other. 3spectral function at the (0,) point for the optimally doped bilayer system (a) for the bonding (solid line) and antibonding (dashed) bands. (b) shows a comparison of the bilayer (dashed) with the monolayer (solid line) spectra spectral function at the (0,) point for the optimally doped bilayer system (a) for the bonding (solid line) and antibonding (dashed) bands. (b) shows a comparison of the bilayer (dashed) with the monolayer (solid line) spectra moreover, it was found that the shape of the quasiparticle peak in the (,0) region of the optimally doped monolayer (bsco-2201) and bilayer material (bsco-2212) are similar. this is also very well reproduced in our data, as can be seen in fig. bilayer splitting has been measured by arpes in several works (see, e.g., [912 ]). in heavily overdoped samples, the splitting is suppressed much more in the superconducting case than in the normal state, contrary to the naive expectation that a global phase coherence below tc will enhance the c - axis coupling, and thus cause larger splitting. we checked these results by plotting the spectral function in the overdoped region2 of the bilayer hubbard model both in the normal and superconducting state. a(k,) as a gray plot in the overdoped (=0.43) region in the superconducting phasefig. 5spectral function a(k,) as a gray plot in the overdoped region in the normal phase spectral function a(k,) as a gray plot in the overdoped (=0.43) region in the superconducting phase spectral function a(k,) as a gray plot in the overdoped region in the normal phase in fig. 6, we focus on details of the energy splitting and plot its k - dependence in the overdoped region. our results suggest a reduction of the splitting at (0,) by about 30 % in the normal and by about 70 % in the superconducting phase with respect to the tight - binding model. moreover, in the superconducting phase, also the shape of the k dependence is modified. this larger suppression in the superconducting phase is in qualitative agreement with experiments. in order to disentangle the effects of correlation from the ones due to the superconducting gap, we also display results obtained for u=0 by introducing by hand a superconducting symmetry breaking field equal to the one obtained variationally at u=8. as one can see from the figure, the superconducting gap only produces a small (about 10 %) reduction, which is uniform in k. the anomalous behavior of fig. 6energy bilayer splitting along the line connecting (0,) and (/2,/2) in the normal and superconducting state in the overdoped region (crosses and lines with errorbars). results are compared to the splitting for u=0 (solid line). in the superconducting phase away from the antinodal region, the excitations for both k z bands develop a two peak structure similar to the peak dip hump that is seen in arpes. our results yield different results for the hump hump and peak peak splittings. for this reason, we display the second value using thin errorbars, shifted to the right for clarity. the errorbars represent the estimated error due to the uncertainty of the peak positions energy bilayer splitting along the line connecting (0,) and (/2,/2) in the normal and superconducting state in the overdoped region (crosses and lines with errorbars). results are compared to the splitting for u=0 (solid line). in the superconducting phase, we also display results obtained for u=0 by introducing by hand a superconducting symmetry breaking field (dashed line, empty squares). away from the antinodal region, the excitations for both k z bands develop a two peak structure similar to the peak dip hump that is seen in arpes. our results yield different results for the hump hump and peak peak splittings. for this reason, we display the second value using thin errorbars, shifted to the right for clarity. the errorbars represent the estimated error due to the uncertainty of the peak positions the values of the splitting for u=8 plotted in fig. 6 are obtained in the following way : in the normal state, there is just one prominent dispersing peak for each kz defining a bonding and antibonding band. the k dependent splitting is defined as the distance between the maxima of these peaks for kz=0,. for the superconducting state, we determine the splitting for the quasiparticle states below the fermi level. we have checked that it very close to the splitting of the mirror states above it. when going away from the antinodal point both in the normal state as well as in the superconducting state, each quasiparticle peak first broadens, which introduces an error in the determination of, and then evolves into a two peak structure, which resembles the peak - dip - hump structure that is observed in arpes. measuring the distance between the second pair of peaks gives a second set of data points, which is also displayed in fig. we have studied the bilayer hubbard model by means of the variational cluster approach, a method appropriate to capture short range correlation in strongly interacting lattice systems. as expected, the interlayer hopping splits the spectrum into a bonding and an antibonding band. this is evident in the overdoped case in both the normal and superconducting phases. in qualitative agreement with arpes measurements, surprisingly, for optimal doping, the bilayer splitting vanishes completely, as found in arpes.	we carry out a theoretical study of the bilayer single - band hubbard model in the undoped and in the superconducting phases by means of the variational cluster approach. in particular, we focus on the splitting between the bonding and antibonding bands induced by the interlayer hopping, as well as its interplay with strong correlation effects. we find that the splitting is considerably suppressed in both the normal and superconducting phases, in qualitative agreement with experiments on bi2sr2cacu2o8+. in addition, in the superconducting phase, the shape of the splitting in k space is modified by correlations.
colorectal cancer is a major health problem worldwide. in the united states, it is the third most common cancer diagnosis and the second leading cause of cancer death. fortunately, this neoplasm is highly suited to screening because of its long preclinical phase, during which it is detectable and curable. prospective randomized trials and observational studies have demonstrated mortality reductions associated with early detection of invasive cancer and removal of adenomatous polyps. based on this evidence, a number of organizations and task forces throughout the world have issued or endorsed guidelines for colorectal cancer screening beginning at age 50 years for individuals at average risk for colorectal cancer. nevertheless, screening programs for colorectal cancer have been only partly successful, largely as a result of poor patient compliance with screening recommendations,. currently only approximately 50% of adults in the united states older than 50 years are receiving any of the recommended colorectal cancer screening tests. the test options for colorectal cancer screening recommended in the most recently published guidelines of the us preventive services task force (uspstf) include annual fecal occult blood test (fobt), flexible sigmoidoscopy every 5 years, and colonoscopy every 10 years. in 2008 the american cancer society, the us multi - society task force on colorectal cancer, and the american college of radiology issued a joint guideline on screening and surveillance for the early detection of colorectal cancer and adenomatous polyps. in this joint guideline double - contrast barium enema (dcbe) every 5 years, computed tomography colonography (ctc) every 5 years, annual fecal immunochemical test (fit), and stool dna test (interval uncertain) were included as screening options in addition to the tests recommended by the uspstf. colorectal cancer screening guidelines issued by the american college of gastroenterology in 2009 included ctc (every 5 years) as an alternative screening test option for persons unwilling to undergo colonoscopy. although two early multi - institutional clinical trials comparing ct colonography with optical colonoscopy showed poor results,, other trials in the united states and europe using state of the art equipment, fluid and fecal tagging, and well - trained readers have demonstrated per patient detection rates of 90% for adenomas 10 mm. a recent study involving 307 subjects compared ctc, colonoscopy, flexible sigmoidoscopy, fobt and fit for the detection of advanced neoplasia in an average risk population. advanced neoplasia is defined as a lesion having one or more of the following characteristics : size 1 cm, high grade dysplasia, > 25% villous histology, or invasive carcinoma. the sensitivities of flexible sigmoidoscopy, fit and fobt (tests that are uniformly recommended as options for colorectal cancer screening and are covered by medicare and other insurers) were 83.3%, 32%, and 20%, respectively. another recent study compared the diagnostic yield from parallel ctc and colonoscopy screening programs which included more than 3000 patients in each arm. ctc and colonoscopy had similar detection rates for advanced neoplasia, but the numbers of polypectomies and complications were considerably smaller in the ctc group. although several insurers provide coverage for colorectal cancer screening with ctc, the vast majority of insurers provide coverage only for diagnostic ctc when colonoscopy is either unsuccessful or contraindicated. despite the recommendation of at least two expert panels to include ctc as a colorectal cancer screening option,, the centers for medicare and medicaid services (cms) in the united states recently decided to deny coverage of ctc for colorectal cancer screening. reasons for the denial that were cited in the cms decision memo were : ctc can not reliably detect polyps < 6 mm. response : although the ability of ctc to demonstrate polyps < 6 mm is limited, the clinical significance of detecting 5 mm and smaller polyps is questionable. fifty percent of colonic polyps 5 mm are non - neoplastic and less than 2% of all polyps 5 mm have advanced histology,. a decision analysis of the relative yield of referring patients with polyps 5 mm to colonoscopic polypectomy demonstrated that 562 such polyps would have to be removed to avoid leaving behind one advanced adenoma. thus, colonoscopy referral for polyps 5 mm likely would do more harm than good, as it would prove to be very costly and would introduce many unnecessary complications.a substantial percentage of patients undergoing ctc may need to be referred to colonoscopy due to identification of polyps 6 mm. response : based on data from the acrin trial, the largest ctc screening trial published to date, if a 6 mm threshold is used to refer patients for colonoscopy, the colonoscopy referral rate after ctc would be 12%. the parallel ctc / colonoscopy screening trial of kim. demonstrated a very similar colonoscopy referral rate of 12.9% based on a threshold of 6 mm.because extracolonic findings are common, the potential impact of extracolonic findings on health outcomes and costs needs to be determined. response : clinically significant extracolonic findings requiring either additional evaluation or urgent care are detected in 4.516% of patients undergoing ctc,. in the majority of cases, the additional diagnostic testing confirms benign findings, but relevant new diagnoses are made in 23% of cases. the mean additional cost per patient for non - surgical procedures is us$2434,29 and for surgical procedures us$6570. thus, extracolonic findings should be handled judiciously to balance the cost of additional evaluation against the early detection of important disease.the radiation exposure from ctc for colorectal cancer screening is a potential concern. response : radiation dose is an important consideration in assessing the risks and benefits of ctc for colorectal cancer screening. the effective radiation dose for ctc in recent clinical trials has been 4.56.0 msv,, which is one half or less that for a standard diagnostic ct of the abdomen and pelvis. several studies have demonstrated that good quality ctc can be performed with even further reductions in radiation dose, with effective mas as low as 10. continuing improvements in ct technology should enhance our ability to provide high quality ctc with very low radiation exposure to the patient. nevertheless, the long - term risk of radiation exposure to individuals undergoing repeated ctc examinations is a factor that must be considered, and all efforts must be made to limit radiation dose as much as possible.no published study has evaluated survival following participation in colorectal cancer screening with ctc. response : it is true that no studies have assessed the effect of ctc screening on mortality from colorectal cancer ; however, at least one study has demonstrated that ctc has a higher sensitivity for detecting advanced neoplasia than flexible sigmoidoscopy and fobt, tests which have been shown to reduce mortality from colorectal cancer.the currently available data from ctc trials is not generalizable to the medicare population (age 65 years and older). response : this is a valid comment, as the mean age of individuals in most ctc clinical trials to date have been in the range of 5758 years. efforts are underway to extract the data on individuals 65 years and older from previously published studies. ctc can not reliably detect polyps < 6 mm. response : although the ability of ctc to demonstrate polyps < 6 mm is limited, the clinical significance of detecting 5 mm and smaller polyps is questionable. fifty percent of colonic polyps 5 mm are non - neoplastic and less than 2% of all polyps 5 mm have advanced histology,. a decision analysis of the relative yield of referring patients with polyps 5 mm to colonoscopic polypectomy demonstrated that 562 such polyps would have to be removed to avoid leaving behind one advanced adenoma. thus, colonoscopy referral for polyps 5 mm likely would do more harm than good, as it would prove to be very costly and would introduce many unnecessary complications. a substantial percentage of patients undergoing ctc may need to be referred to colonoscopy due to identification of polyps 6 mm. response : based on data from the acrin trial, the largest ctc screening trial published to date, if a 6 mm threshold is used to refer patients for colonoscopy, the colonoscopy referral rate after ctc would be 12%. the parallel ctc / colonoscopy screening trial of kim. demonstrated a very similar colonoscopy referral rate of 12.9% based on a threshold of 6 mm. because extracolonic findings are common, the potential impact of extracolonic findings on health outcomes and costs needs to be determined. response : clinically significant extracolonic findings requiring either additional evaluation or urgent care are detected in 4.516% of patients undergoing ctc,. in the majority of cases, the additional diagnostic testing confirms benign findings, but relevant new diagnoses are made in 23% of cases. the mean additional cost per patient for non - surgical procedures is us$2434,29 and for surgical procedures us$6570. thus, extracolonic findings should be handled judiciously to balance the cost of additional evaluation against the early detection of important disease. response : radiation dose is an important consideration in assessing the risks and benefits of ctc for colorectal cancer screening. the effective radiation dose for ctc in recent clinical trials has been 4.56.0 msv,, which is one half or less that for a standard diagnostic ct of the abdomen and pelvis. several studies have demonstrated that good quality ctc can be performed with even further reductions in radiation dose, with effective mas as low as 10. continuing improvements in ct technology should enhance our ability to provide high quality ctc with very low radiation exposure to the patient. nevertheless, the long - term risk of radiation exposure to individuals undergoing repeated ctc examinations is a factor that must be considered, and all efforts must be made to limit radiation dose as much as possible. response : it is true that no studies have assessed the effect of ctc screening on mortality from colorectal cancer ; however, at least one study has demonstrated that ctc has a higher sensitivity for detecting advanced neoplasia than flexible sigmoidoscopy and fobt, tests which have been shown to reduce mortality from colorectal cancer. the currently available data from ctc trials is not generalizable to the medicare population (age 65 years and older). response : this is a valid comment, as the mean age of individuals in most ctc clinical trials to date have been in the range of 5758 years. efforts are underway to extract the data on individuals 65 years and older from previously published studies. colonoscopy perforation rates in the general population are approximately 1/1000 colonoscopies and are as high as 1/500 in those 65 years and older. the risk of perforation with screening ctc in asymptomatic persons is very low, with no perforations reported in 2 large studies, and only one perforation reported in a screening patient in another large study. perforations occur more commonly in symptomatic patients undergoing diagnostic ctc ; however, even in this patient population the perforation rate of ctc is lower than that of colonoscopy. furthermore, up to half of the adverse events that occur after colonoscopy are cardiovascular complications resulting from sedation. if polyps 5 mm are not reported, ctc is the most cost - effective and safest screening option for colorectal cancer. because it is a much less invasive test than colonoscopy and does not require sedation, ctc also has the potential to increase overall compliance with colorectal cancer screening guidelines. in summary, recent trials have demonstrated that ctc has sensitivity comparable to colonoscopy for detecting clinically significant adenomas. ctc is also safer and more cost - effective than colonoscopy when polyps 5 mm detected at ctc are not reported. because it is less invasive than colonoscopy and does not require sedation, it has the potential to increase compliance with colorectal cancer screening guidelines, and thus prevent many colorectal cancers from developing. at least two expert panels recently have endorsed ctc as an option for colorectal cancer screening. with the foregoing in mind, it is time to answer the question is ctc for colorectal cancer screening ready for prime time ? there is no doubt in my mind that ctc is capable of serving as a sensitive, safe, cost - effective and patient - friendly test for widespread colorectal cancer screening ; however, it is incumbent upon radiologists to ensure that those who perform and interpret ctc examinations are properly trained to provide the highest quality of patient care. before ctc screening can be applied in a widespread fashion, large numbers of radiologists (and likely non - radiologist physicians and a variety of physician extenders) need to be trained and possibly certified, a process now beginning to take place in many parts of the world. another obstacle that needs to be overcome before screening ctc can truly become widespread, is the lack of universal insurance coverage for the procedure. until universal coverage for screening ctc is available, only individuals wealthy enough to pay for the procedure themselves will be able to avail themselves of this screening option, thereby severely limiting its considerable potential.	abstractevery year more than one million new patients are diagnosed with colon cancer worldwide. although multiple prospective randomized trials and observational studies have demonstrated that mortality from colon cancer can be reduced with screening and removal of adenomatous polyps, compliance with screening guidelines remains low. recent ct colonography (ctc) trials have shown that ctc is capable of demonstrating adenomatous polyps 10 mm (and in most cases 6 mm) with sensitivities comparable to those for optical colonoscopy. based on these results, at least two expert panels have recommended ctc as an option for colorectal cancer screening. despite these endorsements, the centers for medicare and medicaid services (cms) in the united states recently decided to deny coverage of ctc for colorectal cancer screening. this article addresses the reservations raised by cms and provides a perspective on whether ctc is ready for routine use as a colorectal cancer screening test.
renal colic is a common disease in europe and a common cause of visit to the emergency department. unenhanced computed tomography (ct) is considered the best diagnostic test due to its excellent accuracy detecting ureteral stones. it is a reproducible, non - invasive and non - expensive imaging technique, achieving accurate diagnosis in most cases without the need for radiation. diagnosis is based on the presence of ureteral stones, but indirect findings such as the asymmetry or absence of ureteric jet, an increase of the resistive index or a colour doppler twinkling artefact may help to suggest the diagnosis when the stone is not identified. us should be used as the first imaging modality in patients with renal colic. renal stone disease is common in europe, with a prevalence ranging between 2 and 8 %. it is a condition affecting relatively young individuals with an almost equal sex ratio and high tendency to recur : it is estimated that almost 50 % of stone patients will present recurrence within 10 years. they are a common cause of visits to the emergency department and frequently require imaging evaluation [1, 2 ]. in most institutions non - enhanced multidetector computed tomography (mdct) is considered the gold standard technique to evaluate these patients because of its accuracy in the detection of stones as well as of other pathological conditions mimicking renal colic. it is also considered as the first imaging technique for the evaluation of patients with acute onset of flank pain by the american college of radiology appropriateness criteria. mdct also allows an overall assessment of the stone load, which can help to plan the treatment. however, the vast majority of stones pass spontaneously, and ct imaging in the emergency department rarely alters immediate management. moreover, concerns about the over - utilisation of ct are growing because of increasing health care costs and, more importantly, exposure to ionising radiation [6, 7 ]. the use of low - dose techniques can dramatically reduce the radiation dose, but these low - dose protocols have not been adopted worlwide. on the other hand, ultrasound (us) is a safe, non - invasive and non - expensive technique able to evaluate patients with renal colic. however, its use remains controversial as it has good capability to identify dilatation of the excretory system even in non - experienced hands, but can have difficulties in directly demonstrating the stones, especially in the mid ureters, remaining operator dependent to detect stones and indirect findings that can help in the diagnosis. in addition, the absence of the indirect findings does not exclude ureteral stones. in spite of these difficulties several papers, including a very recent multicentre comparative study between us and ct, have demonstrated the usefulness of us in the diagnosis and management of renal colic patients [1113 ]. the diagnosis of renal colic is usually based on clinical grounds and immediate imaging is not always necessary [5, 14 ]. however, it is now common practice to perform imaging studies in all patients with suspected renal colic admitted to the emergency department. this may be due to fear of missing a life - threatening condition mimicking this condition, such as rupture of an aortic aneurysm, ovarian torsion or appendicitis, or to the need for imaging confirmation of the cause of symptoms before deciding on whether a patient may be discharged. at present, additional strong indications for imaging are the desire of patients to know the cause of their symptoms and the fear of litigation. if not in all patients, immediate imaging modalities are necessary in patients without clinical improvement after treatment, in cases with fever or leukocytosis, or in some special circumstances (i.e., patients with a single kidney and/or renal failure) [14, 15 ] ; furthermore, imaging is also recommended in patients with remission of symptoms who do not eliminate the stone within a few days. ct has become the imaging study of choice for renal colic because of its high sensitivity in the detection of renal and ureteral stones. moreover, when ct is performed with dual energy, it helps to characterise the composition of the renal stones. however, most hospitals do not have this technology, which in addition has very limited usefulness in case of ureteral stones. ct can identify the presence and size of stones with a very high accuracy of > 95 % and is able to detect alternative diagnoses that simulate renal colic in 510 % of patients [16, 17 ]. nonetheless, in spite of its high accuracy, there is increasing concern about the increase of health care costs and radiation risk that accompanies ct scans, since the use of ct rarely changes the treatment plans of these patients. in this way, in a recent retrospective study, westphalen. determined the proportion of patient visits for flank or kidney pain receiving ct or us and calculated the diagnosis and hospitalisation rates for urolithiasis. from 1996 to 2007, the use of ct to assess patients with suspected urolithiasis increased from 4.0 to 42.5 % over the study period, and the use of us remained low, at about 5 %. however, the diagnosis of kidney stones, identification of significant alternate diagnoses or admission to the hospital did not increase. the problem of exposure to radiation is very important in these patients, especially because of the possibility of cumulative radiation that is not usually well assessed when multiple cts are performed in repetitive episodes of renal colic. thus, the use of dedicated low - dose protocols is essential in these patients. in spite of the advances with dedicated low - dose ct protocols, a recent study evaluated renal colic ct studies conducted in 93 institutions in the usa from may 2011 to january 2013 and demonstrated that reduced - dose renal ct protocols are used infrequently. reduced dose of 3 msv, and only 10 % of institutions used an effective dose of 6 msv or less in at least 50 % of patients. in another study performed in a single institution, the mean effective doses for a single study were 6.5 msv for sdct and 8.5 msv for mdct moreover, 4 % of these patients (all with a known history of nephrolithiasis) underwent three or more studies, with estimated effective doses ranging from 19.5 to 153.7 msv. ultrasound is an accurate imaging technique to diagnose renal colic [1113, 20, 21 ]. moreover, this technique also allows the diagnosis of other renal diseases or extrarenal conditions that mimic renal colic (table 1). the diagnosis of renal colic is based on the detection of stones and the consecutive obstruction of the excretory system (fig. 1). although the detection of dilatation of the excretory tract is very useful in the context of renal colic, this sign should be evaluated carefully, as dilatation does not necessarily mean obstruction, and the degree of dilatation does not reflect the severity of obstruction.table 1alternative diagnoses in patients with renal colicentitiesmost common us findingspyelonephritismild disease may demonstrates no abnormalityrenal enlargementintra- or extrarenal fluid collections or abscesses may be presentrenal massrenal tumour (detection depends on tumour size)spontaneous subcapsular or perinephric bleeds may cause flank painadnexal pathology : hemorrhagic ovarian cystsheterogeneous cyst pelvic inflammatory diseasethickened, dilated fallopian tube. abscesses endometriomascyst with diffuse homogenous low - level internal echoes ovarian torsionenlarged hypo or hyperechoic ovary with little or no intra - ovarian venous flow. in some cases ovarian neoplasmsovarian massesappendicitisnoncompressible appendix with diameter > 6 mmdiverticulitisdetection of diverticulumsigns of inflammation of fat (dirty fat / stranding)thickened bowel wall > 45 mmpericolic fluid or collectionsdissection / ruptured aneurysmsthin membrane fluttering in the aortic lumendilatation of the aorta > 3 cm, periaortic fluid collectionfig. 1left proximal ureteral stone (arrow) producing hydronephrosisfeatures of stones in grey - scale and limitations.stones are identified as hyperechogenic foci with posterior shadowing. the most important limitations of us are the detection of small lithiases (0.70 and/or a 10 % difference between the kidneys is considered as diagnostic of obstructive uropathy.colour doppler twinkling artefact [15, 26, 27 ]. this artefact is a mixture of red and blue pixels on colour doppler secondary to the noise produced from rough interfaces composed of sparse reflectors such as urinary stones. it is very useful to confirm findings of grey - scale, especially in doubtful cases due to small size of the stone or located in difficult - to - visualise ureteral portions. in the study by moore., the sensitivity of us improved from 47.6 to 86 % when the twinkling sign was used. in the recent study by ripolles., which analysed the specific value of the twinkling artefact, the sensitivity of us using the twinkling artefact for detecting lithiasis was 90 % and the specificity 100 %. a total of 78 % of the lithiases showed the twinkling artefact, including three stones not identified by b - mode us, and in 68 % of these stones, the artefact was detected before the stone itself with b - mode. alternative diagnoses in patients with renal colic left proximal ureteral stone (arrow) producing hydronephrosis features of stones in grey - scale and limitations. the most important limitations of us are the detection of small lithiases (0.70 and/or a 10 % difference between the kidneys is considered as diagnostic of obstructive uropathy.colour doppler twinkling artefact [15, 26, 27 ]. this artefact is a mixture of red and blue pixels on colour doppler secondary to the noise produced from rough interfaces composed of sparse reflectors such as urinary stones. it is very useful to confirm findings of grey - scale, especially in doubtful cases due to small size of the stone or located in difficult - to - visualise ureteral portions. in the study by moore., the sensitivity of us improved from 47.6 to 86 % when the twinkling sign was used., which analysed the specific value of the twinkling artefact, the sensitivity of us using the twinkling artefact for detecting lithiasis was 90 % and the specificity 100 %. a total of 78 % of the lithiases showed the twinkling artefact, including three stones not identified by b - mode us, and in 68 % of these stones, the artefact was detected before the stone itself with b - mode. an absent, asymmetric and/or reduced ureteric jet from the ureteric orifices evaluated by colour doppler is an additional indicator of obstruction. however, the presence of a positive ureteral jet does not rule out the presence of ureteral stones since ureteral stones quite often only cause partial obstruction. increased resistive index as a sign of acute obstruction, distinguishing between obstructive and non - obstructive dilatation. a renal ri > 0.70 and/or a 10 % difference between the kidneys is considered as diagnostic of obstructive uropathy. this artefact is a mixture of red and blue pixels on colour doppler secondary to the noise produced from rough interfaces composed of sparse reflectors such as urinary stones. it is very useful to confirm findings of grey - scale, especially in doubtful cases due to small size of the stone or located in difficult - to - visualise ureteral portions. in the study by moore., the sensitivity of us improved from 47.6 to 86 % when the twinkling sign was used. in the recent study by ripolles., which analysed the specific value of the twinkling artefact, the sensitivity of us using the twinkling artefact for detecting lithiasis was 90 % and the specificity 100 %. a total of 78 % of the lithiases showed the twinkling artefact, including three stones not identified by b - mode us, and in 68 % of these stones, the artefact was detected before the stone itself with b - mode. ct has become the imaging study of choice for renal colic because of its high sensitivity in the detection of renal and ureteral stones. moreover, when ct is performed with dual energy, it helps to characterise the composition of the renal stones. however, most hospitals do not have this technology, which in addition has very limited usefulness in case of ureteral stones. ct can identify the presence and size of stones with a very high accuracy of > 95 % and is able to detect alternative diagnoses that simulate renal colic in 510 % of patients [16, 17 ]. nonetheless, in spite of its high accuracy, there is increasing concern about the increase of health care costs and radiation risk that accompanies ct scans, since the use of ct rarely changes the treatment plans of these patients. in this way, in a recent retrospective study, westphalen. determined the proportion of patient visits for flank or kidney pain receiving ct or us and calculated the diagnosis and hospitalisation rates for urolithiasis. from 1996 to 2007, the use of ct to assess patients with suspected urolithiasis increased from 4.0 to 42.5 % over the study period, and the use of us remained low, at about 5 %. however, the diagnosis of kidney stones, identification of significant alternate diagnoses or admission to the hospital did not increase. the problem of exposure to radiation is very important in these patients, especially because of the possibility of cumulative radiation that is not usually well assessed when multiple cts are performed in repetitive episodes of renal colic. thus, the use of dedicated low - dose protocols is essential in these patients. in spite of the advances with dedicated low - dose ct protocols, a recent study evaluated renal colic ct studies conducted in 93 institutions in the usa from may 2011 to january 2013 and demonstrated that reduced - dose renal ct protocols are used infrequently.. only 2 % of the studies were conducted with a reduced dose of 3 msv, and only 10 % of institutions used an effective dose of 6 msv or less in at least 50 % of patients. in another study performed in a single institution, the mean effective doses for a single study were 6.5 msv for sdct and 8.5 msv for mdct. moreover, 4 % of these patients (all with a known history of nephrolithiasis) underwent three or more studies, with estimated effective doses ranging from 19.5 to 153.7 msv. ultrasound is an accurate imaging technique to diagnose renal colic [1113, 20, 21 ]. moreover, this technique also allows the diagnosis of other renal diseases or extrarenal conditions that mimic renal colic (table 1). the diagnosis of renal colic is based on the detection of stones and the consecutive obstruction of the excretory system (fig. 1). although the detection of dilatation of the excretory tract is very useful in the context of renal colic, this sign should be evaluated carefully, as dilatation does not necessarily mean obstruction, and the degree of dilatation does not reflect the severity of obstruction.table 1alternative diagnoses in patients with renal colicentitiesmost common us findingspyelonephritismild disease may demonstrates no abnormalityrenal enlargementintra- or extrarenal fluid collections or abscesses may be presentrenal massrenal tumour (detection depends on tumour size)spontaneous subcapsular or perinephric bleeds may cause flank painadnexal pathology : hemorrhagic ovarian cystsheterogeneous cyst pelvic inflammatory diseasethickened, dilated fallopian tube. abscesses endometriomascyst with diffuse homogenous low - level internal echoes ovarian torsionenlarged hypo or hyperechoic ovary with little or no intra - ovarian venous flow. in some cases twisted vascular pedicle ovarian neoplasmsovarian massesappendicitisnoncompressible appendix with diameter > 6 mmdiverticulitisdetection of diverticulumsigns of inflammation of fat (dirty fat / stranding)thickened bowel wall > 45 mmpericolic fluid or collectionsdissection / ruptured aneurysmsthin membrane fluttering in the aortic lumendilatation of the aorta > 3 cm, periaortic fluid collectionfig. 1left proximal ureteral stone (arrow) producing hydronephrosisfeatures of stones in grey - scale and limitations.stones are identified as hyperechogenic foci with posterior shadowing. the most important limitations of us are the detection of small lithiases (0.70 and/or a 10 % difference between the kidneys is considered as diagnostic of obstructive uropathy.colour doppler twinkling artefact [15, 26, 27 ]. this artefact is a mixture of red and blue pixels on colour doppler secondary to the noise produced from rough interfaces composed of sparse reflectors such as urinary stones. it is very useful to confirm findings of grey - scale, especially in doubtful cases due to small size of the stone or located in difficult - to - visualise ureteral portions. in the study by moore., the sensitivity of us improved from 47.6 to 86 % when the twinkling sign was used. in the recent study by ripolles., which analysed the specific value of the twinkling artefact, the sensitivity of us using the twinkling artefact for detecting lithiasis was 90 % and the specificity 100 %. a total of 78 % of the lithiases showed the twinkling artefact, including three stones not identified by b - mode us, and in 68 % of these stones, the artefact was detected before the stone itself with b - mode. alternative diagnoses in patients with renal colic left proximal ureteral stone (arrow) producing hydronephrosis features of stones in grey - scale and limitations.. the most important limitations of us are the detection of small lithiases (0.70 and/or a 10 % difference between the kidneys is considered as diagnostic of obstructive uropathy.colour doppler twinkling artefact [15, 26, 27 ]. this artefact is a mixture of red and blue pixels on colour doppler secondary to the noise produced from rough interfaces composed of sparse reflectors such as urinary stones. it is very useful to confirm findings of grey - scale, especially in doubtful cases due to small size of the stone or located in difficult - to - visualise ureteral portions. in the study by moore., the sensitivity of us improved from 47.6 to 86 % when the twinkling sign was used. in the recent study by ripolles., which analysed the specific value of the twinkling artefact, the sensitivity of us using the twinkling artefact for detecting lithiasis was 90 % and the specificity 100 %. a total of 78 % of the lithiases showed the twinkling artefact, including three stones not identified by b - mode us, and in 68 % of these stones, the artefact was detected before the stone itself with b - mode. an absent, asymmetric and/or reduced ureteric jet from the ureteric orifices evaluated by colour doppler is an additional indicator of obstruction. however, the presence of a positive ureteral jet does not rule out the presence of ureteral stones since ureteral stones quite often only cause partial obstruction. increased resistive index as a sign of acute obstruction, distinguishing between obstructive and non - obstructive dilatation. a renal ri > 0.70 and/or a 10 % difference between the kidneys is considered as diagnostic of obstructive uropathy. this artefact is a mixture of red and blue pixels on colour doppler secondary to the noise produced from rough interfaces composed of sparse reflectors such as urinary stones. it is very useful to confirm findings of grey - scale, especially in doubtful cases due to small size of the stone or located in difficult - to - visualise ureteral portions. in the study by moore., the sensitivity of us improved from 47.6 to 86 % when the twinkling sign was used. in the recent study by ripolles., which analysed the specific value of the twinkling artefact, the sensitivity of us using the twinkling artefact for detecting lithiasis was 90 % and the specificity 100 %. a total of 78 % of the lithiases showed the twinkling artefact, including three stones not identified by b - mode us, and in 68 % of these stones, the artefact was detected before the stone itself with b - mode. the possibility of obtaining a diagnosis using us in patients with suspected renal colic has several advantages including its widespread availability and reduced cost over the use of ct. most importantly, the use of an algorithm in which us is used first can avoid radiation exposure in about 70 % of cases [11, 20, 28 ]., it seems safe to use this diagnostic technique even if it is known to have a lower sensitivity than ct in this field [20, 28 ]. in fact, in both studies, spontaneous passage of the stone within a few days after the acute episode was observed in all patients with a false - negative us examination. in addition, a recent multicentre study clearly described the primary role of us in the investigation of renal colic today, demonstrating that the initial use of us is not associated with more complications, serious adverse events or hospitalisations than the initial evaluation with ct. it must also be remembered that in patients in whom symptoms are not due to a renal colic us also has the ability to identify alternative diagnoses, albeit with a slightly lower sensitivity than ct, and that most of the important, life - threatening situations that may mimic renal colic can be recognised. nonetheless, us also has a few disadvantages : it may take longer to perform than nonenhanced ct and must be performed by an experienced examiner. the first of these limitations can not be justified within the framework of the justification process of diagnostic studies using ionising radiation ; radiologists are strongly advised to use alternative, non - ionising techniques whenever possible. the second limitation may be difficult to overcome, especially if service is to be provided 24/24 h and 7/7 days. however, dalla palma. have shown that high sensitivity results can be obtained in well - hydrated patients by general radiologists on call who are not specifically dedicated to us. the performance of us studies by radiologists also has an impact on the need for additional imaging techniques as demonstrated in the multicentre study of smith - bindman, in which 40.7 % of the patients initially evaluated with us by emergency physicians, and only 27 % of the patients initially evaluated by radiologists underwent additional ct. rethinking the imaging strategies in patients with suspected renal colic taking into account radiation protection considerations is possible and it has started both within the radiological community [15, 30 ] and among emergency physicians [10, 13 ]. urologists also agree on this topic : in the 2014 guidelines on urolithiasis of the european association of urology it is stated that in patients with renal stone disease us should be used as the primary procedure, and ct should be reserved for those patients who do not improve with conservative treatment or on suspicion of a nonurologic process. ultrasound can achieve a high sensitivity and specificity for the depiction of ureteral calculi and acute obstruction and has several advantages including its availability, lower cost and absence of radiation. thus, it should be considered the first imaging modality in patients with renal colic. no more complications, serious adverse events, return emergency department visits or hospitalisations are expected using us first instead of ct. ct should be reserved for patients in whom us does not obtain a diagnosis, if symptoms do not resolve or there is a suspicion of alternative diagnoses.	abstractrenal colic is a common disease in europe and a common cause of visit to the emergency department. clinical diagnosis is usually confirmed by imaging modalities. unenhanced computed tomography (ct) is considered the best diagnostic test due to its excellent accuracy detecting ureteral stones. however, ultrasound (us) should be considered as the primary imaging technique. it is a reproducible, non - invasive and non - expensive imaging technique, achieving accurate diagnosis in most cases without the need for radiation. diagnosis is based on the presence of ureteral stones, but indirect findings such as the asymmetry or absence of ureteric jet, an increase of the resistive index or a colour doppler twinkling artefact may help to suggest the diagnosis when the stone is not identified.main messages renal colic diagnosis is usually confirmed by imaging modalities. imaging diagnosis of renal colic is based on the detection of ureteral stones. ct is the most accurate imaging technique to identify ureteral stones. us allows correct diagnosis in most cases without using radiation. us should be used as the first imaging modality in patients with renal colic.
	direct -alkylation of saturated aldehydes has been accomplished by synergistically combining photoredox catalysis and organocatalysis. photon - induced enamine oxidation provides an activated -enaminyl radical intermediate, which readily combines with a wide range of michael acceptors to produce -alkyl aldehydes in a highly efficient manner. furthermore, this redox - neutral, atom - economical c h functionalization protocol can be achieved both inter- and intramolecularly. mechanistic studies by various spectroscopic methods suggest that a reductive quenching pathway is operable.
the new understanding of obesity and its related disorders has resulted in a renewed interest in finding antiobesity agents from nature, with partial success. an established antiobesity agent, such as green tea polyphenols, is one of the few plants extracts reported to reduce weight in both animals and human subjects [24 ]. others include extracts of nomame herba, cocoa, and chitin / chitosan [57 ]. while these studies yielded significant information on the effect of those plants on diet induced obesity and its biochemical changes, the overall effect on metabolic responses is relatively unknown. this omics technique is concerned with the high throughput identification and quantification of small molecules (1, as metabolites contributing more to the clustering of the different groups (table 5). there are limited studies, which have used a metabolomics approach to identify metabolic changes following intervention with drugs and therapeutics, including the phytochemical strategies for obesity, though the potential is vast. however, there are few metabolomics based reports on weight loss as a result of weight loss intervention, including exercise and surgery. an energy - restricted diet for 8 weeks resulted in an improvement in glucose and lipid metabolism in overweight obese adults. saturated fatty acids such as palmitic acid and stearic acid were significantly decreased as well as branched amino acid, isoleucine. a lifestyle intervention in obese children, obeldicks, resulted in significant weight loss and abdominal obesity, modulated by the role of phosphatidylcholine metabolism. this particular study also highlights the large interindividual variation to lifestyle intervention and the possible need of a more individualised approach to lifestyle interventions. h nmr analysis also showed that while exercise can improve the metabolic disruptions associated with diet induced obesity, the effect can not be cancelled out and diet predicts obesity better with a stronger influence on metabolites ' profiles than exercise alone. one of the few studies reporting the response of natural therapeutic agents in obese subjects assessed the effect of sea buckhorn and bilberry on serum metabolites in overweight women. no significant changes were observed in individual metabolites, though improvements in serum lipids and lipoproteins were observed. the treatment of high fat diet induced hyperlipidemia with xue - fu - zhu - yu decoction was studied using a nmr based metabolomics approach. opls - da analysis revealed the beneficial effects of the decoction, mainly through decrease in ketone bodies production, enhancement of biosynthesis, and modulation of lipid metabolism. dietary intervention of black soybean peptides in overweight human showed an increase in betaine, benzoic acid, pyroglutamic acid, and pipecolic acid, among others. vip analysis showed l - proline, betaine, and lyso - pcs to be more correlated to the discrimination before and after treatment. treatment with mle 60 at 250 mg / kg body weight improved serum levels of lactate, alanine, pyruvate, creatinine, and -glucose, bringing their levels closer to the normal control whereas the level of 3-hydroxyisobutyrate, 3-hydroxybutyrate, and acetate remained unchanged. similar improvements were achieved in the groups receiving 30 mg / kg body weight of orlistat. the relative concentration of -glucose was found to be most reduced, consistent with the actual biochemical measurement done previously where the orlistat treated group had significantly lower plasma glucose (4.97 mmol / l) as compared to the lean group (6.02 mmol / l). this is consistent with the literature reporting that orlistat in a weight loss regimen can significantly improve glucose tolerance and slows down the progression of type 2 diabetes and impaired glucose tolerance in clinical cases of obesity [75, 76 ]. based on the relative quantification of certain metabolites (lactate, pyruvate, and glucose) in the treated groups, it is apparent that treatment with mle 60 improved perturbations in various metabolic pathways, predominantly in the glucose and tca cycle as reflected by positive modulations in lactate, pyruvate, and glucose levels. disruptions in the creatinine and amino acid metabolic pathways were also improved as indicated by a reduction of creatinine and alanine accumulation in the obese groups treated with mle 60. the levels of 3-hydroxyisobutyrate, a metabolite of the gut microbiome metabolism, were unchanged in the treated groups, suggesting that mle 60 did not impact on the obesity - induced disruptions in the gut microbiome. similarly, the levels of 2-hydroxybutyrate, a metabolite of amino acid metabolism, were also unchanged. obesity has been characterised by an elevated tca function in diet induced hepatic insulin and fatty liver as well as decreased brain glucose metabolism, predominantly through the tca cycle [77, 78 ]. treatment with mle 60 improved serum creatinine profiles as shown by h nmr measurement as compared to nonsignificance observed when blood creatinine level was measured. poor creatinine clearance is associated with weight gain and central obesity due to increased metabolic abnormalities as risk factors. an increase in creatine kinase and adenylate kinase 1 activity was observed in obese subjects, attributed to a compensatory effect of the downregulation of muscle mitochondrial function, associated with obesity. hence, antiobesity agent which can positively influence these pathways as well as other parameters such as adipocytes factors and weight loss shows promise for weight management. based on the reported health properties of m. citrifolia, including the antiobesity activities, the aim of this study was to further explore the effect of a leaf extract, mle 60, on obesity using a h nmr metabolomic approach. h nmr spectroscopy and multivariate data analysis revealed clear metabolic differences in the urine and serum samples of the hfd induced obese and lean rats. an opls - da method was chosen to project maximum separation between the groups and to identify discriminating biomarkers. all multivariate models including pls - da and opls - da were duly validated, using permutation tests, ry, qy, and p cv anova values. several metabolites were identified in both the serum and urine samples, which were the basis of difference among the groups. these metabolites were involved in the glucose metabolism and tca cycle (lactate, 2-oxoglutarate, citrate, succinate, pyruvate, and acetate), amino acid metabolism (alanine, 2-hydroxybutyrate), choline metabolism (betaine), creatinine metabolism (creatinine), and gut microbiome metabolism (hippurate, phenylacetylglycine, dimethylamine, and trigonelline). some key metabolites were identified and quantified showing a statistically (p < 0.05) significant improvement in this treated group (500 mg / kg). this study, therefore, confirms the metabolic alteration caused by hfd induced obesity in a rat model and the improvement in certain metabolic pathways, upon treatment with mle 60. it also provides additional information that h nmr metabolomics can be a good approach to study the development of disease and response to treatment in obese subjects.	the prevalence of obesity is increasing worldwide, with high fat diet (hfd) as one of the main contributing factors. obesity increases the predisposition to other diseases such as diabetes through various metabolic pathways. limited availability of antiobesity drugs and the popularity of complementary medicine have encouraged research in finding phytochemical strategies to this multifaceted disease. hfd induced obese sprague - dawley rats were treated with an extract of morinda citrifolia l. leaves (mle 60). after 9 weeks of treatment, positive effects were observed on adiposity, fecal fat content, plasma lipids, and insulin and leptin levels. the inducement of obesity and treatment with mle 60 on metabolic alterations were then further elucidated using a 1h nmr based metabolomics approach. discriminating metabolites involved were products of various metabolic pathways, including glucose metabolism and tca cycle (lactate, 2-oxoglutarate, citrate, succinate, pyruvate, and acetate), amino acid metabolism (alanine, 2-hydroxybutyrate), choline metabolism (betaine), creatinine metabolism (creatinine), and gut microbiome metabolism (hippurate, phenylacetylglycine, dimethylamine, and trigonelline). treatment with mle 60 resulted in significant improvement in the metabolic perturbations caused obesity as demonstrated by the proximity of the treated group to the normal group in the opls - da score plot and the change in trajectory movement of the diseased group towards the healthy group upon treatment.
psoriasis is an immunologically mediated chronic inflammatory and hyperproliferative skin disease affected by both genetic and environmental factors. the prevalence of psoriasis varied among populations with different genetic backgrounds and habitats, from 3% in northern europe and 2% in north america and the uk to 0.10.3% in american indians and east asia [1, 2 ]. psoriasis is proposed to be associated with other immune diseases, such as arthritis and crohn 's disease. initial causative research has identified strong association between psoriasis and the interleukin genes (il4, il10, il12b, il13, and il23r) in the northern european from us and uk [47 ]. furthermore, the snp rs56245420 in the il15 gene has been found to be associated with psoriasis in the chinese han population but not in any of the uk, german, or us caucasian populations investigated [810 ], since the minor allele frequency for this snp and others across il15 differs quite strikingly between the populations, suggesting heterogeneity in the genetic susceptibility to psoriasis. in this study, we aimed to determine if psoriasis is associated with six il genes that have been strongly associated with psoriasis in europeans but not well studied in chinese. the seven included snps are rs2243250 in the il4 gene, rs1800872 in the il10 gene, rs3212227 in the il12b gene, rs1800925 and rs20541 in the il13 gene, rs56245420 in the il15 gene, and rs11209026 in the il23r gene. a total of 200 psoriasis patients and 298 healthy controls were recruited in this study (see table 2). all participants did not suffer from any other diseases and belonged to han nationality in yunnan province, china. the study was performed according to the helsinki declaration with approval of the institutional review boards of the affiliated yan'an hospital of kunming medical college and the kunming institute of botany. the snp rs11209026 in the il23r gene was genotyped by the taqman allelic discrimination method (applied biosystems). new pcr - rflp methods were generated to genotype the snp rs56245420 in the il15 gene. primers 5-ttt ctg tta tta aca aac atc act ctg-3 and 5-caa cac ttg tac ata ttt tta ttc aat at-3 (mismatch is shown in bold lower case) were used for rs56245420. other five snps were genotyped by pcr - rflp methods described previously with slight modification [1115 ]. pcr reaction was carried out in a total volume of 20 l containing 20 ng of genomic dna, 1 pcr buffer, 1.5 mm mgcl2, 200 m of each dntp, 30 ng of each primer, and 1 unit of taq dna polymerase (takara). samples were denatured at 95c for 2 min followed by 30 cycles of 94c for 45 sec, 61c (rs2395029) or 54c (rs56245420) for 45 sec, and 72c for 45 sec and ended with a final extension for 7 min at 72c. pcr products were digested with 4 u of appropriate restriction endonuclease and electrophoresed on 3% agarose gels and stained with ethidium bromide. the restriction endonucleases, pcr product lengths, and restriction patterns are shown in table 1. the frequencies of genotypes and alleles for all the six studied loci were determined assuming codominant inheritance. the hardy - weinberg equilibrium (hwe) for six loci in psoriasis patients and controls was tested by means of chi - square tests. the statistical significance of the genotype and allele frequency variables between the psoriasis patients and control group was evaluated by chi - square test with yates correction for small numbers. relative risk associated with the significant genotype was estimated by the odds ratio (or). or with 95% confidence intervals (95% ci) was tested using a chi - square distribution and the null hypothesis being tested is or = 1. only one of the fourteen hardy - weinberg tests (seven polymorphic loci each in the psoriasis patient and control groups) had p values smaller than 0.05 (p = 0.01 at rs3212227 in controls). all nine remaining genotype frequencies fit hardy - weinberg expectations according to chi - square tests in psoriasis patients and controls (p > 0.05). therefore, there is no meaningful deviation from whe, and our population is derived from random mating. polymorphism (minor allele frequency > 1%) has been found for all studied snps except for rs11209026 in the il23r gene (table 2). table 2 shows that rs3212227 in the il12b gene (p = 0.0218) was associated with psoriasis at genotypic level in the studied population. other snps examined were not associated with psoriasis considered from single locus. as shown in table 3, while the a / c genotype (or = 1.48 ; 95% ci : 0.952.30) and the alleles (or = 0.84 ; 95% ci : 0.631.13) at rs3212227 in the il12b were not a risk factor of psoriasis, the c / c genotype was a protective factor of psoriasis (or = 0.51 ; 95% ci : 0.270.96). genetic factors may play a significant role in the risk of psoriasis in chinese [10, 16 ]. recent genetic studies indicate that the location of these genes varies considerably among populations and families. we are interested to know if psoriasis is associated with the genes that have been strongly associated with psoriasis in europeans but not well studied in chinese. our results showed that the il12b gene was associated with psoriasis in chinese at genotypic level (p < 0.05), which is in line with the findings from european studies. the c / c genotype for rs3212227 in il12b is a protective factor (or = 0.51) from psoriasis. similar result from studying snp rs6887695 in il12b showed that the minor allele c was a protective factor from psoriasis. the nonsynonymous snp in il23r, rs11209026, widely thought to be the primary psoriasis - associated snp in il23r in europeans, was found not to be polymorphic in chinese, which is in agreement with the findings of others. the low frequencies of variant in il23r are accordingly of low risk for psoriasis in chinese. with single snp analysis, no association is found between the psoriasis and the il4, il10, il13, and il15 genes.	psoriasis is a chronic inflammatory and hyperproliferative skin disease affected by both genetic and environmental factors. the aim of the present study was to investigate polymorphisms in a candidate gene family of interleukin (il) in unrelated chinese patients with psoriasis and control subjects without psoriasis. in this case - control study, 200 unrelated chinese psoriasis patients and 298 age- and sex - matched control subjects were enrolled. genomic dna was prepared from peripheral blood obtained from all psoriasis patients and control subjects. we genotyped seven single - nucleotide polymorphisms (snps) in candidate genes of six ils : il4, il10, il12b, il13, il15, and il23r, which have been shown in the literature to be associated with psoriasis in other ethnic groups. among the seven snps in the six il genes studied, only the rs3212227 in the il12b gene was found to be associated with psoriasis at genotypic level in the studied population. the c / c genotype in the il12b gene is a protective factor of psoriasis (p = 0.0218 ; or = 0.51 ; 95% ci : 0.270.96) in chinese. furthermore, the studied chinese population has extremely low minor allele frequency for il23r. together, the data reveal unique genetic patterns in chinese that may be in part responsible for the lower risk for psoriasis in this population.
it is often suggested that technology might be one of the solutions to meet the growing need for health care in the future. advanced technologies are developed constantly, leading to various new and enhanced diagnostic and therapeutic options. advances in health care technology do not only encompass technologies necessary to diagnose or treat patients, but also include assistive technologies such as ehealth, electronic health records and elearning modules regarding health. all these technological developments impact various aspects of care, including the organisation of care, the communication between health care professionals and the communication between health care professionals and their patients.1, 2 systems such as pacs (picture archiving and communication system) and his (hospital information system) are examples. however, research shows that new technology is not always positively perceived by health professionals.3, 4 for example, the occurrence of problems with power supply and lack of knowledge can make health professionals sceptical about the use of new technology.4, 5 furthermore, research indicates that in order for a technological application to be adequately implemented, health care professionals should feel competent to use the technology.3 the speed at which technological developments are introduced is increasing ; especially people working in technologydriven professions are constantly confronted with technological innovations. it is therefore of utmost importance to collect the views and experiences of health professionals regarding technology. by doing so to date, research focused on the considerations of health care professionals regarding technology is scarce.5 the studies that examine the opinions and views of health care professionals were restricted to specific technologies such as ehealth6 and the use of personal health record systems7 and never focused on radiographers, nuclear medicine technologists and radiation therapists (in europe defined as radiographers8). however, it is currently unclear how these health care professionals experience the technology they use in their profession, and maybe more importantly, the constant development of this technology. the first aim of the study was to gain insight into the opinions and experiences of radiographers, nuclear medicine technologists and radiation therapists regarding the technology they currently use in their profession. the second aim focused on their ideas about the role technology might play in their professional future. the results of this study can aid hospitals in supporting and improving the introduction and implementation of (new) technology in health care. hence, the aim of this study is not to find evidence for a hypothesis or a theory, but to provide a basis for new theories or concepts. participants were recruited from five departments in five hospitals in different parts of the netherlands. in order to gain a broad perspective on the subject, all radiographers, nuclear medicine therapists and radiation therapists were invited to participate (for clarity, the term radiographers will be used throughout this article to address these three occupations) and were given an information letter (n = 252). this letter stated the aim of the study and provided detailed information regarding the content and duration of the interview. radiographers who were interested in participation could contact the researcher if they required more information or could contact their department manager in order to schedule an interview. the manager of the departments arranged that the participants did not have to work at the time the interview took place. information regarding the aim and procedure of the study was also given verbally by the researcher. after the participant had signed the informed consent, an audio recorder was turned on and the interview was started. all interviews were conducted in dutch and lasted about 1 h. the interviews were conducted by five researchers (yz, nvdg, pb, ms, wtb). an interview guide was developed based on the available literature.3, 5, 9 the interview guide ensured that the same set of topics (and questions) was covered in all the interviews, namely (1) the role of technology in daily practice, (2) assistance regarding the use of technology and (3) the role of technology in their professional future. the study was conducted in accordance with the ethics policy of all five health departments and of the fontys university ethical committee. data collection and data analysis occurred concurrently;10 after every interview the audio recording was transcribed verbatim. the researchers discussed the initial coding and consulted the senior researchers in (bi)weekly meetings. during these meetings, afterwards, all codes were clustered based on similarity and grouped into themes ; the interpretation of the main themes were discussed with all (senior) researchers. because of the amount of useful quotations, not every interview provided a quotation for the results section. however, every quotation used in the results section came from a different interview (e.g. a different participant). the quotes mentioned in the results section of this article were translated from dutch to english. participants were recruited from five departments in five hospitals in different parts of the netherlands. in order to gain a broad perspective on the subject, all radiographers, nuclear medicine therapists and radiation therapists were invited to participate (for clarity, the term radiographers will be used throughout this article to address these three occupations) and were given an information letter (n = 252). this letter stated the aim of the study and provided detailed information regarding the content and duration of the interview. radiographers who were interested in participation could contact the researcher if they required more information or could contact their department manager in order to schedule an interview. the manager of the departments arranged that the participants did not have to work at the time the interview took place. all interviews were conducted between february and may 2015. before starting the interview, participants were asked if they had any questions regarding the information letter they received. information regarding the aim and procedure of the study was also given verbally by the researcher. after the participant had signed the informed consent, an audio recorder was turned on and the interview was started. all interviews were conducted in dutch and lasted about 1 h. the interviews were conducted by five researchers (yz, nvdg, pb, ms, wtb). an interview guide was developed based on the available literature.3, 5, 9 the interview guide ensured that the same set of topics (and questions) was covered in all the interviews, namely (1) the role of technology in daily practice, (2) assistance regarding the use of technology and (3) the role of technology in their professional future. the study was conducted in accordance with the ethics policy of all five health departments and of the fontys university ethical committee. data collection and data analysis occurred concurrently;10 after every interview the audio recording was transcribed verbatim. the researchers discussed the initial coding and consulted the senior researchers in (bi)weekly meetings. during these meetings, afterwards, all codes were clustered based on similarity and grouped into themes ; the interpretation of the main themes were discussed with all (senior) researchers. because of the amount of useful quotations, not every interview provided a quotation for the results section. however, every quotation used in the results section came from a different interview (e.g. a different participant). the quotes mentioned in the results section of this article were translated from dutch to english. a total of 52 participants were interviewed (20.6% of all radiographers working in these five departments, characteristics of participants (n = 52).a ten radiographers were included per department ; in one department 12 participants were included. a total of 252 radiographers were working in these five departments ; 52 participants were interviewed (20.6%). four major themes emerged : (1) technology as indispensable factor, (2) engagement, support and training in (the usage of) technology, (3) transitions in work and (4) the future of the radiographer. all radiographers mentioned that technology is a key feature ; not only in their work but also in life itself. moreover, they expect that the influence of technology on their daytoday activities will only expand in the upcoming decades.there is a lot of technology in this work [] without technology this profession would not even exist.we can not even live without technology. there is a lot of technology in this work [] without technology this profession would not even exist. we can not even live without technology. radiographers state that all technology should be patientfriendly as well as effective. technology should always improve the quality of care, or at least, not decrease it. according to the participants, the patient should always be the highest priority.the current devices are so sensitive that we can make the same or better pictures with less radiation.there is a risk that you hide behind technology and to neglect the human being.the patient does n't know how technology works and not every patient likes the distance created by technology [] technology should be in favour of the patient, as the patient is more important.sometimes it seems as if we are more technology oriented than patient oriented. the current devices are so sensitive that we can make the same or better pictures with less radiation. there is a risk that you hide behind technology and to neglect the human being. the patient does n't know how technology works and not every patient likes the distance created by technology [] technology should be in favour of the patient, as the patient is more important. sometimes it seems as if we are more technology oriented than patient oriented. radiographers appreciate that technological developments enable fast and efficient communication. several participants mentioned that since there are no handwritten requests any more, the risk of making mistakes has decreased.you can use video conference for meetings [] that is timeefficient.we have a group app []. if somebody wants to discuss something or is ill, we can swap duties easily. that is a really good thing.they (referring to physicians) do not have to come to us. they do not receive a copy. a copy (of a photo) is always inferior to the original. you can use video conference for meetings [] that is timeefficient. we have a group app []. if somebody wants to discuss something or is ill, we can swap duties easily. a copy (of a photo) is always inferior to the original. in this regard, radiographers underscore the importance of warranting the privacy of patients, especially, when using system such as pacs.if you are working with varying patient identification codes, you have to be careful to link the pictures to the right codes.even with maximum security, you still hear of things like hacking. this could be harmful for patients. if you are working with varying patient identification codes, you have to be careful to link the pictures to the right codes. even with maximum security, you still hear of things like hacking. this could be harmful for patients. in was noted that various radiographers expressed feelings of helplessness when using technology ; sometimes they feel they rely too much on the technology they use. [] you get an error message and you can not do anything anymore.if technology works properly, there is no problem whatsoever., you were able to solve the problem in a way, but nowadays that is impossible.it makes you dependent on a digital system. in case of a power failure you are really in trouble. [] you get an error message and you can not do anything anymore. if technology works properly, there is no problem whatsoever. but as soon as technology fails, you are in trouble. before, you were able to solve the problem in a way, but nowadays that is impossible. it makes you dependent on a digital system. in case of a power failure you are really in trouble. according to the radiographers, all new technology needs to be introduced and explained to the employees. in addition, opportunity should be given to evaluate the effects and impact of the technology at hand, as experienced by the radiographers.i would always start a pilot to see if the new procedure works. that everyone is informed and that the new technology is tested for a few weeks [] afterwards people can say what worked and what not regarding the technology.i think that the people that have to use the technology frequently should be involved from the start (of the new technology). i would always start a pilot to see if the new procedure works. that everyone is informed and that the new technology is tested for a few weeks [] afterwards people can say what worked and what not regarding the technology. i think that the people that have to use the technology frequently should be involved from the start (of the new technology). the importance of having knowledge regarding the underlying processes of a technology, was stressed by all radiographers. they stress the importance of education in order to learn how to use technological devices in a safe, efficient and effective manner.i value education, because i want to know what i am doing. i can press a button easily, but i also want to know what is behind the technology [] i want to be able to respond to what happens.i want to know things. i am going to explore that (the technology) in order to know what i am doing. i value education, because i want to know what i am doing. i can press a button easily, but i also want to know what is behind the technology [] i want to be able to respond to what happens. i am going to explore that (the technology) in order to know what i am doing. participants brought up the need for health departments (i.e. the managers thereof) to play a role in facilitating education related to (the use of) technology.more time and energy should be invested. [] in that respect the department could be more active.the keyusers are relatively well educated, but even they do not know all the ins and outs []. i think the entire team should have been better educated.i see it (education) as an employers ' task to facilitate that. more time and energy should be invested. [] in that respect the department could be more active. the keyusers are relatively well educated, but even they do not know all the ins and outs []. i think the entire team should have been better educated. i see it (education) as an employers ' task to facilitate that. participants reported that, by using new technological applications, the work rate has increased ; more patients are seen in 1 h than before. according to the radiographers, the current patient flow would have been unworkable in earlier days.indeed, you do not have to carry patients ' medical folders around the hospital anymore, but on the other hand, you see more patients per time unit.work pressure also increases. of course there are also much more older patients who are less mobile and need more time. indeed, you do not have to carry patients ' medical folders around the hospital anymore, but on the other hand, you see more patients per time unit. work pressure also increases. of course there are also much more older patients who are less mobile and need more time. radiographers stated that technological developments have decreased physical strain in employees. the appreciation for technology such as manual controllers and hoists was expressed by multiple radiographers. especially in light of the fact that employees are still working at an advanced age (i.e. some participants even mentioned a decrease in shoulder problems as a result of this kind of supportive technology.technology can help in decreasing physical burden [] so you can keep your own body in mind. you have to keep yourself mobile.working as a radiographer is less of a physical burden than before. technology can help in decreasing physical burden [] so you can keep your own body in mind. you have to keep yourself mobile. working as a radiographer is less of a physical burden than before. according to the radiographers, the constant development of and changes in technology require flexible employees ; they are expected to keep up with all these developments and changes.you have to keep an open mind and keep up with the developments.it is a lot to learn in a short period.for short periods of time it 's quiet, and then there is a new development [] it always come in waves. you have to keep an open mind and keep up with the developments. it is a lot to learn in a short period. for short periods of time it 's quiet, and then there is a new development [] it always come in waves. radiographers believe that technological developments take over many of their daytoday activities ; they feel that less of their theoretical knowledge is used in performing their profession. many radiographers experience this as a disadvantage.you trust a computer, but can you be sure that it is correct?i think our readytouse available knowledge diminishes, but at the same time, we have more skills that can bring us further [] but in the end i value ready available knowledge as much as skills.in the old times, you were the computer. you were more engaged in education and knowledge.there is less need for knowledge at hand. you trust a computer, but can you be sure that it is correct? i think our readytouse available knowledge diminishes, but at the same time, we have more skills that can bring us further [] but in the end i value ready available knowledge as much as skills. in the old times, you were the computer. you were more engaged in education and knowledge. there is less need for knowledge at hand. concurrently, keeping up with all available knowledge was mentioned to be increasingly difficult ; the pace at which technology develops is just too fast.nowadays you can not keep up with everything and know everything [] you just can not know everything because so much is changing.i think it is difficult to know everything of a technological device. you just can not know everything anymore. nowadays you can not keep up with everything and know everything [] you just can not know everything because so much is changing. you just can not know everything anymore. when asking radiographers about their future work, half of them mentioned to expect to be less autonomous in their work as a result of new technology and more rigid protocols.perhaps we are going to be exchanged for higher educated it personnel.that our influence will diminish, that is what i fear. that our independence will be nonexistent.(i hope) that we are not going to be push the button persons, that you do n't need to think any more. [] for me patient contact is important. there is always that fear that, at a certain point in time [], we are not needed anymore. perhaps we are going to be exchanged for higher educated it personnel. that our influence will diminish, that is what i fear. that our independence will be nonexistent. (i hope) that we are not going to be push the button persons, that you do n't need to think any more. [] for me patient contact is important. actually most important. there is always that fear that, at a certain point in time [], we are not needed anymore. interestingly, other participants reported to expect a more prominent role in the future, because of the increasing complexity of their work.i think we will work in a different manner [] we will make more decisions that were previously made by physicians.i expect that our role will be bigger. because more diagnostic examinations are needed. and those diagnostics need to be of a higher quality. i think we will work in a different manner [] we will make more decisions that were previously made by physicians. i expect that our role will be bigger. because more diagnostic examinations are needed. and those diagnostics need to be of a higher quality. all radiographers mentioned that technology is a key feature ; not only in their work but also in life itself. moreover, they expect that the influence of technology on their daytoday activities will only expand in the upcoming decades.there is a lot of technology in this work [] without technology this profession would not even exist.we can not even live without technology. there is a lot of technology in this work [] without technology this profession would not even exist. we can not even live without technology. radiographers state that all technology should be patientfriendly as well as effective. technology should always improve the quality of care, or at least, not decrease it. according to the participants, the patient should always be the highest priority.the current devices are so sensitive that we can make the same or better pictures with less radiation.there is a risk that you hide behind technology and to neglect the human being.the patient does n't know how technology works and not every patient likes the distance created by technology [] technology should be in favour of the patient, as the patient is more important.sometimes it seems as if we are more technology oriented than patient oriented. the current devices are so sensitive that we can make the same or better pictures with less radiation. there is a risk that you hide behind technology and to neglect the human being. the patient does n't know how technology works and not every patient likes the distance created by technology [] technology should be in favour of the patient, as the patient is more important. sometimes it seems as if we are more technology oriented than patient oriented. radiographers appreciate that technological developments enable fast and efficient communication. sharing several participants mentioned that since there are no handwritten requests any more, the risk of making mistakes has decreased.you can use video conference for meetings [] that is timeefficient.we have a group app []. if somebody wants to discuss something or is ill, we can swap duties easily. that is a really good thing.they (referring to physicians) do not have to come to us. a copy (of a photo) is always inferior to the original. you can use video conference for meetings [] that is timeefficient. we have a group app []. if somebody wants to discuss something or is ill, we can swap duties easily. a copy (of a photo) is always inferior to the original. in this regard, radiographers underscore the importance of warranting the privacy of patients, especially, when using system such as pacs.if you are working with varying patient identification codes, you have to be careful to link the pictures to the right codes.even with maximum security, you still hear of things like hacking. this could be harmful for patients. if you are working with varying patient identification codes, you have to be careful to link the pictures to the right codes. even with maximum security, you still hear of things like hacking. this could be harmful for patients. in was noted that various radiographers expressed feelings of helplessness when using technology ; sometimes they feel they rely too much on the technology they use. [] you get an error message and you can not do anything anymore.if technology works properly, there is no problem whatsoever., you were able to solve the problem in a way, but nowadays that is impossible.it makes you dependent on a digital system. in case of a power failure you are really in trouble. [] you get an error message and you can not do anything anymore. if technology works properly, there is no problem whatsoever., you were able to solve the problem in a way, but nowadays that is impossible. it makes you dependent on a digital system. in case of a power failure you are really in trouble. according to the radiographers, all new technology needs to be introduced and explained to the employees. in addition, opportunity should be given to evaluate the effects and impact of the technology at hand, as experienced by the radiographers.i would always start a pilot to see if the new procedure works. that everyone is informed and that the new technology is tested for a few weeks [] afterwards people can say what worked and what not regarding the technology.i think that the people that have to use the technology frequently should be involved from the start (of the new technology). i would always start a pilot to see if the new procedure works. that everyone is informed and that the new technology is tested for a few weeks [] afterwards people can say what worked and what not regarding the technology. i think that the people that have to use the technology frequently should be involved from the start (of the new technology). the importance of having knowledge regarding the underlying processes of a technology, was stressed by all radiographers. they stress the importance of education in order to learn how to use technological devices in a safe, efficient and effective manner.i value education, because i want to know what i am doing. i can press a button easily, but i also want to know what is behind the technology [] i want to be able to respond to what happens.i want to know things. i am going to explore that (the technology) in order to know what i am doing. i value education, because i want to know what i am doing. i can press a button easily, but i also want to know what is behind the technology [] i want to be able to respond to what happens. i am going to explore that (the technology) in order to know what i am doing. participants brought up the need for health departments (i.e. the managers thereof) to play a role in facilitating education related to (the use of) technology.more time and energy should be invested. [] in that respect the department could be more active.the keyusers are relatively well educated, but even they do not know all the ins and outs []. i think the entire team should have been better educated.i see it (education) as an employers ' task to facilitate that. more time and energy should be invested. [] in that respect the department could be more active. the keyusers are relatively well educated, but even they do not know all the ins and outs []. i think the entire team should have been better educated. i see it (education) as an employers ' task to facilitate that. participants reported that, by using new technological applications, the work rate has increased ; more patients are seen in 1 h than before. according to the radiographers, the current patient flow would have been unworkable in earlier days.indeed, you do not have to carry patients ' medical folders around the hospital anymore, but on the other hand, you see more patients per time unit.work pressure also increases. of course there are also much more older patients who are less mobile and need more time. indeed, you do not have to carry patients ' medical folders around the hospital anymore, but on the other hand, you see more patients per time unit. work pressure also increases. of course there are also much more older patients who are less mobile and need more time. radiographers stated that technological developments have decreased physical strain in employees. the appreciation for technology such as manual controllers and hoists was expressed by multiple radiographers. especially in light of the fact that employees are still working at an advanced age (i.e. some participants even mentioned a decrease in shoulder problems as a result of this kind of supportive technology.technology can help in decreasing physical burden [] so you can keep your own body in mind. you have to keep yourself mobile.working as a radiographer is less of a physical burden than before. technology can help in decreasing physical burden [] so you can keep your own body in mind. you have to keep yourself mobile. working as a radiographer is less of a physical burden than before. according to the radiographers, the constant development of and changes in technology require flexible employees ; they are expected to keep up with all these developments and changes.you have to keep an open mind and keep up with the developments.it is a lot to learn in a short period.for short periods of time it 's quiet, and then there is a new development [] it always come in waves. you have to keep an open mind and keep up with the developments. it is a lot to learn in a short period. for short periods of time it 's quiet, and then there is a new development [] it always come in waves. radiographers believe that technological developments take over many of their daytoday activities ; they feel that less of their theoretical knowledge is used in performing their profession. many radiographers experience this as a disadvantage.you trust a computer, but can you be sure that it is correct?i think our readytouse available knowledge diminishes, but at the same time, we have more skills that can bring us further [] but in the end i value ready available knowledge as much as skills.in the old times, you were the computer. you were more engaged in education and knowledge.there is less need for knowledge at hand. you trust a computer, but can you be sure that it is correct? i think our readytouse available knowledge diminishes, but at the same time, we have more skills that can bring us further [] but in the end i value ready available knowledge as much as skills. in the old times, you were the computer. you were more engaged in education and knowledge. there is less need for knowledge at hand. concurrently, keeping up with all available knowledge was mentioned to be increasingly difficult ; the pace at which technology develops is just too fast.nowadays you can not keep up with everything and know everything [] you just can not know everything because so much is changing.i think it is difficult to know everything of a technological device. you just can not know everything anymore. nowadays you can not keep up with everything and know everything [] you just can not know everything because so much is changing. when asking radiographers about their future work, half of them mentioned to expect to be less autonomous in their work as a result of new technology and more rigid protocols.perhaps we are going to be exchanged for higher educated it personnel.that our influence will diminish, that is what i fear. that our independence will be nonexistent.(i hope) that we are not going to be push the button persons, that you do n't need to think any more. [] for me patient contact is important. actually most important. there is always that fear that, at a certain point in time [], we are not needed anymore. perhaps we are going to be exchanged for higher educated it personnel. that our influence will diminish, that is what i fear. that our independence will be nonexistent. (i hope) that we are not going to be push the button persons, that you do n't need to think any more. [] for me patient contact is important. there is always that fear that, at a certain point in time [], we are not needed anymore. interestingly, other participants reported to expect a more prominent role in the future, because of the increasing complexity of their work.i think we will work in a different manner [] we will make more decisions that were previously made by physicians.i expect that our role will be bigger. because more diagnostic examinations are needed. and those diagnostics need to be of a higher quality. i think we will work in a different manner [] we will make more decisions that were previously made by physicians. i expect that our role will be bigger. because more diagnostic examinations are needed. and the results of this study indicate that radiographers perceive technology as a requisite for their daily work ; without technology their work is simply not feasible. however, the implementation of new technology should always lead to more efficient and effective care ; technology should always be in the patients best interest. these results are consistent with the opinions expressed by other health care professionals, including physicians and nurses.3 the participants valued the positive influences of technological developments (e.g. more effective forms of communication through the use of systems such as pacs) which, according to their opinion, improved the quality of health care. furthermore, technology used to alleviate the physical strain of the profession, including hoists, were very positively received. many participants mentioned that these kind of technological devices have made their profession physically easier. several barriers for the implementation and correct use of technology were also brought forward. participants reported that they sometimes feel they rely too much on the technology at hand. when a technological application is not properly working, their daily work is severely hampered.11 aspects such as crashing computers or power failures bring about feelings of helplessness, which corresponds to the results of previous studies among other health care professionals.3, 4 aspects such as responsibility and privacy were discussed extensively in each interview. participants feel it as their responsibility to use the available technological devices and applications safely, effectively and efficiently. they expressed the need to understand the background and principles of the technology they use, rather than just being button pushers. furthermore, the lack of knowledge regarding a certain technological device or application was mentioned as a major barrier to successfully implement (new) technology. radiographers highly value appropriate education, training and the existence of socalled keyusers in order to stay uptodate regarding the latest technological developments. this is also emphasised in the dutch covenant of hospitals entitled safe use of medical technology in hospitals, which states that health care professionals should have sufficient knowledge of and competence in the technology they use.12 the participants feel that education and training (related to technology usage) should be facilitated by their employers. this is in congruence with the limited available literature that emphasises that health care professionals find it important to be engaged in the technological developments that are implemented.3, 4 it might therefore be recommended that, when a new technology is implemented, radiographers are given the opportunity to attend courses and training in order to safely and effectively use the new technology at hand. the most remarkable finding, and in the authors opinion a very novel one, surfaced when radiographers were asked about the future of their occupation. while some radiographers foresee less autonomy for themselves, because they believe much more processes will be automated and standardised, others expect more autonomy in the future because of these technological developments. these results underscore the already ongoing debate regarding the role of radiographers. in recent years, authors of various studies have proposed role extension for radiographers.13, 14 examples have demonstrated that role extension may benefit the health care professional as well as their managers and, in turn, might also improve the organisation and quality of care. the current findings underscore the need for radiographers and their employers to constantly discuss their experiences, ideas and needs. only when a constant dialogue between health care professionals and their managers exists, new technology can be implemented in health care in a safe and effective manner. although generalisability of the results is not strived for in qualitative research, this sample does resemble the dutch radiographers population, which increases the transferability of the current results. in addition, the variety of context in which this study was conducted (i.e. in various departments around the nation) should be perceived as a strength of this study. to our knowledge, this is the first qualitative research within the field of radiography, nuclear medicine and radiation therapy that provides a broad perspective regarding the role of technology. first, since every interview was conducted by one interviewer, subjectivity could have played a bigger role than when two interviewers were present. however, because of the large sample size (i.e. for a qualitative study) and the frequent meetings to discuss the (analyses of the) interviews, objectiveness was strived for. second, this study addresses the opinions and experiences regarding technology ; the participants were not actually observed when using one or more technological devices or applications. consequently, it can not be ruled out that a discrepancy exists between the ideas and opinions people have and their actual behaviour. since this study was conducted in the netherlands, the question remains if the obtained results are applicable for radiographers in other parts of the world. hence, this study should also be conducted in other countries ; especially in countries in which the role of radiographers might be different compared to the role of radiographers working in the netherlands. why do some radiographers expect more autonomy, while others have an entirely different point of view ? and, perhaps most importantly, what impact does this have on the work experience of radiographers, and, on the care their patients receive ? to our knowledge, this is the first qualitative research within the field of radiography, nuclear medicine and radiation therapy that provides a broad perspective regarding the role of technology. first, since every interview was conducted by one interviewer, subjectivity could have played a bigger role than when two interviewers were present. however, because of the large sample size (i.e. for a qualitative study) and the frequent meetings to discuss the (analyses of the) interviews, objectiveness was strived for. second, this study addresses the opinions and experiences regarding technology ; the participants were not actually observed when using one or more technological devices or applications. consequently, it can not be ruled out that a discrepancy exists between the ideas and opinions people have and their actual behaviour. since this study was conducted in the netherlands, the question remains if the obtained results are applicable for radiographers in other parts of the world. hence, this study should also be conducted in other countries ; especially in countries in which the role of radiographers might be different compared to the role of radiographers working in the netherlands. why do some radiographers expect more autonomy, while others have an entirely different point of view ? and, perhaps most importantly, what impact does this have on the work experience of radiographers, and, on the care their patients receive ? radiographers, nuclear medicine technologists and radiation therapists value technological developments not only to perform their occupations, but also regarding other aspects such as documentation, communication and physical support. according to the participants, all technological developments should be in the best interest of the patient. they want to receive more training aimed at increasing knowledge related to technological developments, preferably facilitated by their respective departments (and the managers thereof). when asked about the future of their profession, participants provided contradictory answers ; while some expect less autonomy in the future through the use of technology, others belief to get more autonomy.	abstractintroductionnew technology is continuously introduced in health care. the aim of this study was (1) to collect the opinions and experiences of radiographers, nuclear medicine technologists and radiation therapists regarding the technology they use in their profession and (2) to acquire their views regarding the role of technology in their future practice.methodsparticipants were recruited from five departments in five hospitals in the netherlands. all radiographers, nuclear medicine therapists and radiation therapists who were working in these departments were invited to participate (n = 252). the following topics were discussed : technology in daily work, training in using technology and the role of technology in future practice. the recorded interviews were transcribed verbatim and analysed using open and axial coding.resultsa total of 52 participants (57.7% radiographer) were included, 19 men and 33 women (age range : 2063). four major themes emerged : (1) technology as an indispensable factor, (2) engagement, support and training in using technology, (3) transitions in work and (4) the radiographer of the future. all participants not only value technological developments to perform their occupations, but also aspects such as documentation and physical support. when asked about the future of their profession, contradictory answers were provided ; while some expect less autonomy, others belief they will get more autonomy in their work.conclusiontechnology plays a major role in all three occupations. all participants believe that technology should be in the best interests of patients. being involved in the implementation of new technology is of utmost importance ; courses and training, facilitated by the managers of the departments, should play a major role. only when a constant dialogue exists between health care professionals and their managers, in which they discuss their experiences, needs and expectations, technology can be implemented in a safe and effective manner. this, in turn, might positively influence quality of care.
inflammation is present in patients with depressive disorders, including recurrent depressive disorder (rdd), and is thought to play an important role in the risk for and pathogenesis of this disease. the presence of inflammation is indicated by the elevated levels of pro - inflammatory cytokines such as interleukin-1b (il-1) and interleukin-8 (il-8), and by increased expression of nod - like receptor family, pyrin domain containing 3 (nlrp3), one of the nod - like receptors, which is a component of inflammasome which is necessary for the release of il-1 and il-18 [36 ]. in depression, inflammation is often accompanied by increased oxidative stress, since increased lipid peroxidation and production of mitochondrial reactive oxygen species (mtros) are found in depressed patients. ros may damage biomolecules, including genetic material, as indicated by elevated levels of 8-oxoguanine (8-oxog), a marker of oxidative dna damage, in patients with clinical depression as well as depression that coexists with other non - mental diseases [813 ]. depression severity may be a factor as well in milder, non - clinical depression, although 8-oxog levels do not differ from controls. our team has confirmed previous study results that showed increased levels of oxidatively modified purines and pyrimidines, as well as dna strand breaks and alkali labile sites in peripheral blood mononuclear cells (pbmcs) isolated from patients with rdd, using comet assay. furthermore, a recent report showed that nrlp3 may regulate the dna damage response, with nrlp3 knockout increasing the expression of proteins involved in base - excision repair (ber) in murine dendritic cells exposed to genotoxic and oxidative stress. in a previous study, we showed that pbmcs of rdd patients cells repaired dna damage induced by hydrogen peroxide (h2o2) less efficiently than in control patients cells. finally, our team genotyped single nucleotide polymorphism (snps) of glycosylases involved in the first step of ber, finding that their polymorphisms may modulate rdd risk. the aforementioned studies indicate that depression may be associated with impairment of the dna damage repair mechanism, more particularly the pathways involved in the repair of oxidative dna damage such as ber. accordingly, we chose to study the relationship between rdd occurrence and snps of genes encoding proteins involved in ber, namely : c.-441g > a (rs174538) of fen1 (flap structure - specific endonuclease 1) ; c.2285t > c (rs1136410) of parp1 (poly [adp - ribose ] polymerase 1) ; c.580c > t (rs1799782) and c.1196a > g (rs25487) of xrcc1 (x - ray repair cross - complementing protein 1) ; c.83a > c (rs4796030) and c.50c > t (rs1052536) of lig3 (dna ligase 3) ; c.-7c > t (rs20579) of lig1 (dna ligase 1) ; and c.-468t > g (rs1760944) and c.444t > g (rs1130409) of apex1 (apex nuclease 1, dna-[apurinic or apyrimidinic site ] lyase). the study included 599 participants : patients with rdd (n=288, 140 women and 148 men ; mean age 49.310.2) and healthy controls (n=311, 153 women and 158 men ; mean age 51.213.3). all patients were hospitalized at the department of adult psychiatry of the medical university of lodz (poland) and were randomly recruited for this study without replacement sampling, based on the inclusion criteria for a current episode of depression and rdd outlined in icd-10 (f32.07.32.2, f33.0f33.8) and a written informed consent to participate in the study. the diagnosis of rdd was established according to icd-10 criteria. in all qualified cases, exclusion criteria included : the presence of axis i and ii disorders other than depressive episodes, a diagnosis of severe and chronic somatic diseases or worsening of symptoms, injuries of the central nervous system, and inflammatory or autoimmune disorders. the control group was selected randomly from respondents with a negative history of mental illness. the control group included community volunteers enrolled in the study following the criteria of the psychiatric cidi interview as well as depressed patients who were not treated for any severe and chronic diseases or worsening of symptoms, injuries of the central nervous system, or inflammatory or autoimmune disorders. an informed, written consent for participation in the study was obtained from each participant, according to the protocol approved by the bioethics committee of the medical university of lodz (no. we selected the studied polymorphisms from the public domain of the national center for biotechnology information, single nucleotide polymorphisms database (ncbi dbsnp) at http://www.ncbi.nlm.nih.gov/snp (bethesda, md, usa). we chose snps with a minor allele frequency larger than 0.05 in a european population (submitter population i d : hapmap - ceu), that are frequently studied in the literature and which are either localized in coding regions causing non - synonymous substitution or in regulatory regions. finally, we selected nine polymorphisms : c.-441g > a localized near 5 end of fen1 ; c.2285t > c localized in exon of parp1 causing valine to alanine substitution in codon 762 ; c.580c > t and c.1196a > g localized in exon of xrcc1 causing arginine to tryptophan substitution in codon 194 and glutamine to arginine substitution in codon 399, respectively ; c.83a > c and c.50c > t localized in untranscribed region at 3 end (utr-3) of lig3 ; c.-7c > t localized in untranscribed region at 5 end (utr-5) of lig1 ; c.-468t > g and c.444t > g localized near 5 end and in exon causing asparagine to glutamic acid in codon 148, respectively, in apex1. blood mini kit (a&a biotechnology, gdynia, poland) was used to extract genomic dna from venous blood. chosen snps were genotyped using taqman snp genotyping assay and taqman fast universal pcr master mix (life technologies, carlsbad, ca, usa) in conditions recommended by the manufacturer. we used bio - rad cfx96 real - time pcr detection system to carry out reactions. the analysis was done with cfx manager software (both from bio - rad laboratories inc., hercules, california, usa). analysis of gathered data was performed in sigmaplot 11.0 and statistica 12 (systat software inc., san jose, ca, usa and statsoft, tulsa, ok, usa, respectively). agreement with hardy - weinberg equilibrium (hwe) of the studied snps genotype frequencies was checked using the chi - square test. an unconditional multiple logistic regression model was used to evaluate the association between case / control and each polymorphism or the gene gene interactions by measuring the odds ratio (or) and its corresponding 95% confidence interval (ci). we used multi - variable logistic regression model (fractional polynomials) to evaluate the independent relationship between the studied variants and the presence of depression disorder adjusting for age covariates to prevent loss of information resulting from age dichotomization. for multiple testing correction, we calculated the effective number of independent tests using single nucleotide polymorphism spectral decomposition (snpspd) method which is based on the spectral decomposition (spd) of matrices of pair - wise linkage disequilibrium values between snps applying significance threshold (p a localized near 5 end of fen1 ; c.2285t > c localized in exon of parp1 causing valine to alanine substitution in codon 762 ; c.580c > t and c.1196a > g localized in exon of xrcc1 causing arginine to tryptophan substitution in codon 194 and glutamine to arginine substitution in codon 399, respectively ; c.83a > c and c.50c > t localized in untranscribed region at 3 end (utr-3) of lig3 ; c.-7c > t localized in untranscribed region at 5 end (utr-5) of lig1 ; c.-468t > g and c.444t > g localized near 5 end and in exon causing asparagine to glutamic acid in codon 148, respectively, in apex1. blood mini kit (a&a biotechnology, gdynia, poland) was used to extract genomic dna from venous blood. chosen snps were genotyped using taqman snp genotyping assay and taqman fast universal pcr master mix (life technologies, carlsbad, ca, usa) in conditions recommended by the manufacturer. we used bio - rad cfx96 real - time pcr detection system to carry out reactions. the analysis was done with cfx manager software (both from bio - rad laboratories inc., hercules, california, usa). analysis of gathered data was performed in sigmaplot 11.0 and statistica 12 (systat software inc., san jose, ca, usa and statsoft, tulsa, ok, usa, respectively). agreement with hardy - weinberg equilibrium (hwe) of the studied snps genotype frequencies was checked using the chi - square test. an unconditional multiple logistic regression model was used to evaluate the association between case / control and each polymorphism or the gene gene interactions by measuring the odds ratio (or) and its corresponding 95% confidence interval (ci). we used multi - variable logistic regression model (fractional polynomials) to evaluate the independent relationship between the studied variants and the presence of depression disorder adjusting for age covariates to prevent loss of information resulting from age dichotomization. for multiple testing correction, we calculated the effective number of independent tests using single nucleotide polymorphism spectral decomposition (snpspd) method which is based on the spectral decomposition (spd) of matrices of pair - wise linkage disequilibrium values between snps applying significance threshold (p a we did not find any statistically significant differences in the distribution of alleles and the genotype of c.-441g > a xrcc1, c.-7c > t lig1, and c.444t > g apex1 between cases and controls. however, genotype a / a and allele a of the c.83a > c lig3 was associated with an increased risk of depression occurrence, whilst allele c was associated with decreased risk. furthermore, genotype c / c and allele c of the c.50c > t lig3 were positively associated with rdd, with allele t being negatively correlated with rdd. apex1 were greater that the statistical significance calculated by the nyholt correction (p=0.012). in order to evaluate if the studied snps were associated with the age of the first rdd episode, we used multivariable logistic regression for analyzing fractional polynomials. we found that lower age of onset of depression was associated with the presence of g / g polymorphic variant of c.-441g > a fen1 polymorphism (=8.47, p=0.042) and g / g variant of c.-7c > t lig1 polymorphism (=6.45, p=0,045). for the other study snps, we did not find statistically significant correlations with age of onset. moreover, we analyzed the relationship between the presence of polymorphic variants and age of onset by dividing the patients into two groups, with first episode before the age 35 years and first episode after the age of 35 years (optimal cutpoint in a univariate analysis) ; results are shown in table 2. thirty patients for whom age of first episode was unknown were not included in the analysis. as with the population of patients as a whole, no statistically significant differences were found in the distribution of genotypes and alleles of the c.-441g > a xrcc1, c.1196a > g xrcc1, c.-7c > t lig1, and c.444t > g apex1 between the controls and either early or late onset depression. genotype a / a of the c.83a > c lig3 was associated with increased risk of both early and late onset depression, whilst allele a was associated with an increased risk of early onset depression only. furthermore, genotype c / c and allele c of the same snp was associated with a decreased risk of early onset depression. in the case of the c.50c > t lig3, genotype c / c and allele c were positively correlated with early onset rdd, and allele t negatively correlated. apex1 increased the risk of early onset depression, while allele t increased the risk of late onset depression, with the g / g genotype and allele g decreasing this risk. additionally, we evaluated whether the gene - gene combinations can modulate the risk of depression. the results of this analysis are shown in table 3 which presents only statistically significant results, and supplementary table 1 which presents all results. we found several significant associations, some of which were for genes that alone did not modulate rdd risk. combined genotype g / t - c / c of the c.444t > g apex1 and the c.1196a > g xrcc1 was positively correlated with rdd, while the combined genotype g / t - c / t negatively correlated with rdd. additionally, the combined genotype a / g - a / g of the c.-7c > t lig1 and the c.-441g > a the distribution of haplotypes of the studied snps are presented in table 4 and supplementary table 2. we only found statistically significant results for the haplotypes of c.50c > t lig3 and c.83a > c lig3 haplotype ca, which were associated with an increased rdd risk, while haplotype tc was associated with a decreased rdd risk. the distribution of all genotypes was in agreement with hwe, with the exception of the c.-441g > a we did not find any statistically significant differences in the distribution of alleles and the genotype of c.-441g > a xrcc1, c.-7c > t lig1, and c.444t > g apex1 between cases and controls. however, genotype a / a and allele a of the c.83a > c lig3 was associated with an increased risk of depression occurrence, whilst allele c was associated with decreased risk. furthermore, genotype c / c and allele c of the c.50c > t lig3 were positively associated with rdd, with allele t being negatively correlated with rdd. apex1 were greater that the statistical significance calculated by the nyholt correction (p=0.012). in order to evaluate if the studied snps were associated with the age of the first rdd episode, we used multivariable logistic regression for analyzing fractional polynomials. we found that lower age of onset of depression was associated with the presence of g / g polymorphic variant of c.-441g > a fen1 polymorphism (=8.47, p=0.042) and g / g variant of c.-7c > t lig1 polymorphism (=6.45, p=0,045). for the other study snps, we did not find statistically significant correlations with age of onset. moreover, we analyzed the relationship between the presence of polymorphic variants and age of onset by dividing the patients into two groups, with first episode before the age 35 years and first episode after the age of 35 years (optimal cutpoint in a univariate analysis) ; results are shown in table 2. thirty patients for whom age of first episode was unknown were not included in the analysis. as with the population of patients as a whole, no statistically significant differences were found in the distribution of genotypes and alleles of the c.-441g > a xrcc1, c.1196a > g xrcc1, c.-7c > t lig1, and c.444t > g apex1 between the controls and either early or late onset depression. genotype a / a of the c.83a > c lig3 was associated with increased risk of both early and late onset depression, whilst allele a was associated with an increased risk of early onset depression only. furthermore, genotype c / c and allele c of the same snp was associated with a decreased risk of early onset depression. in the case of the c.50c > t lig3, genotype c / c and allele c were positively correlated with early onset rdd, and allele t negatively correlated. apex1 increased the risk of early onset depression, while allele t increased the risk of late onset depression, with the g / g genotype and allele g decreasing this risk. additionally, we evaluated whether the gene - gene combinations can modulate the risk of depression. the results of this analysis are shown in table 3 which presents only statistically significant results, and supplementary table 1 which presents all results. we found several significant associations, some of which were for genes that alone did not modulate rdd risk. combined genotype g / t - c / c of the c.444t > g apex1 and the c.1196a > g xrcc1 was positively correlated with rdd, while the combined genotype g / t - c / t negatively correlated with rdd. additionally, the combined genotype a / g - a / g of the c.-7c > t lig1 and the c.-441g > a the distribution of haplotypes of the studied snps are presented in table 4 and supplementary table 2. we only found statistically significant results for the haplotypes of c.50c > t lig3 and c.83a > c lig3 haplotype ca, which were associated with an increased rdd risk, while haplotype tc was associated with a decreased rdd risk. as indicated in the introduction, depression is accompanied by inflammation and increased oxidative stress, both of which may be important in its pathogenesis. oxidative stress may induce dna damage, and our team and others have found increased levels of oxidatively modified dna bases, especially 8-oxog, dna breaks, and an alkali labile site in depressed patients [813,15 ]. pbmcs isolated from cells of rdd patients repaired hydrogen peroxide - induced oxidative dna damage less efficiently than from the cells of controls, which may indicate impairments in this repair pathway. furthermore, recent studies have shown that some polymorphic variants of the ber genes may negatively impact the repair of oxidative dna damage, and our team found that glycosylase snps can modulate rdd risk. therefore, in this study, nine snps of the six genes encoding proteins involved in later steps of ber were genotyped ; and to our knowledge, none of them have been studied in the context of mental illnesses. one of the selected genes was lig3, which encodes ligase, an essential component of ber, sealing in place the new base in the final step of this pathway. it is mainly utilized in short - patch ber, but it can be used as a back - up ligase in the long - patch sub - pathway. lig3 also plays a major role in mitochondrial ber (mtber), since its depletion by sirna leads to the reduction of mitochondrial dna (mtdna) copy number and elevations in dna single - strand breaks. we examined the c.50c > t and the c.83a > c, both localized in utr-3, where they can alter gene expression by affecting mrna stability, half - life, and degradation. indeed, the c.83a > c affects the binding site of microrna, modulating the risk of bladder cancer, whilst the c.50c > t modulates the risk of young - onset lung cancer. here, both snps showed a significant association with rdd (table 1). we found that the aa genotype of the c.83a > c and the cc genotype of c.50c > t increased the risk of depression, with these genotypes also increasing cancer risk. these snps are also associated with rdd onset with the c.50c > t variant modifying the risk of early onset rdd only, with the c.83a > c variant also being more associated with early rather than late onset depression (table 2). such data indicates that these snps are linked to early incidence of rdd, possibly being less related to non - genetic factors, which may have a greater role in late - life depression. finally, we found that the haplotype c / a of the c.50c > t and the c.83a > c were associated with a significantly increased rdd risk, in contrast to the haplotype t / c, which was associated with decreased risk (table 4). apex1 encodes endonuclease, which recognizes the apurinic / apyrimidinic (ap) site arising from the actions of glycosylases after the first ber step, but is also responsible for rna processing and the regulation of transcription. this protein takes part in mtber, although its role is still the subject of investigation. on the one hand, overexpression of mitochondrial apex1 in human umbilical vein endothelial cells however, on the other hand, expression of exonuclease iii (with which apex1 has significant homology) in the mitochondria of a malignant breast epithelial cell line caused cells to be more sensitive to oxidative stress due to deficient mtdna repair. we genotyped two polymorphisms in this gene : the c.-468t > g and the c.444t > g. the latter, located near the 5 end of the gene, was found to affect the promoter strength and by this may modulate the risk of lung cancer. the former causes amino acid substitution of asparagine to glutamic acid in codon 148 and although it did not alter the in vitro activity of apex1, it modulated the risk of cancer and was associated with parkinson s disease, probably by affecting interactions of the enzyme with other ber proteins [3942 ]. data in our study showed that the heterozygote of the c.-468t > g increased rdd risk (table 1). this snp was more associated with late onset rather than with early onset depression, which could mean that the c.-468t > g has lower penetration than the lig3 polymorphisms and needs other factors to induce rdd later in life (table 2). although the heterozygote increased the risk only of early onset depression, the a allele homozygote and the a allele alone decreased late onset rdd risk, while the t allele increased this risk. these results are consistent with the work of lo and colleagues that showed that the g allele increased luciferase reported gene expression in several adenocarcinoma cell lines. the hypothesis that this polymorphism had less impact on depression occurrence was confirmed by the fact that, only together with heterozygotes of lig3 snps genotype gg, was the risk for the disease decreased (table 3). in the case of c.444t > g as well as haplotypes of these two apex1 polymorphisms, we did not find any statistically significant results (tables 1, 4). two snps were also genotyped in xrcc1, which encodes a protein that is not only an important component of ber, but also a component of other dna repair pathways, such as single - strand break repair and non - homologous end joining. in ber, xrcc1 is involved in each of these pathway steps due to its function as a scaffold protein, which physically associates with repair enzymes. its polymorphisms are associated with cancers and neurodegenerative diseases such as alzheimer s disease [4648 ]. again two snps were genotyped : the c.580c > t, which is localized in a region that coordinates protein integrations, whereas the c.1196a > g is positioned in the breast cancer 1 c terminus (brct1) domain, which is responsible for interactions with parp [4850 ]. no significant associations of depression with either genotypes, alleles, nor haplotypes of xrcc1 snps were found (tables 1, 2, and supplementary table 1). however, combined genotypes of this gene with genes encoding proteins interacting with xrcc1 provided some statistically significant results. the most interesting being the combination of the c.1196a > g xrcc1 and the c.444t > g apex1, which alone were not rdd associated. in this gene - gene interaction, the latter snp is more important due to changes of this polymorphism s genotype causing either a decrease (homozygote cc) or an increase (heterozygote) in rdd risk (table 3). moreover, this heterozygote increased the risk of rdd in combination with the tg genotype of the c.-468t > g apex1 and aa genotype of the c.83a > c lig3. such results suggest that this snp may have little if any impact on the development of depression. similarly, the two polymorphisms of fen1 and lig1, although neither alone influenced rdd risk, the combined genotype consisting of heterozygotes decreased rdd risk (tables 1, 3). fen1 encodes structure specific endonuclease that removes the 5-flap structures arising during long - patch ber and is involved in maturation of okazaki fragments, as well as releasing stalled replication forks [5153 ]. a polymorphism of this gene, the c.-441g > a, reduced expression of fen1, elevated levels of dna damage, and increased risk of lung cancer in individuals carrying the gg genotype. in our study, this snp was associated with depression in combination with other snps, most notably the combination of its gg genotype with the homozygote aa of the c.83a > c lig3 or the homozygote cc of the c.50c > t the product of lig1 is also involved in long - patch ber and maturation of okazaki fragments. the snp located in the 5-utr of this gene is associated with lung cancer in heterozygote carriers, although its influence on gene expression is unknown. we found that its heterozygote in combination with the homozygote aa of the c.83a > c lig3 or the homozygote cc of the c.50c > t it must be noted that this association was weaker than in the case of the c.-441g > a fen1, which could indicate that the latter snp is more involved in the pathogenesis of rdd than the former. finally, we genotyped one snp in parp1, which encodes protein involved not only in dna damage, including the ber, but also in the inflammation response, transcription, and apoptosis. however, no significant correlation was found between this polymorphisms and depression (tables 1, 2). the results showing a strong association between depression and some polymorphism variants of lig3, their haplotypes, or their interactions with other snps of ber genes are especially interesting in the context of recent work demonstrating impairments in oxidative dna damage repair in rdd patients. dna damage repair kinetics in the aforementioned study revealed that the differences between controls and cases were more likely to be present in the later stages of ber, as initial dna damage caused by the actions of dna glycosylases in both groups occurred at the same time. as such, it can be speculated that this difference may arise, at least partly, by the more frequent occurrence of a specific variant of lig3 and others genes involved in the later steps of ber in depressed patients versus controls. furthermore, as indicated earlier, lig3 plays an important role in the maintenance of the mitochondrial genome and a growing number of reports indicate the importance of mitochondrial dysfunctions in depression. depressed patients have lower mitochondrial atp production, lower coq10 levels and increased mitochondrial ros, all of which indicate mitochondrial damage [6063 ]. the mitochondrial impairments may be due to increased mtdna damage, and elevated levels of mtdna deletions are found in depressed patients muscles and pbmcs. such deletions can be triggered by dna damage, and one of the most frequently occurring deletions, called common deletion, is triggered by a single - strand break following incomplete dna damage repair, when ligase does not seal the nick in the last step of ber. given the classical decrease in serotonin in depression, and its role as a precursor for melatonin and a significant regulator of mitochondrial functioning as well as an inhibitor of oxidative damage and inflammation, it is not unlikely that variations in melatonin availability will interact with genes regulating the ber. one limitation was a relatively small sample size, although comparable studies concerning depression had similar study group size. in addition, our results can not be generalized to the world population, due to ethnic homogeneity of the studied population. this study is concordant with the extant literature in showing that snps of genes involved in oxidative dna damage repair may modulate the risk of rdd. further studies are needed to elucidate the role of nuclear dna as well as mitochondrial dna damage and repair in pathogenesis of depression, and the effect on clinical outcome. aging effects on telomerase, which can be offset by melatonin, would be expected to interact with the genetic susceptibility to rdd, driven partly by ber snps. gene - gene interactions of the studied polymorphisms and the risk of rdd. or adjusted for sex. p<0.05 along with corresponding ors are in bold ; p<0.012 along with corresponding ors are in bold and italic.	backgrounddepressive disorder, including recurrent type (rdd), is accompanied by increased oxidative stress and activation of inflammatory pathways, which may induce dna damage. this thesis is supported by the presence of increased levels of dna damage in depressed patients. such dna damage is repaired by the base excision repair (ber) pathway. ber efficiency may be influenced by polymorphisms in ber - related genes. therefore, we genotyped nine single - nucleotide polymorphisms (snps) in six genes encoding ber proteins.material/methodsusing taqman, we selected and genotyped the following snps : c.-441g > a (rs174538) of fen1, c.2285t > c (rs1136410) of parp1, c.580c > t (rs1799782) and c.1196a > g (rs25487) of xrcc1, c.83a > c (rs4796030) and c.50c > t (rs1052536) of lig3, c.-7c > t (rs20579) of lig1, and c.-468t > g (rs1760944) and c.444t > g (rs1130409) of apex1 in 599 samples (288 rdd patients and 311 controls).resultswe found a strong correlation between rdd and both snps of lig3, their haplotypes, as well as a weaker association with the c.-468t > g of apexi which diminished after nyholt correction. polymorphisms of lig3 were also associated with early onset versus late onset depression, whereas the c.-468t > g polymorphism showed the opposite association.conclusionsthe snps of genes involved in the repair of oxidative dna damage may modulate rdd risk. since this is an exploratory study, the results should to be treated with caution and further work needs to be done to elucidate the exact involvement of dna damage and repair mechanisms in the development of this disease.
although laryngoscopy is a safe procedure and complications rarely occur, intraoral manipulation can produce damage to soft and hard tissues of the oral cavity, patient discomfort, and postoperative pain. dental injury has been reported as the most common anesthetic - related incident. patients with pathological changes, especially if involving pathosis of the incisors, are considered to be at the greatest risk of oral tissue trauma following laryngoscopy and endotracheal intubation. most complications during anesthesia are related to unrecognized problems. recognizing and understanding ; oral and dental pathological changes ; and the presence of dental prosthesis are thus important. an 8-year - old boy reported to the department of pedodontic and preventive children dentistry complaining of a missing upper front permanent tooth since 1 month. the child 's parents gave a history of missing tooth during the tonsillectomy procedure under general anesthesia and the same was mentioned in the discharge summary. on examination, the patient was moderately built, well nourished, conscious, and cooperative. on intraoral examination an upper left central incisor and a mesially erupting upper left lateral incisor were not seen [figure 1 ]. an orthopantomograph was taken to confirm the finding, which revealed a missing upper left central incisor and a mesially - erupting upper left lateral incisor with a developing root [figure 2 ]. patient showing a missing 21, mesialy erupted 22 we decided to give a removable anterior space maintainer, i.e. removable partial denture (rpd) to prevent the mesial drifting of 22. both the child and the parents were pleased with the new look of the child, which gave him a normal appearance [figure 3 ]. his mother was advised to maintain good oral hygiene for the child ; recall visits to make any necessary adjustments for the rpd, if needed ; and to go for an implant, fixed prosthetic appliance, or orthodontic correction in future. the loss of maxillary incisors in childhood is problematic, especially up to 10 - 12 years of age, and predictable treatment methods are difficult. children in the age group of 6 - 8 years are susceptible to the loss of maxillary teeth during endotracheal intubation due to the prevalence of immature roots, increased overjet, ectopic eruptions, dilacerated roots, or pathology. the principle treatment options are : (a) re - implantation of the avulsed tooth immediately within a certain time period, (b) maintaining the space by giving a space maintainer or reopening the space for auto - transplantation using a premolar ; future prosthodontic restoration of missing teeth or single tooth implant, (c) total orthodontic space closure, followed by prosthodontic modification of the lateral incisor to imitate the central incisor. it is advisable to re - implant the avulsed tooth into the socket within the specified time interval in order to preserve the vitality of the tooth and also to promote root completion. if dental injury occurs in children during laryngoscopy and intubation an evaluation should be done, as soon as possible, by a pediatric dentist to determine the extent of the injury, to confirm the location and ensure successful retrieval of the avulsed or broken tooth. in the case of any tooth fragment avulsion of a primary tooth does not require treatment, as replacement into the socket can damage the underlying permanent successor. when a permanent tooth is avulsed, it should be stored in normal saline or cool fresh milk until it can be splinted or re - implanted. after a traumatic intubation, the success of the treatment is primarily determined by the time elapsed since injury. if the young permanent tooth is avulsed during general anesthesia, a pediatric dentist must be informed immediately to replace the tooth in the socket and splint the tooth. dental damage in the operating room may also be caused by surgeons such as otorhinologists by inadvertent injury during laryngoscopy. traumas to a patient 's dentition have also been reported following endoscopic and bronchoscopic interventions. in the case of such a trauma, the patient this should include a clear apology and a description of the events that led to damage and the efforts made to minimize any complications. all actions and discussions should be clearly documented in patient 's records. during the preoperative assessment, the anesthesiologist should enquire about loose teeth, unstable crowns, veneers, bridgework, and any intraoral prosthesis (dentures or orthodontic appliances). minimizing dental injuries begins with the anesthesiologist 's preoperative assessment of the patient 's oral health including dentition. documentation of the patient 's preoperative dental condition and informing the patient about the potential dental damage will diminish costs for any related postoperative dental treatment. upon discovery of a potentially hazardous dental condition, whilst there is no standardized method for recording this, a simple diagram and a brief written description may be satisfactory. all risk factors, both anesthetic and dental, should be identified and explained to the patient. the flange of the macintosh blade is responsible for much damage, and alternative equipment or techniques of endotracheal intubation should be considered, particularly when risk factors are present. custom mouth guards can be useful to decrease the force of a laryngoscope affecting the upper central incisors. oral tissue trauma, a common anesthetic complication, should be considered a recognized hazard of endotracheal intubation during general anesthesia. before administering anesthesia, understanding and recognizing the oral anatomical conditions and pathological changes may help the anesthesiologists prevent oral and dental complications, thus avoiding legal suits.	anesthesiologists consistently work in the mouth of patients but are not exposed to comprehensive education of teeth, the surrounding structures, and intraoral prosthesis. one of the most common adverse events related to anesthesia is perioperative dental damage. to minimize these dental injuries, a preoperative assessment of patient 's dentition and intra - oral tissues should be undertaken.
pharmacological substances carry an intrinsic risk of toxicity as the result of either idiosyncrasy or overdose. for example, there were 2,188,013 cases of human exposures to various toxic substances resulting in 20,749 cases of serious adverse reactions and 1,552 deaths in 2013. in such cases, hemodialysis was used in more than 2,290 cases. in the year 2014, pharmaceutical toxicities were responsible for 61.4% of cases and nonpharmacological exposures accounted for 14.1% of registered cases in 2014. the goal of this article is to review the data and evidence on the use of rrt in the management of certain pharmacologic overdoses. first, we review and discuss the different factors that would affect dialyzability of drugs and toxins. second, we discuss different extracorporeal treatment modalities with focus on hemodialysis and hemofiltration treatments. third, we review the role of rrt in the management of specific drugs and poisons including toxic alcohols, salicylate, lithium, metformin, valproic acid, and dabigatran. lastly, we discuss the role of rrt in the management of less common miscellaneous cases of intoxication. it is important to mention that the management of the toxicities mentioned above is complex and usually requires measures in addition to dialysis. the use of extracorporeal techniques to remove toxins is justified if there is an indication of severe toxicity. the extent to which a drug is affected by extracorporeal therapies is determined primarily by several physicochemical characteristics of the drug which are summarized in table 1. these include molecular size, protein binding, volume of distribution, water solubility, and endogenous clearance. in addition to these properties of the drug, technical aspects of the procedure may also determine the extent to which a drug is removed [3, 4 ]. dialysis is dependent upon the use of a synthetic dialytic membrane with fixed pore size. the movement of drugs or other solutes is largely determined by the size of these molecules in relation to the pore size of the membrane. as a general rule, smaller molecular weight substances will pass through the membrane more easily than larger molecular weight substances. another important factor determining drug removal during dialysis is the concentration gradient of unbound (free) drug across the dialysis membrane. because the primary binding proteins for most drugs (mainly albumin) are of large molecular size, the drug protein complex is often unable to cross the dialysis membrane. drugs with a high degree of protein binding will have a low plasma concentration of unbound drug available for dialysis and therefore lower clearance. the efficacy of toxin removal is also influenced by its theoretical volume of distribution (vd). a drug with a large vd is distributed widely throughout tissues and is present in relatively small amounts in the blood. factors that contribute to a large vd include a high degree of lipid solubility and low plasma protein binding. drugs with a large volume of distribution (> 1 l / kg) are likely to be minimally dialyzed. the dialyzate used for hemodialysis is an aqueous solution. in general, drugs with high water solubility highly lipid - soluble drugs tend to be distributed throughout tissues, and therefore only a small fraction of the drug is present in plasma and is accessible for dialysis. dialysis will have a limited impact if the rate of drug removal is significantly faster by endogenous routes (> 4 ml / kg / min). it is generally accepted that use of extracorporeal treatment is justified, if at least 30% can be added to total body clearance by such treatment. the extracorporeal techniques most frequently employed for the removal of toxins are intermittent hemodialysis, continuous renal replacement therapy, and hemoperfusion. there are a few reports on the use of molecular adsorbent recirculating system (mars) in poisoning, specifically for those toxins that are strongly protein bound ; however, the use of mars is limited by its availability, technical applicability, and high costs. during hemodialysis (hd), toxins and other solutes are cleared from the blood by diffusion against a steep concentration gradient through a semipermeable membrane into dialyzate. in addition to its specific properties (table 1), the clearance of a toxic substance during hd depends also on membrane surface area and type, as well as on blood and dialyzate flow rates. hd comes in standard as well as high - efficiency or high - flux modalities. the major difference is the pore size of the membrane, the type of membrane, and the amount of dialyzate flow that occurs. increasing blood and dialyzate flow rates can increase the concentration gradient between blood and dialyzate, thus optimizing the rates of diffusion and elimination. larger - solute removal can be enhanced by increasing dialyzer flux when intermittent hd is used (for toxins > 500 d and up to 10,000 d) or by switching to hemofiltration, which is usually applied continuously as discussed later. the major drawback of hd is the risk of rebound toxicity after cessation of the treatment, due to redistribution of the toxin between body compartments. extending the hd session beyond 4 hours can to some extent an alternative or adjunctive solution is to increase dialysis session frequency or switch to continuous therapy after initial hd treatment specifically for substances with higher volume of distribution. intermittent hd is usually the first - choice extracorporeal modality because of its common availability, the rapidity of toxin removal, and the low molecular weight of the common agents of poisoning. the use of crrt has become common practice in intensive care unit (icu) settings during the last 2 decades for treatment of acute kidney injury. continuous venovenous hemofiltration (cvvh) is the most commonly used of the crrt modalities, where dialysis occurs by convective transport. in continuous venovenous hemodiafiltration (cvvhdf), diffusive transport of molecules is combined with convective removal in order to mainly improve the clearance of small solutes. the main advantage of crrt is its applicability in hemodynamically unstable patients. it can be easily set up and run by regular icu staff, thereby avoiding the need for specially trained dialysis nurses and technicians. the membranes used in crrt are typically more permeable compared to standard intermittent hd membranes. most high - flux hd membranes allow for the clearance of molecules up to 10,000 da. crrt membranes allow for the clearance of molecules as large as 20,00040,000 da and therefore would be the preferred modality for larger toxins removal. another advantage of crrt is the ability to avoid rebound of toxins removed from intravascular space, due to continuous nature of the procedure and slower rate of clearance, leading to less dramatic decreases in plasma drug levels and slower reequilibration of toxins between intracellular and intravascular spaces [5, 7 ]. although crrt gives better longer - term solute clearances (over the course of several days), it is less efficient in the short term and does not provide the rapidity of elimination afforded by intermittent hd when minimizing toxin exposure is a high priority. other disadvantages of crrt include the requirement for intensive anticoagulation which can place a patient at risk for bleeding and it is more associated with electrolyte disturbances. finally, crrt is not available at many smaller hospitals, possibly due to high equipment, training, and staffing costs [4, 8 ]. there are abundant case reports as well as a few small case series in the medical literature documenting the use of crrt in the treatment of poisonings, but specific techniques and the clinical outcomes vary considerably. some patients, particularly those who are hemodynamically unstable and are not candidates for conventional hd, may warrant a trial of crrt. if it is logistically possible, an ideal combination may be initial use of intermittent hd for rapid reduction of toxin levels followed by continuous therapy to ameliorate any postdialysis rebound when this is predicted. controlled trials to better clarify the role of crrt in treatment of poisonings would be beneficial, though such studies would be extremely difficult to conduct in this field. hemoperfusion consists of the passage of anticoagulated blood through a cartridge containing an adsorbent material such as activated charcoal or a resin. in order to be removed by hemoperfusion, the toxic substance must have binding affinity to the sorbent in the cartridge and a low volume of distribution (table 1). water - soluble and lipid - soluble substances with molecular weights ranging from 100 to 40,000 daltons are well adsorbed with hemoperfusion. in general, hemoperfusion is preferred to hemodialysis for the removal of chemicals that are lipid - soluble or are highly protein bound. however, the advantage of hemoperfusion over hd has lessened with the advent of high - flux dialysis membranes. additionally, there is generally greater expertise and availability with respect to hemodialysis than hemoperfusion. methanol, ethylene glycol, diethylene glycol, and isopropyl alcohol (also known as isopropanol) are alcohols commonly used in household solutions such as various cleaners, disinfectants, solvents, and antifreeze solutions as well as machine fluids [4345 ]. there were 52,430 exposures to alcohols resulting in 174 fatalities in 2013. the vast majority of methanol, ethylene glycol, and isopropyl alcohol toxicities arise either as a result of suicidal attempts or after drinking the toxic alcohol as a substitute for ethanol. however, the vast majority of diethylene glycol toxicities are the result of the introduction of diethylene glycol into various pharmacologic substances as a substitution for more expensive and less toxic substances. to understand the basic pathogenesis of methanol, ethylene glycol, diethylene glycol, and isopropyl alcohol toxicities, it is important to briefly review the metabolism in vivo. when ingested, both methanol and ethylene glycol undergo an initial biochemical reaction catalyzed by alcohol dehydrogenase (the same enzyme metabolizing ethanol), which converts the parent alcohol into formaldehyde and glycolaldehyde, respectively. the final products of methanol and ethylene glycol metabolism are formic acid and oxalic acid, respectively. the metabolism of methanol and ethylene glycol disrupts cellular energy metabolism leading to cellular damage [47, 48 ]. these end products result in classic features of toxicity such as retinal toxicity caused by methanol and renal injury mediated by oxalic acid. the first step of diethylene glycol metabolism also involves the alcohol dehydrogenase enzyme which converts diethylene glycol into 2-hydroxyethoxyacetaldehyde. the pathogenesis of diethylene glycol toxicity was first believed to involve the in vivo formation of ethylene glycol as the result of metabolism. however, further animal studies showed that the major toxic metabolite is 2-hydroxyethoxyacetic acid and that the metabolic conversion of diethylene glycol into ethylene glycol does not occur in vivo. the first step of isopropyl alcohol in vivo metabolism also involves the enzyme alcohol dehydrogenase, which converts it into acetone. acetone, in turn, undergoes several intermediate metabolic steps with the end product being glucose. it is important to note that, in the vast majority of cases, isopropyl alcohol appears to be less toxic than methanol and ethylene glycol which are associated with greater toxicities and mortality rates [44, 47, 48 ]. the clinical presentations of methanol, ethylene glycol, and isopropyl alcohol overlap and include cns depression, altered mental status, and seizures. retinal toxicity and blindness are more specific for methanol intoxication, and acute kidney injury and hypocalcemia are more typical for ethylene glycol intoxication. laboratory testing and diagnosis of methanol and ethylene glycol are based on the presence of a high anion gap metabolic acidosis, presence of a serum osmolal gap (a difference between measured osmolality and calculated osmolality 10), and measuring the levels of the toxic alcohols which is used for confirmation (typically these tests are not time sensitive, and treatment should not be withheld in any patient suspected of having toxic alcohol ingestion). isopropyl alcohol laboratory findings include the presence of a high serum osmolal gap, presence of ketone bodies in the blood and/or urine (because of acetone), and typically the absence of a high anion gap metabolic acidosis. a brief overview of the in vivo metabolism of methanol and ethylene glycol is presented in figure 1 and an overview of isopropyl alcohol metabolism is presented in figure 2. the management of methanol and ethylene glycol poisoning includes supportive care, respiratory support if needed (mechanical ventilation), the use of cofactors to stimulate formation of less toxic metabolites (see figures 1 and 2), and the use of either an alcohol dehydrogenase inhibitor (fomepizole) or ethanol, which work by displacing the toxic alcohol and preventing it from being metabolized by alcohol dehydrogenase. it is important to remember that fomepizole and ethanol have no effect on the metabolism and clearance of toxic metabolites such as formic acid and glycolic acid. therefore, inhibition of alcohol dehydrogenase will not translate into an improved outcome once the parent alcohol has been metabolized. rrt should be considered in cases of ongoing hemodynamic instability despite appropriate management and especially in the presence of severe metabolic acidosis, acute kidney injury, and target organ damage (retinal toxicity in methanol and acute kidney injury in ethylene glycol toxicity) [45, 47 ]. the small size, low vd, and low protein binding for these alcohols make them readily dialyzable, making standard hemodialysis the first - line therapy for extracorporeal elimination except in cases where hemodialysis is not available or in the setting of significant hemodynamic compromise where crrt would be indicated. consensus guidelines recommend hemodialysis when the levels of parent alcohols exceed 50 mg / dl, although some patients without evidence of target organ damage, acute kidney injury, and metabolic acidosis may be managed without hemodialysis. hemodialysis should also be considered in patients with ethylene glycol poisoning that have a persistent hyperosmolar state (despite appropriate management) and levels of glycolic acid above 8 mmol / l. it is important to mention that, in cases of methanol poisoning, hemodialysis enhances the clearance of methanol (the endogenous clearance of methanol is slow after alcohol dehydrogenase inhibition), but it only marginally increases the clearance of formic acid. also, hemodialysis may be less costly than therapy with fomepizole ; however, it is essential to remember that hemodialysis is associated with more complications and should be limited to patients with clear indications. end goals of hemodialysis in these patients should be normalization of acid base status, resolution of hyperosmolar states, and a decreased blood level of parent toxic alcohols (less than 25 mg / dl). redistribution of methanol and ethylene glycol can occur after hemodialysis and the serum electrolytes, osmolality, and acid base status should be monitored for additional 1236 hours after the last hemodialysis treatment [47, 48 ]. literature on the role of hemodialysis in the management of diethylene glycol is scant, and it is unclear whether the active toxic metabolite is removed by hemodialysis. nevertheless, hemodialysis should be considered in patients with progressive clinical deterioration despite appropriate care and persistent high anion gap metabolic acidosis. in cases of isopropyl alcohol toxicity, isopropyl alcohol intoxication generally has a more favorable outcome compared to methanol and ethylene glycol poisonings and the vast majority of patients will improve with supportive therapy and alcohol dehydrogenase inhibition. in the rare patient with hemodynamic instability and an isopropyl alcohol level above 4,000 mg / dl (which is usually due to a massive ingestion), hemodialysis may be considered. when considering renal replacement therapy in these patients, it is important to note that both fomepizole and ethanol are cleared by hemodialysis and the doses of fomepizole and ethanol should be adjusted accordingly. an overview of the clinical presentations, major laboratory findings, general principles of management, and indications for hemodialysis among patients with toxic alcohol ingestion is presented in table 2. a summary of toxic alcohol pharmacokinetics and the utility of hemodialysis is presented in table 3. salicylates are a group of pharmacologic agents which includes aspirin, bismuth salicylate, and local skin preparations such as salicylic acid and methyl salicylate (topical preparations occasionally cause toxicity if used excessively or in patients with skin damage leading to increased absorption) [10, 52, 53 ]. analgesics including aspirin are the most common etiology of all drug poisonings in the usa, and salicylate poisoning caused 34 out of 2,113 deaths due to poisonings reported in 2013. the major mechanism of action of aspirin is via inhibition of cyclooxygenase enzyme resulting in decreased production of thromboxane a2 and various prostaglandins. however, with higher dosages, other biochemical alterations may occur such as uncoupling of oxidative phosphorylation in the electron transport chain resulting in heat release and stimulation of the respiratory center in the medulla. a decrease in blood ph will favor formation of lipid - soluble salicylic acid which easily penetrates the blood brain barrier and undergoes renal reabsorption. when used therapeutically, aspirin has a high degree of protein binding which significantly decreases in cases of overdose and poisoning. patients with salicylate toxicity typically present with tinnitus, gastrointestinal complications (nausea, vomiting, bleeding, and liver toxicity), hyperthermia (via uncoupling of oxidative phosphorylation), pulmonary edema, and a mixed acid - base disorder (high anion gap metabolic acidosis and respiratory alkalosis via stimulation of respiratory center in the brainstem) [54, 55 ]. recent consensus panel guidelines on the management of severe salicylate toxicity recommend intermittent hemodialysis over other modalities of extracorporeal removal. hemodialysis should be strongly considered in patients with an altered mental status (which may be reflective of high salicylate content in the cns), pulmonary edema, hypoxemia, fluid overload states or presence of a medical condition limiting the administration of sodium bicarbonate (such as congestive heart failure), presence of either acute or chronic kidney failure (since it will limit the amount of sodium bicarbonate administration and endogenous salicylate clearance), salicylate levels > 90 mg / dl in acute toxicity and normal renal function and levels > 80 mg / dl in acute toxicity, and impaired renal function and in cases of failure of appropriate management. a summary of aspirin pharmacokinetics and the utility of hemodialysis is presented in table 3. the exact mechanism of action of lithium is not clear, but it may involve modulation of intracellular signaling pathways. the major route of lithium elimination is renal excretion, but it is important to note that about 80% of filtered lithium is reabsorbed. several clinical scenarios of lithium toxicity can occur such as acute overdose in a suicidal patient, acute on chronic toxicity in patients taking lithium whose renal function has declined (e.g., patients with gastroenteritis, decreased oral intake, and patients concomitantly taking other medications such as nonsteroidal anti - inflammatory drugs and angiotensin converting enzyme inhibitors), and chronic toxicity in patients who slowly accumulate the medication and develop toxicity [14, 57 ]. lithium was responsible for 6,610 cases of toxicities including 5 fatalities in the year 2013. patients with chronic lithium poisoning typically develop nephrogenic diabetes insipidus and urinary concentrating defects, neurologic symptoms (ataxia, tremors, and altered mental status), hyperparathyroidism, hypothyroidism, and weight gain. patients with more acute presentations tend to have more pronounced gastrointestinal symptoms such as nausea, vomiting, diarrhea, cardiac arrhythmias, and neurologic symptoms. laboratory findings of acute lithium intoxication may include a negative anion gap and an osmolal gap. also it is important to assess kidney function since acute kidney insults often precipitate lithium toxicity. the management of lithium intoxication includes stopping the offending medication, supportive care, and, in selected cases, renal replacement therapy such as hemodialysis. hemodialysis should be considered in patients with lithium levels > 4 meq / l regardless of symptomatology and in patients with lithium levels > 2.5 meq / l who either are symptomatic or have some clinical factors (advanced kidney disease and decompensated congestive heart failure) limiting the use of intravenous hydration. the end points of hemodialysis in patients with lithium toxicity are resolution of clinical symptoms of toxicity and lithium levels however, it is important to monitor lithium levels after the cessation of hemodialysis, since lithium tissue stores can be redistributed into the bloodstream [15, 16 ]. most cases of lithium intoxication treated with hemodialysis require at least a second session of hemodialysis following rebound. this rebound can be avoided by use of crrt as described above preferably after the initial hemodialysis session for rapid reduction of lithium level [8, 15 ]. a summary of lithium pharmacokinetics and the utility of hemodialysis is presented in table 3. valproic acid is used for the management of epilepsy, bipolar disorder, migraine headaches, and peripheral neuropathy. the mechanism of action includes modulation of gamma aminobutyric acid activity and sodium channel blockade. valproic acid is a fatty acid and its toxicity is believed to involve the inhibition of mitochondrial beta oxidation. valproic acid has a favorable molecular weight and volume of distribution to be cleared by hemodialysis, though the degree of protein binding is high at therapeutic concentrations. however, the degree of protein binding decreases with extra therapeutic concentrations due to protein saturation, thus making it amenable for hemodialysis. clinical manifestations may include altered mental status, tremors, myoclonus, hypotension, tachycardia, and respiratory depression. classic laboratory abnormalities include hyperammonemia, presence of an osmolal gap, hypernatremia, high anion gap metabolic acidosis, and elevated liver enzymes. management of acute valproic acid intoxication includes supportive care, administration of naloxone to help against respiratory depression, and antidote therapy with carnitine supplementation to offset the inhibitory effects on mitochondrial fatty acid oxidation. rrt should be strongly considered in patients with severe toxicity including those with cerebral edema (patients with papilledema, focal neurologic deficits, altered mental status, imaging findings, etc.) and hemodynamic instability and in those with valproic acid levels > 1300 mg / l. mg / l, respiratory depression, hyperammonemia, and severe metabolic acidosis (ph < 7.1). if hemodialysis is not available, then intermittent hemoperfusion or continuous renal replacement therapy is an acceptable alternative., it is important to monitor valproic acid levels after the cessation of hemodialysis, since redistribution of the medication can cause reemergence of toxicity. a summary of valproic acid pharmacokinetics and the utility of hemodialysis is presented in table 3. besides treatment of type 2 diabetes mellitus and prediabetes, it is often used in the management of polycystic ovarian syndrome. metformin 's mechanism of action includes decreased hepatic and intestinal gluconeogenesis, enhanced glucose utilization, and modulation of mitochondrial oxidation of fatty acids [20, 59 ]. the majority of overdose cases occur in patients with renal disease (either acute or chronic), advanced liver disease, and acute concurrent illness. the pharmacokinetics of metformin are generally favorable for hemodialysis and extracorporeal elimination such as a low molecular weight and minimal protein binding except with high volume of distribution [20, 21 ]. clinical manifestations of metformin poisoning are nonspecific and may include gastrointestinal symptoms such as nausea, vomiting, diarrhea, abdominal pain, altered mental status, and hemodynamic instability. laboratory features of metformin poisoning include a high anion gap metabolic acidosis due to accumulation of lactic acid. the low molecular weight, negligible plasma protein binding, and rapid transport of drug from cells to serum allow for drug removal by hemodialysis despite a relatively large vd. furthermore, its efficacy may be suboptimal in patients who present after tissue redistribution occurs as that leads to a large volume of distribution. nevertheless, hemodialysis should be strongly considered in patients with advanced renal failure, decompensated congestive heart failure, severe metabolic acidosis (ph < 7.1), and hemodynamic and clinical decline despite supportive care. whenever possible, prolonged sessions of hemodialysis should be undertaken ; alternatively crrt can be considered. a summary of metformin pharmacokinetics and the utility of hemodialysis is presented in table 3. dabigatran is a non - vitamin k oral anticoagulant used in the management of nonvalvular atrial fibrillation, venous thromboembolism, and postprocedural deep venous thrombosis prophylaxis. dabigatran represents an alternative to vitamin k antagonists in the vast majority of patients with the above - mentioned conditions. dabigatran is predominantly excreted by the kidneys and is contraindicated in patients with advanced renal disease typically defined as creatinine clearance < 30 ml / min. the major adverse effect related to the use of dabigatran is bleeding which may be minor or life - threating, such as in case of intracranial hemorrhage. until recently however, recently, a monoclonal antibody, idarucizumab, has been shown to be effective in the management of patients with dabigatran related bleeding. before the availability of idarucizumab several case reports highlighted the efficacy of rrt in the management of patients with dabigatran related bleeding. in a small study, intermittent hemodialysis enhanced elimination of dabigatran more efficiently than crrt, though dabigatran levels may rebound after cessation of hemodialysis via the effect of redistribution. dabigatran levels should be repeated and repeat hemodialysis should be considered in patients with a rebound increase in dabigatran concentration. alternatively, longer duration hemodialysis sessions or crrt may be considered specifically that most patients with dabigatran toxicity are critically ill with life - threatening bleeding or are in need for an emergent surgery, where crrt would be more tolerated. however, it is important that the vast majority of patients with dabigatran poisoning do not have advanced renal disease and do not receive renal replacement therapy. therefore, a dialysis vascular catheter must be inserted which may be difficult in an overanticoagulated patient and result in more bleeding and other complications. thus, rrt should be considered in patients with severe bleeding and patients on dabigatran requiring emergent surgery when idarucizumab is not available. it is important to note that rrt does not have a role in the management of other non - vitamin k anticoagulants. a summary of dabigatran pharmacokinetics and the utility of hemodialysis is presented in table 3. rrt has been effective in the management of various medication related toxicities [2841, 6066 ]. it is important to note that scientific data is limited to case reports and case series. a summary of some of these medications and the role of hemodialysis in the management of these toxicities is presented in table 4. the use of rrt should be considered in patients with toxic alcohol poisoning, salicylate toxicity, lithium overdose, and metformin poisoning as well as valproic acid toxicity. the role of rrt in the management of dabigatran toxicity is likely limited to cases with severe bleeding when idarucizumab is not available. rrt use should also be considered in the management of other drug toxicities on a case - by - case basis.	pharmacologic toxicities are common and range from mild to life - threatening. the aim of this study is to review and update the data on the role of renal replacement therapy (rrt) in the management of various pharmacologic poisonings. we aim to provide a focused review on the role of rrt in the management of pharmacological toxicities. relevant publications were searched in medline with the following search terms alone or in combination : pharmacologic toxicity, hemodialysis, hemofiltration, renal replacement therapy, toxicology, poisonings, critical illness, and intensive care. the studies showed that a pharmacologic substance should meet several prerequisites to be deemed dialyzable. these variables include having a low molecular weight (< 500 da) and low degree of protein binding (< 80%), being water - soluble, and having a low volume of distribution (< 1 l / kg). rrt should be strongly considered in critically ill patients presenting with toxic alcohol ingestion, salicylate overdose, severe valproic acid toxicity, metformin overdose, and lithium poisoning. the role of rrt in other pharmacologic toxicities is less certain and should be considered on a case - by - case basis.
acetaminophen (n - acetyl-4-aminophenol) also known as apap or paracetamol is one of the most widely used medicines in the united states. according to the data from ims health, recent data has demonstrated a 28% growth in the market sales of acetaminophen between 2004 and 2008 with sales approaching approximately $ 2.6 billion in 2008 alone (data from ims health). acetaminophen exhibits both analgesic and antipyretic properties and has been widely used as an active ingredient in many approved drugs. according to the u.s. food and drug administration (fda), 479 drugs contain acetaminophen (table 1). as of august 2011, 235 out of the 479 acetaminophen - containing drugs exhibit active approval status, which include 214 prescription drug products owned by 31 companies and 21 over - the - counter (otc) drug products produced by nine companies (tables 1 and 2). given its wide use and easy availability, scientists have recently begun to examine acetaminophen for off - label applications. herein, we will investigate these potential applications, and attempt, where possible, to highlight how these findings may lead to new directions of inquiry and clinical relevance of other disorders. approved drug products containing acetaminophen as an active ingredient. over - the - counter drug products (otc) containing acetaminophen. although a toxic dose of acetaminophen (3 h following 500 mg apap / kg body weight) can rapidly induce hyperglycemia (hinson., 1984) and acute liver failure secondary to clinical acetaminophen overdose can further impair the peripheral uptake of glucose (clark., 2001), recent animal studies have demonstrated that acetaminophen, when taken at lower dosages (2030 mg / kg body weight) exhibits an ability to lower blood glucose levels in several animal disease models, including diabetes, high - fat (hf) diet induced obesity, and aging. using a streptozotocin (stz)-induced diabetic mice model, shertzer. (2008) showed that acetaminophen at 20 mg / kg body weight is able to normalize stz - induced increases in blood glucose levels. this effect appears to be associated with protection against stz - induced destruction of pancreatic beta - cells given the finding that acetaminophen injection appears to be associated with the maintenance of pancreatic insulin synthesis (shertzer., 2008). like that observed in the stz model, acetaminophen at 20 or 30 mg / kg body weight has also been reported to prevent hyperglycemia in animals fed a hf diet (shertzer., 2008, 2010). in addition to preventing hyperglycemia, acetaminophen ingestion is also associated with the restoration of fasting insulin levels, improvements in glucose tolerance, and an increased ability of the hf - fed mice to boost blood insulin levels after a glucose challenge (shertzer., 2008, 2010). although the exact mechanism(s) remains unclear, these effects, like that seen in the rat stz model, have been postulated to arise from improvements in pancreatic insulin synthesis / secretion. research using both humans and animal models have demonstrated that the incidence of insulin resistance increases with age (houmard., 1995 wu. (2009a) using fisher 344 brown norway (f344bn) aging rat model have shown that age - associated increases in blood glucose can be corrected by chronic acetaminophen treatment at the dosage of 30 mg / kg body weight. it has been postulated that this effect was predicated, at least in part, on the ability of acetaminophen to diminish age - related losses in amount of muscle glucose transporter-4 (glut4) expression (wu., 2009a). further study demonstrated that acetaminophen treatment was also associated with an attenuation of muscle reactive oxygen species (ros) levels and in the amount of proteins that become oxidatively modified (wu., 2009a). it has been shown that prolonged mitogen activated protein kinase (mapk) activation is associated with decreased glut4 expression (fujishiro., 2001 ; carlson., 2003) and (2009a) showed that acetaminophen treatment could normalize the marked increases in p38- and erk mapk activation seen in aged muscle. taken together, these findings suggests that acetaminophen might function to improve blood glucose levels by employing multiple mechanisms including decreases in intracellular ros levels, diminished aging - associated mapk hyperphosphorylation and by increasing muscle glut4 expression (wu., 2009a). in addition to the beneficial effect of acetaminophen on relieving muscle soreness and pain (prior., 2011), recent studies have suggested that acetaminophen can improve aged skeletal muscle structure and function (sarcopenia). 2009b) firstly reported that chronic acetaminophen treatment at 30 mg / kg body weight is able to decrease the amount of aging - associated myocyte apoptosis and increase myocyte size (muscle fiber cross sectional area), with this latter effect occurring most likely associated with an increase in myosin and actin expression in aged muscle. these effects are believed to be mediated, at least in part, via reductions in the amount of oxidative and nitrosative stress as acetaminophen intervention lowers the amount of superoxide and the abundance of oxidatively modified proteins (wu., 2009a). it also appears that acetaminophen treatment reduces the phosphorylation of eukaryotic initiation factor 2a (eif2a ; wu., 2010), a key protein translational factor which when phosphorylated in response to stress leads to the inhibition of protein translational initiation (kimball, 1999). other data has demonstrated that acetaminophen can decrease muscle reactive nitrogen species (rns) as evidenced by reduced expression of inducible nos (inos) and s - nitrosylation of akt (wu., 2009b). the akt / protein kinase b is critical regulator of cellular homeostasis and functions to control cellular anabolism (protein synthesis, glucose uptake, and metabolism) and cell fate (proliferation, apoptosis ; wu., 2011). akt s - nitrosylation impairs akt kinase activity (carvalho - filho., 2005 ; yasukawa., 2005 ; wu., 2009b), which if not corrected can lead to a dysregulation of akt signaling. importantly, the restoration of akt function by acetaminophen is associated with improvements in protein translational signaling [increased phosphorylation / activation of s6 ribosomal protein (ser235/236) and translation initiation factor eif4e (ser209) ], increases in the amount of muscle glut4, myosin, and actin, along with a decrease in myocyte apoptosis and the prevention of age - associated hyperglycemia (wu. the positive effects of acetaminophen on skeletal muscle structure and function have also been proved in other experimental sitting. (2008) reported that acetaminophen is able to prevent hf diet induced decreases in lean muscle volume and body water retention. (2011) demonstrated during 12 weeks of knee extensor progressive resistance exercise training that acetaminophen (4 g / day) can increase exercise - induced muscle (quadriceps) hypertrophy and strength in older adults when compared to that seen with placebo, although muscle (vastus lateralis) protein content, muscle water content, and myosin heavy chain distribution did not increase. interestingly, increased muscle volume and strength by acetaminophen appears not to be mediated by cyclooxygenase (cox), as the expression of cox-1 and cox-2 was not changed with acetaminophen consumption (trappe., 2011). importantly, acetaminophen at 4 g / day for 12 weeks when used in combination with resistance training did not alter blood creatinine and alt levels (trappe., 2011), a finding which suggests that this dosage does not cause liver or kidney damage. interestingly, the effect of acetaminophen and exercise combination appears dependent on type of exercise as acetaminophen has been reported to suppress the protein synthesis response in skeletal muscle after eccentric resistance exercise (trappe., 2002), which appears to be mediated through decreasing exercise - induced pgf(2alpha) expression and hence affecting the anabolic response of muscle to eccentric resistance exercise (trappe., 2001). whether a similar finding exists for other types of exercise modalities (e.g., aerobic) is currently unclear in addition to the beneficial effect of acetaminophen on relieving muscle soreness and pain (prior., 2011), recent studies have suggested that acetaminophen can improve aged skeletal muscle structure and function (sarcopenia). wu. (2009b) firstly reported that chronic acetaminophen treatment at 30 mg / kg body weight is able to decrease the amount of aging - associated myocyte apoptosis and increase myocyte size (muscle fiber cross sectional area), with this latter effect occurring most likely associated with an increase in myosin and actin expression in aged muscle. these effects are believed to be mediated, at least in part, via reductions in the amount of oxidative and nitrosative stress as acetaminophen intervention lowers the amount of superoxide and the abundance of oxidatively modified proteins (wu., 2009a). it also appears that acetaminophen treatment reduces the phosphorylation of eukaryotic initiation factor 2a (eif2a ; wu., 2010), a key protein translational factor which when phosphorylated in response to stress leads to the inhibition of protein translational initiation (kimball, 1999). other data has demonstrated that acetaminophen can decrease muscle reactive nitrogen species (rns) as evidenced by reduced expression of inducible nos (inos) and s - nitrosylation of akt (wu., 2009b). the akt / protein kinase b is critical regulator of cellular homeostasis and functions to control cellular anabolism (protein synthesis, glucose uptake, and metabolism) and cell fate (proliferation, apoptosis ; wu., 2011). akt s - nitrosylation impairs akt kinase activity (carvalho - filho., 2005 ; yasukawa., 2005 ; wu., 2009b), which if not corrected can lead to a dysregulation of akt signaling importantly, the restoration of akt function by acetaminophen is associated with improvements in protein translational signaling [increased phosphorylation / activation of s6 ribosomal protein (ser235/236) and translation initiation factor eif4e (ser209) ], increases in the amount of muscle glut4, myosin, and actin, along with a decrease in myocyte apoptosis and the prevention of age - associated hyperglycemia (wu. the positive effects of acetaminophen on skeletal muscle structure and function have also been proved in other experimental sitting. (2008) reported that acetaminophen is able to prevent hf diet induced decreases in lean muscle volume and body water retention. (2011) demonstrated during 12 weeks of knee extensor progressive resistance exercise training that acetaminophen (4 g / day) can increase exercise - induced muscle (quadriceps) hypertrophy and strength in older adults when compared to that seen with placebo, although muscle (vastus lateralis) protein content, muscle water content, and myosin heavy chain distribution did not increase. interestingly, increased muscle volume and strength by acetaminophen appears not to be mediated by cyclooxygenase (cox), as the expression of cox-1 and cox-2 was not changed with acetaminophen consumption (trappe., importantly, acetaminophen at 4 g / day for 12 weeks when used in combination with resistance training did not alter blood creatinine and alt levels (trappe., 2011), a finding which suggests that this dosage does not cause liver or kidney damage. interestingly, the effect of acetaminophen and exercise combination appears dependent on type of exercise as acetaminophen has been reported to suppress the protein synthesis response in skeletal muscle after eccentric resistance exercise (trappe., 2002), which appears to be mediated through decreasing exercise - induced pgf(2alpha) expression and hence affecting the anabolic response of muscle to eccentric resistance exercise (trappe., 2001) whether a similar finding exists for other types of exercise modalities (e.g., aerobic) is currently unclear. clinical studies have suggested that the risk for major cardiovascular events will be increased if acetaminophen is taken frequently (22 days / month) or at high doses (15 tablets / week ; chan., 2006). nonetheless, when used properly, acetaminophen has been shown to exhibit cardioprotective effects. using an isolated guinea pig heart model, several studies have shown that acetaminophen (0.35 mm) can increase coronary vascular resistance and positive inotropy (merrill., 2001), attenuate ischemia - reduced monophasic action potentials (merrill and goldberg, 2001), and improve left ventricular contractility (rate of developed pressure) during post ischemia reperfusion (merrill., 2001 ; merrill, 2002). further examination using electron microscopy has suggested that acetaminophen can preserve left ventricular myofibrillar ultrastructure in the reperfused myocardium, including protection against ischemia / reperfusion - induced diffused and blurred z lines, the presence of contraction bands, and swollen, sparsely packed mitochondria (merrill., 2001). using two dog models of ventricular arrhythmias induced by regional myocardial ischemia / reperfusion or ouabain (25 g / kg), merrill. (2007) demonstratedan anti - arrhythmic effect for acetaminophen, and found that acetaminophen (15 mg / kg, i.v.) significantly reduce the number of ventricular ectopic beats during ischemia and reperfusion, the amount of ouabain - induced ventricular premature beats, ventricular salvo, ventricular bigeminy, and non - sustained ventricular arrhythmia. in the iron - overloaded gerbil, walker. (2007) have demonstrated that acetaminophen is able to prevent iron overload - induced cardiac structural and functional changes, including alterations in cardiac rhythm, ventricular distension, reductions in left ventricular ejection fraction, decreases in fractional shortening, and decreases in mortality (walker., 2009). (2010) reported that ingestion of acetaminophen (1.5 g) can improve the performance of a 10-mile cycle time trial (tt) with no difference in exertion or perceived pain, and that cyclists who ingested acetaminophen had a higher mean power output and heart rate. merrill. (2004) using a myocardial infarction dog model showed that acetaminophen at 30 mg / kg body weight is able to decrease infarct size. these researchers also showed acetaminophen treatment can reduce cardiac damage, including swollen mitochondria and fragmented nucleus (merrill., 2004). conversely, others using different animal models did not show beneficial effects on infarct size in non - preconditioned rats (dai and kloner, 2003) or in coronary artery occlusion / reperfusion rabbits (hale and kloner, 2004), however all suggest that acetaminophen is a safe drug in the postmyocardial infarction setting (dai and kloner, 2003 ; hale and kloner, 2004 ; leshnower., 2006). further studies defining detailed conditions are needed to verify the protective effect of acetaminophen on infarct size. (2004) reported that acetaminophen (0.251 mm) treatment ex vivo can inhibit cyanide - induced superoxide anion generation and lipid peroxidation in rat brain homogenates. further animal study has suggested the acetaminophen (100 mg / kg / day, i.p.) can inhibit quinolinic acid (qa)-induced lipid peroxidation, superoxide anion generation, and cell damage in the rat hippocampus (maharaj., 2006). (2009) also reported acetaminophen (5100 mg / kg) can reduce brain and microsomal lipid peroxidation, while it also increases brain vitamin e levels and microsomal glutathione peroxidase (gsh - px) activity. (2002) used rat primary hippocampal neurons and rat pheochromocytoma cells demonstrated that acetaminophen (100 m) can protect against amyloid beta - fragment - induced impairment of mitochondrial redox activity, increases in phospholipid peroxidation, and apoptotic nuclear fragmentation, suggesting a possible therapeutic effect of acetaminophen on alzheimer s disease. it is well known that acetaminophen overdose can lead to oxidative stress and induce hepatic and renal damage (ghosh., 2010 ; agarwal., acetaminophen is initially metabolized in the liver, and generates the toxic metabolite, n - acetyl - p - benzoquinone imine (napqi). napqi can be efficiently detoxified by glutathione (gsh) which is an important cellular antioxidant for detoxification of drugs and foreign chemicals. when overdosed with acetaminophen, intracellular glutathione can be depleted within 14 h (al - turk and stohs, 1981 ; porubek., 1987 ; lores arnaiz., 1995), resulting in accumulation of intracellular reactive oxygen and nitrogen species (ros / rns), and oxidative / nitrosative stress will occur. the over - produced napqi can covalently bind to the cysteinyl thiol groups of cellular proteins and form protein-(cystein - s - yl)-apap adducts (hoffmann., 1985), which may impair protein function. given this mechanism, antioxidant therapy using n - acetylcysteine (nac) is commonly used to attenuate acetaminophen - induced hepatotoxicity. interestingly, extensive animal and in vitro studies have suggested that acetaminophen possesses remarkable antioxidant properties when used within the therapeutic dosage. acetaminophen is phenolic in structure with a substituent at the para position relative to the hydroxyl group (figure 1) which allows it to react with reactive species (dinis., 1994 ; shertzer., 2008) 2008) using cell - free assay systems demonstrated that acetaminophen at a concentrations of 210 m is able to directly scavenge reactive oxygen. (2009) found that acetaminophen has higher reactivity with peroxyl radicals than many widely used phenolic antioxidants, including ubiquitous butylated hydroxytoluene (bht). other in vitro studies showed that acetaminophen can significantly inhibit hemoprotein - induced lipid peroxidation by its ability to reducing ferryl heme to its ferric state and the quenching globin radicals (boutaud., 2010). acetaminophen has also be shown to reduce the degree of low - density lipoproteins (ldl) hydroperoxides induced by cu ions (ozsoy and pabuccuoglu, 2007) or myeloperoxidase in presence of nitrite and hydrogen peroxide (chou and greenspan, 2002). several studies have also demonstrated that acetaminophen can directly scavenge peroxynitrite (van dyke., 1998 ; rork. 2008), a highly reactive oxidant and nitrating agent that can oxidize lipids, proteins, and nucleic acids. (2009) showed that when nitrogen is incorporated into the phenolic ring the antioxidant activity of acetaminophen is greatly reduced and that this finding seems to be associated with increased o however, this structural change increases the efficacy of acetaminophen to act as an inhibitor of lipid hydroperoxide biosynthesis by soybean lipoxygenases-1 (slox-1) (nam., 2009) and further evidences suggested that altered acidity of the phenolic o h may lead to chelation of the catalytic non - heme iron atom in slox-1 (nam.,, it appears that the structure of the acetaminophen phenolic ring is critical for its pharmacological and antioxidant properties. the phenolic structure with a substituent at the para position relative to the hydroxyl group allows acetaminophen to react with reactive species and possesses antioxidant activity. ex vivo and in vivo animal studies have suggested that acetaminophen can effectively reduce ros / rns in multiple tissue types. back in 1983, dubois. (2008) also showed that acetaminophen (20 mg / kg) can decrease liver mitochondrial h2o2 formation in both control and hf diet fed mice. (2009a, b, 2010) demonstrated that acetaminophen treatment at 30 mg / kg for 6 months can attenuate aging - increased skeletal muscle ros content, the amount of proteins that are oxidatively modified, and protein s - nitrosylation, suggesting acetaminophen can decrease oxidative / nitrosative stress during aging. using a rhabdomyolysis - induced renal failure animal model, boutaud. (2010) showed that acetaminophen (100 mg / kg, i.p.) can significantly decrease myoglobin - derived radical species and that this finding was associated with reduced renal damage and improved renal function. usinga hf fed animal model, shertzer. (2010) demonstrated that acetaminophen (30 mg / kg / day) was able to inhibit production of reactive oxygen species (at least partially via inhibition of nadph oxidase activity) and lipid peroxidation in white adipose tissue (wat) which improved glucose tolerance and insulin sensitivity in hf animals. although not yet well understood, the cardioprotective effects of acetaminophen seem to relate to its antioxidant property (merrill and goldberg, 2001 ; merrill. (2007) reported that acetaminophen (0.35 mm) can reduce mitochondrial swelling, and inhibit the mitochondrial permeability transition pore - induced apoptotic pathway and mitochondrial cytochrome c release in heart with induced low - flow global myocardial ischemia. (2001) demonstrated that acetaminophen can significantly attenuate the burst of hydroxyl radicals during the first 10 min of reperfusion, and block 3-morpholinosydnominine (sin-1)-induced peroxynitrite generation. (2010) have found that chronic acetaminophen treatment at 30 mg / kg body weight is able to attenuate aging - associated increases in cardiac oxidative (superoxide) and nitrosative (protein nitrotyrosylation) stresses, caspase-3 activation, and apoptosis in f344bn rats. (2006) reported that acetaminophen (0.35 mm) can reduce peroxynitrite in the isolated guinea pig myocardium and that this finding was associated with an attenuated activation of mmp-2 and decreased cleavage of troponin i (tni) following ischemia / reperfusion. therefore, cardioprotective effects of acetaminophen are at least partially mediated by reducing tissue reactive oxygen and nitrogen species. recent experimental data suggests that acetaminophen may have several remarkable effects other than its well known analgesic / antipyretic properties. thus far, acetaminophen has been shown to improve blood glucose control, improve skeletal muscle structure and function in the aged, and that this agent exhibits cardioprotective and neuroprotective effects (figure 2). current laboratory and pre - clinical studies have revealed that many of these findings can be linked to its incredible antioxidant properties. it is also worth noting that since acetaminophen overdose or ingestion with alcohol can cause hepatotoxicity and death, well controlled clinical studies must be conducted to ensure the safety and efficiency of acetaminophen before its clinical application for off - label application. in addition to the clinically proven analgesic / antipyretic properties, laboratory and pre - clinical studies demonstrated that acetaminophen has other beneficial effects that would increase clinical application of acetaminophen. however, clinical studies are needed to ensure its safety, efficiency, and proper dosage. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	acetaminophen, also known as apap or paracetamol, is one of the most widely used analgesics (pain reliever) and antipyretics (fever reducer). according to the u.s. food and drug administration, currently there are 235 approved prescription and over - the - counter drug products containing acetaminophen as an active ingredient. when used as directed, acetaminophen is very safe and effective ; however when taken in excess or ingested with alcohol hepatotoxicity and irreversible liver damage can arise. in addition to well known use pain relief and fever reduction, recent laboratory and pre - clinical studies have demonstrated that acetaminophen may also have beneficial effects on blood glucose levels, skeletal muscle function, and potential use as cardioprotective and neuroprotective agents. extensive laboratory and pre - clinical studies have revealed that these off - label applications may be derived from the ability of acetaminophen to function as an antioxidant. herein, we will highlight these novel applications of acetaminophen, and attempt, where possible, to highlight how these findings may lead to new directions of inquiry and clinical relevance of other disorders.
in recent years there has been a growing interest in the therapeutic use of herbal medicines or phytopharmaceutical products [1, 2 ]. the reasons of such interest can be referred mainly to the fears of abuse and misuse of synthetic drugs, the side effects or inefficacy of conventional medicine, the access to conventional pharmacological treatment is not available to whole population, and the suggestions regarding the safety of natural products. ivy - thyme cough syrup is an herbal medicine that combines the extract of hedera helix (english ivy, family : lamiaceae) leaves along with the extract of thymus vulgaris (thyme, family : araliaceae) herb for the purpose of cough treatment. as a medicinal plant, h. helix leaf extract is approved by the german commission e for its efficacy against chronic inflammatory bronchial conditions and productive coughs due to its actions as an expectorant and its spasmolytic effect among children and adults [5, 6 ]. these effects are attributable, in particular, to the therapeutically important constituents of the ivy leaf extract, which belong to the class of triterpene saponins such as hederacoside c (figure 1), a marker and an active ingredient of ivy leaf extract [710 ]. on the other hand, thyme is a medicinal herb whose leaf extract is approved by commission e in the treatment of bronchitis, whooping cough, and upper respiratory inflammation. thyme and its various extracts are well known for their bronchospasmolytic, expectorant, and antibacterial effects. these latter effects are most likely attributed to the phenolic components (thymol and carvacrol) of the plant volatile oil [12, 13 ]. to evaluate the quality of herbal medicines, reliable analytical methods have to be applied to the quantitative determination of the constituents with known therapeutic activity or to the markers in final products [1, 14 ]. for the quality control of ivy - thyme cough syrup, hederacoside c and thymol, the active markers of ivy and thyme, respectively, are needed to be quantified by a validated method in order to determine the contents of their corresponding plant extracts in the finished product. accordingly, the objective of the present study was to develop a simple, reliable, and validated analytical method for the quantification of hederacoside c, the marker of ivy, by the use of high - performance liquid chromatography (hplc) with uv detection, a method that can be used efficiently for routine quality control and analysis of such herbal product. actually, the challenge in the development of such a method is attributed to the presence of hederacoside c in a complex matrix of the cough syrup. it seems that the presence of other saponins from other extracts (e.g. thyme extract) used in cough preparations as well as other ingredients of the cough syrup may interfere with the chromatographic peak of hederacoside c and, thus, its quantification. these factors increase the complexity of analysis and necessitate the development of a new analytical method for the accurate quantification of hederacoside c, the quality marker of ivy. the hplc system used was a shimadzu class - vp system equipped with a model series lc-10 advp pump, fcv-10alvp low pressure gradient flow controller valve, scl-10 advp controller, 50 l loop injector, cto-10as column oven, and a spd - m10avp diode array detector. the standardized raw material of hedera helix leaves (standardized to contain 14.8% of hederacoside c) was supplied by fenzilberg (germany), hederacoside c standard with purity of more than 99% was purchased from phytoplan (germany), orthophosphoric acid (h3po4) 85% from merck (germany), filter membranes were purchased from vivid (ptfe/0.45 m/25 mm diameter), ivy - thyme cough syrup (potency : 0.7 mg hederacoside c and 0.2 mg thymol per 1 ml syrup), and placebo were generously provided by sana pharmaceutical research co. (jordan). all solvents used were of hplc grade ; acetonitrile and methanol gradient grade were purchased from scharlau (spain), bidistilled water was produced via a bi - dest 2302 gfl (germany). the column was a reversed - phase phenomenex - gemini c18 (250 4.6 mm i.d. ; 5 m) equipped with a phenomenex security guard column (4.0 3.0 mm i.d.). for hplc separation, the mobile phase consisted of a binary mixture of solvent - a (h2o : acn : h3po4) in the ratio of (860 : 140 : 2) and solvent - b (acn : h3po4) in the ratio of (998 : 2) with a gradient program as follows : 060% b (040 min), 60100% b (4041 min), 100% b isocratic (4155 min), and return to 0% b (55 - 56 min), and finally, reconditioning the column with 0% b isocratic (5570 min). the uv detector was set at 205 nm and separation was performed isothermally at 40c. a reference solution of hederacoside c was prepared at a concentration of 1.0 mg / ml by transferring 10 mg of hederacoside c to a 10 ml volumetric flask, methanol was added, and the solution was sonicated for 10 min then completed to volume. hederacoside c stock solution containing 1.0 mg / ml of hederacoside c was prepared by transferring 3.378 g of standardized ivy raw material powder (containing 14.8% hederacoside c) in 500 ml volumetric flask, filled up to 80% of its volume with methanol, then it was sonicated for 30 minutes in the ultrasonic bath to dissolve hederacoside c. after cooling to room temperature, the volume was filled up to the mark with methanol. calibration standard solutions with different concentrations were prepared by appropriate dilutions from the stock using methanol as the diluent. 1.7 g of ivy - thyme cough syrup was weighed in 10 ml volumetric flask, filled with 80% of the volume with methanol, sonicated for 15 min to homogenize the solution in ultrasonic bath, and after cooling to room temperature the flask was filled up to the mark. according to this procedure, the final solution is supposed to have a concentration of 0.1 mg / ml hederacoside c. challenges in the development of an hplc method with efficient separation of hederacoside c in cough syrup include its separation from a saponin mixture coming from ivy extract along with its separation from other saponins present in the thyme extract. in addition, hederacoside c, like almost all other saponins, lacks a chromophore ; it absorbs uv - light at wavelengths below 210 nm [8, 16 ], which makes its detection in a complex matrix of a herbal product along with its analysis by the use of gradient elution not an easy task. to the best of our knowledge, the present study is the first to report a validated method for the analysis of ivy extract in pharmaceutical products (syrup) and the first on ivy - thyme mixture. in literature, analysis of ivy plant and extracts, using hederacoside c as a marker, has been generally performed by means of rp - hplc and uv detection (wavelengths below 210 nm) based mainly on c18 columns under gradient elution [6, 17, 18 ]. for the analysis of other types of saponins, some of which from other natural sources, comparable methods with modified conditions, solvent systems or detector were also developed and reported [16, 19 ]. unfortunately, reproducing these methods to the analysis of ivy - thyme syrup under study was always unsuccessful. in the present study, the developed hplc method ensured sufficient chromatographic separation (figure 2(a)), and accurate and precise quantification of hederacoside c. moreover, the use of uv detector, the most common type of detectors used with hplc instruments [14, 20 ], makes the developed method a simple and suitable method for routine quality control of ivy - based cough syrups. the developed method was validated according to the guidelines of the international conference on harmonization (ich) for validation of analytical procedures and usp for its linearity, precision, accuracy, and specificity. the calibration range over which the analysis should be validated is determined according to the purpose of the analysis. for the quantification of an active ingredient in final product, hederacoside c in cough syrup ; the suggested range to be covered according to ich guidelines is 80%120% of the test concentration. in our experiment the chosen range was (0.030.15) mg / ml of hederacoside c, which corresponds to a range of 30%150% of the test concentration (0.1 mg / ml). the linearity of the hplc method for determination of hederacoside c was evaluated by analyzing a series of different concentrations of hederacoside c. according to ich guidelines, at least five concentrations must be used covering the specified range. in the present experiment, seven different concentrations of hederacoside c were chosen, covering the range of (0.030.15) mg / ml of hederacoside c. each concentration was injected in duplicate. the method was found linear over the specified range with a correlation coefficient (r) value of 0.9992 (table 1). for the evaluation of method precision, repeatability and intermediate precision were performed at 100% of the test concentration as guided by ich. six samples of the drug, at 0.1 mg / ml concentration level of hederacoside c, were prepared and analyzed by the developed method at the same day for repeatability assessment, and then repeated 24 hours apart for the evaluation of intermediate precision. coefficient of variation (or relative standard deviation, rsd) has been calculated, the results indicated that the developed method was with acceptable precision (table 2). injection of sample placebo (consisting of all matrix components with the exception of hederacoside c) in duplicate under the conditions of the developed hplc method showed that there was no interference from sample matrix (figure 2(b). in addition, peak purity testing of hederacoside c showed that the peak refers only to one component with peak - purity coefficient of 0.996. this test was performed by the addition of known amounts of the hederacoside c standard to cough syrup placebo. the resulting mixtures, at three different concentration levels covering the method range, were assayed by the developed method analyzing 6 replicates at each concentration. the good recovery values obtained and their low rsd (less than 2%, table 3) suggested that the accuracy of the proposed method was acceptable. based on the values of standard deviation of the response (sd), calculated as the standard deviation of y - intercepts of regression lines, and slope (s) obtained from the linear regression equation described above, the lod and loq values were calculated, according to the ich guidelines, as follows:(1)lod=3.3(sds),loq=10(sds). from these data, shown in table 4, the developed method proved to be suitable for detection and quantitative determination of even very low drug contents (lod = 0.011 mg / ml, and loq = 0.032 mg / ml). a validation of the calculated loq, as according to ich guidelines, was also performed by the analysis, in duplicate, of 6 samples containing hederacoside c at the concentration of 0.03 mg / ml. the method proved to be with good repeatability (cv = 1.88) and intermediate precision (cv = 1.93) at the calculated loq. on the other hand, linearity and accuracy parameters were not tested because the calculated loq already occurs within the tested drug range (0.030.15 mg / ml) for these two parameters conducted as above. a simple and reliable reversed phase high - performance liquid chromatographic method with uv detection for the quantification of hederacoside c, in the cough syrup has been developed and validated for its linearity, precision, specificity, and accuracy, providing a suitable and practical analytical method for the routine quality control analyses necessary for providing herbal medicines with high safety and efficacy.	a simple reversed phase high - performance liquid chromatographic (rp - hplc) method coupled with a photodiode array detector (pad) has been developed and validated for the analysis of hederacoside c, the marker of ivy plant, in ivy - thyme cough syrup. separation of hederacoside c was achieved using a phenomenex - gemini c18 column isothermally at 40c. a mobile phase system constituted of solvent a (water : acetonitrile : orthophosphoric acid (85%), 860 : 140 : 2 v / v) and solvent b (acetonitrile : orthophosphoric acid (85%), 998 : 2 v / v) was used, at gradient conditions, at a flow rate of 1.5 ml / min. analysis was performed using uv - detection (205 nm). the method was linear over the range (0.030.15) mg / ml of hederacoside c (r = 0.9992). repeatability and intermediate precision were acceptable (rsd < 2%). limits of detection (lod) and quantitation (loq) were 0.011 and 0.032 mg / ml, respectively. percentage recovery was found to lie between 99.69% and 100.90% (rsd < 2%). the method was also proved to be specific (peak - purity coefficient = 0.996).
reproductive rights are one of the sub - domains of reproductive health and act as the foundation for the development in a society. in a paragraph of these rights, which is associated with the domain of marriage and familial relationship, men are asked to accept responsibilities and to have commitments in provision of reproductive health equal to women and to cooperate with their spouses in different stages of their marital life. based on this issue, socially approved and accepted behaviors of women and men in their family life through which they acquire their gender - based roles can be suggested as an indicator to manifest this dimension of reproductive rights. gender roles are normative behaviors and attitudes that are expected from individuals based on their biological sex. these roles have a vast concept based on which numerous responsibilities and assignments in the family are defined, as the meaning of gender role is the definition of manhood and womanhood in a society. gender role attitudes reflect individuals attitudes concerning appropriate role activities for men and women and contain the general concept from gender - based roles such as gender - related duties in the family. numerous researchers have investigated the importance of gender role attitudes and their effect on various dimensions of human behaviors such as domestic violence and health promotion behaviors. research showed that having egalitarian attitudes in gender roles can lead to some outcomes like increased feeling of self - efficacy in women and men, more adaptation in marital life, decreased number of requests by men for divorce, increased participation of spouses in taking care of children, increased employment and income of women, decreased suicidal thoughts, and decreased inter - generational gaps. meanwhile, some of the gender role attitudes in the family are associated with couples sexual affairs. sexual affairs are considered as one of the most important domains of reproductive health and quality of life in the family. in islam, sexual relationship has been considered as a part of human 's identity, so achieving mutual satisfaction among the couples has been emphasized. achievement of sexual satisfaction is of great importance for the couples, and increased sexual satisfaction is accompanied with increased satisfaction among the couples in their marital life globally. ability to have an informed pleasant and safe sexual life is based on positive expression of sex specifications and reciprocal respect of men and women in their sexual relationship. non - sexual interactions between sexual partners and inappropriate attitudes and beliefs toward sexual issues have notable effect on the incidence of sexual dysfunctions. gender role attitudes in the domain of sexual affairs represent appropriate role activities for men and women during their sexual relationship. research showed that having egalitarian gender role attitudes can lead to improvement of sexual function, increase of satisfaction in marital life among men and women, promotion of mental health, and a better relationship with the sexual partner. existing international instruments that are used to measure gender role attitudes often pay less attention to sexual affairs domain. attitudes toward women scale and sex - role egalitarianism scale, which have considered these roles in some items, indicate the sexual relationship out of the marriage, which is not acceptable in iranian islamic culture. there is consensus on the fact that gender role attitudes are absolutely culture based and different in each society ; therefore, the instrument to measure them should be designed accordingly. a measurement tool in research should coincide with the mental realities of its respondents, so as to be perceived the same way the researcher intends. considering the fact that gender role attitudes are culture oriented and due to the importance of these attitudes in couples sexual affairs domain and lack of a questionnaire adapting to iranian culture, the researchers decided to primarily explore various aspects of partners gender - based roles in sexual affairs in iranian families in a qualitative study, and then generate the items to measure gender role attitudes in this domain and assess these attitudes in the study population quantitatively. the present study is a part of a greater exploratory mixed methods study which aimed to explore and assess gender role attitudes in all domains of iranian family life. the present article reports gender role attitudes in the domain of iranian couples sexual affairs. in an exploratory mixed methods study, the goal is that the primary results, obtained by the first method (qualitative), can develop and form the second method (quantitative). this design is especially helpful when the researcher needs to develop an instrument, or needs to detect important variables of a domain due to unavailability of an appropriate instrument or the unknown variables effective on an outcome. in this type of research, the quantitative phase is consequently conducted after ending the qualitative phase and based on its findings. with regard to ethical considerations of the project, this study firstly received the approval of the related university ethical committee (no. 89.d.130/676), and based on their recommendations, qualitative interviews and quantitative sampling were administered by interviewers of the same gender as that of the participants. in the first qualitative phase of the present study, researchers explored participants perceptions and experiences about gender - based roles in the domain of couples sexual affairs through the content analysis method. in this phase, purposeful sampling with maximum variations was conducted on iranian women and men 20 years who were married at the time of the study. in order to achieve maximum variations among the participants, they were selected so as to have the highest variations concerning their age, education level, job categories, duration of marital life, and the number of children. men and women were selected in every other gender, in order to have a balance in the number of male and female participants and to have various perceptions and experiences of both genders. participants were firstly selected among the individuals accessible to the researchers ; then, snowball method was adopted to select the rest of participants. data collection goes on until the participants yield either little new data or repeat their previous ideas. in this phase, semi - structured individual interviews and the interviewers of the same gender as that of the participants (two principal researchers) conducted the interviews and group discussions. focus group discussions were held in five - member groups with an identical gender, and the participants, as mentioned before, were selected with maximum variations. at the beginning of the present study, individual interviews were conducted, and then, in order to be sure that the existing interaction in group discussions could lead to collection of new data, focus group discussions were adopted. the interviews were held in the participants houses, researchers offices in the faculty, health care centers, or a research center, based on participants agreement and preference. after getting a written informed consent from the participants, conventional content analysis was used to analyze the qualitative data. in this type of analysis, the researcher inductively and just based on research data begins coding process of the interviews transcriptions. in the qualitative phase of the present study, data analysis was conducted concurrently with data collection. in order to verify the rigor of data in this phase of study, criteria of credibility and dependability of the data were used. for credibility of the findings, we used member checking ; and for dependability, two principal researchers (a female and a male) conducted the interviews with the women and men, respectively. they independently coded all of the transcriptions, and the cases concerning the lack of consensus were modified through consultation. a part of the transcriptions was independently recoded by an external evaluator out of research team, and the cases concerning the lack of consensus were modified through face - to - face discussions. after the qualitative phase ended, the findings of this phase were used to generate some items to assess the research subject quantitatively. these items underwent stages of face validity (through a pretest on female and male individuals selected from general population of tehran), content validity (with approval of 10 experts in courses of reproductive health, maternal and child health, psychology, psychiatry, and health education and promotion) and structure validity (through testing in general population of tehran). as the stages of scientific validity and reliability of the instrument have been conducted for our broader study to explore and assess gender role attitudes in all domains of family life, and the details related to instrument developing are out of the objectives of the present article, they are not mentioned in this paper. in the quantitative phase of the study, which was conducted on the general population of tehran, (iranian women and men 20 years), the sample size was calculated to be 385 subjects based on the following formula and significance level of 0.05 (z = 1.96, = 66, d = 6.6), and thus firstly, 80 blocks were selected through quota sampling from the city, which were assigned to women (n = 40 blocks) and men (n = 40 blocks). in each of the blocks, using systematic sampling, five families residing in the block were selected and the respondent in each family was selected through quota sampling, so that age distribution of the subjects matched the general population. in the qualitative phase of the present study, 21 participants (11 women and 10 men) residing in three provinces of iran who were accessible to the research team due to either the researchers location of study or their working in these provinces (tehran, semnan, or khorasan razavi) were selected during 2010 - 2011 and sampling ended by data saturation. the participants were from different job categories (workers, employees, managers, homemakers, retired, self - employed, students, etc.). their marital life duration ranged 1 - 36 years, and their number of children ranged 0 - 3. after categorizing the codes obtained by content analysis of the qualitative data of the present study, based on their similarities and dissimilarities, four categories emerged for couples roles in the domain of sexual affairs, i.e. decision making, relationships, care, and supervision and control. for instance, one of the participants whose narrations led to emergence of the category of decision making stated : their decision making about when to have sexual intercourse and when not to have is very important. you see, there are some books recommending when (a certain time) to be conceived and when not to be. he should consider his spouse 's physical and psychological status, and then does that ; of course if his spouse desires. (21-year - old female student with no children) another participant whose narrations were placed in the category of relationships said : the husband and wife should not be in disguise in their sexual affairs. they should absolutely express the things, which bother them or can better their relationship. (39-year - old female employee with one child) another participant whose narrations led to emergence of the category of care stated thus : it is not right that one partner emotionally cares about his / her spouse and the other one not. for example, one says i do this as i like my husband to be satisfied or to enjoy more. if so, one can upmost tolerate something against her desire for one year, two years or six months but not longer. it leads to problems in long term we (the spouses) should be careful about each other. (48-year - old male employee with two children) the emerged codes from another participant 's narrations were categorized in supervision and control category. i like they honestly declare anything about genital infections to each other, the husband to his wife or the wife to her husband. after being honest to each other and understanding their sexual problems, they should check it whether they followed treatment correctly or not, or check whether the partner follows up the physician 's order or not. if they should not have an intercourse or consume any medication (49-year - old female homemaker with two children) after ending the qualitative phase of the study, the emerged codes were written in the form of items of a questionnaire. out of 21 items that emerged from the qualitative phase of the study, 7 items remained after item reduction stages, which were used in the quantitative questionnaire. individuals attitudes concerning the gender - based division of these roles with a six - option spectrum of responses [entirely feminine, preferably feminine, masculine and feminine with equal role and responsibility, preferably masculine, entirely masculine, and neither feminine nor masculine : (no one should do it as it is not a correct action) ] were investigated [table 1 ]. items related to couples gender - based sexual roles after administration of the questionnaire to the general population residing in 22 urban districts of tehran in 2012, 390 questionnaires were collected to enter data analysis stage. their age ranged 20 - 76 years (mean age 38 years and sd = 13.25) and their education level varied from illiteracy to a university degree. a total of 270 subjects (69.2%) were married and their number of children ranged 0 - 7. the findings of the quantitative phase of the present study showed that most of the subjects (78.9%) believed that the spouses should mutually play all the sexual role in their marital life. in five cases of these roles, the subjects second most dominant attitude was superiority or monopoly of masculine role (71.43%), while in one case of these roles, the item of respect to genital health issues, the attitude of being entirely feminine ranked second among the respondents attitudes (14.28%). frequency distribution of gender role attitudes in the domain of couples sexual affairs with regard to investigation of the association between respondents demographic characteristics and their type of gender role attitude in sexual affairs, the analysis of the data through chi - square test (in significance level of 0.05) showed that subjects attitudes concerning the roles of talking with the spouse about sexual issues were associated with the subjects gender (p = 0.01) and level of education (p = 0.02). subjects attitude about the role of paying attention to spouse 's sexual needs and desires was associated with their level of education (p = 0.01). the research on gender differences in sexual affairs domain approved four issues related to the difference between women and men in their sexual affairs, i.e., higher sexual desire among men compared to women, more emphasis by women on having sexual relationship based on commitment to the sexual partner, more sexual aggression and invasion of men, and higher flexibility of women 's sexual affairs in response to various cultural, social, and situational factors. another research showed more sexual activities and more qualified sexual life among men compared to women, and such differences increase by age. the present study is in line with the previous studies in the detection of gender - based differences among women and men in their sexual affairs, exploring gender - based roles related to such affairs in iranian families, and through this exploration, suggesting and validating some items to be added to the existing instruments for measuring gender role attitudes. the existing international instruments used to measure gender role attitudes have often paid little attention to the division of gender - based roles associated with the domain of sexual affairs. attitudes toward women scale and sex - role egalitarianism scale, which have considered these roles in some items, pointed to the sexual relationship out of marital range, which is not acceptable in iranian islamic culture. in the qualitative phase of the present study, seven items emerged to measure gender role attitudes in sexual affairs domain, i.e., showing desire to the spouse to start sexual intercourse, selection of the kind of sexual activities with the spouse, decision making about the time and frequency of sexual intercourse with the spouse, talking with the spouse about sexual issues, paying attention to spouse 's sexual needs and desires, decision making about the way of confronting and treating the sexual matters, and respect to genital health issues. in the study of refaie shirpak. conducted in tehran (2007), based on the findings of the qualitative phase, a questionnaire was developed for need assessment among women in the domain of sexual health education. the third part of their questionnaire, which contained questions on attitudes toward sexual health, consisted of six attitude domains. we believe that three of these six domains were comparable and consistent with the items emerged in our study. their three obtained domains included : talking and discussing with the spouse about sexual issuesthe possibility of suggesting a sexual intercourse from the side of womenshared sexual satisfaction and pleasure between spouses. talking and discussing with the spouse about sexual issues the possibility of suggesting a sexual intercourse from the side of women shared sexual satisfaction and pleasure between spouses. the latter domain seemed to be similar to our paying attention to spouse 's sexual needs and desires item. (2004) conducted a study on female and male university students in ghazvin, iran, and reported some gender differences in the domain of sexual behaviors. they reported gender to be the most independent factor effective on teenage university students sexual behavior. also, in their study, a gender difference was observed in subjects perception of intensity and outcomes of venereal diseases and aids, and its effect on their life, work, and social communication. these findings seems to be comparable with respect to genital health issues obtained in the present study. no items similar to the remaining items obtained in the present study were found in the available research. with regard to the findings of the quantitative phase of the present study, although in the investigation of all questionnaire items, most of the subjects believed in mutual responsibility of the women and men to play various gender - based roles, in five items out of seven, related to sexual affairs, the second dominant attitude among the respondents was the belief in entirely or preferably masculine role in couples sexual affairs [table 2 ]. with regard to the role of showing desire to the spouse to start a sexual intercourse, peplau (2003) reported that men typically pioneer to express the sexual desire to women. in the study of hashemi. (2013) conducted on married women referring to taleghani health care center in tehran, it was declared that in 66% of the subjects, men either always or often started the sexual activity. in the study of ziaee and khoury (2006) on working married women who had at least one child residing in gorgan, iran, it was reported that only 0.6% of women always started sexual activity, while 38% of them only sometimes suggest having sex. (2007) in tehran reported that women had a positive attitude about the reciprocality of sexual affairs. they revealed the fact that women can initiate a sexual relationship, but the result of their qualitative phase of research (2010) showed that the studied women did not believe that their initiation of sexual activities was applicable and claimed that their spouses might have an unexpected reaction to this issue. as our study investigated the women 's and men 's attitude concurrently and revealed that most of the subjects from both genders believed in equality of women 's and men 's responsibility and role to manifest their desire to start a sexual activity, it seems that efficient steps should be taken to modify unrealistic concerns of some women in relation with their spouses inappropriate reaction toward their sexual desire expression. with regard to talking with the spouse about sexual issues, although in our study, most of the subjects believed in an equal role for women and men, a significant association was observed between subjects gender and their attitudes ; so that men believed this role to be more masculine compared to women. in this regard, impett and paplau (2003) reported that sexual interactions need both sexual partners to coordinate their preferences and functions with each other. in the qualitative study of refaie shirpak. in tehran (2010), all participants (both reproductive health providers and clients) emphasized on the inadequacy of couples communication concerning sexual issues. (2004) also reported that unfortunately in iranian culture, sexual issues are a taboo and it is a rare occurrence that the couples talk about them. ziaee and khoury in a study in gorgan (2006) on married working women showed that 14% of the subjects never talked about sexual issues with their spouses. lack of communication among the couples can lead to misunderstandings and dissatisfaction with their relationships. this issue can prevent talking about and reaching an agreement on the manner and quality of sexual relationship among the couples, which is consistent with the finding of the present study (selection of the kind of sexual activity with the spouse and decision making about the time and frequency of sexual intercourse with the spouse). on the other hand, this issue is important, as one of the characteristics of healthy sexual behavior is mutual agreement on sexual activity. talking with the spouse concerning sexual issues can help her to express her reason for not doing the activities she does not like or desire to do. it seems that the struggle to change the attitudes of some men related to talking about sex and empowerment of women to negotiate with their spouses on sexual affairs is one of the important interventions for which the present study can create an appropriate background. with regard to the role of paying attention to spouse 's sexual needs and desires, our findings revealed that most of the subjects believed in women and men having equal responsibility in this role. in this, in a qualitative study in tehran (2010), revealed that in some women 's viewpoint, sexual pleasure is just for men and women have no rights in this issue. as the findings of a qualitative study can not be generalized, there is no inconsistency between the present study and their study. in addition, in our study, 9.2% of the subjects also believed in dominancy or monopoly of women 's role concerning paying attention to spouse 's sexual needs and desires, which reflects the priority to provide the men with satisfaction in sexual affairs from their viewpoint. however, it seems that this problem exists in iranian society more or less and appropriate educational interventions are needed to modify these attitudes. with regard to the role of respect to genital health issues, as the second dominant attitude among the subjects in our study, the priority or monopoly was for women (11.9%). this finding is a notable point of the present study as it seems that the mutual nature of sexually transmitted infections is sometimes forgotten, and due to more overt disease manifestations among women, the prevention or treatment of the disease may be considered more as a feminine responsibility. education of organized principles of sexual health seems to solve this problem. with regard to the association between subjects gender role attitudes in the domain of sexual affairs and their level of education, our findings revealed a significant association between these two variables, so that individuals higher level of education was accompanied with the increase of their belief in women 's and men 's shared responsibility in roles such as paying attention to spouse 's sexual needs and desires and talking with the spouse about sexual issues. arends - toth. (2007), hoffman and kloska (1995), and kiani. (2009) approved the association between higher level of education and subjects more egalitarian gender role attitudes. this study extended previous research on gender role attitudes by exploring gender - based roles in the domain of sexual affairs in iranian families, and through this exploration, suggesting and validating some items to be added to the existing instruments for measuring gender role attitudes. finally, the researchers of the present study hoped to define the existing capacities to achieve some dimensions of reproductive rights in iranian families. in addition, they tried to make a background for further research related to gender roles in iranian and other similar cultures. with regard to the limitations of the study, firstly, this study explored gender - based roles in sexual affairs domain just among healthy couples, while if one or both spouses suffer from a chronic physical or mental disease, their accepted and expected roles are likely to change and new role domains can be explored. secondly, in the present study, the age difference between couples was not considered in the exploration of gender - based sexual roles, but the range of age difference among the couples may be effective on these roles. it is suggested to include these points in future studies to be able to reveal other possibly existing gender - based sexual role domains.	background : gender role attitudes toward sexual matters may define suitable and appropriate roles for men and women during a sexual relationship. this study aimed to explore and assess gender - based sexual roles in iranian families.materials and methods : this was an exploratory mixed methods study in which perceptions and experiences of 21 adult iranian participants about gender - based sexual roles have been explored in three provinces of iran in 2010 - 2011, to generate items for developing a culture - oriented instrument to assess gender role attitudes. the developed and validated instrument, then, was applied to 390 individuals of general population of tehran, iran in 2012.results:in content analysis of the qualitative phase data, four categories emerged as the main gender - based sexual roles : decision making, relationships, care, and supervision and control. after passing the stages of item reduction, seven items remained for the instrument. in the quantitative phase, results showed that most of the participants (78.9%) believed in shared sexual roles for both genders. consideration of a sexual role as entirely masculine or preferably masculine was the second prevalent attitude in 71.43% of gender - based sexual roles, whereas entirely or preferably feminine role was the second next most dominant attitude (14.28%).conclusions : the results of the present study have revealed some new gender - based sexual roles within iranian families ; which may be applicable to show the capacity for achieving some domains of reproductive rights in iran.
lymphoplasmacytic sclerosing pancreatitis (lsp) or autoimmune pancreatitis is a rare condition characterised by diffuse fibroinflammatory infiltrates that can involve both the pancreatic ducts and acinar parenchyma. it is also known as primary inflammatory sclerosis of the pancreas, sclerosing pancreatitis, autoimmune sclerosing pancreatitis, duct destructive chronic pancreatitis, and sclerosing pancreatico - cholangitis [1, 2 ]. since the condition was first described in 1961 by sarles., several cases have been reported, and in recent years, lsp has become increasingly recognised as an important and unique cause of chronic pancreatitis. of particular note, it presents with a pancreatic mass that can be extremely difficult to differentiate from pancreatic carcinoma (which it may resemble both clinically and radiologically). whipple resections are relatively common in high - volume centers (9.2%) and that lsp represents 23.4% of whipple resections performed for benign disease. the occasional association of lsp with other autoimmune conditions is well documented, including sjogren 's syndrome, primary sclerosing cholangitis, inflammatory bowel disease, and systemic lupus erythematosus. although cases of lsp associated with idiopathic retroperitoneal fibrosis have also been reported, the association is rare. to our knowledge, not more than 14 cases have been reported in the literature [411 ]. a 74-year - old afro - caribbean man presented with a 4-week history of obstructive jaundice, weight loss, and fatigue. nineteen months earlier, symptoms of polyuria and nocturia had led to a diagnosis of retroperitoneal fibrosis : renal function was impaired, and both kidneys were poorly perfused on ct scan, with evidence of retroperitoneal fibrosis obstructing both ureters. ercp showed an irregular stricture in the lower cbd suggestive of malignancy (although the biliary brushings later showed no malignant cells), and an endobiliary stent was inserted. ct confirmed that the pancreas was markedly bulky with no focal mass lesion or abnormal calcifications. there was thickening of soft tissue around the porta hepatis suggestive of lymphadenopathy. although the main concern was pancreatic carcinoma, lsp was also considered to be a possible diagnosis. an autoimmune screen was negative for ana, cytoplasmic antibodies (hep-2), anca iif, mitochondrial antibodies, smooth muscle antibodies, liver / kidney microsomal antibodies, anti - gpc, rheumatoid factor, and striated muscle antibodies. g / l), particularly igg4 at 15.10 g / l (0.01.3 g / l). although, there have been reports of an association with a raised igg4 level and lsp, the diagnosis in this case was still unclear. to confirm the diagnosis, an exploratory laparotomy was considered to be most appropriate (and preferable to eus fna) as it offered the opportunity to obtain multiple biopsies from a diffusely enlarged pancreas, whilst also offering a therapeutic option. at laparotomy, the entire pancreas was hard, swollen, and actively inflamed, most particularly the body of the gland. the appearances were not typical for pancreatic cancer or chronic pancreatitis. a shave biopsy of a nodule on the body of pancreas plus three core biopsies with a trucut needle and a fourth from the head of pancreas were submitted to frozen - section histopathology examination ; all showed florid chronic inflammation but no cancer. a portion of thickened retroperitoneum tissue in front of the aorta was also removed and this was shown to be benign fibrotic tissue. since the pathology was consistent with a diagnosis of lsp, an internal biliary bypass was performed using a roux loop of jejunum. the patient made a good postoperative recovery and was referred to gastroenterology for further management. lsp primarily affects middle - aged men, with a mean age of 56 years. the recently increasing number of articles on lsp is an indication of the current interest in this subject. its similarity to pancreatic carcinoma, both clinically and radiologically, presents a diagnostic challenge. in distinguishing lsp from pancreatic carcinoma, the japanese pancreas society proposed diagnostic criteria for lsp in 2002 (see table 2) [14, 17 ]. patients with lsp also have elevated serum igg4 levels which return to normal after administration of oral steroids. only a limited number of conditions are associated with elevated igg4 (such as atopic dermatitis, some parasitic diseases, pemphigus vulgaris, and pemphigus foliaceus). hamano and associates found high serum igg4 concentrations specifically in patients with lsp and not in those with chronic pancreatitis, primary biliary cirrhosis, primary sclerosing cholangitis, or sjogren 's syndrome. not only does this finding suggest that lsp is immunologically different, but it may also follow that igg4 would be a useful marker. the association of lsp with other autoimmune conditions, including retroperitoneal fibrosis, is well recognised. our patient developed retroperitoneal fibrosis prior to lsp, but review of the 14 previous cases indicates that the onset of lsp is variable. further, hydronephrosis secondary to ureteric obstruction (as in our case) was not uncommon [411 ]. the aetiology of retroperitoneal fibrosis is unclear, but the presence of autoantibodies and the response to steroids in some patients may suggest an autoimmune mechanism. infiltration with igg4-positive plasma cells has been shown in both the pancreas and the retroperitoneal mass of patients with lsp and retroperitoneal fibrosis, indicating a likely common aetiology. lsp can be treated with medical therapy (steroids in reducing dosage) or surgically. the prognosis is usually good with either form of management but an important factor is the difficulty in diagnosing lsp, which can so often mimic a malignant intra - abdominal mass. preoperative or even intraoperative diagnosis can be problematic, particularly in patients for whom there are no clinical grounds to suspect lsp. it has been suggested that surgical resection may be an effective treatment for lsp especially when immunological tests are normal or when malignancy is highly suspected (this may be the only option to exclude malignancy). however, surrounding inflammation can make surgical resection hazardous, so a trial of steroids is worthwhile if a confident diagnosis can be made without laparotomy.	although cases of lymphoplasmacytic sclerosing pancreatitis (lsp) associated with idiopathic retroperitoneal fibrosis have been reported, the association is rare. we describe a 74-year - old man who presented with obstructive jaundice and weight loss. nineteen months earlier, he had been diagnosed with idiopathic retroperitoneal fibrosis and treated with bilateral ureteric stents. initial investigations were suggestive of a diagnosis of lsp, however, a malignant cause could not be ruled out. he underwent an exploratory laparotomy and frozen sections confirmed the diagnosis of lsp. an internal biliary bypass was performed using a roux loop of jejunum, and the patient made an uneventful recovery. this case illustrates the difficulty in distinguishing lsp from pancreatic carcinoma preoperatively.
coarctation of the aorta is one of the most common diagnoses in congenital cardiac defects. it comes in an isolated form or in association with various obstructive lesions along the left side of the heart or with a perimembranous ventricular septal defect (vsd). the diagnosis is mostly established in the neonatal period and hence the great majority of corrections are performed within two or three days after birth. an extended resection [1, 2 ] is almost always possible at that time due to the particular elasticity of the aortic tissue allowing extensive mobilization of both segments of the thoracic aorta. the risk of paraplegia is minimal as long as the cross - clamping of the aorta does not exceed 30 minutes. later in life, the repair may require an assisted circulation (a left heart bypass between the left atrial appendage and the thoracic aorta) to the lower body to avoid such a complication or other visceral ischemic lesions [4, 5 ]. coarctation associated with a long, hypoplastic distal aortic arch is also repaired with a left thoracotomy without circulatory assistance in our center, but is preceded by an enlargement plasty of the roof of the distal arch. the enlargement is performed first, with a patch of tanned pericardium, and with the use of ductal perfusion to avoid any visceral ischemia. the resection - anastomosis of the coarctation is performed after the plasty. since the establishment of a minimally invasive, muscle sparing and extrapleural approach to repairing coarctations, we have abandoned the single stage repair when a perimembranous vsd was associated. for us, the only consideration for a coarctation repair with a sternotomy is when enlargement of the distal aortic arch is not possible with regular cross - clamping (in the case of common carotid truncus, in some cases of arteria lusoria and very rarely in the case of particularly unfavorable anatomy of the arch). banding of the pulmonary artery is not routinely performed in the regular cases, and the vsd is closed when signs of heart strain arise, usually a little before the regular time. the extrapleural approach has furthermore reduced the development of subsequent collaterals between the thoracic wall and the left upper lobe, and has proved a significant advantage in cyanotic univentricular hearts requiring subsequently a fontan circulation. recoarctations refractory to balloon dilatation [8, 9 ] are handled in our center anatomically, with surgery. the choice of approach between a sternotomy and a posterior thoracotomy depends on the exact location of the residual stenosis. in a few patients, our preferred approach is through a sternotomy with enlargement of either the superior or the inferior part of the aortic arch, depending on the anatomy. in children, we have often used the anterior wall of the (dilated) pulmonary artery to enlarge the inferior part of the aortic arch. this autologous patch has excellent elastic characteristics and holds the systemic pressure well, with no degeneration. we have simply used a regular patch for the arch enlargement. in many patients, the anatomy is favorable for a roof plasty of the aortic arch (a technically easier repair). we commonly transect the left common carotid artery to facilitate this enlargement, and reimplant it at the end of the reconstruction. it is only in multiple redo procedures, or in very complex arch anatomy that we consider an extra - anatomic bypass between the ascending and the coeliac aorta [11, 12 ].	the surgical treatment of an aortic coarctation requires a resection of the stenotic area and direct suture of the aorta. an extended mobilization allows an enlargement of a hypoplastic distal aortic arch. in ductal dependent circulation, the distal aortic arch can be enlarged with a patch before tackling the coarctation itself. postsurgical aortic arch stenoses often require a surgical intervention. our preferred method is an anatomic correction with an enlargement plasty either on the concavity or on the convexity of the arch, depending on the local anatomy. an extra - anatomic bypass is also an option.
acute kidney injury (aki), formerly known as acute renal failure, continues to represent a very common and potentially devastating problem in critically ill children and adults. the reported incidence of aki in this population varies greatly due to the lack of a standard consensus definition. for example, aki affects between 5% and 50% of critically ill patients in reported series. unfortunately, the mortality and morbidity associated with aki remain unacceptably high (up to 80% mortality in critically ill children and adults with multiple organ dysfunction syndrome (mods)). while this dismal prognosis is partly attributable to other comorbid conditions, recent studies have revealed that aki may be an independent risk factor for mortality in both critically ill children and adults. however, it is well known that creatinine is an unreliable and insensitive indicator during early acute changes in kidney function. first, serum creatinine concentrations may not change until about 50% of kidney function has already been lost. second, serum creatinine does not accurately depict kidney function until a steady state has been reached, which may require several days. neutrophil gelatinase - associated lipocalin is a 25 kda protein that is expressed at very low concentrations in several human tissues, including the kidney, lungs, and gastrointestinal tract. the aim of this study was to detect the extent of serum neutrophil gelatinase - associated lipocalin (ngal) to early detect acute kidney injury (aki) in critically ill children and to evaluate its sensitivity and specificity in aki detection. this study had been carried out in pediatric intensive care unit (picu) and clinical pathology department, faculty of medicine, zagazig university hospital. they were classified into group i (15), apparently healthy volunteers, and their ages ranged between 1 and 29 months with the median age of 6 months, and group ii (60), children who were admitted at picu, and their ages ranged between 1 and 108 months with the median age of 9 months. for group i routine investigations as liver function, kidney function, and complete blood count were done to confirm their healthy state. for group ii, serum creatinine was measured in the first day of admission, and then another sample was measured in the 3rd day of admission to the picu. this group of patients was further subdivided according to rifle criteria into two other categories : group a, patients who developed aki (aki) and group b, patients who did not develop aki (naki). the rifle criteria include (risk of acute renal failure, injury to the kidney, and failure of renal function). then, the patients who developed aki are further divided into two other groups according to the severity (dialysis dependence) as follow : aki with dialysis (aki + d) and aki without dialysis (aki d). all studied subjects included in this study were subjected to the following : full history taking, including family history of underlying kidney disease, complete clinical examination, routine laboratory investigations, and serum ngal assay. 1 ml was delivered into edta containing polypropylene tube and then gently inverted, then cbc was performed.4 ml were collected in plain tube for serum separation. after clotting, samples were centrifuged at 1000 g for 15 minutes, and sera were separated and divided into two aliquots. a fresh serum aliquot from each individual was used for assay of serum creatinine, blood urea nitrogen (bun), and liver function tests. the other aliquot was stored at 20c until the assay of neutrophil gelatinase - associated lipocalin. hemolyzed samples were discarded, and repeated freezing and thawing was avoided. 1 ml was delivered into edta containing polypropylene tube and then gently inverted, then cbc was performed. after clotting, samples were centrifuged at 1000 g for 15 minutes, and sera were separated and divided into two aliquots. a fresh serum aliquot from each individual was used for assay of serum creatinine, blood urea nitrogen (bun), and liver function tests. the other aliquot was stored at 20c until the assay of neutrophil gelatinase - associated lipocalin. assay of ngal was carried out by a sandwich enzyme - linked immunosorbent assay (elisa) technique using reagents provided by quantikine r&d international inc. standards and samples are pipetted into the wells, and any ngal present is bound by the immobilized antibody. after washing away any unbound substances, an enzyme - linked monoclonal antibody specific for ngal is added to the wells. following a wash step to remove any unbound antibody enzyme reagent, a substrate solution is added to the wells, and color develops in proportion to the amount of ngal bound in the initial step. the color development is stopped, and the intensity of the color is measured. reagents are as follows ngal microplate : 96-well polystyrene microplate (12 strips of 8 wells) coated with a rat monoclonal antibody against ngal;ngal conjugate : 21 ml of monoclonal antibody against ngal conjugated to horseradish peroxidase with preservatives;ngal standard : 100 ng of recombinant human ngal in a buffer with preservatives lyophilized. this reconstitution produces a stock solution of 100 ng / ml. the standard was mixed to ensure complete reconstitution, and the standard was allowed to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. ngal microplate : 96-well polystyrene microplate (12 strips of 8 wells) coated with a rat monoclonal antibody against ngal ; ngal conjugate : 21 ml of monoclonal antibody against ngal conjugated to horseradish peroxidase with preservatives ; ngal standard : 100 ng of recombinant human ngal in a buffer with preservatives lyophilized. this reconstitution produces a stock solution of 100 ng / ml. the standard was mixed to ensure complete reconstitution, and the standard was allowed to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. serial dilution of standard was done to obtain the following concentrations : 10, 5, 2.5, 1.25, 0.625, 0.312, and 0.156 ng / ml. 900 l of calibrator diluent were pipetted into the 10 ng / ml tube. the calibrator diluent served as the zero standard (0 ng / ml);(iv) assay diluent rd1 - 52 : 11 ml of a buffer with preservatives;(v) calibrator diluent rd5 - 24 concentrate : 21 ml of a 5-fold concentrated solution containing buffered protein base with preservatives;(vi) wash buffer concentrate : 21 ml of a 25-fold concentrated solution of buffered surfactant with preservatives;(vii) color reagent a : 12.5 ml of stabilized hydrogen peroxide;(viii) color reagent b : 12.5 ml of stabilized chromogen (tetramethylbenzidine);(ix) stop solution : 6 ml of 2 n sulfuric acid ; (x) plate covers : 4 adhesive strips. assay diluent rd1 - 52 : 11 ml of a buffer with preservatives ; calibrator diluent rd5 - 24 concentrate : 21 ml of a 5-fold concentrated solution containing buffered protein base with preservatives ; wash buffer concentrate : 21 ml of a 25-fold concentrated solution of buffered surfactant with preservatives ; color reagent a : 12.5 ml of stabilized hydrogen peroxide ; color reagent b : 12.5 ml of stabilized chromogen (tetramethylbenzidine) ; stop solution : 6 ml of 2 n sulfuric acid ; plate covers : 4 adhesive strips. assay procedure 100 l of assay diluent rd1 - 52 were dispensed into each well.50 l of standards, controls, and samples were added to each well. the plate was covered and incubated for 2 hours at 28c.aspiration of each well was done and this was followed by a washing step which was repeated three times.200 l of ngal conjugate were added into each well, covered with a new adhesive strip and incubated for 2 hours at 28c.aspiration/washing was repeated as in step (iii).200 l of substrate solution were dispended into each well.plate was covered and incubated for 30 minutes at room temperature.50 l of stop solution were added into each well. determination of the optical density of each well within 30 minutes was done, using a microplate reader set to 450 nm. 50 l of standards, controls, and samples were added to each well. aspiration of each well was done and this was followed by a washing step which was repeated three times. 200 l of ngal conjugate were added into each well, covered with a new adhesive strip and incubated for 2 hours at 28c. determination of the optical density of each well within 30 minutes was done, using a microplate reader set to 450 nm. a standard curve was constructed by plotting the mean absorbance for each standard on the y - axis against the concentration on the x - axis on log - log graph, and a best fit line was drawn through the points on the graph. concentration of lipocaline-2 in each sample was determined by finding the mean absorbance value of each one. from the y - axis of the standard curve graph, a vertical line was extended from this absorbance value to the standard curve. at the point of intersection, a horizontal line was extended to the x - axis and the corresponding concentration was read. the data were tabulated and statistically analyzed using statistical package for social sciences (spss inc., comparison of continuous data was performed using student t - test and mann - whitney test for two groups, while for more than two groups, anova test was used. roc curves analysis (an efficient way to display the relationship between sensitivity (true positive rate) and specificity (true negative rate) for tests with continuous outcomes ; a point in the curve moves down and to the left showing lower sensitivity and high specificity, while it moves up and to the right showing higher sensitivity and lower specificity) was used. the cutoff value was selected from the roc curve by choosing a point having the best sensitivity and considerable specificity. positive predictive value (ppv) is probability of disease in a patient with an abnormal test. negative predictive value (npv) is probability of a patient not having the disease when the test result is negative. we found that range of ngal at day zero was (34210) with median of 88.5 and at 3rd day was (34550) with median 114, urea at day zero ranged from 15 to 116 with median of 35.5 and at 3rd day ranged from 27 to 310 with median of 41.5, while creatinine at zero day was (x sd : = 0.68 0.22) while at 3rd day (x sd : 1.18 1.08). so, there were statistical significance between ngal, urea, and creatinine at day zero and 3rd day (p = 0.000) as shown in table 2. a significant increase between two groups with and without aki regarding ngal, urea, and creatinine at admission and at 3rd day (p < 0.05) was shown in table 3. there was no statistical significance among the 3 groups regarding ngal at admission and urea at 3rd day (p = 0.24 and 0.76, resp.). however, there was statistical significance among the 3 groups regarding ngal at 3rd day, urea at admission and creatinine at admission, and at 3rd day (p = 0.07, 0.01, 0.004, and 0.000 resp.) as presented in table 4. table 5 described that applying the receiver operating characteristic (roc) curve of ngal to early detection of aki revealed area under the curve of 0.63 with 95% confidence interval (ci) of 0.500.77. a cutoff of 89.5 ng / ml was chosen for early detection of aki presented in figure 1. ngal acts as a sensitive marker rather than a specific one. at the same time, it presents a negative predictive value more valuable than being a positive predictive value in detecting aki. we described in table 6 that, at cutoff of 89.5 ng / ml, ngal was positive at 11 out of 13 patients with aki ; the sensitivity of ngal in diagnosis of aki was 84.6%. the positive predictive value (11/(11 + 19)) was 36.7%, and the negative predictive value (28/(2 + 28)) was 93.3%. acute kidney injury is a common and serious condition, and the diagnosis of which depends on serum creatinine urea which is a delayed and unreliable indicator of aki. fortunately, understanding the early stress response of the kidney to acute injuries has revealed a number of potential biomarkers. neutrophil gelatinase - associated lipocalin is a small protein of the lipocalin superfamily and is expressed by immune cells, hepatocytes, and renal tubular cells. in particular, ngal is emerging as an excellent biomarker in the urine and plasma for several processes such as early prediction of aki, monitoring clinical trials in aki, and for the prognosis of aki in several common clinical scenarios. the aim of the present work was to assess serum levels of ngal in critically ill children affected by aki and to evaluate its clinical significance in diagnosis as well as assessment of disease severity in comparison with other markers as serum creatinine and urea ratio in critically ill children. we prospectively measured serum ngal concentrations during day zero and after the 3rd day of admission to the picu in 60 critically ill children (28 males and 32 females) and 15 healthy children (8 males and 7 females). in our prospective study, there were no statistical significant differences between controls and patients group regarding demographic data including age and gender, p = 0.15, and p = 0.64, respectively. the liver function (total bilirubin, direct bilirubin, and alt) showed that there was no statistical significance difference between critically ill children and control group. however, there was a significant increase in the level of ast and significant decrease in the levels of albumin and total protein among the two groups. regarding kidney function, there was significant increase in the level of urea in patients group compared to control group (p = 0.000). however, creatinine value increased in critically ill children group showing no statistical significance with control group (p = 0.69). (2006) who reported that urea is not produced at a constant rate, and the rate can be influenced by extrarenal factors. urea production can be increased by diet, critical illness, burns, trauma, gastro intestinal bleeding, and sepsis. also, in patients with decreased circulating blood volume due to volume depletion or low cardiac output, resorption of urea increases because of the relationship between urea levels and water conservation mechanisms. therefore, urea can be influenced by multiple factors and does not represent real - time changes in gfr. in the present study, the level of serum ngal concentration for control group ranged from 44 to 141 ng / ml with median of 54 ng / ml and critically ill children group ranged from 34 to 210 ng / ml with median of 88.5 ng / ml at admission. there was significant increase in the level of ngal in the critically ill children group compared to control group, p < 0.01. (2007) who reported that there was a significant difference in serum ngal between healthy children and critically ill children. critically ill children who developed aki (by doubling of serum creatinine from baseline, according to rifle classification) had significantly elevated serum ngal, urea, and creatinine levels at 3rd - day compared to serum ngal, urea, and creatinine at day zero. also, critically ill children who developed aki had significantly elevated serum ngal, urea, and creatinine levels at day zero and at 3rd day when compared with critically ill children who did not develop aki. (2008) who demonstrated that serum ngal concentrations within 24 hours of picu admission were significantly increased in these children who developed aki compared to children who did not develop aki. a second sample was obtained on the third day of admission to the picu, and the difference between critically ill children with aki and critically ill children without aki remained significant (p < 0.001). aki developed in 13 out of 60 (21.7%) critically ill children included in this study all but 5 of these critically ill children (8.3%) had a greater severity of illness and need to dialysis. patients who developed aki and need to dialysis showed higher level of ngal at day 0 compared to patients who developed aki without need to dialysis and patients who did not develop aki, but with no statistical significance, while at 3rd day, it markedly increased (median = 350 ng / ml) and became highly significant (p = 0.04). (2003) who found that aki developed in 32 out of 69 (46.3%) critically ill children all but 8 of these critically ill children (11.5%) had a greater severity of illness and need to dialysis. in our study, the receiver operating characteristic curve of ngal for early detection of aki revealed area under the curve of 0.63 with 95% (ci of 0.500.77). at cutoff value of 89.5 ng / ml, the sensitivity of ngal was 84.6%, the specificity was 59.6%, positive predictive value was 36.7%, and negative predictive value was 68.4%. (2007) who had shown that at cutoff of 139 ng / ml, the receiver operating characteristic curve of ngal revealed area under the curve of 0.677 with 95% (ci of 0.5570.786). the sensitivity of ngal was 86%, specificity was 39%, positive predictive value was 39%, and negative predictive value was 94%. our results of early predictive, sensitive, nonspecific serum ngal, although of clear statistical significance, will certainly need to be validated in a larger trial, including patients with preexisting chronic kidney disease and co morbid conditions that normally accumulate with impaired renal function. the ability of biomarkers, such as ngal, to discern both the onset and resolution of aki will further validate their use in the clinical setting and greatly enhance our understanding of aki in the pediatric population. in conclusion, we found that ngal acts as a sensitive marker rather than a specific for one aki. at the same time, it presents a negative predictive value more valuable than being a positive predictive value in detecting aki.	introduction. the mortality and morbidity associated with acute kidney injury (aki), unfortunately, remain unacceptably high. we aimed to detect the extent of serum neutrophil gelatinase - associated lipocalin (ngal) to early detect aki in critically ill children. subjects and methods. this is a case control study. it included 75 subjects that include 15 as controls and 60 critically ill children. patients were further subdivided according to rifle criteria into two other categories : patients who developed aki and patients who did not develop aki. serum ngal assayed on admission and after 3 days. results. there was significant increase in the level of ngal among patients group when compared with control group. also, 21.7% of children admitted to picu developed aki from which 8.3% needed dialysis. the receiver operating characteristic curve of ngal at day 0 revealed auc of 0.63 with 95% ci of 0.500.77. at a cutoff value of 89.5 ng / ml, the sensitivity of ngal was 84.6%, while specifcity was 59.6%, positive predictive value was 36.7%, negative predictive value was 68.4%, and accuracy was 93.3% in diagnosis of aki. conclusion. we found that ngal acts as a sensitive marker rather than a specific one for aki. at the same time, it presents as a negative predictive value more valuable than being a positive predictive value in detecting aki.
leprosy is a disease of public health concern because of the case load and the social stigma attached to the disease. leprosy is a disease known to be a great scourge for the suffering humanity from time immemorial. in the year 1955, the government of india first launched a national leprosy control program. those were the days when dapsone was the sole cure for leprosy. during the 1970s multidrug therapy (mdt) was identified as having potential to cure leprosy and subsequently in 1982, mdt came into use.. initially started in a phased manner, it took 13 years for mdt to be available countrywide. mdt has proven to be a powerful tool in the control of leprosy, especially when patients report early and start prompt treatment. unfortunately, due to a number of personal, psychosocial, economic, medical and health service factors, a significant number of patients become irregular and default from mdt. the success of the current who key strategy for leprosy elimination (i.e. multidrug therapy [mdt ] regimen) depends largely on the efficiency of health care delivery services and patient compliance. a high rate of noncompliance with this regimen has serious implications for the leprosy control program because it can set the stage for the emergence of drug resistance, eventually resulting in treatment failure and failure of the program. research works on drug compliance have indicated that if a patient understands his /her disease and its treatment well, he /she is more likely to be motivated to take the whole prescribed course of treatment properly. it is widely believed that the understanding and behavior of patients in relation to drug compliance are largely influenced by their socio - economic condition and level of knowledge. in 1981, a who study group recommended that multibacillary (mb) leprosy patients should be given multidrug therapy (mdt) for at least two years and, wherever possible, until skin - smear becomes negative. to improve operational efficiency as well as to improve patient compliance in leprosy programs, danish development assistance (danida) introduced blister - calendar packs (bcp) to deliver mdt in four mdt districts in india in 1987. the study was undertaken to assess the adherence to who - mdt therapy and its successful completion by leprosy patients and the extent of such defaulting, its correlates and reasons. this retrograde cohort analysis was conducted with no interventions during the first quarter of 2007 from the cases registered for who - mdt treatment during 2002 to 2005 in kamrup district of assam, india, based on both quantitative as well as qualitative parameters with review of relevant documents, records and literature. it was decided to interview patients with treatment compliance, and defaulters, using a pre - designed and pre - tested schedule. information about type of disease, duration of disease, duration of treatment, type of treatment and patient compliance was collected from patients o.p.d. main outcome measures were the correlates of treatment compliance and defaulters of leprosy patients. the sample was selected from the cases discharged from treatment during 2002 to 2005 in kamrup district of assam ; the total number was 1020 among which 362 were defaulters. year wise, 460 cases were discharged during 2002 - 03 of which 181 (39.35%) were defaulters. similarly during 2003 - 04, among 302 cases discharged 73 (24.17%) were defaulters and during 2004 - 05, among 258 cases discharged 108 (41.86%) were defaulters. only those defaulters who could not be traced back during treatment for a period of 6 months were finally discharged from treatment and reflected as discharged otherwise. taking an average of the defaulter rate which was 35.13%, the sample size was calculated. among 362 defaulters, pre - tested close - ended questionnaires that contained questions linking to correlates of treatment compliance and default of leprosy patients in relation with the socio - demographic situation prevailing in india. by initial translation, back - translation, re - translation followed by pilot study, the pilot study was carried out on the general patients of other diseases from the same area following which some of the questions from the interview schedule were modified. the data collection tool used for the study was an interview schedule that was based on at the institute with the assistance from the faculty members and other experts developed on information provided by the global experts prior to the study for ensuring feasibility, acceptability, time management, validity and reliability. all the patients or their caregivers were explained the purpose of the study and were ensured strict confidentiality. written informed consents the collection of the data was from the january 15 till the march 30, 2007. on an average, information on leprosy was disseminated to the patients their and caregivers in health education sessions to complement the findings of study. the data collected were thoroughly cleaned and entered into ms - excel spread sheets for analysis. among the total number of cases discharged in the period from 2002 to 2005, the percentage of defaulters was very high. defaulter rate was higher in urban areas 91.7%, 94.5% and 100% in respective years of study even in presence of more accessible health services and more educated and stabilized communities. a total of 254 leprosy cases reflected the treatment seeking behavior of the registered cases during study period. urban - rural distribution of study population on treatment outcome the study group consisted of 60.63% males and 39.37% females whereas the control group had 75.59% males and 24.41% females. both the compliance and default was higher in the age group of 16 to 30 years. the distribution of defaulters on basis of literacy status, per capita monthly income and socioeconomic status in comparison to control group reflected that majority (32.28%) had passed the high school leaving certificate examination and per capita monthly income between rs 500- 749 (30.71%) and belong to social class iv (33.86%) and v (30.71%). there is significant statistical association between literacy status, per capita income per month and socioeconomic status with treatment outcome [table 2 ]. analysis of treatment outcome with various parameters on analysis of the reasons of defaulting treatment, majority (33.07%) defaulted treatment due to loss of occupational hours when they had to receive drugs at the health center, 25.98% defaulted due to adverse reactions of drugs, 18.11% feared social stigma, 10.24% were unable to continue due to difficult transportation from their residing area, 4.72% could not come due to physical inability and rest 1.57% could not give any valid reasons [table 3 ]. a total of 254 leprosy cases reflected the treatment seeking behavior of the registered cases during the study period. majority of the cases were from urban areas and defaulter rate was higher in urban areas. the study group consisted of 60.63% males and 39.37% females. both the compliance and default was higher in age group - 16 to 30 years. majority of the defaulters (32.28%) had passed the high school leaving certificate examination and per capita monthly income between rs 500- 749 (30.71%) and belong to social class iv (33.86%) and v (30.71%). significant statistical association was found between gender, literacy status, per capita income per month and socioeconomic status with treatment outcome. on analysis for the reasons of defaulting treatment, majority (33.07%) defaulted treatment due to loss of occupational hours when they come for receiving drugs at health centre. 25.98% defaulted due to adverse reactions of drugs and 18.11% feared social stigma among major causes. according to researchers from new delhi, in six leprosy mission hospitals, nearly half of the patients closer to the hospitals defaulted as compared to 60% who stayed beyond. patients from outside the district had significantly higher default rate for all types of leprosy cases as compared to patients living close by to the centers. a community - based descriptive study conducted in 12 leprosy endemic areas in cebu, philippines, showed that the noncompliance rate with the who - mdt regimen among 233 study subjects was 30%. the causes of noncompliance are drug - related, health care provider - triggered, or patient - inducted, or some combination of these. in a non - intervention study carried - out in dhanusha - a district in nepal, among a total of 57 non - compliant leprosy cases, majority were illiterate, laborers by occupation and from poor economic class family background (73.7%). there were 183 male (68.3% on mb - mdt) and 90 female (61.1% mb - mdt) leprosy patients. the study found that 79.2% of male patients completed treatment, while 34.4% female patients did not complete within the given time frame. the study found significant associations between treatment completion status and gender (adjusted or 2.05, 95% ci : 1.07 - 3.94), educational status (adjusted or 2.37, 95% ci : 1.12 - 4.99. a study from two districts in cabo delgado province in northern mozambique conducted during the period from 1993 to 1997 found that 548 (59.2%) of 926 mb patients completed treatment and 378 (40.8%) defaulted during the period. of the 378 defaulters, 57.7% defaulted treatment within six months and 83.1% within one year of starting treatment. it was observed that patients tend to default early rather than late in the treatment period and that this pattern is maintained over time despite a fall in defaulter rates. patients established early into a treatment routine were more likely to complete treatment. a study from mumbai followed smear - positive leprosy cases registered at an urban leprosy center for three years to study the drop - out pattern in them and judge the utility of some corrective measures for the same. drop - out in smear - positive cases registered at the same centre in 1989, 1990, 1992 and 1993. by introduction of the special measures, the drop - out rate was significantly reduced from 52% (for other years) to 36% (1991). the causes of non - compliance explored by us ranged from the relatively longer course of treatment to irregular supply of the drugs, health caregiver prompted, receiver inducted or an amalgamation of these. in our study we presented an analysis of primary data collected by recall method and therefore short memory and forgetfulness of the study population during interview may fail to give accurate information. again, collection of secondary data was difficult to some extent because of reasons like frequent transfer of government officials and dependence on government records. the success of the current who key approach for leprosy elimination schedule depends for the most part on the competence of health care delivery services and patient conformity. research on post intervention knowledge and practice among patients and caregivers has to be repeatedly explored. the causes of non - compliance explored by us ranged from the relatively longer course of treatment to irregular supply of the drugs, health caregiver prompted, receiver inducted or an amalgamation of these. in our study we presented an analysis of primary data collected by recall method and therefore short memory and forgetfulness of the study population during interview may fail to give accurate information. again, collection of secondary data was difficult to some extent because of reasons like frequent transfer of government officials and dependence on government records. the success of the current who key approach for leprosy elimination schedule depends for the most part on the competence of health care delivery services and patient conformity. research on post intervention knowledge and practice among patients and caregivers has to be repeatedly explored. who - mdt has proven to be a commanding tool in control of leprosy, particularly when patients report early and start treatment without delay. observance to and its successful completion are uniformly imperative. unfortunately, due to a number of individual, psycho - social, financial, therapeutic and health service factors, a considerable quantity of patients become irregular and defaulting from who - mdt. any short term solution may not help us to reach the goal of eradication of leprosy in india in near future. recommendations on strategic interventions to obviate the cause for noncompliance and to solve the problem, points to holistic responsibility of professionals, health services, governments and teaching institutions. health education system needs to improve knowledge about leprosy among the people with lesser educational level. it could be done by means of improving educational tools preferably based on audiovisual techniques. the system should create wider awareness about the importance of continuation of full course of therapy for cure of leprosy. the information education and communication system should have some productive advertisements to motivate the general public for leprosy. advertisements need to help clear the myths and misconceptions about leprosy. to obviate the cause for noncompliance we have to train and re train health care workers of all levels with periodic random evaluation by health service research, and above the political will to remove this menace from the globe. there should be provision of incentives like help diagnosing patients in his / her wards/ relations/ friends. a non - monetary incentive in the form of certificates of recognition patient - friendly health services, spreading awareness about the advantages of eradicate leprosy not only for the patient but also for the citizen, could be motivating factors, making people aware of recent findings like association of who - mdt with a lower risk of relapse. any short term solution may not help us to reach the goal of eradication of leprosy in india in near future. recommendations on strategic interventions to obviate the cause for noncompliance and to solve the problem, points to holistic responsibility of professionals, health services, governments and teaching institutions. health education system needs to improve knowledge about leprosy among the people with lesser educational level. it could be done by means of improving educational tools preferably based on audiovisual techniques. the system should create wider awareness about the importance of continuation of full course of therapy for cure of leprosy. the information education and communication system should have some productive advertisements to motivate the general public for leprosy. advertisements need to help clear the myths and misconceptions about leprosy. to obviate the cause for noncompliance we have to train and re train health care workers of all levels with periodic random evaluation by health service research, and above the political will to remove this menace from the globe. there should be provision of incentives like help diagnosing patients in his / her wards/ relations/ friends. a non - monetary incentive in the form of certificates of recognition patient - friendly health services, spreading awareness about the advantages of eradicate leprosy not only for the patient but also for the citizen, could be motivating factors, making people aware of recent findings like association of who - mdt with a lower risk of relapse.	objectives : the study was undertaken to assess the adherence to world health organization (who)-multidrug therapy (mdt) and its successful completion by the leprosy patients and the extent of such defaulting, its correlates and reasons.design:retrograde cohort analysis was conducted during the first quarter of 2007 from the cases registered for who - mdt treatment during 2002 to 2005 in kamrup district of assam, india.results:a total of 254 leprosy cases reflected the treatment seeking behavior of registered cases during the study period. majority of the cases were from urban areas and defaulter rate higher in urban areas. the study group consisted of 60.63% males and 39.37% females.. both the compliance and default was higher in the age group of 16 to 30 years. majority of defaulters (32.28%) had passed the high school leaving certificate examination had per capita monthly income between rs 500 - 749 (30.71%) and belonged to social class iv (33.86%) and v (30.71%). significant statistical association was found between gender, literacy status, per capita income per month and socioeconomic status with treatment outcome. on analysis for the reasons of defaulting treatment ; majority (33.07%) defaulted treatment due to loss of occupational hours when they come for receiving drugs at health center, 25.98% defaulted due to adverse reactions of drugs and 18.11% feared social stigma among major causes.conclusions:the causes of defaulting treatment were related to gender, educational status, income as well as social class, or some combination of these. recommendations, on strategic interventions to obviate the cause for noncompliance, were presented.
memory impairment is one of the most common cognitive complaints during the course of aging. mild cognitive impairment is a condition in which people experience memory problems more often than expected for their age. however, the symptoms do not prevent them from daily activities and are not as severe as those of alzheimer 's disease. symptoms of memory decline or mild cognitive impairment usually represent a transitional state between healthy aging and alzheimer 's disease. although individuals with memory complaints do not meet criteria for dementia, they may perform poorly on episodic memory tests. in some older people, alzheimer 's disease and neurodegenerative dementias are growing health problems that affect all ethnic groups worldwide. alzheimer 's disease is characterized by widespread cortical changes, loss of neurons, and presence of senile plaques and neurofibrillary tangles. although definitive diagnosis is based on pathological examination, recent advances in imaging techniques may contribute to early diagnosis of mild cognitive impairment and alzheimer 's disease. increasing evidence from structural and functional mri studies suggests that alzheimer 's disease and mild cognitive impairment may target specific brain networks. proton magnetic resonance spectroscopy appears to be a valuable means of tracking brain metabolic changes due to cognitive impairment. structural imaging can detect the time course of brain atrophy in patients with alzheimer 's disease and may serve as a surrogate marker for pathological changes in people with suspected alzheimer 's disease. emerging magnetic resonance techniques such as diffusion tensor imaging and proton density weighted imaging, and advances in image analysis software, provide us with an efficient tool for early detection of subtle microstructural, perfusion and metabolic changes in the brain. in this review, we highlight the body of literature on brain abnormalities detected by imaging in mild cognitive impairment and alzheimer 's disease. in particular, we address the viability of mri techniques to discriminate between dementias and to measure disease progression. volumetric mri is becoming an increasingly important tool in the early detection and monitoring of people suspected to have mild cognitive impairment or alzheimer 's disease. previously, the detection of atrophy depended on the measurement of regional brain volumes, using volumetric imaging techniques. patients with alzheimer 's disease may have typical pathologic changes in cortical gray matter, characterized by the accumulation of amyloid beta plaques, formation of neurofibrillary tangles, and neuronal and synaptic loss. the vast majority of structural brain imaging studies have been based on histopathological evidence that the entorhinal cortex and hippocampus are among the first sites affected by mild cognitive impairment. similarly, in patients with alzheimer 's disease, atrophy has been found to occur in the hippocampal formation and entorhinal cortex, as demonstrated by several volumetric mri studies. to date, many imaging methods have been developed to monitor disease progression and understand the pathogenesis of dementia. mri is extensively used for the diagnosis of mild cognitive impairment and alzheimer 's disease. t1-weighted mri are useful for the assessment of the topographic distribution of cortical and subcortical atrophy. recently, three - dimensional gradient - echo sequences that allow calculation of volumes and coregistration of images during follow - up examinations have been used in clinical practice. unlike t1-weighted imaging, t2 relaxometry allows the quantitative measurement of signal changes on t2-weighted images. however, the ability of t2-weighted imaging to differentiate between patients with mild cognitive impairment or alzheimer 's disease and healthy subjects is very limited because various confounding pathologies such as brain edema, demyelination and axonal loss may also result in changes similar to those seen in alzheimer 's disease. elevated t2 values are found in the hippocampus, and these values correlate strongly with the severity of functional and cognitive impairment in patients with alzheimer 's disease. early reports used serial manual delineating of the anatomical boundaries of the hippocampus and entorhinal cortex. patients with mild cognitive impairment have a smaller entorhinal cortex and hippocampus than healthy age - matched subjects. however, patients with alzheimer 's disease may have a prominent reduction in the entorhinal cortex and hippocampus. volume reductions in patients with mild cognitive impairment appear to be intermediate, between those of healthy subjects and patients with alzheimer 's disease. mri volumetry of the entorhinal cortex, the superior temporal sulcus and the anterior cingulate cortex may also differentiate normal subjects from patients with mild cognitive impairment. although mild cognitive impairment can present with a variety of symptoms, when memory loss is the predominant symptom it is termed amnestic mild cognitive impairment and is frequently seen as a prodromal stage of alzheimer 's disease. in previous studies, patients with amnestic mild cognitive impairment were examined using high - resolution functional mri during a continuous recognition task. the authors found that structural and functional changes in the ca3/dentate region of the hippocampus contributed to the deficits in episodic memory that were observed in patients with amnestic mild cognitive impairment. another study suggested that patients with amnestic mild cognitive impairment were selectively impaired on a functional mri task that emphasized pattern separation ; the response distribution was strikingly similar for lures and repetitions. mri studies using morphometric techniques have demonstrated that the ca1 region of the hippocampus is structurally compromised early in the course of amnestic mild cognitive impairment. in addition to the examination of the hippocampi and entorhinal cortices, there is a growing interest in white matter changes in mild cognitive impairment and alzheimer 's disease. vascular risk factors such as hypertension, hypercholesterolemia, and the apolipoprotein e4 allele may occur in patients with alzheimer 's disease. although it is sometimes difficult to differentiate between vascular dementia and vascular lesions in alzheimer 's disease, vascular lesions are generally less severe in patients with alzheimer 's disease than those with vascular dementias. there are a variety of mri techniques by which to measure changes in water content. the mri t2 signal decay is sensitive to water content and has been used to measure white matter damage in patients with mild cognitive impairment and alzheimer 's disease. however, conventional mri techniques yield insufficient contrast to provide information on the microstructural integrity of white matter. diffusion tensor imaging is a promising technique that allows documentation of hydrogen - based alterations in mri signal at the microstructural level. this makes it possible to measure the restricted diffusion of water in tissue and thus produce neural tract images, instead of using the data solely for the purpose of assigning contrast or colors to pixels in a cross - sectional image. by applying diffusion weighted gradients in at least six non - collinear gradients, the technique sensitizes the mri signal to the movement of hydrogen in the micron range. using diffusion tensor imaging, it is also possible to measure the direction and magnitude of hydrogen movement and discriminate fiber tracts in the white matter. the application of diffusion tensor imaging allows examination of diffusion characteristics, irrespective of head position. cortical neuronal loss in patients with alzheimer 's disease is associated with axonal degeneration in specific white matter pathways. in patients with mild cognitive impairment, diffusion tensor imaging abnormalities are seen in various brain areas such as the hippocampus, thalamus and posterior white matter. however, variability in region of interest placement may hinder the consensus identification of white matter in alzheimer 's disease. the fiber tractography can easily parcellate the white matter tracts with the use of high - field - strength mri scanners and modern gradient coils. reduced fractional anisotropy values were detected in white matter regions using region of interest analysis in diffusion tensor imaging studies. sometimes, the splenium of the corpus callosum might also be involved, as demonstrated by diffusion tensor imaging fractional anisotropy reductions. during the past few years, the widespread application of advanced mri techniques such as diffusion tensor imaging and functional mri has detected a rapid increase in neurodegenerative disorders. a great variety of innovative methods of extracting diffusion tensor imaging data have been employed in the study of white matter changes in alzheimer 's disease and mild cognitive impairment. for most studies employing diffusion tensor imaging technology, most studies are all multiple - subject group comparison studies, usually one group of experimental subjects compared with a matched group of control subjects. region of interest and whole brain voxelwise analyses are the most commonly used approaches to analyzing mri data. the process includes either hand - drawn region of interests from negative images of individual scans, or template - based region of interests applied to scans that have been warped into a common coordinate system. it is highly operator - dependent, and a skilled operator may provide superior delineation of cerebral structures. because the operator has great control over which voxels to include, the drawing of multiple region of interests for many subjects can be laborious. more importantly, if more than one operator is constructing the region of interests, inter - operator reliability must be established. template - based region of interests have been applied to normalized scans to improve efficiency. because of individual differences in brain morphology, the accuracy of this approach is dependent upon the normalization process and relies on the alignment of different brain regions across individuals. partial voluming (in which a voxel represents more than one tissue type), in either native space or normalized space, is relatively common in both region of interest approaches. the partial voluming effect means that the values extracted from the region of interests are, to some extent, averaged for the entire region of interest. compared with the region of interest approach, the whole - brain voxelwise approach has the advantage of being extremely time efficient. large numbers of scans can be processed quickly, thereby potentially increasing the power of statistical analyses. because most diffusion tensor imaging studies with statistical testing were performed in one or more of the aforementioned ways, a fast measuring approach such as whole - brain voxelwise analysis would be a great advantage. however, whole - brain voxelwise analysis also has potential weaknesses, such as mismatching between the native and normalized image due to imperfect warping algorithms, alteration of the diffusion tensor imaging values due to the effects of normalization, and possible alteration of the diffusion tensor imaging values caused by smoothing. in clinical practice, the structural properties of white matter are being increasingly investigated by diffusion tensor imaging and voxel - based approaches. diffusion tensor imaging is a relatively sensitive technique that reveals group differences between patients with alzheimer 's disease and those with mild cognitive impairment. although diffusion tensor imaging meets the requirements of many basic and clinical research purposes, the technique has several limitations. for example, water diffusion is an indicator of the underlying neuroanatomy, and there are numerous microscopic structures that may affect the diffusion. therefore, different histopathological conditions may result in similar alterations of diffusion tensor imaging - derived parameters. structural and functional mri may allow the prediction of future conversion from mild cognitive impairment to alzheimer 's disease. previous studies have suggested that alzheimer 's disease is associated with reduced anisotropy and increased diffusivity compared with healthy controls. it is prominent in widespread brain regions, most notably in the frontal and temporal lobes, corpus callosum, and posterior cingulum. recent studies have suggested that hippocampal volume predicts conversion from mild cognitive impairment to alzheimer 's disease with high accuracy. a recent study compared volumetric mri with clinical measures predicting progression from mild cognitive impairment to alzheimer 's disease. the authors measured the whole brain, and ventricular, hippocampal, and entorhinal cortex volumes, and participants were followed up with clinical and cognitive evaluations until formal criteria for alzheimer 's disease were met. of the four mri measures evaluated, only changes in ventricular or hippocampal volumes were associated with progression to alzheimer 's disease. maximal predictive accuracy using only mri measures was obtained by hippocampal volumes. in a recent study using deformation - based morphometry and principal component analysis, patterns of regional brain atrophy including the medial temporal lobes, neocortical association areas, thalamus, and basal ganglia, as well as concurrent ventricle widening, were detected. these findings may provide a clue by which to discriminate mild cognitive impairment converters from nonconverters. the vast majority of studies that employ diffusion tensor imaging to investigate white matter integrity in alzheimer 's disease and mild cognitive impairment have greatly increased during the past decade. a large number of these studies have used mean diffusivity and fractional anisotropy as markers of cerebral integrity. mean diffusivity, which is a scalar measure of the total diffusion within a voxel, is commonly used in the clinic to localize white matter lesions. although the mean diffusivity does not provide information about the directionality of diffusion, it gives essential information regarding measure of translational diffusion. previous results have suggested that mean diffusivity increases may have a significant negative correlation with cognitive performance measures. increased mean diffusivity has been observed in many parts of the brain in patients with alzheimer 's disease, including frontal lobes, temporal lobes, parietal lobes and occipital lobes. the regional distribution of increased mean diffusivity has been documented in fiber tracts that were involved in intercerebral communications. these areas include the superior longitudinal fasciculus, corpus callosum, hippocampus, parahippocampus, and cingulum. fractional anisotropy is a scalar value that describes the degree of anisotropy of a diffusion process that could provide an in vivo marker of cerebral integrity. it is commonly used in diffusion imaging and it is thought to reflect fiber density, axonal diameter, and myelination in the white matter. fractional anisotropy is an extension of the concept of eccentricity of conic sections in three dimensions and provides a measure of the directionality of diffusion. high fractional anisotropy is seen in highly organized tissue with parallel structure in white matter. therefore, any damage to the white matter may break down the organization of the anatomical structure, leading to a decrease in fractional anisotropy. fractional anisotropy changes have been reported in multiple regions including the frontal and parietal lobes in patients with mild cognitive impairment and alzheimer 's disease. however, no differences in fractional anisotropy were observed in the occipital lobes between subjects with alzheimer 's disease or mild cognitive impairment, or healthy controls. despite some conflicting findings of fractional anisotropy in alzheimer 's disease and mild cognitive impairment, there have been frequent reports of decreased fractional anisotropy in the medial temporal lobe including the hippocampus, entorhinal cortex, parahippocampal white matter, and posterior cingulum. although the substrates of mean diffusivity and fractional anisotropy may differ, alterations of these two indices can be secondary to changes in diffusion, either parallel or perpendicular to the principal direction of the tensor. tract - based spatial statistics are also useful in diffusion tensor imaging of alzheimer 's disease and mild cognitive impairment. in a recent study, significant decreases were observed in fractional anisotropy values in patients alzheimer 's disease, compared with that of controls, whereas patients with mild cognitive impairment had fractional anisotropy values between those of controls and alzheimer 's disease patients. therefore, voxel - based analysis with tract - based spatial statistics is a promising method for examining the degeneration of neurofiber tracts in alzheimer 's disease and mild cognitive impairment patients. diffusion tensor imaging is also an important tool in differentiating alzheimer 's disease from other neurological disorders. diffusion tensor imaging can be used to estimate white matter lesions in dementia patients. in a recent study, the value of diffusion tensor imaging in the diagnosis and differential diagnosis of patients with subcortical ischemic vascular dementia and alzheimer 's disease were studied. compared with normal controls and patients with alzheimer 's disease, patients with subcortical ischemic vascular dementia had lower fractional anisotropy values and higher diffusion coefficients in the genu and splenium of the corpus callosum, and in the superior longitudinal fasciculus. patients with alzheimer 's disease had lower fractional anisotropy values in the anterior frontal lobe, temporal lobe, and hippocampus, and higher diffusion coefficients in the temporal lobe and hippocampus compared with controls and patients with subcortical ischemic vascular dementia. the potential of using diffusion tensor imaging in conjunction with machine learning algorithms to automate the classification of healthy older subjects and those with mild cognitive impairment has also recently been examined. when diffusion tensor imaging measures were then used together with support vector machines, greater than 90% sensitivity and specificity was achieved using this method. in patients with mild cognitive impairment and alzheimer 's disease, a reduction in size of the hippocampal formation it is well established that the temporal lobe has higher atrophy rates in patients with mild cognitive impairment than in healthy people. a recent study suggested that diffusion tensor imaging changes in temporal lobe white matter correlate well with episodic memory, frontal changes with executive function, and parietal changes with general cognition. furthermore, patients with mild cognitive impairment may mark diffusion tensor imaging abnormalities in multiple locations along the cingulum fiber bundle, including the posterior cingulate and parahippocampal regions. recently, magnetization transfer imaging has also been used for imaging of patients with cognitive impairment. it is based on the exchange of magnetization between immobile and free protons, and is an innovative imaging method that provides essential information regarding the underlying histopathologic changes in brain tissue. furthermore, magnetization transfer imaging allows the assessment of ongoing global and regional brain damage, independent of atrophy, in patients with alzheimer 's disease. it is dependent on the concentration, surface chemistry, and biophysical characteristics of macromolecules. during the past 20 years, magnetization transfer imaging has been used regularly in the evaluation of subjects with multiple sclerosis. a decrease in the white matter magnetization a reduction in magnetization transfer ratio has also been reported in the hippocampus of early dementia patients, compared with control subjects. such studies suggest that measurements of magnetization transfer ratio may be valuable in the detection of structural damage in the hippocampus of alzheimer 's disease patients. interestingly, magnetization transfer is particularly sensitive to gray matter abnormalities and provides complementary information to conventional mri in the characterization of alzheimer 's disease by quantifying gray matter atrophy. the characteristic histopathological findings in the hippocampus of alzheimer 's disease patients include a loss of pyramidal cells accompanied by an increase in the number of astrocytes, microglia, and oligodendrocytes. although the exact mechanism for the reduction in magnetization transfer in the hippocampus of patients with alzheimer 's disease is not yet clear, such pathologic changes may decrease magnetization transfer ratio because of a decrease in the bound - proton fraction. results of studies in patients with clinically diagnosed alzheimer 's disease suggest that quantitative measurement of certain parameters using magnetization transfer imaging may serve as a potential biomarker of the disease. with the growing prevalence of cognitive impairments, functional imaging for neurodegenerative diseases proton magnetic resonance spectroscopy is a viable imaging method for tracking brain metabolic changes due to neurodegenerative diseases. in addition, proton magnetic resonance spectroscopy may also play a role in discriminating between dementias, and measuring disease progression. the amyloid plaques and neurofibrillary tangles in alzheimer 's disease present in a characteristic pattern, with early involvement of the medial temporal lobes and hippocampus. mild cognitive impairment is a transitional phase between normal cognitive aging and alzheimer 's disease. patients with mild cognitive impairment may have subjective complaints of memory loss and objective impairment on memory testing compared with normal age - matched individuals, while their activities of daily living may be generally preserved. a recent study suggested that mild cognitive impairment may be associated with widespread reduction in brain volume and changes in regional function. the technique allows monitoring of metabolic ratios such as myoinositol / creatine - phosphocreatine, choline / creatine - phosphocreatine, and n - acetyl aspartate / creatine - phosphocreatine. in patients with mild cognitive impairment or alzheimer 's disease, such metabolic ratio changes are most apparent in the left temporal lobe, the posterior cingulate cortex, and the medial occipital lobe. however, significantly lower ratios of myoinositol / creatine - phosphocreatine were seen in patients with mild cognitive impairment than in those with alzheimer 's disease. patients with mild cognitive impairment may also have a significant increase in choline / creatine - phosphocreatine ratio in the right frontal cortex and posterior cingulate, as demonstrated by proton magnetic resonance spectroscopy studies. another prominent feature of mild cognitive impairment is reduced n - acetyl aspartate / creatine - phosphocreatine ratio in the left medial temporal lobe and the right hippocampus. cross - sectional magnetic resonance spectroscopy further confirmed that metabolic abnormalities in mild cognitive impairment are transitional between normal older adults and patients with alzheimer 's disease. the observed progression of metabolic changes generally corresponds to the known early neuropathology of alzheimer 's disease. proton magnetic resonance spectroscopy studies indicate that patients with alzheimer 's disease have a reduced n - acetyl aspartate / creatine - phosphocreatine ratio, most prominently in the hippocampus and medial temporal lobe, but also in the temporal - parietal area, frontal and occipital lobes. furthermore, whole brain magnetic resonance spectroscopy has demonstrated reductions in n - acetyl aspartate in alzheimer 's disease, primarily observed in posterior gray matter. these findings of widespread reductions in n - acetyl aspartate are consistent with the progression of neurofibrillary tangles in alzheimer 's disease. in general, proton magnetic resonance spectroscopy can reproducibly distinguish between early alzheimer 's disease patients and normal subjects as well as patients with mild cognitive impairment. further studies will be needed to confirm whether changes in precuneus / posterior cingulate metabolite ratios reliably correlate with measures of function, and whether they may yet prove useful as longitudinal biomarkers in clinical trials of disease - modifying therapies. the cerebral metabolic rate of glucose consumption is a parameter that measures glucose metabolism within the brain. [18f]-fluorodeoxyglucose - position emission tomography can assist with the diagnosis of alzheimer 's disease at an early stage. the utility of [18f]-fluorodeoxyglucose - position emission tomography in alzheimer 's disease has greatly improved early diagnosis and may also be used in monitoring drug effects in the future. it is well established that by the time a patient presents with clinical symptoms of alzheimer 's disease, the cerebral metabolic rate of glucose consumption is severely reduced in some cortical regions. in patients with mild cognitive impairment, interestingly, while position emission tomography measures were not sensitive for people with early stage mild cognitive impairment, reports using [18f]-fluorodeoxyglucose - position emission tomography have suggested that the cerebral metabolic rate of glucose consumption was reduced in patients with more advanced mild cognitive impairment, particularly in the limbic structures, including the hippocampus, medial thalamus and posterior cingulate. a recent study suggested that combining [18f]-flutemetamol position emission tomography with structural mri may provide additional information for categorizing disease and potentially predicting time to progression from mild cognitive impairment to alzheimer 's disease. during the past few years, the widespread application of advanced mri techniques such as diffusion tensor imaging and functional mri has detected a rapid increase in neurodegenerative disorders. a great variety of innovative methods of extracting diffusion tensor imaging data have been employed in the study of white matter changes in alzheimer 's disease and mild cognitive impairment. for most studies employing diffusion tensor imaging technology, most studies are all multiple - subject group comparison studies, usually one group of experimental subjects compared with a matched group of control subjects. region of interest and whole brain voxelwise analyses are the most commonly used approaches to analyzing mri data. the process includes either hand - drawn region of interests from negative images of individual scans, or template - based region of interests applied to scans that have been warped into a common coordinate system. it is highly operator - dependent, and a skilled operator may provide superior delineation of cerebral structures. because the operator has great control over which voxels to include, the drawing of multiple region of interests for many subjects can be laborious. more importantly, if more than one operator is constructing the region of interests, inter - operator reliability must be established. template - based region of interests have been applied to normalized scans to improve efficiency. because of individual differences in brain morphology, the accuracy of this approach is dependent upon the normalization process and relies on the alignment of different brain regions across individuals. partial voluming (in which a voxel represents more than one tissue type), in either native space or normalized space, is relatively common in both region of interest approaches. the partial voluming effect means that the values extracted from the region of interests are, to some extent, averaged for the entire region of interest. compared with the region of interest approach, the whole - brain voxelwise approach has the advantage of being extremely time efficient. large numbers of scans can be processed quickly, thereby potentially increasing the power of statistical analyses. because most diffusion tensor imaging studies with statistical testing were performed in one or more of the aforementioned ways, a fast measuring approach such as whole - brain voxelwise analysis would be a great advantage. however, whole - brain voxelwise analysis also has potential weaknesses, such as mismatching between the native and normalized image due to imperfect warping algorithms, alteration of the diffusion tensor imaging values due to the effects of normalization, and possible alteration of the diffusion tensor imaging values caused by smoothing. in clinical practice, the structural properties of white matter are being increasingly investigated by diffusion tensor imaging and voxel - based approaches. diffusion tensor imaging is a relatively sensitive technique that reveals group differences between patients with alzheimer 's disease and those with mild cognitive impairment. although diffusion tensor imaging meets the requirements of many basic and clinical research purposes, the technique has several limitations. for example, water diffusion is an indicator of the underlying neuroanatomy, and there are numerous microscopic structures that may affect the diffusion. therefore, different histopathological conditions may result in similar alterations of diffusion tensor imaging - derived parameters. structural and functional mri may allow the prediction of future conversion from mild cognitive impairment to alzheimer 's disease. previous studies have suggested that alzheimer 's disease is associated with reduced anisotropy and increased diffusivity compared with healthy controls. it is prominent in widespread brain regions, most notably in the frontal and temporal lobes, corpus callosum, and posterior cingulum. recent studies have suggested that hippocampal volume predicts conversion from mild cognitive impairment to alzheimer 's disease with high accuracy. a recent study compared volumetric mri with clinical measures predicting progression from mild cognitive impairment to alzheimer 's disease. the authors measured the whole brain, and ventricular, hippocampal, and entorhinal cortex volumes, and participants were followed up with clinical and cognitive evaluations until formal criteria for alzheimer 's disease were met. of the four mri measures evaluated, only changes in ventricular or hippocampal volumes were associated with progression to alzheimer 's disease. maximal predictive accuracy using only mri measures was obtained by hippocampal volumes. in a recent study using deformation - based morphometry and principal component analysis, patterns of regional brain atrophy including the medial temporal lobes, neocortical association areas, thalamus, and basal ganglia, as well as concurrent ventricle widening, were detected. these findings may provide a clue by which to discriminate mild cognitive impairment converters from nonconverters. the vast majority of studies that employ diffusion tensor imaging to investigate white matter integrity in alzheimer 's disease and mild cognitive impairment have greatly increased during the past decade. a large number of these studies have used mean diffusivity and fractional anisotropy as markers of cerebral integrity. mean diffusivity, which is a scalar measure of the total diffusion within a voxel, is commonly used in the clinic to localize white matter lesions. although the mean diffusivity does not provide information about the directionality of diffusion, it gives essential information regarding measure of translational diffusion. previous results have suggested that mean diffusivity increases may have a significant negative correlation with cognitive performance measures. increased mean diffusivity has been observed in many parts of the brain in patients with alzheimer 's disease, including frontal lobes, temporal lobes, parietal lobes and occipital lobes. the regional distribution of increased mean diffusivity has been documented in fiber tracts that were involved in intercerebral communications. these areas include the superior longitudinal fasciculus, corpus callosum, hippocampus, parahippocampus, and cingulum. fractional anisotropy is a scalar value that describes the degree of anisotropy of a diffusion process that could provide an in vivo marker of cerebral integrity. it is commonly used in diffusion imaging and it is thought to reflect fiber density, axonal diameter, and myelination in the white matter. fractional anisotropy is an extension of the concept of eccentricity of conic sections in three dimensions and provides a measure of the directionality of diffusion. high fractional anisotropy is seen in highly organized tissue with parallel structure in white matter. therefore, any damage to the white matter may break down the organization of the anatomical structure, leading to a decrease in fractional anisotropy. fractional anisotropy changes have been reported in multiple regions including the frontal and parietal lobes in patients with mild cognitive impairment and alzheimer 's disease. however, no differences in fractional anisotropy were observed in the occipital lobes between subjects with alzheimer 's disease or mild cognitive impairment, or healthy controls. despite some conflicting findings of fractional anisotropy in alzheimer 's disease and mild cognitive impairment, there have been frequent reports of decreased fractional anisotropy in the medial temporal lobe including the hippocampus, entorhinal cortex, parahippocampal white matter, and posterior cingulum. although the substrates of mean diffusivity and fractional anisotropy may differ, alterations of these two indices can be secondary to changes in diffusion, either parallel or perpendicular to the principal direction of the tensor. tract - based spatial statistics are also useful in diffusion tensor imaging of alzheimer 's disease and mild cognitive impairment. in a recent study, significant decreases were observed in fractional anisotropy values in patients alzheimer 's disease, compared with that of controls, whereas patients with mild cognitive impairment had fractional anisotropy values between those of controls and alzheimer 's disease patients. therefore, voxel - based analysis with tract - based spatial statistics is a promising method for examining the degeneration of neurofiber tracts in alzheimer 's disease and mild cognitive impairment patients. diffusion tensor imaging is also an important tool in differentiating alzheimer 's disease from other neurological disorders. diffusion tensor imaging can be used to estimate white matter lesions in dementia patients. in a recent study, the value of diffusion tensor imaging in the diagnosis and differential diagnosis of patients with subcortical ischemic vascular dementia and alzheimer 's disease were studied. compared with normal controls and patients with alzheimer 's disease, patients with subcortical ischemic vascular dementia had lower fractional anisotropy values and higher diffusion coefficients in the genu and splenium of the corpus callosum, and in the superior longitudinal fasciculus. patients with alzheimer 's disease had lower fractional anisotropy values in the anterior frontal lobe, temporal lobe, and hippocampus, and higher diffusion coefficients in the temporal lobe and hippocampus compared with controls and patients with subcortical ischemic vascular dementia. the potential of using diffusion tensor imaging in conjunction with machine learning algorithms to automate the classification of healthy older subjects and those with mild cognitive impairment has also recently been examined. when diffusion tensor imaging measures were then used together with support vector machines, greater than 90% sensitivity and specificity was achieved using this method. in patients with mild cognitive impairment and alzheimer 's disease, a reduction in size of the hippocampal formation it is well established that the temporal lobe has higher atrophy rates in patients with mild cognitive impairment than in healthy people. a recent study suggested that diffusion tensor imaging changes in temporal lobe white matter correlate well with episodic memory, frontal changes with executive function, and parietal changes with general cognition. furthermore, patients with mild cognitive impairment may mark diffusion tensor imaging abnormalities in multiple locations along the cingulum fiber bundle, including the posterior cingulate and parahippocampal regions. recently, magnetization transfer imaging has also been used for imaging of patients with cognitive impairment. it is based on the exchange of magnetization between immobile and free protons, and is an innovative imaging method that provides essential information regarding the underlying histopathologic changes in brain tissue. furthermore, magnetization transfer imaging allows the assessment of ongoing global and regional brain damage, independent of atrophy, in patients with alzheimer 's disease. it is dependent on the concentration, surface chemistry, and biophysical characteristics of macromolecules. during the past 20 years, magnetization transfer imaging has been used regularly in the evaluation of subjects with multiple sclerosis. a decrease in the white matter magnetization a reduction in magnetization transfer ratio has also been reported in the hippocampus of early dementia patients, compared with control subjects. such studies suggest that measurements of magnetization transfer ratio may be valuable in the detection of structural damage in the hippocampus of alzheimer 's disease patients. interestingly, magnetization transfer is particularly sensitive to gray matter abnormalities and provides complementary information to conventional mri in the characterization of alzheimer 's disease by quantifying gray matter atrophy. the characteristic histopathological findings in the hippocampus of alzheimer 's disease patients include a loss of pyramidal cells accompanied by an increase in the number of astrocytes, microglia, and oligodendrocytes. although the exact mechanism for the reduction in magnetization transfer in the hippocampus of patients with alzheimer 's disease is not yet clear, such pathologic changes may decrease magnetization transfer ratio because of a decrease in the bound - proton fraction. results of studies in patients with clinically diagnosed alzheimer 's disease suggest that quantitative measurement of certain parameters using magnetization transfer imaging may serve as a potential biomarker of the disease. with the growing prevalence of cognitive impairments, functional imaging for neurodegenerative diseases is increasingly important in clinical and research settings. proton magnetic resonance spectroscopy is a viable imaging method for tracking brain metabolic changes due to neurodegenerative diseases. in addition, proton magnetic resonance spectroscopy may also play a role in discriminating between dementias, and measuring disease progression. the amyloid plaques and neurofibrillary tangles in alzheimer 's disease present in a characteristic pattern, with early involvement of the medial temporal lobes and hippocampus. mild cognitive impairment is a transitional phase between normal cognitive aging and alzheimer 's disease. patients with mild cognitive impairment may have subjective complaints of memory loss and objective impairment on memory testing compared with normal age - matched individuals, while their activities of daily living may be generally preserved. a recent study suggested that mild cognitive impairment may be associated with widespread reduction in brain volume and changes in regional function. the technique allows monitoring of metabolic ratios such as myoinositol / creatine - phosphocreatine, choline / creatine - phosphocreatine, and n - acetyl aspartate / creatine - phosphocreatine. in patients with mild cognitive impairment or alzheimer 's disease, such metabolic ratio changes are most apparent in the left temporal lobe, the posterior cingulate cortex, and the medial occipital lobe. however, significantly lower ratios of myoinositol / creatine - phosphocreatine were seen in patients with mild cognitive impairment than in those with alzheimer 's disease. patients with mild cognitive impairment may also have a significant increase in choline / creatine - phosphocreatine ratio in the right frontal cortex and posterior cingulate, as demonstrated by proton magnetic resonance spectroscopy studies. another prominent feature of mild cognitive impairment is reduced n - acetyl aspartate / creatine - phosphocreatine ratio in the left medial temporal lobe and the right hippocampus. cross - sectional magnetic resonance spectroscopy further confirmed that metabolic abnormalities in mild cognitive impairment are transitional between normal older adults and patients with alzheimer 's disease. the observed progression of metabolic changes generally corresponds to the known early neuropathology of alzheimer 's disease. proton magnetic resonance spectroscopy studies indicate that patients with alzheimer 's disease have a reduced n - acetyl aspartate / creatine - phosphocreatine ratio, most prominently in the hippocampus and medial temporal lobe, but also in the temporal - parietal area, frontal and occipital lobes. furthermore, whole brain magnetic resonance spectroscopy has demonstrated reductions in n - acetyl aspartate in alzheimer 's disease, primarily observed in posterior gray matter. these findings of widespread reductions in n - acetyl aspartate are consistent with the progression of neurofibrillary tangles in alzheimer 's disease. in general, proton magnetic resonance spectroscopy can reproducibly distinguish between early alzheimer 's disease patients and normal subjects as well as patients with mild cognitive impairment. further studies will be needed to confirm whether changes in precuneus / posterior cingulate metabolite ratios reliably correlate with measures of function, and whether they may yet prove useful as longitudinal biomarkers in clinical trials of disease - modifying therapies. the cerebral metabolic rate of glucose consumption is a parameter that measures glucose metabolism within the brain. [18f]-fluorodeoxyglucose - position emission tomography can assist with the diagnosis of alzheimer 's disease at an early stage. the utility of [18f]-fluorodeoxyglucose - position emission tomography in alzheimer 's disease has greatly improved early diagnosis and may also be used in monitoring drug effects in the future. it is well established that by the time a patient presents with clinical symptoms of alzheimer 's disease, the cerebral metabolic rate of glucose consumption is severely reduced in some cortical regions. in patients with mild cognitive impairment, interestingly, while position emission tomography measures were not sensitive for people with early stage mild cognitive impairment, reports using [18f]-fluorodeoxyglucose - position emission tomography have suggested that the cerebral metabolic rate of glucose consumption was reduced in patients with more advanced mild cognitive impairment, particularly in the limbic structures, including the hippocampus, medial thalamus and posterior cingulate. a recent study suggested that combining [18f]-flutemetamol position emission tomography with structural mri may provide additional information for categorizing disease and potentially predicting time to progression from mild cognitive impairment to alzheimer 's disease. interest is growing in the early investigation of mild cognitive impairment and alzheimer 's disease. early identification of mild cognitive impairment is extremely important for the counseling of patients, making therapeutic decisions, and planning clinical trials. recent advances in mri scanning techniques have allowed the examination of structural changes of the hippocampus, entorhinal cortex, and gray matter structures in the medial temporal lobe. diffusion tensor imaging allows accurate depiction of white matter microstructural integrity based on the directionality of diffusion in the brain. measurements of magnetization transfer ratio may be valuable for the detection of structural damage in the hippocampus of alzheimer 's disease patients. proton magnetic resonance spectroscopy has emerged as an increasingly important tool in discriminating between dementias and measuring disease progression. cross - sectional magnetic resonance spectroscopy confirmed that metabolic abnormalities in mild cognitive impairment are transitional between normal healthy older adults and patients with alzheimer 's disease. the utility of such functional imaging techniques has greatly improved the early diagnosis of mild cognitive impairment and alzheimer 's disease. technical advances in multiple imaging modalities allow us to assess both anatomical and functional changes in alzheimer 's disease and mild cognitive impairment, thereby advancing our understanding of the pathophysiological evolution of these diseases.	the rapidly increasing prevalence of cognitive impairment and alzheimer 's disease has the potential to create a major worldwide healthcare crisis. structural mri studies in patients with alzheimer 's disease and mild cognitive impairment are currently attracting considerable interest. it is extremely important to study early structural and metabolic changes, such as those in the hippocampus, entorhinal cortex, and gray matter structures in the medial temporal lobe, to allow the early detection of mild cognitive impairment and alzheimer 's disease. the microstructural integrity of white matter can be studied with diffusion tensor imaging. increased mean diffusivity and decreased fractional anisotropy are found in subjects with white matter damage. functional imaging studies with positron emission tomography tracer compounds enable detection of amyloid plaques in the living brain in patients with alzheimer 's disease. in this review, we will focus on key findings from brain imaging studies in mild cognitive impairment and alzheimer 's disease, including structural brain changes studied with mri and white matter changes seen with diffusion tensor imaging, and other specific imaging methodologies will also be discussed.
the acquired immunodeficiency syndrome (aids) epidemic is continuing to grow and global estimates indicated that over 40 million people are infected. the fact that the number of human immunodeficiency virus (hiv)-infected patients under dental care is expected to increase highlights the importance of providing healthcare, part of which is dental treatment, to all individuals indiscriminately. the reports indicated that about 90% of the hiv infections among healthcare workers occurs in developing countries where occupational safety is a neglected issue. the aids epidemic is one of the most destructive health crisis of modern times, ravaging families and communities throughout the world. in india, a semiautonomous body called national aids control organization (naco) was established under ministry of health and family welfare to control the hiv epidemic. according to joint united nations (un) programme on hiv / aids (unaids) and world health organization (who), approximately 34 million people are currently living with hiv and about 30 million people have died of aids - related causes since the beginning of epidemic. according to new estimates released by naco supported by unaids and who, an estimated 23.9 lakh people are infected with hiv in india by 2009 - 2010. according to naco, till date very less work has been done to assess the knowledge and attitude among indian dental students, hence a sincere attempt has been made on this front. the purpose of this study was to assess the dental student 's knowledge of hiv / aids and attitude toward them and their willingness to treat patients living with hiv and aids (plwha). a cross - sectional questionnaire survey was conducted among 600 students studying in third and fourth years of dental colleges located in national capital region (ncr). the students were given a predesigned questionnaire [table 1 ] during a regular theory class. all the students participated voluntarily in the study and were informed about the confidentiality of their response. all students were asked to report about their age, gender and year of study. the questionnaire included 25 questions out of which 15 questions represent knowledge of the subjects and 10 questions represent attitude of the subject towards hiv / aids. each and every question was explained to them before they answered to prevent any ambiguity. later the data were subjected to statistical analysis by using statistical package for social sciences (spss). one - way anova (analysis of variance) was used to compare the mean level of knowledge and attitude toward hiv / aids between the genders, years of study and age groups. to calculate mean level of knowledge and attitude, 0 later the data were subjected to statistical analysis by using statistical package for social sciences (spss). one - way anova (analysis of variance) was used to compare the mean level of knowledge and attitude toward hiv / aids between the genders, years of study and age groups. to calculate mean level of knowledge and attitude, 0 a total of 600 dental students returned a completed questionnaire giving an overall response of 100%. about 60% respondents fell into 21 - 23 years age group and majority of dental students were unmarried (89.5%). male to female ratio was approximately 1:2 [table 2 ]. according to the survey, only 28% subjects had excellent knowledge and 54% subjects had good knowledge regarding hiv and aids. in all, 7% responded poorly to the questionnaire [table 3 ]. when the subjects were asked where they will refer the hiv / aids patients for the treatment, 86% responded for hospital but 21.5% subject said they will refer them to traditional healers and 16% to miracle center (p value : 0.00) [table 4 ]. demographic characteristics there were more misconceptions regarding the mode of transmission among the subjects, 78.5% said the mode of transmission is unprotected sex, whereas 34 and 25% said breast feeding and kissing, respectively [table 5 ]. the most important source of information about hiv / aids was electronic media (63.5%) followed by newspaper (57%) and text books (57%) [table 6 ]. the study showed that there were very less involvement of parents (16.6%) regarding sharing the knowledge about aids / hiv [table 7 ]. source of information interpersonal communication only 43% subjects were aware about their own hiv status and only 58% were willing for hiv testing [table 8 ]. when asked about their attitude towards plwha, 43% subjects responded that they have the right to refuse the treatment and 29% said they should be quarantined [table 9 ]. five parameters were given regarding the risk perception, 67.6% subjects said that dentists are at high risk group, whereas 44.4% are not worried about hiv infection [table 10 ]. most of the survey has included less than 200 subjects, but in our study 600 subjects were involved. there was disproportionate gender distribution that was similar to the findings in study conducted by soukaina t raylat on jordanian dental students. we found that students showed moderate knowledge with respect to modes of hiv transmission and infection control practices. a similar finding nearly one - third (34%) of the students thought that hiv / aids could be acquired by the breast feeding and one - fourth (25%) thought that hiv could be contracted by kissing an infected person, which is a misconception. the attitude and willingness to treat hiv / aids patient was assessed and found an overall negative attitude of students toward hiv / aids patients., also reported dental students having negative attitude toward hiv / aids. according to our study, whereas in study conducted by ajayi yo and ajayi eo, main source of information was health workers and textbooks. this negative attitude of the dental students will have a direct impact on the treatment of the plwha. in this study, only 43% subjects were aware of their hiv status and 58% were willing for hiv testing. kopacz., in their study reported that 84% were aware of their hiv status and only 20% were willing for hiv testing. it is quite alarming that more than 50% of the students are not aware of their hiv status. the results of this survey can be interpreted as true representation of hiv / aids knowledge and attitude among dental students of ncr. dental students have repeatedly reported good knowledge regarding hiv / aids with some misconception that was also supported by our survey findings. from the present study, we conclude that knowledge regarding hiv / aids should be included from first year of dentistry or from school level, so that they are well trained in treating the plwha. therefore, students must be made well aware of the importance of treating hiv / aids patients and help the society from this drastic disease. it is recommended that a comprehensive training of the dental students be done, to promote a good delivery of accurate information on hiv / aids to the public and to provide proper patient care. dental students should work with different non governmental organizations (ngos) working for aids patients. a separate dental unit should be established in a hospital specially for treating plwha and dental students should be posted there for more exposure to the hiv / aids patients. emphasis must be placed on in - depth discussion on hiv / aids issue by experienced health workers and lectures with dental students in order to clarify existing misconceptions and discourage discriminatory behaviour.	background : india is estimated to have third highest number of human immunodeficiency virus (hiv) infection in world with about 2.4 million people currently living with hiv / acquired immunodeficiency syndrome (aids). there is a possibility of hiv transmission in the oral health care setting and thus adequate knowledge and proper attitude among dental students is vital to prevent the chances of transmission and for proper care of the patient.aims and objectives : the present study aimed to investigate the knowledge of dental students about hiv infection and their attitude toward treating hiv / aids patients and behaviour practiced.materials and methods : a cross - sectional survey was conducted among 600 dental students of different colleges present in national capital region (ncr). the students were from third and fourth year and they completed a predesigned questionnaire assessing the knowledge, attitude and willingness to treat hiv / aids patients. one - way analysis of variance (anova) was applied to compare mean level of knowledge and attitude toward hiv / aids.result : the results showed that only 28% students have excellent knowledge regarding hiv / aids. certain misconceptions were prevalent regarding mode of transmission. it also shows that around 43% of the dental students have an overall negative attitude.conclusion:the findings suggest that the students had adequate knowledge about hiv / aids and their attitude toward this group of people was significantly negative. there is need and scope to provide correct and detailed information on hiv / aids for dental students.
the high - resolution analysis of 1% of the human genome by the encode project has shown that up to 90% of the genome is being transcribed while only about 1.5% of these transcripts correspond to protein coding exons. therefore, it was suggested that the majority of the transcripts might serve as a source for regulatory non coding rnas (ncrnas) [2, 3 ], with the predicted number of ncrnas present in the human genome reaching up to 0.5 million transcripts. however, most of these transcripts still remain of unknown function, and their functionality is even debated. these novel exciting aspects of the cellular transcriptome content thus require novel methods for profiling ncrnas expression in a high - throughput manner. lately, the most widely used expression profiling technique has become high - throughput sequencing or rna - seq [5, 6 ], with numerous advantages. rna - seq provides full genome coverage and allows detection of single nucleotide polymorphisms as well as rna editing events, independently of hybridization artifacts. however, rna - seq drawbacks and artifacts are not completely absent, generally linked to reverse transcription or library generation protocols [6, 7 ]. in addition, analysis of sequencing datasets is still rather time consuming and requires a strong bioinformatic expertise, which does not make it suitable for rapid diagnostic or clinical profiling so far. recently, novel microarray technologies have evolved to efficiently profile mirna expression [8, 9 ] or detect single nucleotide polymorphisms by employing locked nucleic acid (lna) arrays. lnas are synthetic rna analogs characterized by increased thermostability of nucleic acid duplexes, allowing increased hybridization temperatures and thus improved mismatch discrimination. with the recent interest in ncrnas as biomarkers [1214 ], ncrna microarrays might represent a suitable tool to profile ncrna expression for diagnostic purposes. however, an lna platform would not be generally financially affordable for these applications. here, we describe a mixed dna / lna microarray platform that allows the hybridization of directly and simultaneously labeled small and longer ncrnas onto microarrays consisting of both lna - modified and custom - designed dna capture probes, respectively. this method enables a sensitive and specific analysis of a complex and heterogeneous rna population on a unique array in one experiment, complying with nowadays most criteria in biomedical diagnostics in terms of cost and sample requirements. the mircury lna mirna array ready - to - spot probe set (reference 208010) was purchased from exiqon (denmark) as an lna capture probe set for short ncrnas detection. this set comprises 2,056 capture probes designed to have a uniform tm of 72c and covers all mirnas of mirbase (version 9.2). they were 5-c6 amino - modified, designed so as to comply with a 72c tm, desalted and diluted in 3xssc, 1.5 m betaine buffer to a final concentration of 20 m. the lna - based capture probe set for short ncrnas as well as the self - designed dna - based capture probe set for long ncrnas was spotted on hisens epoxy - coated glass slides (nexterion) using the microgrid ii microarray spotter (zinsser analytic). every probe (antisense, mismatch, deletion, and sense) was spotted twice on the slide in four replicates (local separation) to ensure quality assurance and reliability. hybridizations have been performed using the tecan hs400 hybridization station according to the exiqon protocol for hybridization with the mirna lna platform. total mouse brain rna was extracted from c57/bl6 mice (48 weeks old) and total mouse embryonic stem cell rna from e14 stem cells with trireagent (sigma - aldrich) following the manufacturer 's protocol. total mouse brain rna (0.25 g5 g) and total mouse embryonic stem cell rna (2 g) were directly labeled employing the ncode rapid mirna labeling system (invitrogen), following the manufacturer 's protocol with the following modifications : (i) prior to poly - a tailing, rna was denatured at 90c for 3 min, centrifuged, and cooled on ice for 2 min and (ii) the reaction buffer provided with the kit was replaced by a custom reaction buffer containing 50 mm tris - hcl (ph 8.0), 250 mm nacl, and 10 mm mgcl2. for differential expression, 2 biological replicates of total mouse brain rna and total mouse embryonic stem cell rna were used. post - processing steps, including verification of probes specificity or processing events coverage, were added. for a full description, were used : snora71 5-tatcaatgaccagggcacccgcagccc-3, snord55 5-gtcgggagtgtgcagcatacccaggtg-3 and 5-gcaattcacattaattctcgcagctagc-3. total rna was isolated from mouse es cells and mouse brain of c57/bl6 mice, 46 weeks old, with tri reagent (sigma - aldrich, vienna, austria) according to the manufacturer 's protocol. five hundred nanograms of total rna were poly - a tailed and reverse transcribed to cdna using the microrna 1st strand synthesis kit (agilent technologies, bblingen, germany) following the manufacturer 's protocol. the cdna was used as template for the real - time pcr. the universal reverse primer provided with the kit was used together with the following forward primers : mmu - mir-125 - 5p 5-tccctgagaccctaacttgtga-3, mmu - mir-293 5-agtgccgcagagtttg - tagtgt-3, snord113 5-gggtgctgtatgagtcgtgtattatga-3, 7sk 5-ccattgtaggagaacgtagggtag-3, snoz39 5-tgatgaagcaaatcagtatgaataaaatg-3, snora18 5-tgactcacaggactgactgttaggcctg-3, snord55 5-cacctgggtatgctgcacactcc-3, snora71 5-ctgccggtgccctggtcattg-3, u6 5-ctcgcttcggcagcaca-3. primers were purchased from sigma - aldrich. real - time pcr was performed using power sybr green pcr master mix (applied biosystems, darmstadt, germany). all results from three technical replicates were normalized to u6 and expressed as ct values. five independent biological samples from either mouse brain or mouse es cells contributed to the data set. we investigated whether a combined dna and lna platform, dedicated to the expression analysis of long ncrnas as well as small ncrnas, respectively, could be used for the expression profiling of a heterogeneous population of ncrnas. to that end, we generated a custom microarray spotted with (i) dna capture probes for trnas, 7sk rna as well as c / d and h / aca box snornas (supplementary table 1) and (ii) the commercially available mircury lna mirna ready - to - spot probe set from exiqon. to generate a mixed dna / lna microarray, all probes spotted had to exhibit the same melting temperature. we opted for a fixed hybridization temperature to avoid elevated background due to unspecific hybridization, as observed when using temperature gradients (see the supplementary material available online at doi:10.1155/2012/283560). as the lna capture probe set melting temperature corresponds to 72c for an optimal hybridization temperature of 64c, dna capture probes were designed to comply with this criterion, independently of their sizes. dna capture probes were designed to hybridize to conserved regions of ncrnas, spanning regions of 30 to 60 nt (see section 2). 7sk rna and trnas were chosen to test hybridization capabilities for highly structured ncrnas and snornas to check for the system sensitivity. two or more dna capture probes were designed per ncrna if the length of the target was sufficient (supplementary table 1). additionally, in order to test the specificity of the system, probes bearing one or two nucleotides mismatches were designed in addition to the perfect matching antisense probes. finally, for more structured ncrnas, probes with one or two nucleotides deletions were designed (supplementary table 1). indeed, for microarray assays, small rnas are generally directly labeled while longer rnas are generally reverse transcribed into cdna and labeled through incorporation of aminoallyl - modified nucleotides. we employed a commercially available dual fluorescent dye rna labeling kit based on poly - a tailing and ligation of fluorophore - bearing dendrimers (see section 2). we used 5 g total mouse brain rna for our initial proof of concept experiments. to exclude dye bias effects, alexafluor3 and alexafluor5 labeled total mouse brain rna replicas were self - self hybridized on the custom dna / lna chip. analysis of the results showed that neural mirnas such as mir-9 and mir-9 were well detected (figure 1(a)) in contrast to snornas which were almost undetectable (figure 1(b)). 7sk rna was only marginally detectable (supplementary figure 1(a)) while trnas were almost not detectable at all. as insufficient detection of these longer ncrnas could be linked to secondary structure - related inefficient polyadenylation and labeling, we introduced a denaturation step prior to poly - a tailing. additionally, as mn cations were reported to stimulate unspecific activity of poly - a polymerase activity in vitro, we tested a mg custom poly - a tailing buffer (see section 2), which increased efficiency of labeling, most likely by stimulating polyadenylation. the improved labeling protocol enabled enhanced snornas, trnas, and 7sk rna detection (cf. supplementary figures 1(b) and 1(a), figures 2(a) and 2(b) and supplementary figure 1(c)) without altering detection of mirnas (cf. figures 1(a) and 1(c)). finally, posttranscriptional rna modifications, such as pseudouridylation or 2o - methylation [22, 23 ], might interfere with labeling of ncrnas ; we did not investigate, however, the extent of this parameter. together with rna labeling it was also necessary to optimize rna quantity used for labeling. as few as 30 ng total rna are generally sufficient for hybridization on lna microarrays, while dna microarrays require at least 1025 g of total rna as starting material for cdna labeling through reverse transcription. amounts of total rna ranging from 0.25 to 1 g per labeling reaction were used, with a first hybridization temperature of 56c. under these conditions, labeling of total rna quantities below 1 g provided insufficient results (data not shown) while labeling of 1 g of rna resulted in satisfying results (figures 2(a) and 2(b)). next, the hybridization temperature was raised to 64c to better comply with the lna platform. in order not to compromise the sensitivity at this temperature, the quantity of labeled total rna was raised from 1 g to 2 g. in these conditions, we observed similar results regarding mirna lna probes, but improved detection in the case of the 7sk rna and snornas (figures 3(a) and 3(b)). we generally observed improved detection at 64c for the dna probes compared to the 56c condition, while lna probes remained unaffected (figure 3(b)). however, as for tissue profiling, large quantities of total rna might not be available, we opted for 2 g of total rna per labeling reaction, with self - self hybridizations performed at 64c, which appeared as the best compromise. for diagnostic purposes with lower amounts of material, further optimization of the protocol might be needed. we next tested the influence of ncrna structure on the sensitivity and specificity of hybridization on the dna / lna combined platform. at 56c, expression of highly structured rnas, such as trnas and 7sk rna, could be detected (figures 2(a) and 2(b)). for example, trna was detected with a mean intensity of 7555 with the 60 nt long capture probe trnaphe_16 - 75, but with a reduced mean intensity of 1564 with the 30 nt long probe trnaphe_47 - 76 (figure 2(c)). on the other hand, detection of 7sk rna was almost 2-fold higher with the 36 nt long capture probe 7sk_126 - 162 compared to the probes 7sk_1763 and 7sk_5591 of 46 and 36 nucleotides in length, respectively, (figure 2(c)). thus, detection of highly structured ncrnas appears rather independent of the capture probe 's length, and employing multiple probes complementary to one particular rna therefore increases sensitivity of detection. at 64c, the results showed that antisense snorna capture probes detected efficiently snornas (figure 4(a)) with similar intensities compared to the condition where higher amounts of labeled total rna and lower hybridization temperatures were employed (figure 1(d)). moreover, almost all antisense snorna capture probes detected their specific snorna but with different intensities (figure 4(b)), while the detection levels of mir-9 and mir-9 remained identical (figure 4(c)). therefore, we wanted to test how specific the detection with dna capture probes can be with the dna / lna platform. the specificity was therefore checked employing also probes with mismatches at one (mm1) or two (mm2) positions. at 64c, the snorna snoz39 was only detected by the antisense and one nucleotide mismatch capture probes, while the signal with the two nucleotides mismatch probe snoz39_6 - 60mm2 was falling below threshold (figure 5(a)), indicating that discrimination was already possible with two nucleotides mismatches. however, the comparison of the mean intensity values between the perfect matching and one nucleotide mismatch probes showed a reduction of 40% for the mm1 probe for detection of snoz39 (figure 5(b)). in some cases though, a signal was still detectable with mm2 probes (7sk rna or snorna snord55, figure 5(c)), but with reduced intensities compared to the perfect matching capture probes. for instance, 7sk_1763mm2 and 7sk_5591mm2 showed a further 20% reduction in intensity levels compared to the mm1 capture probes (figure 5(c)). we next applied our dna / lna platform to expression profiling, employing dye swap experiments with 2 g of total mouse brain rna and 2 g of total mouse embryonic stem cells rna. as expected, differences in ncrna expression between mouse adult brain rna and mouse embryonic stem cell rna could be detected (figure 6). for example, stem cell specific mirnas of the mir-290 family (mir-291295,) were detected to be about 25-fold overexpressed in mouse embryonic stem cells, while brain - specific mir-124 and mir-9 were about 14-fold overexpressed in mouse brain (supplementary table 2). also let-7 was overexpressed in brain compared to es cells, which was expected, since mature let-7 is expressed upon stem cell differentiation into neural cells (data not shown) [25, 26 ]. regarding dna probes, overexpression of snord55 by about 2-fold and snora71 by about 5-fold in mouse embryonic stem cells (figure 6 and supplementary table 2) could be observed. this differential expression was confirmed by northern blots (figure 6(b)) where snord55 and snora71 appear overexpressed in embryonic stem cells in comparison to mouse brain. we employed real - time pcr to verify the differential expression observed and validate the dna / lna platform. as expected from the microarray data, we verified that mir-125b-5p and mir-293 were significantly overexpressed in mouse brain and mouse es cells, by 20.7-fold and 6.5-fold, respectively (figure 7). the snornas snora71 and snord55 were verified to be significantly overexpressed in mouse es cells in comparison to mouse brain as well, by 3.3- and 2.5-fold, respectively (figure 7). according to the dna / lna microarray platform data, we did not observe any differential expression for snord113 or 7sk rna between mouse es cells and mouse brain (figure 7). finally, while they did not appear to be differentially expressed in the microarray data, snora18 and snoz39 seemed to be overexpressed in mouse es cells or in mouse brain, respectively, according to real - time pcr results (figure 7). while this differential expression was not significant for snora18, it reached 3.6-fold for snoz39, highlighting the necessity to validate microarray data, as well as deep - sequencing data, by additional means like northern blotting or real - time pcr. nevertheless, our observation of differential expression of canonical snornas constitutes an exciting aspect, especially regarding their recently described noncanonical functions as mirna precursors or regulators of alternative splicing [2731 ]. ncrnas are now widely considered as excellent disease biomarkers. for instance, mirnas [14, 33 ], snornas or long interspersed noncoding rnas (lincrnas) can be employed to determine the origin of various cancers. noncoding rnas have also been shown to be involved in chromatin regulation or in neurological diseases [36, 37 ]. for diagnostic purposes, microarrays still appear as a less expensive method compared to high - throughput sequencing and, additionally, microarray analysis requires significantly less time. however, a microarray platform enabling simultaneous analysis of both small and long molecules for ncrna - based diagnostic or expression profiling was lacking. here we developed a microarray platform where both small and long ncrnas can be profiled on the same chip. the size limitation of small rnas prompted us to employ the already available lna platform for mirnas, to combine it with custom dna capture probes for longer ncrnas and to allow detection of all ncrnas with a universal direct labeling procedure. hence, long, structured ncrnas and mirnas could be detected with the dna / lna platform, and this detection was independent of the capture probe length but rather depending on secondary structure. we observed that capture probes were more efficient when designed to hybridize to less structured regions. in case of highly structured trnas, the mixed microarray is sensitive and specific and requires relatively low amounts of directly labeled total rna. problems due to ncrna structure can be solved if probes are designed to span low structured regions and if a denaturation step is introduced prior to rna direct labeling. hence, this platform might become a very attractive tool for combined expression profiling of small and long ncrnas as well as in biomedical diagnostic.	mammalian transcriptomes mainly consist of non protein coding rnas. these ncrnas play various roles in all cells and are involved in multiple regulation pathways. more recently, ncrnas have also been described as valuable diagnostic tools. while rna - seq approaches progressively replace microarray - based technologies for high - throughput expression profiling, they are still not routinely used in diagnostic. microarrays, on the other hand, are more widely used for diagnostic profiling, especially for very small ncrna (e.g., mirnas), employing locked nucleic acid (lna) arrays. however, lna microarrays are quite expensive for high - throughput studies targeting longer ncrnas, while dna arrays do not provide satisfying results for the analysis of small rnas. here, we describe a mixed dna / lna microarray platform, where directly labeled small and longer ncrnas are hybridized on lna probes or custom dna probes, respectively, enabling sensitive and specific analysis of a complex rna population on a unique array in one single experiment. the dna / lna system, requiring relatively low amounts of total rna, which complies with diagnostic references, was successfully applied to the analysis of differential ncrna expression in mouse embryonic stem cells and adult brain cells.
	essentially pure (97%) alveolar macrophages were isolated by bronchoalveolar lavage of rats with warm (37 degrees c) pbs solution. these cells were allowed to adhere to the inside walls of open - ended glass cylinders which were closed off at each end by three - way stopcocks. the adhering cells were perifused with rpmi-1640 medium supplemented with 5% fetal bovine serum for 18 hr at the rate of 1 ml / hr, and the effluent medium was collected automatically in 2-ml aliquots. cell recoveries and viabilities did not differ from those found for petri cultures treated similarly, indicating that the perifusion method under study offered an adequate milieu for short - term primary cultures. the alveolar macrophages in culture were subjected to the presence of particulate (chrysotile asbestos) and soluble (phorbol myristate) toxicants, and their response was monitored in the effluent medium by measuring the release of prostaglandins (pge) by radioimmunoassay. a significant increase in the sequential release of pge was observed in the presence of asbestos (100 micrograms / ml) or phorbol myristate (200 ng / ml). treatment of the cells with indomethacin (20 microm) completely abolished the release of pge stimulated with phorbol myristate. a cumulative response to the toxicants was also observed when cells were harvested manually from the chambers : asbestos caused a 2-fold increase in cell mortality relative to control, while phorbol myristate brought about a 3-fold increase in the number of dead cells. this effect was not prevented by the presence of indomethacin. cell aggregation was also observed when cells were perifused in the presence of phorbol myristate, whether indomethacin was present or absent. our results indicate that the cell perifusion system combines the advantages of conventional adherent cell cultures (viability, aggregation) with those of perifusion techniques (sequential metabolism studies).imagesfigure 1.figure 2.figure 3.figure 6.
regional anesthesia is gaining popularity as a sole anesthetic technique and is also performed in conjunction with general anesthesia for a large diversity of surgical procedures.1 it offers superb postoperative analgesia, has preventive effects in regards to the occurrence of postoperative complications, and may play a role in enhanced recovery programs.24 furthermore, an increasing number of anesthesiologists are trained and educated in performing regional anesthesia during their residency, resulting in wider application of regional anesthetic techniques with or without ultrasound (us) guidance.5 in daily practice, however, some regional anesthetic possibilities may be easily overlooked. this is illustrated in the following cases of two very old patients with considerable comorbidities in whom, eventually, a mental nerve blockade was used as the sole anesthetic technique. by presenting these two cases, we would like to bring the mental nerve field block under renewed attention as a safe alternative to general anesthesia in high - risk patients and to achieve broader application of this easy - to - perform nerve block. an 84-year - old man was scheduled for surgical excision of an ulcerative lesion at the lower lip under general anesthesia. he suffered from chronic obstructive pulmonary disease and had had a pneumonectomy at the age of 68 years resulting in a forced vital capacity of 1.78 l and a forced expiratory volume of 1.4 l after 1 second. his exercise tolerance was estimated to correspond to 48 metabolic equivalents.6 according to the consultant pulmonologist, the chronic obstructive pulmonary disease was optimally treated. because of the patient s age and comorbidity, blockade of the mental nerve on both sides as an alternative anesthetic technique was considered superior to general anesthesia and informed consent was obtained from the patient. on the day of surgery, a 23-gauge needle was directed into the buccal mucosa until it approximated the mental foramen. after careful aspiration to exclude intravascular placement, a dose of 2 ml of lidocaine 2% with adrenaline 1:100.000 was given and the same procedure repeated on the other side. after a few minutes, the anesthesiologist stimulated the lower lip and chin with an ice cube to determine the onset and area of desensitization. before incision, the surgeon confirmed effective analgesia of the lower lip and chin by giving a sharp stimulus with a surgical forceps. a squamous cell carcinoma was surgically removed from the lower lip uneventfully followed by same - day discharge to home. a 91-year - old man was also scheduled for surgical excision of an ulcerative lesion at the lower lip under general anesthesia. in addition, a transient ischemic attack and vascular encephalopathy had resulted in an abnormal gait. preoperative consultation by the cardiologist revealed a preexisting, silent anteroseptal myocardial infarction that had resulted in a hypokinetic anteroseptal wall with a satisfactorily contracting left ventricle (dimensions diastolic / systolic : 46 mm/24 mm). his exercise tolerance was estimated to correspond to 24 metabolic equivalents.6 the patient gave consent to undergo the procedure receiving regional anesthesia only. a bilateral mental nerve block was performed in a similar fashion as with patient a, using 2 ml of lidocaine 2% with adrenaline 1:100.000 for each mental nerve. after an uneventful procedure, the patient was discharged from our hospital the same day. an 84-year - old man was scheduled for surgical excision of an ulcerative lesion at the lower lip under general anesthesia. he suffered from chronic obstructive pulmonary disease and had had a pneumonectomy at the age of 68 years resulting in a forced vital capacity of 1.78 l and a forced expiratory volume of 1.4 l after 1 second. his exercise tolerance was estimated to correspond to 48 metabolic equivalents.6 according to the consultant pulmonologist, the chronic obstructive pulmonary disease was optimally treated. because of the patient s age and comorbidity, blockade of the mental nerve on both sides as an alternative anesthetic technique was considered superior to general anesthesia and informed consent was obtained from the patient. on the day of surgery, a 23-gauge needle was directed into the buccal mucosa until it approximated the mental foramen. after careful aspiration to exclude intravascular placement, a dose of 2 ml of lidocaine 2% with adrenaline 1:100.000 was given and the same procedure repeated on the other side. after a few minutes, the anesthesiologist stimulated the lower lip and chin with an ice cube to determine the onset and area of desensitization. before incision, the surgeon confirmed effective analgesia of the lower lip and chin by giving a sharp stimulus with a surgical forceps. a squamous cell carcinoma was surgically removed from the lower lip uneventfully followed by same - day discharge to home. a 91-year - old man was also scheduled for surgical excision of an ulcerative lesion at the lower lip under general anesthesia. in addition, a transient ischemic attack and vascular encephalopathy had resulted in an abnormal gait. preoperative consultation by the cardiologist revealed a preexisting, silent anteroseptal myocardial infarction that had resulted in a hypokinetic anteroseptal wall with a satisfactorily contracting left ventricle (dimensions diastolic / systolic : 46 mm/24 mm). his exercise tolerance was estimated to correspond to 24 metabolic equivalents.6 the patient gave consent to undergo the procedure receiving regional anesthesia only. a bilateral mental nerve block was performed in a similar fashion as with patient a, using 2 ml of lidocaine 2% with adrenaline 1:100.000 for each mental nerve. after an uneventful procedure, the patient was discharged from our hospital the same day. in both cases, a carcinoma of the lower lip was successfully removed using a mental nerve block. basal and squamous cell carcinoma are common skin cancers in caucasians, and sun (ultraviolet light) exposure is the most prominent risk factor. due to the strong relationship with solar radiation, the majority of skin cancers occur on the face and head. unawareness of and inexperience in orofacial, regional anesthetic techniques for the removal of lower lip cancer were the reasons that these techniques were originally not considered in the preoperative period. the mental nerve is a terminal branch of the mandibular nerve, which in turn is part of the fifth cranial, trigeminal nerve and innervates the ipsilateral side of the lower lip, mucosa, and skin of the chin. the mental nerve leaves the mental foramen, which can be palpated intraorally just caudal to the lower first and second premolar, midway between the tooth and inferior mandibular border. the mental foramen can be localized extraorally, in a vertical plane with the ipsilateral pupil in mid - position. after eversion of the lip, the mental foramen can be reached intraorally by directing a 23-gauge needle with a 5 ml syringe attached into the buccal mucosa. however, to prevent nerve injury, it is advised not to enter the mental foramen. infiltration with 2 ml of anesthetic solution will suffice to produce effective analgesia of the lower lip and chin for operative procedures ; in our clinic, oral and maxillofacial surgeons use this volume for intraoral mental nerve blocks. the time to onset of analgesia and the duration of the nerve block were comparable in both patients in this report, ie, less than 3 minutes and approximately 60 minutes, respectively. we performed our nerve blocks using the intraoral approach in a blind fashion, but us guidance for percutaneous mental nerve block seems recommendable for several reasons. first, anatomic variations in the position of the mental foramen, related to age and dentition, are known. the mental foramen is more caudal on the mandibular ramus in youth and closer to the alveolar margin of the mandible in the edentulous aged person.1 therefore, us anatomy becomes more important than surface anatomy landmarks. second, the more widespread use of us by a growing number of anesthesiologists confirms a trend of questioning not if us guidance is useful, but rather where it can be applied. finally, us guidance is suitable for educational purposes as well as improving learning curves. recently, us guidance for mental nerve block was used with success in three cases of postherpetic neuralgia.7 in both of the present cases, a regional anesthetic technique was considered preferable and superior to general anesthesia, because the patients were very old with an estimated increased frailty due to their serious comorbidities. additionally, the procedure in patient a was performed in a beach - chair position to keep the closing capacity of the lung below the functional residual capacity, thereby preventing atelectasis and optimizing the ventilation / perfusion ratio of the single lung. this beach - chair position would be more challenging under general anesthesia, leading to a greater risk of complications. to our knowledge, this case report is the first to describe successfully performed mental nerve blocks in clinical prac - tice in two very old patients for the surgical removal of lower lip carcinoma. an older study compared the percutaneous versus intraoral technique in terms of pain of administration and effectiveness of anesthesia, but all subjects were healthy volunteers aged 22 to 33 years.8 lower lip surgery can be successfully performed under a mental nerve block, which is an easy - to - perform field block	purposeregional anesthesia is gaining popularity with anesthesiologists as it offers superb postoperative analgesia. however, as the sole anesthetic technique in high - risk patients in whom general anesthesia is not preferred, some regional anesthetic possibilities may be easily overlooked. by presenting two cases of very old patients with considerable comorbidities, we would like to bring the mental nerve field block under renewed attention as a safe alternative to general anesthesia and to achieve broader application of this simple nerve block.patients and methodstwo very old male patients (84 and 91 years) both presented with an ulcerative lesion at the lower lip for which surgical removal was scheduled. because of their considerable comorbidities and increased frailty, bilateral blockade of the mental nerve was considered superior to general anesthesia. as an additional advantage for the 84-year - old patient, who had a pneumonectomy in his medical history, the procedure could be safely performed in a beach - chair position to prevent atelectasis and optimize the ventilation / perfusion ratio of the single lung. the mental nerve blockades were performed intraorally in a blind fashion, after eversion of the lip and identifying the lower canine. a 5 ml syringe with a 23-gauge needle attached was passed into the buccal mucosa until it approximated the mental foramen, where 2 ml of lidocaine 2% with adrenaline 1:100.000 was injected. the other side was anesthetized in a similar fashion.resultsboth patients underwent the surgical procedure uneventfully under a bilateral mental nerve block and were discharged from the hospital on the same day.conclusiona mental nerve block is an easy - to - perform regional anesthetic technique for lower lip surgery. this technique might be especially advantageous in the very old, frail patient.
solitary plasmacytoma (sp) is defined as a solitary mass of neoplastic plasma cells, and can be classified into 2 types according to location : skeletal and non - skeletal plasmacytoma. the clinical outcome of sp varies greatly ; many patients are cured with the appropriate therapy but some patients develop disseminated multiple myeloma (mm) years later. radical radiotherapy and alternative surgery are treatment modalities producing sufficient local control12,14). however, despite these treatments, 50 - 60% of patients with sp progresses to mm6,17). regarding time to progression, knobel.13) reported that median time to mm development from skeletal sp was 21 months with a 5-year probability of 51% and bertanha.1) analyzed the average time was 41 months. thus, identifying the predictors associated with plasma cell malignant proliferation, in addition to the primary detection of aggravation of the sp are crucial in the management and survival of patient. in general, clinicians assume that patients with sp who do not have profiles consistent with progression to multiple myeloma will be eventually cured, or will progress to mm only slowly. this report describes a case of sp of the lumbar vertebra without progression factors, which developed into mm less than two months after initial diagnosis despite appropriate treatment. he had experienced low back pain and hyperesthesia in the right leg for six weeks without a trauma history. neurological evaluation revealed right hip flexion (g3/5) and extension weakness (g4/5), and the patient showed an abnormal increased sensitivity in the right leg l3 dermatome, and he had continuous lower back pain with tenderness. computed tomography (ct) scanning and magnetic resonance imaging (mri) showed a geographic osteolytic lesion involving the right mid - posterior element of the l3 vertebra and the right psoas muscle, and an epidural mass with dural sac compression (fig. subsequently, the patient underwent f-18 fdg whole body pet / ct examination, which revealed a destructive bone lesion and a paravertebral soft tissue mass with mild increased fdg tracer uptake (suvmax=4.6)(fig. we performed a total corpectomy of l3 to remove the destructive column and dural sac mass and stabilized the vertebrae with an expandable interbody cage (synex system, synthes, usa) and posterior pedicle screw fixation l1/2/4 (varian medical system, varians, usa) (fig. the immunohistochemical (ihc) staining of the neoplastic plasma cells revealed only weak immunoglobulin (ig) kappa chain restriction and cd138 positive expression (fig. bone marrow aspiration biopsy was performed when the plasmacytoma was diagnosed, and normal marrow proliferation was observed. serologic studies of immunofixation and protein electrophoporesis revealed weak monoclonal gammopathy, igg - kappa type. the serum free light chain (sflc) ratio was 1.16 (normal reference range ; 0.26 - 1.65). the patient had no rapid progression factors and underwent local fractional radiotherapy (rt) at a dose of 45 gy. less than two months after pathologic diagnosis, he noted a painful, soft mass, 33 cm in diameter in the left wrist. a repeat pet / ct also demonstrated multiple bony masses with moderately increased fdg uptake in the left distal ulna, right parietal skull, right and left clavicle, right and left humerus, right radius, sternum, left 8th rib, right and left ilium, left acetabulum, and left proximal femur, due to spread of malignant tumor. a soft tissue mass with increased fdg uptake was noted in the right proximal thigh with lymph node enlargement seen in the right and left external iliac, left pericolic, and right inguinal lymph nodes (fig. the right inguinal mass was excised for evaluation for multiple myeloma, and ihc staining of the mass revealed a strong kappa chain positive reaction. the patient was diagnosed with mm and underwent adjuvant chemotherapy, and thalidomide and dexamethasone were administered. solitary plasmacytoma of bone (spb) represents only 5% of all plasma cell malignancies and is a heterogenous condition4,5,9). the usual presentation of this is with bone pain ; however, 25% develop neurological dysfunction in the form of cord or nerve root compression19,21). the diagnosis of spb requires the presence of a solitary bone lesion confirmed by skeletal survey including bone scan or pet / ct, abnormal plasma cell proliferation proven by bone marrow or tissue biopsy and lack of proof of organ dysfunction1,3,25,30). knobel.13) confirmed favorable local disease control with radiotherapy alone in their review of 206 patients with spb. local relapse occurred in 21 (14%) out of 148 patients who received radiotherapy alone compared with 4 (80%) out of 5 patients who were treated with surgery and chemotherapy. previous studies recommend radiotherapy for spb encompassing the tumor volume shown on mri with a margin of at least 2 cm and treating to a dose of 40 gy in 20 fractions with a higher dose of 50 gy in 25 fractions being considered for spb>5 cm24). recently researchers evaluated the outcomes of over 125 patients with sp receiving only radiation therapy as initial treatment. recurrences occurred in only 20% of patients who showed a disappearance of immunoglobulin (m - protein) following treatment with radiation. patients who did not show a decrease in m - protein levels following radiation therapy had a 60% chance of recurrence. this result was compared to other reports evaluating the outcome of patients with sp who received both chemotherapy and radiation as initial treatment. the addition of chemotherapy to radiation therapy revealed no overall benefit regarding the rate of progression to mm5,6). surgery (" radiotherapy " versus " surgery and radiotherapy ") did not affect the 10-year probability of local control5,6). therefore, surgical resection is not indicated for spb, but some patients may require neural decompression, spinal stabilization because of their neurologic compromise or structural instability8,17,21). this patient complained of right leg monoparesis with difficulty in ambulation and lumbar mri revealed epidural mass with dural sac compression of l3. he underwent surgical decompression with stabilization of l1/2/4 using pedicle screws. according to the mostly used durie and salmon (ds) staging system, the international myeloma staging system (iss) was announced which divides ds stage i patients into three further stages9). this patient belonged to ds stage ia and iss stage i ; this stage is defined as showing mildly increased serum beta 2-microglobulin and normal serum albumin. the natural course of stage i when traced over a 10-year follow - up period demonstrates four patterns of prognosis. these are development of mm (65%), local recurrence (12%), dissemination to a new solitary lesion (15%), and cured. the average median time is 24 to 36 months5,9,13,18,25), knobel.13) reported it is 21 months (range : 2 - 135), and bertanha.1) announced it is 41 months (range : 1.5 - 120). less than two months, rapid progression to mm in sp of ds stage ia and iss stage i without potential dissemination factors is a rare condition. for the difficulty in predicting prognosis, most patients need further follow - up to detect possible progression of sp and monitoring with regular serum or urine immunologic studies. there are several reports on the potential risk factors that influence the frequency of progression to mm. knobel.13) found that younger patients, especially with vertebral localization, had the better outcome when treated with moderate - dose (30 gy) rt. kilciksiz.12) reported that younger age was an independent good prognostic factor for progression to mm. lesion size was reported as one of the prognostic features for conversion of spb into mm. tsang.27) showed that patients with a mass size < 5 cm had a local control rate of 100%, while for patients with larger tumors, the rate reached was almost 40%. in this case, the disrupted soft mass was about 4 cm in diameter. the optimum rt dose of treatment of spb has not been established20) but local failure has not been observed in the patients who received 45 gy or more to the isolated lesion15,27). following rt application, in most spb patients, the myeloma protein level has a key role in the primary detection of mm13,16,19,29) pathological factors have been investigated in some studies. kumar.16) examined whether increased angiogenesis may help to identify the patients likely to progress to myeloma. recently, an abnormal sflc ratio has been shown to be a powerful prognostic factor in determining the risk of progression to mm at the time of the initial diagnosis in patients with sp7,22,23). to assess clonality, dingli.7) used the ratio of kappa - lambda light chain levels and reported that patients with an abnormal flc had a higher incidence of monoclonal protein in the urine and a larger serum m spike. the surgical procedure itself reduces immunity and diminishes natural killer (nk) cell activity 2,10,26). angiogenesis is the process by which new blood vessels are formed during wound healing process after surgery. this may not only benefit local recurrence and the formation of metastatic disease, but also result in activation of dormant micrometastases16,28). in abundant angiogenesis condition, isolated plasma cell tumors that break away from the primary spb must breach the connective tissue and then enter a blood vessel or lymphatic system28). the patient in our case was diagnosed with spb during his first admission. though he had no progression risk factors, less than two months later the spb of the lumbar vertebra we supposed that the possibility of rapid dissemination caused by surgery itself may be considered in this patient without any progression factors. though patients presenting with solitary plasmacytoma may have no risk factors for developing mm, we suggest that surgeons should be always be aware of the potential for rapid progression to mm from the time of the initial diagnosis.	the prognosis of solitary plasmacytoma varies greatly, with some patients recovering after surgical removal or local fractional radiation therapy, and others progressing to multiple myeloma years later. primary detection of progression to multiple myeloma is important in the treatment of solitary plasmacytoma. there have been several analyses of the risk factors involved in the early progression to multiple myeloma. we describe one case of solitary plasmacytoma of the lumbar vertebra that was treated with surgical decompression with stabilization and additional radiotherapy. the patient had no factors associated with rapid progression to multiple myeloma such as age, size, immunologic results, pathological findings, and serum free light chain ratio at the time of diagnosis. however, his condition progressed to multiple myeloma less than two months after the initial diagnosis of solitary plasmacytoma. we suggest that surgeons should be vigilant in watching for rapid progression to multiple myeloma even in case that the patient with solitary plasmacytoma has no risk factors for rapid progression to multiple myeloma.
mycotic aortic pseudoaneurysm is a rare condition, but it is life - threatening due to its rapid progression. it has been associated with a range of infectious conditions, including psoas abscesses, from which salmonella, staphylococcus, and escherichia coli are commonly isolated. however, cases of psoas abscess caused by methicillin - resistant staphylococcus aureus (mrsa) following spinal fusion are extremely rare. in the present report, we describe a case of mycotic abdominal aortic pseudoaneurysm due to a psoas abscess caused by mrsa after spinal fusion. to our knowledge, this is the first such case to be reported in the literature. a 36-year - old man presented to wonkwang university hospital with a one - month history of left flank pain and a one - week history of fever. he had undergone thoracoscopic anterior spinal fusion with the plate system and posterior screw fusion of t12 to l1 for the treatment of a t12 burst fracture three months previously. computed tomography (ct) indicated the presence of a psoas muscle abscess (fig. laboratory test results showed an elevated erythrocyte sedimentation rate (56 mm / hr) and c - reactive protein level (118 mg / l). after the empirical systemic administration of first - generation cephalosporin, ct - guided percutaneous catheter drainage (pcd) and biopsy were performed. mrsa was identified in blood and pus culture, and the treatment was changed to vancomycin. we therefore performed repeat ct and identified a pseudoaneurysm in the abdominal aorta around the abscess. we confirmed the presence of a large abdominal aortic pseudoaneurysm immediately below the diaphragm on aortography (fig. after a thoracoabdominal incision was made and laparotomy was performed, we approached the pseudoaneurysm via the retroperitoneum. 2). in situ reconstruction of the aorta was performed with a prosthetic vascular graft (18-mm vascutek, gelseal ; terumo, ann arbor, mi, usa). vancomycin was therefore administered, with the serum drug level maintained at > 10 g / ml. the patient gradually recovered, but fever reoccurred 33 days postoperatively. moreover, the vancomycin dose was increased, with the serum drug level maintained at 15 g / ml. after 11 weeks of systemic vancomycin administration, the patient was discharged and has undergone two years of follow - up ; at present, he remains asymptomatic. due to its rich blood supply, the psoas muscle is believed to be predisposed to primary abscess formation by hematogenous spread, primarily among children and young individuals. secondary psoas abscess is generally observed in industrialized countries and primarily affects patients aged 1050 years primary psoas abscess with s. aureus as the primary pathogen only accounts for 20% of all cases of psoas abscess ; the remaining 80% of cases of psoas abscess have been reported to be secondary psoas abscesses from adjacent infectious sources. a previous study found that skeletal infections (48%, 29/61), such as vertebral osteomyelitis (33%), pelvic osteomyelitis (8%), and septic arthritis (7%), were the most frequent source of secondary infections, followed by intra - abdominal infections (23%). although rare, several cases of psoas abscess after spinal fusion have been reported in the literature. alonso. reported that mrsa has recently become the predominant pathogen involved in cases of psoas abscess, accounting for 32.5% (13/40) of all cases of psoas abscess with a definitive pathologic diagnosis. therefore, although only one case of psoas abscess due to mrsa after spinal surgery has been reported in the english - language literature, an increase in the incidence of psoas abscesses with mrsa after spinal surgery is expected in the future. such infectious conditions may cause bacteremia and the embolization of infectious agents that are implanted in diseased or atherosclerotic arterial walls or mural thrombi. alternatively, as observed in the present case, the infectious agent may directly penetrate an adjacent vascular structure, resulting in necrosis and mycotic pseudoaneurysm formation. mycotic aortic pseudoaneurysm is a life - threatening condition that exhibits relatively rapid progression in comparison with noninfectious aortic aneurysms. most mycotic aneurysm patients are symptomatic and complain of back, abdominal, or thoracic pain depending on the location of the aneurysm. fever, back pain, leukocytosis, and elevated inflammatory marker levels are common symptoms and signs of psoas abscess and mycotic pseudoaneurysm. although the psoas sign is helpful in the diagnosis of psoas abscess, it is difficult to distinguish between psoas abscess and mycotic pseudoaneurysm due to their similar symptoms and signs. the optimal surgical management of mycotic aneurysm and graft selection (antibiotic - bondage grafts, silver - coated dacron grafts, or cryopreserved arterial homografts, among others) remain controversial. however, for infrarenal lesions, extra - anatomic bypass grafts are the standard treatment used to avoid graft contamination. in contrast, for suprarenal lesions, in situ prosthetic graft reconstruction following mycotic aneurysm resection is the preferred method of revascularization. overall, the removal of the instrumentation combined with extensive debridement, irrigation, administration of long - term antibiotics, and in situ revascularization using a prosthetic graft is considered the treatment of choice [46 ]. in cases involving infection after spinal surgery, as occurred in our patient, however, if the infection develops before the maturation of the fusion, the implant can be left in place and irrigation and debridement can be performed successfully. in the present case, the abscess recurred despite debridement, irrigation, and wrapping of the prosthetic graft with omentum. however, the abscess was successfully treated with pcd and long - term antibiotic administration, and instrumentation removal was not necessary. we believe that insufficient debridement, the presence of mrsa, and the remaining metal instrumentation influenced the recurrence in the present case. however, graft wrapping prevented contamination and facilitated successful treatment with pcd and long - term (three - month) antibiotic administration. thus, intensive antibiotic therapy may be crucial for the successful treatment of such cases. the initial use of broad - spectrum antibiotics and the parenteral administration of culture - specific antibiotics for more than six weeks is recommended. muller. suggested that postoperative antibiotics should be administered for at least three months and should be discontinued only when the patient does not exhibit any further signs of infection. in conclusion, complete resection of the infected tissue and removal of instrumentation prior to in situ graft reconstruction is necessary. however, in patients who can not undergo complete removal of the infected tissue and the metal instrumentation, omental wrapping may provide a suitable barrier to prevent the propagation of contamination, facilitating successful treatment.	a 36-year - old man, who had undergone thoracoscopic anterior spinal fusion using the plate system and posterior screw fusion three months previously, presented to our hospital with left flank pain and fever. computed tomography indicated the presence of a psoas muscle abscess. however, after two days of percutaneous catheter drainage, a mycotic abdominal aortic pseudoaneurysm was detected via computed tomography. we performed in situ revascularization using a prosthetic graft with omental wrapping. methicillin - resistant staphylococcus aureus was identified on blood and pus culture, and systemic vancomycin was administered for one month. although the abscess recurred, it was successfully treated with percutaneous catheter drainage and systemic vancomycin administration for three months, without the need for instrumentation removal. the patient remained asymptomatic throughout two years of follow - up.
plyometric exercise involves the use of fast eccentric loading to produce increased concentric force, also known as the stretch - shortening cycle (ssc) (12). plyometric exercises that utilize the ssc can be used as specific training for athletes involved in sports that require fast explosive movements (4, 18). upper extremity plyometrics have also been suggested as an integral part of terminal rehabilitation for overhead throwing athletes (4, 13, 18). most research concerning plyometrics has focused on the lower extremity (1). in contrast, there is a lack of research on upper extremity plyometrics (4, 11). of the studies examining upper extremity plyometrics, many focus on open - chain exercises. ballistic theraband (the hygenic corporation, akron oh, usa) exercises and overhead medicine ball throws (2) have also been included in training studies examining the effects of plyometrics on power and shoulder internal rotator strength. electromyography (emg) and kinetics during medicine ball drops have also been examined with regards to plyometric effects on traditional strength training methods (5). additionally, ballistic bench press throws (7, 11), have been utilized to examine the effects of load on kinematics and kinetics. research concerning closed kinetic chain upper extremity plyometric exercises, specifically various types of plyometric push - ups is limited. 18), and davies (3) suggest the use of plyometric push - ups as an upper extremity plyometric exercise but do not report any quantitative data concerning exercise intensity or progression. a training study by vossen. (16) assessed the efficacy of plyometric push - ups on upper extremity power and strength measures when compared to traditional push - ups, but their use of female subjects performing push - ups from the knees does not allow for comparison to push - ups performed from the toes by males. (6) assessed various types of traditional and plyometric push - ups, including clap and alternating ball pushups, mainly examining spinal loading and trunk muscle emg during the push - up variations. while research on varying aspects of plyometric push - ups has been conducted, to date, only two studies have selectively examined kinetic or kinematic variables regarding plyometric push - ups. (8) studied surface emg and vertical ground reaction force (vgrf) of countermovement push - ups, fall pushups, and jump push - ups in an attempt to quantify the intensity of the three variations. the authors recommend utilizing the various push - up types depending on the goal of the training session due to the musculoskeletal demands established through examination of emg and vgrf data. (10) examined five characteristics of vgrf during box drop and clap push - ups. the results of this study revealed no significant differences in peak vgrf between clap push - ups and box drop push - ups from various heights. theoretically, a fall from a greater height would result in greater vertical forces, but as these results were not obtained by koch. (10) the purpose of the present study was to examine various upper extremity kinematic variables during clap push - ups (cpu) and box drop push - ups (bd) in physically active males to potentially explain the lack of peak vgrf differences revealed in the previous study. kinematic variables including peak flight, elbow flexion at ground contact, elbow flexion displacement, and hand separation at ground contact during cpu and bd from various heights were examined. in addition we hypothesized that peak vgrf would be similar between all conditions because of increased elbow flexion displacement as the box drop heights increased, with cpu revealing the greatest elbow flexion displacement. additionally, we expected that peak flight height would increase as the box height increased with the cpu condition having the highest peak flight. further, we hypothesized that elbow flexion at ground contact and hand separation would be similar between all conditions. twenty - one recreationally active adult males participated in this study (24.5 3.7 yrs, 1.82.05 m, 83.2 11.7 kg). recreationally active was defined as participating in moderate- to vigorous - intensity exercise at least three times a week, 20 minutes per session. subjects were included in the study if they could perform four repetitions of the cpu and bd from 11.4 cm without any body part other than the hands and feet touching the ground and with minimal torso flexion or extension. all subjects were free of any upper extremity pathology or musculoskeletal injury within the past six months. each participant received a full description of study procedures and signed an approved armstrong atlantic state university institutional review board informed consent document prior to study participation. this included a five - minute warm up on an upper - body ergometer (cybex aerobic ube, medway, ma, usa) at a self - selected pace, as well as arm circles and a static stretch for the chest, shoulders and triceps held for 2030 seconds each. arm circles constitute a dynamic shoulder stretch and the static stretches were included to ensure that the involved musculature would be prepared for the high - intensity push - ups. upon completion of the warm up and stretches, subjects were instructed on the procedures for the push - up variations. for all four variations, subjects positioned each hand on a separate force plate using a self - selected hand placement width. additionally, for all variations, subjects started in an up push - up position with their hands on the force plates, their elbows fully extended, torso in a straight line, legs extended and their toes on a platform of equal height to the force plate (figure 1). bd plyometric push - ups used 3.8 cm height plyometric boxes. bd1 used one box (3.8 cm), bd2 used two boxes stacked (7.6 cm), and bd3 used three boxes stacked (11.4 cm) (figures 24). to perform the box drop push - ups, subjects lowered themselves towards the force plates then forcefully pushed themselves up, landing with their hands on the boxes. subjects then pushed themselves up off the boxes and landed with their hands on the force plates. pushing off from the plates, landing on the boxes and landing back on the plates counted as one repetition. in keeping with the explosive nature of plyometrics, subjects were not required to complete a full push - up while their hands were in contact with the boxes but instead were encouraged to flex their elbows enough to catch themselves, and then fully extend their elbows to push themselves off the boxes before landing on the force plates. to perform the clap pushups (cpu), subjects lowered themselves towards the force plates then forcefully pushed themselves into the air and performed a clap before returning their hands to the separate force plates (figure 5). during the cpu, subjects were instructed to push off the force plates as explosively as possible. this protocol is consistent with directions given when instructing someone to perform these two different types of plyometric push - ups. data collection began after subjects were given time to practice and qualitative observation of the push - ups indicated proper technique ; in addition, this served as a specific warm - up for the plyometric push - ups. kinematic and vgrf data were collected while the subjects completed four repetitions of each push - up variation (bd1, bd2, bd3, and cpu) in a counterbalanced order, resting no less than 90 seconds between variations. an electromagnetic tracking system (motion monitor, innovative sports training, inc chicago, il, usa) collected (100 hz) kinematics of the trunk, dominant elbow and both hands. separate sensors were attached to subjects seventh cervical vertebra s spinous process, the upper arm just distal to the deltoid insertion, the forearm (over the ulna to minimize sensor movement from forearm musculature), and to the dorsal side of both hands. during subject set - up, joint centers of the shoulder, elbow and wrists were calculated by taking midpoints between contralateral points at each respective joint using an additional electromagnetic sensor attached to a customized calibrated stylus. from the collected kinematic data, peak flight, elbow flexion at ground contact, elbow flexion displacement, and hand separation at ground contact were computed. peak flight was calculated as the maximal vertical trunk position during push - up flight based on the position of the sensor on c7. elbow flexion at ground contact was calculated as the angle of the elbow at ground contact. elbow flexion displacement was calculated using the difference between elbow flexion at ground contact and peak elbow flexion. vgrf data was collected (1000 hz) using two force plates (bp400600nc 2000 advanced mechanical technology, inc., watertown, ma, usa) with body weight normalized peak vgrf computed under the dominant extremity only. because of the novel nature of this study, we chose to only initially examine dominant extremity kinematics and vgrf data. for the bd push - ups, kinematic and vgrf data were computed only from the push - up phase involving impact onto and propulsion from the force plates. chicago, il, usa) using separate repeated measures analysis of variance for peak flight, elbow flexion at ground contact, elbow flexion displacement, hand separation and vgrf. twenty - one recreationally active adult males participated in this study (24.5 3.7 yrs, 1.82.05 m, 83.2 11.7 kg). recreationally active was defined as participating in moderate- to vigorous - intensity exercise at least three times a week, 20 minutes per session. subjects were included in the study if they could perform four repetitions of the cpu and bd from 11.4 cm without any body part other than the hands and feet touching the ground and with minimal torso flexion or extension. all subjects were free of any upper extremity pathology or musculoskeletal injury within the past six months. each participant received a full description of study procedures and signed an approved armstrong atlantic state university institutional review board informed consent document prior to study participation. this included a five - minute warm up on an upper - body ergometer (cybex aerobic ube, medway, ma, usa) at a self - selected pace, as well as arm circles and a static stretch for the chest, shoulders and triceps held for 2030 seconds each. arm circles constitute a dynamic shoulder stretch and the static stretches were included to ensure that the involved musculature would be prepared for the high - intensity push - ups. upon completion of the warm up and stretches, subjects were instructed on the procedures for the push - up variations. for all four variations, subjects positioned each hand on a separate force plate using a self - selected hand placement width. additionally, for all variations, subjects started in an up push - up position with their hands on the force plates, their elbows fully extended, torso in a straight line, legs extended and their toes on a platform of equal height to the force plate (figure 1). bd1 used one box (3.8 cm), bd2 used two boxes stacked (7.6 cm), and bd3 used three boxes stacked (11.4 cm) (figures 24). to perform the box drop push - ups, subjects lowered themselves towards the force plates then forcefully pushed themselves up, landing with their hands on the boxes. subjects then pushed themselves up off the boxes and landed with their hands on the force plates. pushing off from the plates, landing on the boxes and landing back on the plates counted as one repetition. in keeping with the explosive nature of plyometrics, subjects were not required to complete a full push - up while their hands were in contact with the boxes but instead were encouraged to flex their elbows enough to catch themselves, and then fully extend their elbows to push themselves off the boxes before landing on the force plates. to perform the clap pushups (cpu), subjects lowered themselves towards the force plates then forcefully pushed themselves into the air and performed a clap before returning their hands to the separate force plates (figure 5). during the cpu, subjects were instructed to push off the force plates as explosively as possible. this protocol is consistent with directions given when instructing someone to perform these two different types of plyometric push - ups. data collection began after subjects were given time to practice and qualitative observation of the push - ups indicated proper technique ; in addition, this served as a specific warm - up for the plyometric push - ups. kinematic and vgrf data were collected while the subjects completed four repetitions of each push - up variation (bd1, bd2, bd3, and cpu) in a counterbalanced order, resting no less than 90 seconds between variations. an electromagnetic tracking system (motion monitor, innovative sports training, inc chicago, il, usa) collected (100 hz) kinematics of the trunk, dominant elbow and both hands. separate sensors were attached to subjects seventh cervical vertebra s spinous process, the upper arm just distal to the deltoid insertion, the forearm (over the ulna to minimize sensor movement from forearm musculature), and to the dorsal side of both hands. during subject set - up, joint centers of the shoulder, elbow and wrists were calculated by taking midpoints between contralateral points at each respective joint using an additional electromagnetic sensor attached to a customized calibrated stylus. from the collected kinematic data, peak flight, elbow flexion at ground contact, elbow flexion displacement, and hand separation at ground contact were computed. peak flight was calculated as the maximal vertical trunk position during push - up flight based on the position of the sensor on c7. elbow flexion at ground contact was calculated as the angle of the elbow at ground contact. elbow flexion displacement was calculated using the difference between elbow flexion at ground contact and peak elbow flexion. vgrf data was collected (1000 hz) using two force plates (bp400600nc 2000 advanced mechanical technology, inc., watertown, ma, usa) with body weight normalized peak vgrf computed under the dominant extremity only. because of the novel nature of this study, we chose to only initially examine dominant extremity kinematics and vgrf data. for the bd push - ups, kinematic and vgrf data were computed only from the push - up phase involving impact onto and propulsion from the force plates. an electromagnetic tracking system (motion monitor, innovative sports training, inc chicago, il, usa) collected (100 hz) kinematics of the trunk, dominant elbow and both hands. separate sensors were attached to subjects seventh cervical vertebra s spinous process, the upper arm just distal to the deltoid insertion, the forearm (over the ulna to minimize sensor movement from forearm musculature), and to the dorsal side of both hands. during subject set - up, joint centers of the shoulder, elbow and wrists were calculated by taking midpoints between contralateral points at each respective joint using an additional electromagnetic sensor attached to a customized calibrated stylus. from the collected kinematic data, peak flight, elbow flexion at ground contact, elbow flexion displacement, and hand separation at ground contact were computed. peak flight was calculated as the maximal vertical trunk position during push - up flight based on the position of the sensor on c7. elbow flexion at ground contact was calculated as the angle of the elbow at ground contact. elbow flexion displacement was calculated using the difference between elbow flexion at ground contact and peak elbow flexion. vgrf data was collected (1000 hz) using two force plates (bp400600nc 2000 advanced mechanical technology, inc., watertown, ma, usa) with body weight normalized peak vgrf computed under the dominant extremity only. because of the novel nature of this study, we chose to only initially examine dominant extremity kinematics and vgrf data. for the bd push - ups, kinematic and vgrf data were computed only from the push - up phase involving impact onto and propulsion from the force plates. chicago, il, usa) using separate repeated measures analysis of variance for peak flight, elbow flexion at ground contact, elbow flexion displacement, hand separation and vgrf. bd3 had greater peak flight than bd2 (p <.001, d=.74) and bd1 (p <.001, d= 1.85). additionally, bd2 was revealed to have significantly greater peak flight than bd1 (p<.001, d=1.28). peak flight for cpu was significantly greater than both bd1 (p <.001, d=2.04) and bd2 (p=.001, d=1.01). elbow flexion at ground contact for bd1 was significantly greater than bd2 (p <.001, d=.68) and bd3 (p <.001, d=.84). cpu demonstrated significantly more elbow flexion at ground contact than both bd2 (p=.003, d=.97) and bd3 (p=.013, d=1.13). cpu had significantly greater elbow flexion displacement than bd1, (p <.001, d=1.58), bd2 (p <.001, d=1.98), and bd3 (p <.001, d=1.67). peak vgrf was significantly greater during cpu than bd1 (p=.001, d=.66), bd2 (p <.001, d=.53), and bd3 (p=.001, d=.51). the purpose of this study was to measure various kinematic variables and peak vgrf during box drop and clap push - ups in recreationally active males. specifically, peak flight, elbow flexion at ground contact, elbow flexion displacement, hand separation at ground contact and peak vgrf under the dominant extremity were measured during clap push - ups and box drop push - ups from 3.8 cm (bd1), 7.6 cm (bd2), and 11.4 cm (bd3) heights. the results refuted our hypothesis with regards to what we predicted would be a step - wise progression of peak flight and increased elbow flexion displacement from bd1 to cpu. additionally, our hypothesis of similar peak vgrf and elbow flexion at ground contact between variations was also refuted. an important outcome of this study was the relationship between peak flight, elbow flexion displacement and peak vgrf with regards to cpu. contrary to our hypothesis, peak flight heights were not significantly different between bd3 and cpu. however, the cpu had greater elbow flexion displacement as well as significantly greater peak vgrf than all other conditions. peak flight for cpu was 14% greater than bd1 and 6% greater than bd2 ; cpu elbow flexion displacement was 104%, 159% and 103% greater than bd1, bd2, and bd3. cpu peak vgrf was 13% greater than bd1 and 10% greater than both bd2 and bd3. based on peak flight height, elbow flexion displacement and vgrf results, the cpu appears to have the greatest intensity of all conditions tested. peak flight during the push - up variations is related to the force exerted by the subjects to push themselves up into the air during the cpu or pushing off the boxes during bd push - ups. our results indicate that subjects had significantly higher peak flight during the cpu and bd3 than during both bd2 and bd1. during the cpu, subjects were instructed to push - up as forcefully as possible, ensuring elbow extension. during the bd conditions, subjects were instructed to reach full extension when leaving the boxes before landing on the force plates. this was to ensure that subjects were actively pushing up and exploding off of the boxes, rather than just dropping down onto the force plates with already flexed elbows. essentially, by requiring full extension pushing off the boxes, we hoped to establish the same relative arm position leaving the boxes between subjects and trials to accurately assess the peak flight as well as the amount of elbow flexion displacement upon landing. based on our observations during the data collection, we expected peak flight to increase in a sequential manner from bd1 to cpu. lack of significant differences in the flight heights of bd3 and cpu are possibly attributed to muscle power limitations. in other words, some subjects may not have had adequate ability to propel themselves to a flight height that was greater than the flight height of bd3. further analysis of our results revealed that 7 subjects cpu flight height was indeed lower than their bd3 flight height. additionally, as these pushup variations were novel for most subjects, differences in motor - unit recruitment patterns or motor learning differences may have contributed to the results obtained. elbow flexion at ground contact was recorded as the elbow angle at initial ground contact on the force plate. both bd1 and cpu had significantly greater elbow flexion at ground contact when compared to bd2 and bd3. this contradicts our hypothesis that all conditions would have similar elbow flexion at ground contact. this may be explained in part due to the timing required by the participant to complete the bd1 and cpu variations. the bd1 condition was the lowest height of all conditions, meaning that the subjects were closer to the force plates after leaving the boxes and therefore had less time to plan for the loading (pre - stretch) phase. similarly, even though greater peak flight heights were achieved for the cpu, subjects had to perform a clap while in the air. the time it took during flight to perform the clap as well as return the hands to a ready position before landing may explain the greater elbow flexion at ground contact during the cpu condition. the required clap put the subjects arms into an already elbow flexed position before landing whereas the subjects were preparing for landing in the bd conditions (specifically bd2 and bd3) with more elbow extension because no clap was required. during the bd2 and bd3 conditions, the subjects hands began from a higher vertical position than with bd1, therefore allowing more time to ready themselves for the loading phase, which may explain why they landed with less elbow flexion. elbow flexion displacement during the landing phase was initially hypothesized to increase as the heights of the boxes increased, with cpu having the greatest elbow flexion displacement under the premise that a greater elbow displacement would occur to absorb the greater peak vgrf incurred after presumably falling from a greater height. elbow flexion displacement was significantly greater during cpu compared to all the bd conditions, but contrary to our hypothesis, elbow flexion displacement was not significantly different between the three box drop conditions. this is likely attributable to the relatively small difference in height of the three boxes. even though the box heights increased by 3.8 cm with each bd condition, the range of heights may not be large enough to elicit a change in elbow flexion displacement. bd push - ups from heights greater than 11.4 cm may require a larger elbow flexion displacement to absorb the landing impact force, but this was not tested. interestingly, the cpu had a greater displacement than all the bd conditions, although peak flight height between cpu and bd3 was not significantly different. this is possibly explained by the instructions of the cpu versus those of bd3 to be as explosive as possible. to perform the cpu as explosively as possible it is possible that subjects inherently went through more elbow flexion displacement (pre - stretch) upon landing on the force plates to prepare for the subsequent concentric phase. because subjects were essentially aiming for a target (push up to the boxes) for bd3, it is possible they did not go through as much elbow flexion displacement to prepare for their next push - up. in other words, subjects may have allowed their elbows to flex more between cpu repetitions, creating a greater pre - stretch of the muscles and thus a more explosive push - up, whereas for bd3, the presence of a target to reach may have caused subjects to subconsciously only elbow flex as much as needed to successfully land the next pushup to the boxes. hand separation was measured to eliminate the possible confounding effect that hand separation distance may have had on elbow flexion upon landing on the force plates. unlike the bd variations, the cpu did not require having the hands inside the boxes. because hand separation during cpu was not constrained, we thought that a wider or narrower hand separation distance might occur and change the kinematics at the elbow for the cpu. no significant differences between the four push - up variations were noted for hand separation. the lack of significant differences is likely due to subjects self - selecting their beginning hand separation for all push - up positions. because the subjects selected their own hand separation it is likely they chose a comfortable amount of separation and it remained consistent for all conditions including the cpu. this was despite similar flight height between bd3 and cpu, as well as greater elbow flexion displacement during the cpu than for all other conditions. based on these results, the cpu condition appears to have a greater intensity than any of the box drop pushups. revealing a significant difference in peak vgrf contradicts a previous study by koch. (10) that revealed no difference in peak vgrf between the same four pushup variations. based on the results of koch. (10), we expected similar peak vgrf results ; however, our hypothesis was not supported. multiple factors may be responsible for the difference in peak vgrf results between our investigation and the former. first, over half of the subjects in koch. (10) study were active duty marines who performed a variety of pushups on a weekly basis. our study included physically active subjects ; however we did not require subjects to have extensive experience with various push - up types. we sought physically active males, with the intent to generalize results to recreational athletes or physically active males who might be required to perform plyometric push - ups as part of a rehabilitation or training program. subjects qualified for the study based upon activity level and the researchers qualitative analysis of the subject s ability to perform the various plyometric push - ups. some literature focusing on lower extremity plyometrics suggest a base level of strength for the lower extremities should be attained prior to engaging in lower extremity plyometric training (14, 17) however, there is little evidence for how much strength is sufficient in the upper extremities to effectively perform plyometrics (4). we did not assess isotonic strength of our subjects and it is possible that even though our subjects were recreationally active, some may have had less than adequate strength levels necessary to effectively eccentrically decelerate during the loading phase when compared to the marines in the previous study. this lack of strength to decelerate may have additionally impacted elbow flexion and elbow flexion displacement causing increases in both variables across the variations. 10) did not examine kinematic variables, so the kinematic differences between subject groups can not be determined. in the present study it is possible that subjects adopted an asymmetrical loading strategy during the more difficult plyometric push - up conditions. subjects may have preferentially loaded the dominant upper extremity, which could explain the significantly different peak vgrf between bd3 and cpu conditions despite no difference in peak flight. the investigation by koch.(10) revealed the dominant limb demonstrating significantly greater peak vgrf than the non - dominant across all four push - up conditions. al (10) and those of the present study regarding peak vgrf suggest that future research is needed to increase the understanding of underlying kinematic variables of closed chain upper - extremity plyometrics. future plyometric push - up research comparing a variety of populations may help in determining whether the differences in peak vgrf between the different variations revealed in the current study are atypical. evaluation of kinematic data including the glenohumeral and scapulothoracic joints may also help explain variances in peak vgrf during various plyometric push - up conditions. in addition, research involving higher box drop conditions with experienced subjects could aid in the development of a step - wise progression of plyometric push - up intensity. the purpose of this study was to assess kinematic variables and peak vgrf patterns during various plyometric pushups in recreationally active males. based on our results, cpu appear to be the most intense of all conditions tested, however box drop push - ups from boxes higher than 11.4 cm were not assessed in this study. understanding the demands and intensities of various plyometric push - up variations will aid physical therapists and sports performance coaches in the prescription of these exercises in returning an athlete to play or in improving explosive upper - body power.	plyometric research in the upper extremity is limited, with the effects of open - chain plyometric exercises being studied most. kinematic and ground reaction force data concerning closed - chain upper extremity plyometrics has yet to be examined. twenty - one recreationally active male subjects performed four variations of plyometric push - ups in a counterbalanced order. these included box drop push - ups from 3.8 cm, 7.6 cm, 11.4 cm heights, and clap push - ups. kinematics of the trunk, dominant extremity and both hands were collected to examine peak flight, elbow flexion at ground contact, elbow displacement, and hand separation. additionally peak vertical ground reaction force was measured under the dominant extremity. the 11.4 cm and clap push - ups had significantly higher peak flight than the other variations (p<.001). at ground contact, the elbow was in significantly greater flexion for the 3.8 cm and clap push - up compared to the other variations (p<.001). the clap push - up had significantly more elbow displacement than the other variations (p<.001) while hand separation was not significantly different between variations (p=.129). peak vertical ground reaction force was significantly greater for the clap push - ups than for all other variations (p <.001). despite similar flight heights between the 11.4 cm and clap push - ups, the greater peak vertical ground reaction force and elbow displacement of the clap push - ups indicates the clap push - up is the most intense of the variations examined. understanding the kinematic variables involved will aid in the creation of a closed chain upper - extremity plyometric progression.
rheumatoid arthritis (ra) is characterized by an abnormal immune response, leading to progressive synovial inflammation and joint destruction6). the cervical spine is often affected, and cervical pain is reported by 40 - 88% of ra patients. cervical subluxation occurs primarily as anterior atlantoaxial subluxation (20%), representing the most common cervical lesion in ra11). atlantoaxial subluxation develops via multiple mechanisms, including inflammation of the transverse cruciate ligament around the dens and erosion of the dens itself8). compression of the second cervical dorsal root ganglion (drg) may also occur in patients with atlantoaxial subluxation, leading to headaches in the occipital region. we report a case in which pulsed radiofrequency (prf) therapy was successful in controlling posterior neck pain and radiating occipital pain in a ra patient with atlantoaxial subluxation. a 74-year - old female patient visited our pain clinic complaining of right posterior neck pain and occipital radiating pain occurring for the past 2 - 3 years. the patient was being treated with 5 mg / day prednisolone, 1300 mg / day acetaminophen, 25 mg / week etanercept and 10 mcg / week buprenorphine (patch). the posterior neck pain was continuous with a dull quality, and the pain intensity was rated as 6/10 on the visual analog scale (vas). for pain intensity, the vas ranges from " no pain " (score of 0) to " pain as bad as it could be " or " worst imaginable pain " (score of 10). the patient complained of right occipital radiating, electric shock - like paroxysmal pain during neck movement, with a pain intensity of 10/10 on the vas. radiating pain did not occur without neck motion or when the patient was in a supine position. physical examination confirmed the occurrence of pain in the right occipital area precipitated by cervical movement and severe tenderness in the right 4th, 5th, and 6th cervical facet joint regions. the atlantodental interval (adi) was measured as 4.5 mm, based on a simple lateral cervical radiographic examination (fig. study revealed a fracture of the odontoid process (type ii), myelopathy associated with instability, limited flexion and extension associated with focal sagittal segmental instability in the atlantoaxial joint, and severe cervical spondylosis (fig. initial treatment consisted of right 3rd occipital and right 4th, 5th, and 6th cervical medial branch blocks under fluoroscopic guidance. levobupivacaine (0.3 ml ; 0.75%) and triamcilonone (1 mg) were injected at each level. although the posterior neck pain intensity was reduced to a vas score of 3, the radiating occipital pain was not affected. a 20-gauge touhy needle was inserted into the right side interlaminar space of the 5th cervical spine and 6th cervical spine under fluoroscopic guidance. we confirmed the location of the epidural space using a contrast medium (iopamidol 1 ml) after feeling loss of resistance (lor) and subsequently injected lidocaine (0.5% ; 6 ml) and betamethasone (2 mg). the intensity of the radiating occipital pain was reduced to a vas score of 3 for 2 days after each block. we next performed a c2 drg block after suspecting that the pain could be associated with c2 radiculopathy, owing to atlantoaxial joint instability. lidocaine (1% ; 3 ml) and betamethasone (2 mg) were injected using a 22-gauge ciba needle, after confirming the location of the right c2 drg under flouroscopic guidance using 0.5 ml iopamidol. following this treatment, the pain disappeared for 2 days but increased thereafter. after confirming the positive result, the patient was scheduled for prf therapy. prf therapy was performed in the prone position using a c - arm for appropriate needle positioning. a 21-gauge 10-cm insulated needle was inserted between the lamina of atlas and the lamina of axis, by referring to lateral images, and was advanced toward the center of the c2 pedicle, by referring to anterior - posterior images. the needle tip did not cross the medial side of the pedicle during the procedure. the c2 nerve ganglion was identified by applying a 50-hz, 0.6-v electrical stimulation, using a radiofrequency generator, and injecting contrast medium (iopamidol ; 0.5 ml). prf therapy was performed at 42 for 120 seconds and repeated three times (fig. the patient has not complained of any occipital radiculopathy for 6 months, and the posterior neck pain has since been reduced to a vas score of 3. ra patients with atlantoaxial subluxation complain of occipital and posterior neck pain, which is aggravated by cervical locomotion and is sometimes accompanied by crepitation. additional neuralgia of the face, ear and occiput can occur as a result of c2 root compression10). a positive result of the sharp - pruser test is described as a clunking sensation associated with spontaneous atlantoaxial reduction during neck extension. lhermitte 's phenomenon, characterized by electric shock - like sensations of the extremities in response to forward flexion of the head, may also be present6). these symptoms are caused by irritation of the c2 nerve ganglia and nerve root, since the second cervical drg is exposed to the atlantoaxial zygapophygeal joint16). atlantoaxial instability is defined as loss of the stable configuration of the anterior c1 - 2 synovial articulation, resulting in abnormal movement. although most patients with cervical spine involvement are asyptomatic for years, they show approximately 50% radiological progression during follow - up1). a positive diagnosis is made if the adi exceeds 3 mm8), as assessed by lateral cervical radiographic examination. the average onset time for myelopathy is 16.6 years in ra patients, but it can be detected earlier in males, particularly in cases associated with the peripheral joints and long - term use of steroids9). myelography or mri can accurately diagnose neural compression, spinal stenosis, bony erosion, and others ; however, simple x - ray images are a cost - efficient means of predicting neurological complications by measuring the anterior adi or posterior adi5). the pathophysiology of anterior atlantoaxial subluxation is similar to that observed in other peripheral joints and involves proliferation of fibroblasts and inflammatory cells, resulting in rheumatoid pannus. this pannus destroys cartilage, ligaments and bones by generating collagenase and other proteolytic enzymes. the resulting destructive synovitis induces ligament laxity and bony erosion, which leads to cervical spine instability and subluxation7). diverse methods, beginning with palliative treatment, have been applied to treat pain caused by irritation of the c2 nerve ganglion and nerve root associated with anterior atlantoaxial subluxation. surgical approaches include micro decompression, neurectomy and atlantoaxial fusion, while other interventions, such as rf therapy have also been used14). however, limited data exist regarding the effects of conventional rf (crf) and prf on pain caused by anterior atlantoaxial subluxation. shim and shim13) reported pain relief lasting 6 months after crf in two ra patients with anterior atlantoaxial subluxation. similarly, zhang.6) reported pain relief lasting 6 months after prf in patients with cervicogenic headaches. prf provides a long - term reduction in headaches with minimal procedural risks in selected patients with medically intractable occipital neuralgia4). these studies suggest that prf therapy may be an effective alternative treatment for patients with posterior neck pain due to anterior atlantoaxial subluxation. although the exact mechanism of prf action is not known, it is believed to involve changes in neurotransmission at the site of stimulation2). unlike crf, which can cause nerve and tissue injuries because of the relatively high temperature involved, prf uses a pulsating high - frequency stimulation at a temperature below 42 for 0.5 s, repeated at 20-ms intervals. due to the relatively short procedural duration and lower risk, prf can be applied to regions known to be at a relatively high risk of complications2). prf provides a shorter duration of pain relief than does crf, but it has fewer complications and is safer. it has also been shown that prf is effective in cases where the needle approach would be difficult technically2). prf can also be used to treat neuropathic pain, because it does not destroy nerves or block their transmission. conversely, crf damages nerves and therefore is not used frequently for neuropathic pain3). facet joint pain due to severe c - spine spondylosis was considered to be the cause of the patient 's continuous neck pain. we suspected that the radiating occipital pain experienced during neck movement was associated with c2 radiculopathy and therefore performed a c2 drg block, which successfully reduced radiating pain for 2 days. the patient subsequently received additional prf therapy and was pain - free for more than 6 months. although our study is limited by its size and the absence of an objective measurement of pain, we suggest that c2 prf may be an effective treatment for pain in ra patients with anterior atlantoaxial subluxation.	rheumatoid arthritis (ra) is a chronic inflammatory disease involving multiple joints. the cervical spine is often affected, and cases involving atlantoaxial joint can lead to instability. anterior atlantoaxial subluxation in ra patients can lead to posterior neck pain or occipital headache because of compression of the c2 ganglion or nerve. here, we report the successful treatment of a ra patient with occipital radiating headache using pulsed radiofrequency therapy at the c2 dorsal root ganglion.
thyroid hormones directly affect endothelial and vascular smooth muscle cells, and high levels of thyroid hormones are known to cause hypertrophy and stiffness of the vessels.12)20) some authors have reported that coronary vasospasms occur as a result of hyperthyroidism accompanied by myocardial infarction.11)16)22) however, the reverse causal relationship has rarely been reported. furthermore, cerebral vasospasm (cv) associated with hyperthyroidism has never been reported to cause cerebral infarction. we therefore report here a rare and first of its kind case of severe cv that was caused by cerebral infarction in a patient with mild head injury and hyperthyroidism. a 30-year - old woman was transferred to our hospital in a stuporous state with right hemiparesis. she had earlier been admitted to another hospital for two weeks after she was injured in a traffic accident. on admission to our hospital initial brain computed tomography (ct) and magnetic resonance (mr) images taken at the other hospital showed no abnormal lesions in the intracranial space (fig. she was diagnosed with hyperthyroidism 2 months ago, and has been taking 15 mg of methimazole and 40 mg of propranolol daily. she had no past history of other diseases. on admission, laboratory investigations showed low levels of thyroid stimulating hormone (tsh) (0.011 iu / ml, reference range : 0.27 - 4.2 iu / ml), low levels of free t4 (0.644 ng / dl, reference range : 0.93 - 1.7 ng / dl), elevated levels of triiodothyronine (t3) (2.58 ng / ml, reference range : 0.8 - 2.0 ng / ml) and elevated levels of thyrotropin - binding inhibitory immunoglobulin (tbii) (62.80 mr angiography demonstrated cerebral infarction in the posterior cerebral artery (pca) territories and multiple stenotic lesions in both middle cerebral arteries (mcas), anterior cerebral arteries (acas), and pcas (fig. cerebral angiography also revealed severe vasospasms in all the mca, aca and pca territories (fig. therefore, intra - arterial chemical angioplasty with verapamil was performed for two consecutive days. a total of 25 mg of verapamil was injected slowly : 10 mg each for the left and right internal carotid artery and 5 mg for the left vertebral artery. after the procedures, the patient was given antithyroid medication and " triple - h " therapy (hypervolemia, hypertension, and hemodilution) to increase cerebral perfusion. follow - up angiography performed at 6 weeks demonstrated complete recovery of the vasospasm (fig., she was alert with mild weakness and cortical blindness (modified rankin scale score, 4). cv is common in patients with aneurysmal subarachnoid hemorrhage (sah), and it is a leading cause of morbidity and mortality.8) cv in traumatic sah is characterized by earlier onset and milder clinical symptoms than that in aneurysmal sah.10) post - traumatic vasospasm without traumatic sah has a brief duration of moderate spasms with an average time course of 1.25 days.15) however, this is the first report on cv in a patient with mild head injury and hyperthyroidism. in our case, cv could not be concluded from a simple mild head trauma without hemorrhage, so other risk factors associated with cv needed to be investigated. hyperthyroidism was confirmed 2 months ago, and there was no other medical history except for elevation of t3 and tbii. cerebrovascular changes in hyperthyroidism have been reported to be limited to moyamoya disease (mmd). a recent study of patients with mmd reported a high incidence of elevated thyroid function and thyroid autoantibody levels.2)13) thyrotoxicosis may be the trigger of the vascular attack in mmd patients that results in the increase in sympathetic nervous system sensitivity.9) choi. have reported about coronary vasospasms with thyrotoxicosis in 6,923 subjects ; they showed that the hyperthyroid state enhances the sensitivity of the vascular contractile responses to either norepinephrine (ne) or 5-hydroxytryptamine (5-ht). catecholamines can induce vasospasm in patients with coronary vasospasm.3) in general, many studies have shown that the thyroid hormone is an important regulator of endothelial nitric oxide (no) production and vascular tone.7) moreover, hyperthyroidism was shown to enhance the sensitivity of resistant vessels to the vasoconstrictive action of ne.17) cerebral vascular reactivity is not very different from typical vascular reactivity. cerebral arteries are innervated by noradrenaline (na)- and 5-ht - containing nerves,14) and ne is known to play a role in inducing a dose - mediated vasoconstrictive effect on cerebral vasculature.5)23) we therefore hypothesize that vasoconstriction may be exacerbated by hyperthyroidism and that stressful situations may result in ne secretion and then cerebral infarction after cv. we have reported only one case for which there is no clear evidence of the association between hyperthyroidism and cv. nonetheless, it seems that mild head trauma was induced by a stressful event in her condition, and the absence of medication in the hyperthyroid state enhanced the sensitivity of the cerebral vascular contractile responses. in this situation in general, management of traumatic brain injury - related cv can be quite different from treatment of aneurysmal sah - related cv because of the severity of the injury and associated comorbidities.10) however, in our case, hemorrhage did not occur, and triple - h therapy and endovascular chemical angioplasty were performed at the same time as in cases of aneurysmal sah - related cv. endovascular therapy for cv has a safety efficacy profile that shows neurological improvement.1)6) papaverine, verapamil, nimodipine and nicardipine have been used as intra - arterial vasodilators to treat patients.21) therefore, intra - arterial verapamil was administered in our case. previously published cases about coronary vasospasm and mmd with hyperthyroidism demonstrated that restoration of euthyroidism was a priority.4)18)19) according to ni., a sudden increase in the thyroid hormone levels should be avoided to prevent cerebrovascular accidents, because cerebrovascular hemodynamic changes due to thyrotoxicosis might be responsible for cv.18) the treatment of hyperthyroidism was accompanied by neurological improvement. in the case of our patient, we administered medication for hyperthyroidism at the endocrinology department., a sudden increase in thyroid hormone levels in the clinical setting should be avoided to prevent cerebrovascular accidents. when neurological deterioration is noticed without primary cerebral parenchymal lesions, an evaluation of thyroid function	cerebral vasospasm associated with hyperthyroidism has not been reported to cause cerebral infarction. the case reported here is therefore the first of cerebral infarction co - existing with severe vasospasm and hyperthyroidism. a 30-year - old woman was transferred to our hospital in a stuporous state with right hemiparesis. at first, she complained of headache and dizziness. however, she had no neurological deficits or radiological abnormalities. she was diagnosed with hyperthyroidism 2 months ago, but she had discontinued the antithyroid medication herself three days ago. magnetic resonance imaging and angiography showed cerebral infarction with severe vasospasm. thus, chemical angioplasty using verapamil was performed two times, and antithyroid medication was administered. follow - up angiography performed at 6 weeks demonstrated complete recovery of the vasospasm. at the 2-year clinical follow - up, she was alert with mild weakness and cortical blindness. hyperthyroidism may influence cerebral vascular hemodynamics. therefore, a sudden increase in the thyroid hormone levels in the clinical setting should be avoided to prevent cerebrovascular accidents. when neurological deterioration is noticed without primary cerebral parenchyma lesions, evaluation of thyroid function may be required before the symptoms occur.
the video presents a patient with involuntary arrhythmic abdominal contractions consistent with belly dancer syndrome.	key clinical messagein our patient presenting with abdominal myoclonus, it is important to understand its pathophysiology. various etiologies need to be taken into consideration before coming to a conclusion. the finding on magnetic resonance imaging (mri)spine disclosing cervical lesion may just be an incidental finding.
unruptured middle cerebral artery (mca) aneurysms can be optimally treated by surgical clipping and endovascular coiling. we report an emergency intracranial in situ bypass surgery case which was performed as a rescue procedure after aneurysmal neck laceration during mca large aneurysm clipping. a 76-year - old woman presenting with a one - month standing headache visited our outpatient clinic. on neurological examinations, no focal neurological deficits were observed. outside brain magnetic resonance imaging revealed two unruptured intracranial aneurysms : a right mca aneurysm and a distal a2 - 3 aneurysm. the mca aneurysm measured 11.39.0 mm, and the a2 - 3 aneurysm measured 7.13.1 mm (fig. a right mca aneurysm clipping operation was planned because of the headache location (right temporal area) and aneurysmal size (the mca aneurysm was larger than the a2 - 3 aneurysm). during the clipping procedure, the lacerated length was too long to use a clipping technique with cotton or bemsheet material coverage. consequently, primary closure of the aneurysmal neck was performed with 8 - 0 nylon, and correct re - clipping was done. the temporary trapping time during primary closure was fourteen minutes, and the waves on the motor evoked potential (mep) and somatosensory evoked potential (ssep) decreased after eight minutes of trapping (fig. moreover, the flow of the inferior m2 trunk was not detected after primary closure of aneurysmal neck, and the mep / ssep change was not recovered. thus, we planned to perform emergency bypass surgery, in situ m2-m2 side - to - side anastomosis as a rescue procedure, because the parietal branch of the superficial temporal artery (sta) was sacrificed when the skin incision was made. but, it was not possible to perform in situ m2-m2 bypass, because of limited mobility (can not be fully pulled to be sutured). in addition, limited vessel mobility also disturbed in situ m3-m3 bypass, so we planned to do m3-sta interposition graft - m3 anastomosis with harvesting the short pedicle of the sta frontal branch. after harvesting the frontal branch of the sta, the first anastomosis between the m3 of the inferior m2 trunk and the sta interposition graft was made. a little back - flow from the m3 was detected when an incision was made in it. a second anastomosis between the m3 of the superior m2 trunk and the sta interposition graft was made. after performing the in situ m3-sta interposition graft - m3 bypass procedure, the inferior m2 trunk flow resumed, and the flattened wave on the sep changed into a normal wave pattern (fig. immediate postoperative computed tomography (ct) angiography demonstrated good flow of the inferior m2 trunk with good patency of the bypass graft (fig. 3). however, she was not awakened well, and additional ct on postoperative day one revealed some low density around the inferior m2 trunk territory. emergency tfca demonstrated occlusion of the inferior m2 trunk, and no visible bypass pedicle was detected (fig., she was awakened, but the motor power of her left upper arm was grade ii. however, to our surprise, her left side weakness improved very rapidly, and nearly complete recovery was achieved at postoperative two weeks. follow - up ct and tfca at postoperative two weeks revealed much decreased low density in the m2 inferior trunk territory and complete recanalization of the m2 inferior trunk with an intact sta interposition graft (fig. generally, in mca aneurysm surgery clipping, aneurismal neck laceration is a well known complication, and its result usually has catastrophic cerebral ischemia in many cases1,3 - 5). so, bypass procedures have been reported to be helpful in clipping of complex middle cerebral artery aneurysms2,8). in our case, an aneurismal neck laceration happened during the clipping procedure due to severe atherosclerotic change in the aneurysm, and emergency suture of aneurismal neck was performed. finally, we performed an m3-sta interposition graft - m3 anastomosis, and the flow of the m2 inferior trunk and mep / ssep were recovered. intracranial - intracranial (ic - ic) bypass is known as third - generation bypass surgery7) and has some benefits over extracranial - intracranial (ec - ic) bypass surgery, such as similar caliber of donor and recipient arteries, no need to harvest ec donor vessels, and less vulnerability to neck torsion or trauma6,7). however, in situ bypass between two mca branches requires a more challenging side - to - side anastomosis between m3 with limited morbidity and has the co - sacrifice risk involving two mca branches. in this case, we used the frontal branch of the sta as an interposition graft because the parietal branch of the sta was sacrificed when the initial skin incision was made. the mep change was recovered, and the normal wave patterns had been preserved until the end of surgery. additionally, immediate postoperative ct angiography demonstrated good m2 trunk flow with good patency of the bypass graft. however, the patient was not easily roused, and acute cerebral infarction due to occlusion of the inferior m2 trunk developed. however, the occluded m2 inferior trunk was recanalized, and the low density at basal ganglia on ct disappeared almost at postoperative two weeks. first, we think that the cause of delayed occlusion of the m2 inferior trunk after surgery is probably due to the direction of the free sta graft. namely, the direction of flow is m3 of superior m2 trunk - sta interposition graft - m3 of inferior m2 trunk, but we performed an anastomosis with an acute angle between the m3 of superior m2 trunk and the sta graft with an obtuse angle between the sta graft and the m3 of m2 inferior trunk. thus, the flow direction is not " natural ", and the flow burden of the m2 superior trunk might have increased. if in this situation a small m2 inferior trunk flow around the mca bifurcation resumed, flow " collision " might have occurred, and transient occlusion or low flow would have been sustained, leading to cerebral infarction. second, we think that the small amount of flow through the m3-sta graft - m3 might have been sustained, and delayed spontaneous thrombolysis might have occurred. finally, the origin of the mca inferior trunk around the mca bifurcation might have been open. in any event, we obtained a final good result in this case with an emergency ic - ic bypass method ; thus, if a sta - mca anastomosis is not available under mca flow obstruction, we can consider an emergency in situ mca - mca bypass procedure with or without an sta interposition graft. in addition, we must consider the " anastomosis angle " between the parent artery and free graft to get the natural flow direction. we report an emergency in situ m3-m3 bypass procedure using an sta interposition graft as a rescue procedure in the case of an m2 trunk flow obstruction during clipping of an mca aneurysm. if an sta - mca anastomosis is not available under mca flow obstruction, we can consider an emergency in situ mca - mca bypass procedure with or without an sta interposition graft.	many reports have been published on complications related to middle cerebral artery (mca) aneurysm surgical clipping procedures. we report an emergency intracranial in situ bypass surgery case which was performed as a rescue procedure after aneurysmal neck laceration during clipping of an mca large aneurysm. in this case, we performed in situ m3-superficial temporal artery (sta) interposition graft - m3 bypass procedure. if a sta - mca anastomosis is not available under mca flow obstruction, we can consider an emergency in situ mca - mca bypass procedure with or without an sta interposition graft.
approximately 20,000 reproductive - aged american women carry a diagnosis of leukemia. for women less than 45 years old at the time of diagnosis, the 5-year survival rate is approximately 50% and 40% for women with aml and all. cancer patients are not only focused on survival, but on quality of life and long - term plans, such as family building. health care providers have recognized the importance of fertility to cancer patients and the impact of cancer treatment on fertility. the american society of clinical oncology recommends that fertility preservation be discussed at the time of diagnosis [2, 3 ]. fluorouracil, vincristine, bleomycin, and dactinomycin are associated with a low or no risk of amenorrhea, which is used as a surrogate marker for infertility, while alkylating agents are more likely to cause ovarian damage and amenorrhea. alkylating agents, like cyclophosphamide, may lead to early menopause by damaging primordial follicles [4, 5 ]. anthracyclines, used for induction remission and postremission chemotherapy in young adults with acute myeloid leukemia (aml) and acute lymphocytic leukemia (all), also likely lead to ovarian damage [6, 7 ]. ovarian reserve refers to the number of primordial ovarian follicles in the ovaries and is related to fertility potential. ovarian reserve can be measured by menstrual cycle day 3 follicle stimulating hormone (fsh) and estradiol, inhibin b (inb), ovarian antral follicle count (afc), or antimullerian hormone (amh). in the current study, we examined ovarian reserve in women in first complete remission of leukemia who underwent treatment with standard chemotherapy, but who did not require stem cell transplantation. we hypothesized that premenopausal women with a history of chemotherapy for leukemia, even if stem cell transplantation is not performed and menstrual cycles are regular, have decreased ovarian reserve. we conducted a cross - sectional study of premenopausal women, aged 1845, with a history of chemotherapy treatment for all, aml, or acute promyelocytic leukemia (apml). women with infertility and those with medical conditions that may compromise fertility or affect ovarian reserve markers such as a history of hysterectomy or oophorectomy were excluded. women using hormonal therapy were excluded or required to stop it for one month prior to study participation. tests for ovarian reserve included serum amh, estradiol, fsh, inb, afc and ovarian volume. ovarian volume was calculated by multiplying the longest dimension of the ovary (in cm) by the two orthogonal dimensions by a factor of 0.523 : ovarian volume = length width thickness 0.523. serum estradiol was measured by the access chemiluminescence immunoassay (bechman coulter, fullerton, ca) ; coefficient of variation (cv) was between 2150% for levels of estradiol 12050 pg / ml. serum fsh was measured by the access chemiluminescence immunoassay (bechman coulter, fullerton, ca), cv 12 months and one of these women was 35 years old at the time of treatment. in this age - matched study of ovarian reserve in premenopausal women who underwent chemotherapy without transplantation for acute leukemia, there were no statistically significant differences in ovarian reserve markers between the groups. however, there appeared to be a trend towards decreased ovarian reserve in leukemia survivors. survivors may be falsely reassured that their regular menses represents normal ovarian reserve and may not seek consultation for fertility preservation until near ovarian failure. our findings suggest that patients treated with chemotherapy for leukemia who continue to menstruate should be tested for decreased ovarian reserve after treatment. furthermore, they should not delay consultation with an infertility specialist if they have difficulty conceiving. in the oncology literature, ovarian failure is described in a number of ways : treatment - induced amenorrhea, menopausal levels of fsh or estradiol, menopausal symptoms, or infertility. in our study, women with longer treatment durations had lower ovarian reserve. similarly influenced by their age at treatment and the stage of cancer, approximately 5080 percent of women that received chemotherapy for hodgkin 's lymphoma had secondary amenorrhea [13, 14 ] however, 75 percent of women with a history of treatment for hodgkin 's lymphoma achieved pregnancy without assisted reproductive technologies. of note, the mean age of diagnosis was 23 years old, and those treated after the age of 30 were less likely to achieve posttreatment pregnancy. aisner. found a similar pregnancy rate in their female hodgkin 's survivors, and as the length of time after therapy increased so did the probability of pregnancy. given the overwhelming evidence of diminished ovarian function following treatment for other hematologic malignancies, a reproductive - aged woman with a new hematologic cancer diagnosis should be aware of her potential options to retain her fertility and ovarian function. such options include oocyte, ovarian tissue, or embryo cryopreservation, which are selected based on the patient 's clinical and social situation. however, there are a small number of epidemiologic studies examining parenthood after a history of cancer treatment. compared to the norwegian general population of women, of whom 79% become parents, significantly fewer women (66%) with a history of cancer moreover, the pregnancies of women with a history of cancer were complicated by low birth weight and premature deliveries. of note, 31% of these women had malignant melanoma (none specified as having leukemia). these findings are similar to a finnish study that measured probability of parenthood in women diagnosed with cancer between 2034 years old compared to their siblings. the probability of parenthood in survivors compared to siblings was 38% versus 73%. only a small proportion (3.6%) of those studied had leukemia. there are more data on pregnancy outcomes of women diagnosed with leukemia as children than women diagnosed as adults, which reflects the improved survival rate in this age group compared to adults with acute leukemia. among participants in the childhood cancer survivor study, the chance of live birth was significantly less (rr 0.52, 95% ci 0.360.76) for women treated with chemotherapy compared to their healthy siblings. for women who did deliver, offspring were more likely to be of low birth weight. signorello. also found that survivors were nearly twice as likely to have premature deliveries. the aforementioned studies describe pregnancy outcomes and parenthood for many types of cancer, but leukemia patients are not well represented. leukemia survivors are a large part of the childhood cancer survivor study participants, but these females were diagnosed when younger than age 22. thus, the oncology and fertility literature lacks information on ovarian reserve after adult diagnosis and treatment of leukemia. another aim of our study was to determine which ovarian reserve markers performed best in this population. the optimal ovarian reserve testing may be different for cancer survivors than for women with other infertility etiologies. in a study of childhood cancer survivors, women with normal fsh levels (< 10 mui / ml) and normal menstrual cycles were found to have a lower afc and ovarian volumes compared to age - matched controls. similarly, amh levels were lower in women treated as children with chemotherapy for hodgkin 's lymphoma, even in women with normal fsh and inb levels. thus, amh appears to be an earlier and more sensitive marker of ovarian reserve in women with a history of chemotherapy. our study also showed a trend towards low amh and afc, with normal fsh in the leukemia group. unlike fsh or estradiol, the interpretation of amh is not menstrual cycle - day dependent. during and after treatment, she may be amenorrheic from the chemotherapy or gnrh agonist use, and her cycle day unknown. also, she often can not postpone urgent chemotherapy for a menstrual cycle day 3 fsh evaluation. thus, the ability to evaluate ovarian reserve regardless of menstrual status suggests that amh is an excellent ovarian reserve marker for the cancer population. current options for fertility preservation in cancer patients receiving chemotherapy include embryo cryopreservation after in vitro fertilization (ivf), oocyte cryopreservation, and ovarian suppression with gonadotropin releasing hormone (gnrh) agonists or antagonists. in addition, ovarian tissue cryopreservation, currently experimental, may be considered more in the future. aside from ivf potentially being cost or time prohibitive for young women, especially those needing urgent chemotherapy for acute leukemia, ivf and embryo cryopreservation may not be desirable to single women, as they would need donor sperm. they may consider oocyte cryopreservation ; however, this is still thought to be experimental, requires ovarian stimulation, and may not be feasible in the acute leukemia population because of the need to delay chemotherapy. many providers offer women gnrh agonists, as it may be covered by insurance and can be given without delaying chemotherapy. however, in 2006, the american society of clinical oncology stated there was insufficient evidence regarding the safety and effectiveness of gnrh analogs and other means of ovarian suppression on female fertility preservation. the primary limitation of this study was its size and we encourage providers to consider this when interpreting the results. although our participants were from an academic center in a large city, we still had difficulty with recruitment. low numbers of eligible patients may reflect that very few women with leukemia are able to be cured with chemotherapy alone. for those who have a good prognosis, and may not need transplantation, our study suggests that there may be long - term implications regarding fertility. due to the lack of data in the field regarding the impact of leukemia treatment on menstrual cycles and infertility, we feel that our study is a worthwhile contribution and may assist with patient counseling. we believe that women treated with chemotherapy for leukemia should be counseled that their ovarian reserve may be negatively impacted. in conclusion, we determined that premenopausal women who have received chemotherapy as adults for leukemia may have compromised ovarian reserve, even in the setting of regular menses. oncologists, infertility specialists, and patients should consider our results when discussing the impact of chemotherapy and fertility preservation. further, women and providers may be able to have a more realistic conversation about the options for fertility preservation.	purpose. it is well known that chemotherapy regimens may have a negative effect on ovarian reserve, leading to amenorrhea or premature ovarian failure. there are little data regarding the effects of leukemia chemotherapy on ovarian reserve, specifically in women who received the chemotherapy as adults and are having regular menstrual periods. our primary objective was to determine if premenopausal women with a history of chemotherapy for leukemia, without subsequent stem cell transplantation, have decreased ovarian reserve. materials and methods. we measured ovarian reserve in five women who had been treated for acute lymphocytic leukemia (all) or acute myeloid leukemia (aml) and compared them to age - matched control women without a history of chemotherapy. results. there appeared to be a trend towards lower antimullerian hormone and antral follicle counts and higher follicle - stimulating hormone levels in the leukemia group. conclusion. our results indicate that chemotherapy for aml or all without stem cell transplantation may compromise ovarian reserve. although our results should be confirmed by a larger study, oncologists, infertility specialists, and patients should be aware of the potential risks to ovarian function and should be counseled on options for fertility preservation.
the optimal surgical approach for cervical spondylotic myelopathy (csm), especially multilevel csm, continues to be debated. the goal of surgical intervention for multilevel csm is to decompress the spinal cord and maintain stability of the cervical spine. in many cases, when stenotic pathology can not be found at the disk level alone, or is accompanied by ossification of posterior longitudinal ligament (opll), a posterior strategy can provide satisfactory cord decompression., the satisfactory clinical outcomes achieved with laminoplasty in the treatment of multilevel csm have received increasing attention, but there are potential postoperative risks associated with this procedure, such as lamina reclosure, which may cause restenosis of the spinal canal, and prolonged operative duration. posterior laminectomy with decompression is a technique that is relatively simple and safe compared with laminoplasty. however, it can also be impeded by late complications of kyphosis and instability. in 1983, gui. introduced lateral laminectomy for the treatment of cervical myelopathy to avoid the complications that can result from posterior laminectomy, but its ability to achieve canal decompression is limited compared with the posterior approach. expansive hemilaminectomy was introduced by xu. in 1999 to treat patients with spinal cord injury, and we have been using this technique for more than 10 years to treat multilevel csm. the present study compared functional and radiological outcomes of two posterior techniques in the treatment of multilevel csm. forty - four patients with multilevel csm were treated with posterior cervical surgery in department of orthopedic surgery, beijing army general hospital from march 2011 to june 2012 and were recruited for this study. inclusion criteria consisted of : (1) consecutive or nonconsecutive multifocal (affecting more than three levels) spinal cord compression on magnetic resonance imaging (mri) ; (2) preoperative cervical lordosis > 10 ; (3) clinical symptoms matching radiographic imaging and mri ; and (4) operated on by one of two appointed surgeons. exclusion criteria were : (1) cervical kyphosis ; (2) previous cervical injury or operation ; (3) other pathological conditions, such as spine deformity, tumor, tuberculosis, infection, or metabolic bone disorders ; and (4) mental disorder or heavy opioid or alcohol use. patients were divided into two groups according to the type of surgery performed : laminoplasty (group l) and hemilaminectomy (group h). perioperative parameters were also recorded, including age, gender, duration of symptom, operative duration, axial pain, and intraoperative blood loss. posterior open - door laminoplasty with titanium mini - plate fixation the surgery was performed following the hirabayashi technique. general anesthesia was induced, and the patient was placed on the operating table in 30 reverse - trendelenburg position. the head was immobilized with a mayfield head clamp. a midline incision was made along the spinous processes of c3c7 and the paracervical muscles were stripped off the exposed c3c7 laminae bilaterally. levels were identified by palpation and visualization of the prominent, bifid c2 spinous process. on the opening side, a high - speed cutting burr was used to create a trough along the lamina - lateral mass junctions. burring continued until both the dorsal and ventral cortices of the laminae were completely excised. the muscles attached to c2 were preserved as much as possible to minimize the risk of postoperative kyphotic deformity. on the hinge side, another trough was created along the lamina - lateral mass junctions from c3 to c7 by decorticating the posterior aspects of the laminae. after the adhesions between the dura and ventral surfaces of the laminae were removed with an elevator, a greenstick osteotomy was made by carefully pressing the spinous processes toward the hinge side while elevating the laminae on the open side with a nerve hook. adequate expansion of the spinal canal, with pulsatile flow in the dura, could usually be achieved with an opening width of 810 mm. skipped levels on the open side were stabilized using mini plates from the centerpiece plate fixation system (medtronic sofamor danek, memphis, tn, usa). posterior expansive hemilaminectomy patient position and anesthesia were the same as for the open - door laminoplasty procedure. standard posterior exposure of the cervical spine was achieved. the number of segments and the operated side depended upon the severity of spinal cord compression. the paracervical muscles were stripped from the laminae between c3 and c7 on one side only. the entire posterior element, from the base of the spinous process to the lamina - facet joint junction, was carefully removed unilaterally, over the side of interest, with a high - speed burr. at the base of the spinous process at each operated level, a cross - over, which removed both the base of the spinous processes on the operative side and part of the inner cortex of laminae on the opposite side, was made in order to expand the spinal canal as much as possible, until the opposite nerve roots were noted. after the decompression was complete, an anti - adhesion membrane (activematrix, skye biologics, redondo beach, ca, usa) was applied to prevent tissue adhesion on the decompressed side of the spinal canal postoperatively [figure 1 ]. a cervical collar was worn for 3 months. the range of decompression with expansive hemilaminectomy technique. assessment of neurological function clinical outcomes were assessed using the japanese orthopedic association (joa) scoring system. rate of joa score improvement (jsi) was calculated as follows : (postoperative score preoperative score)/(17 preoperative score) 100%. surgical outcome was defined as excellent (jsi 75%), good (75% > jsi 50%), fair (50% > jsi 25%), and poor (jsi 10 ; (3) clinical symptoms matching radiographic imaging and mri ; and (4) operated on by one of two appointed surgeons. exclusion criteria were : (1) cervical kyphosis ; (2) previous cervical injury or operation ; (3) other pathological conditions, such as spine deformity, tumor, tuberculosis, infection, or metabolic bone disorders ; and (4) mental disorder or heavy opioid or alcohol use. patients were divided into two groups according to the type of surgery performed : laminoplasty (group l) and hemilaminectomy (group h). perioperative parameters were also recorded, including age, gender, duration of symptom, operative duration, axial pain, and intraoperative blood loss. posterior open - door laminoplasty with titanium mini - plate fixation the surgery was performed following the hirabayashi technique. general anesthesia was induced, and the patient was placed on the operating table in 30 reverse - trendelenburg position. the head was immobilized with a mayfield head clamp. a midline incision was made along the spinous processes of c3c7 and the paracervical muscles were stripped off the exposed c3c7 laminae bilaterally. levels were identified by palpation and visualization of the prominent, bifid c2 spinous process. on the opening side, a high - speed cutting burr was used to create a trough along the lamina - lateral mass junctions. burring continued until both the dorsal and ventral cortices of the laminae were completely excised. the muscles attached to c2 were preserved as much as possible to minimize the risk of postoperative kyphotic deformity. on the hinge side, another trough was created along the lamina - lateral mass junctions from c3 to c7 by decorticating the posterior aspects of the laminae. after the adhesions between the dura and ventral surfaces of the laminae were removed with an elevator, a greenstick osteotomy was made by carefully pressing the spinous processes toward the hinge side while elevating the laminae on the open side with a nerve hook. adequate expansion of the spinal canal, with pulsatile flow in the dura, could usually be achieved with an opening width of 810 mm. skipped levels on the open side were stabilized using mini plates from the centerpiece plate fixation system (medtronic sofamor danek, memphis, tn, usa). posterior expansive hemilaminectomy patient position and anesthesia were the same as for the open - door laminoplasty procedure. standard posterior exposure of the cervical spine was achieved. the number of segments and the operated side depended upon the severity of spinal cord compression. the paracervical muscles were stripped from the laminae between c3 and c7 on one side only. the entire posterior element, from the base of the spinous process to the lamina - facet joint junction, was carefully removed unilaterally, over the side of interest, with a high - speed burr. at the base of the spinous process at each operated level, a cross - over, which removed both the base of the spinous processes on the operative side and part of the inner cortex of laminae on the opposite side, was made in order to expand the spinal canal as much as possible, until the opposite nerve roots were noted. after the decompression was complete, an anti - adhesion membrane (activematrix, skye biologics, redondo beach, ca, usa) was applied to prevent tissue adhesion on the decompressed side of the spinal canal postoperatively [figure 1 ]. a cervical collar was worn for 3 months. the range of decompression with expansive hemilaminectomy technique. assessment of neurological function clinical outcomes were assessed using the japanese orthopedic association (joa) scoring system. rate of joa score improvement (jsi) was calculated as follows : (postoperative score preoperative score)/(17 preoperative score) 100%. surgical outcome was defined as excellent (jsi 75%), good (75% > jsi 50%), fair (50% > jsi 25%), and poor (jsi < 25%). the proportion of patients with excellent and good results at 6-month follow - up was calculated. assessment of spinal canal expansion magnetic resonance imaging scan of the cervical spine was performed on a 1.5 t system (magnetom avanto, siemens, germany) preoperatively and at the 6-month follow - up visit. for each pathological level, the t2-weighted coronal slice (repetition time / echo time : 3800/115 ; flip angle : 150 ; excitations : 3 ; slice thickness : 2 mm ; gap : 0 mm ; field of view : 300 mm, image matrix : 256 512) in which the most severe compression cord area could be confirmed was chosen to represent the involved level., san rafael, ca, usa) were used as follows : the dicom format of mri can not be recognized by autocad, a software application for two- or three - dimensional computer - aided design and drafting. therefore, prior to comparing the expansion of the spinal canal in both groups, the mris were converted to png format using adobe illustrator cs6 ; autocad was then used to measure the area of the spinal canal. when the selected mri was opened in autocad, an enclosed region could be defined by outlining the border of the compressed spinal canal with the mouse pointer. the area and perimeter of the enclosed region appeared at the bottom of the command - line window. the postoperative spinal canal area was defined as the enclosed region between the border of the compression and the dural sac on the decompressed side. for each patient, an average spinal canal area for all involved levels was calculated before the operation and at follow - up [figures 2 and 3 ]. (a) preoperative and (b) postoperative magnetic resonance imaging images showing measurement of spinal canal areas in group h using autocad software. (a) preoperative and (b) postoperative magnetic resonance imaging images showing measurement of spinal canal areas in group l using autocad software. comparisons of quantitative data (operative duration, intraoperative blood loss, and spinal canal area) between groups were analyzed by student 's t - test. the chi - squared test was performed to compare postoperative recovery of neurological function between groups. statistical analysis was conducted using graphpad prism 5.01 (graphpad software, inc., la jolla, ca, usa). a p < 0.05 was considered as statistically significant. there were 23 patients in group l (15 males, eight females ; mean age, 66.1 14.8 years [range, 4572 years ]) and 21 patients in group h (17 males, four females ; mean age 68.4 18.1 years [range, 5481 years ]). duration of symptom was 3115 months (mean, 28.7 13.2 months) in group l and 6102 months (mean, 24.1 11.5 months) in group h. in group l, there was a significant improvement in joa score from 9.78 6.62 preoperatively to 14.75 3.33 at 6-month follow - up (p < 0.001), and 78.2% of group l patients had excellent (n = 9) or good (n = 9) results at 6-month follow - up. there was also significant improvement in joa score from preoperatively (9.6 3.4) to 6-month follow - up (14.7 3.4) (p < 0.05) in group h, and 66.7% of these patients had excellent (n = 6) or good (n = 8) results at 6-month follow - up. there was no significant difference in percentage of patients with excellent or good follow - up joa scores between group l and group h. two patients in group l (8.6%), but none in group h, had axial pain at the 6-month follow - up. operative duration and intraoperative blood loss operative duration was significantly longer in group l (139.3 35.1 min) than in group h (100.4 27.1 min) (p < 0.05). mean intraoperative blood loss was significantly greater in group l (335.7 50.1 ml) than in group h (221.3 22.5 ml) (p < 0.05). assessment of spinal canal area mean spinal canal area at 6-month follow - up was significantly greater in both groups compared with before surgery. mean expansion ratio was significantly greater in group p (77.83 6.41%) than in group h (62.72 3.86% in group h which was significantly larger in group comparison of spinal canal area preoperatively and at 6-month follow - up p<0.01 compared with mean expansion ratio in group h. l : laminoplasty ; h : hemilaminectomy. in group l, there was a significant improvement in joa score from 9.78 6.62 preoperatively to 14.75 3.33 at 6-month follow - up (p < 0.001), and 78.2% of group l patients had excellent (n = 9) or good (n = 9) results at 6-month follow - up. there was also significant improvement in joa score from preoperatively (9.6 3.4) to 6-month follow - up (14.7 3.4) (p < 0.05) in group h, and 66.7% of these patients had excellent (n = 6) or good (n = 8) results at 6-month follow - up. there was no significant difference in percentage of patients with excellent or good follow - up joa scores between group l and group h. two patients in group l (8.6%), but none in group h, had axial pain at the 6-month follow - up. operative duration and intraoperative blood loss operative duration was significantly longer in group l (139.3 35.1 min) than in group h (100.4 27.1 min) (p < 0.05). mean intraoperative blood loss was significantly greater in group l (335.7 50.1 ml) than in group h (221.3 22.5 ml) (p < 0.05). assessment of spinal canal area mean spinal canal area at 6-month follow - up was significantly greater in both groups compared with before surgery. mean expansion ratio was significantly greater in group p (77.83 6.41%) than in group h (62.72 3.86% in group h which was significantly larger in group comparison of spinal canal area preoperatively and at 6-month follow - up p<0.01 compared with mean expansion ratio in group the primary goals of surgical treatment for multilevel csm are relief of neurological compression, stabilization of the cervical spine, and restoration of cervical lordosis. posterior cervical decompression is an accepted treatment of csm and is the procedure of choice for the patients with multilevel csm. since laminoplasty was introduced in 1973 to decrease the rate of late complications associated with laminectomy, various methods for performing cervical laminoplasty, in addition to open - door laminoplasty, have been developed. all these variations were designed to widen the spinal canal while retaining the dorsal elements. the development of cervical fixation systems appears to have lowered the incidence of complications in recent years. open - door laminoplasty has become the preferred posterior procedure for treating multilevel csm. nevertheless, there are shortcomings associated with this procedure, such as high cost, long operative duration, potential for reclosing of the door, restriction of range of motion, and lack of a truly stable fusion. cervical laminectomy, which permits adequate decompression of the spinal cord, has long been the treatment for multilevel cervical spondylosis. however, it has been shown that laminectomy may cause instability owing to damage to the posterior elements of the spinal column. hemilaminectomy is advantageous in preserving spinal structures but provides relatively limited decompression of the spinal canal. therefore, a modified laminectomy procedure, the expansive hemilaminectomy, was developed to obtain better spinal canal decompression. because it could widen the spinal canal while preserving the interspinous ligaments and paracervical muscles on the contralateral side, it began to be indicated for the treatment of multilevel csm. in this study, we found that there was no significant difference in postoperative neurological improvement, as measured using joa score, between expansive hemilaminectomy and laminoplasty with mini titanium plate. this suggests that both procedures can achieve similar clinical outcome in the treatment of multilevel csm. we also found that operation time was shorter, and intraoperative blood loss less, in expansive hemilaminectomy than in laminoplasty with titanium mini - plate fixation. less invasive surgery, reconstruction of the extensor musculature, avoiding detachment of the semispinalis cervical muscles, and early removal of external immobilization have been shown to be effective in preventing axial pain after these procedures. in the present study, no patients in group h, and 8.6% of patients in group l, were observed to have postoperative axial pain, suggesting that expansive laminectomy may reduce the incidence of postoperative axial pain compared with laminoplasty. however, because axial pain is influenced by a number of factors, high - quality studies are necessary to investigate this further. in the present study, mri, rather than computed tomography or radiographic images, was used to measure the area of the spinal canal because it allows a more detailed view of the spinal cord and compression. because the shapes of the remnant vertebral canal area were mostly irregular or polygonal, autocad software was used for precise measurement of spinal canal area. by comparing the areas of the spinal canal before surgery and at 6-month follow - up in both groups, we found that both posterior approaches were able to widen the spinal canal significantly and that, although the mean expansion ratio in group l was larger than that in group h, sufficient space for the spinal cord to drift backward was created by both procedures. our results suggest that laminoplasty may be a better choice for patients with a severely stenotic spinal canal, especially in cases where the spinal canal area is less than half that of the bony spinal canal or in cases of opll, because of its ability to achieve a greater degree of enlargement. a variety of factors may affect the surgical outcomes of patients with csm, and each surgical approach also has its own distinct advantages and disadvantages. the retrospective design and small sample size of the study group are the main limitations of the present study. because of the very small sample, we were unable to divide the patients into subgroups according to the severity of the spinal canal stenosis. the lack of mid- and long - term follow - up is another drawback of the study. in summary, both surgical approaches are safe and effective in the treatment of multilevel csm ; both allow the spinal cord to drift posteriorly in the enlarged spinal canal, and both provide satisfactory neurological improvement at 6-month follow - up. compared with laminoplasty with mini - titanium plate fixation, expansive hemilaminectomy has the advantages of a shorter operation and less intraoperative blood loss, which theoretically can speed postoperative recovery. in addition, unilateral soft - tissue stripping and preservation of the posterior elements as much as possible can facilitate maintenance of spinal alignment.	background : posterior cervical decompression is an accepted treatment for multilevel cervical spondylotic myelopathy (csm). each posterior technique has its own advantages and disadvantages. in the present study, we compared the functional and radiological outcomes of expansive hemilaminectomy and laminoplasty with mini titanium plate in the treatment of multilevel csm.methods:forty-four patients with multilevel csm treated with posterior cervical surgery in department of orthopedic surgery, beijing army general hospital from march 2011 to june 2012 were enrolled in this retrospective study. patients were divided into two groups by surgical procedure : laminoplasty (group l) and hemilaminectomy (group h). perioperative parameters including age, sex, duration of symptoms, operative duration, and intraoperative blood loss were recorded and compared. spinal canal area, calculated using autocad software (autodesk inc., san rafael, ca, usa), and neurological improvement, evaluated with japanese orthopedic association score, were also compared.results:neurological improvement did not differ significantly between groups. group h had a significantly shorter operative duration and significantly less blood loss. mean expansion ratio was significantly greater in group l (77.83 6.41%) than in group h (62.72 3.86%) (p < 0.01).conclusions : both surgical approaches are safe and effective in treating multilevel csm. laminoplasty provides a greater degree of enlargement of the spinal canal, whereas expansive hemilaminectomy has the advantages of shorter operative duration and less intraoperative blood loss.
samples : a total of 62 formalin fixed organs (49 spleens and 13 livers) were collected from the slaughterhouses in iwate prefecture, japan. most chickens were around 60 days old ; however, the age of some chickens was unidentified. gross examination : the diameters and weight of the organs were recorded. the severity of the gross lesions, based on the size and distribution of the lymphomatous lesions in the affected organs, was scored as follows : () score for the absence of lymphomatous lesions and no changes in the anatomical architecture of the organs ; (+) for solitary distributed mild lymphomatous lesions of sizes in the range of 23 mm and without changes in the anatomical architecture of the organs ; (2 +) for solitary distributed moderate lymphomatous lesions with sizes in the range of 35 mm and with slight changes in the anatomical architecture of the affected organs ; (3 +) for diffusely distributed severe lymphomatous lesions of > 5 mm in size and with noticeable changes in the anatomical architecture of the affected organs. histopathological examination : for histopathological examination, the organs were cut into slices and fixed with 10% neutral buffered formalin for 2 days, following which they were dehydrated and embedded in paraffin wax. all the sections were cut approximately 4 m thick and stained with hematoxylin and eosin. tsukamoto (national institute of animal health, tsukuba, japan) and was used to detect avian leukosis virus (alv) infected neoplastic cells by detecting the alvgs - antigen. cav antiserum was used to detect cav antigen ; it was prepared by intramuscular inoculation of 0.1 ml of mdcc - msb1 culture supernatant containing 10 mean tissue culture infective dose of the msb1-tk5803 strain into 3-week - old specific pathogen free chicks, followed by a second inoculation 2 weeks later. the sera were then collected 4 weeks after the second inoculation inactivated in a water bath for 30 min at 56c and stored at 80c. four m thick paraffin sections were deparaffinized in xylene and rehydrated in graded alcohol. antigen retrieval was performed by autoclaving the sections in a 10 mm citrate buffer solution, ph 6. the endogenous peroxidase was blocked by treating the sections with 0.3% h2o2 solution in methanol for 30 min at room temperature. subsequently, the sections were treated with diluted normal goat serum (vector lab., peterborough, u. k.) in phosphate - buffered saline (1:10) for 30 min to block nonspecific reactions. the sections used to detect the alvgs - antigen were covered by the primary antibody, rabbit anti - rav2 (1:10,000), while the sections used to detect cav were covered with cav antisera (1:100). the sections were kept overnight in a humid chamber at 4c. after washing with tris - buffered saline, the sections were covered with a secondary antibody. the sections used to detect cav were covered with goat polyclonal anti - chicken igg conjugated to horseradish peroxidase (hrp) enzyme (bethyl lab. the labeled streptavidin - biotin - peroxidase complex technique using the lsab system - hrp kit (dako north america, inc., carpinteria, ca, u.s.a.) was used to detect the alvgs - antigen. the peroxidase was activated by using 3, 3 -diaminobenzidine (dako north america, inc.). gross findings : the tested organs were of various weights, shapes and sizes ; the weight of the spleens ranged from 2.9150 g, and the weights of the livers ranged from 58214 g. there were white nodules of variable sizes in 49% (24/49) of the tested spleens and 39% (5/13) of the tested livers (fig. 1.the gross pathology of the spleen (a) and liver (b) of a 60-day - old chicken shows alteration in the anatomical architecture due to the presence of variable sized nodules. on cut surface, the spleen (a) shows a clear marbling appearance due to the presence of white to grayish creamy nodular masses which are embedded within the parenchyma. the liver (b) surface appears uneven due to the presence of raised white nodular mass.). the gross pathology of the spleen (a) and liver (b) of a 60-day - old chicken shows alteration in the anatomical architecture due to the presence of variable sized nodules. on cut surface, the spleen (a) shows a clear marbling appearance due to the presence of white to grayish creamy nodular masses which are embedded within the parenchyma. the liver (b) surface appears uneven due to the presence of raised white nodular mass. on the cut surface, 61% (30/49) of the tested spleens showed a clear marbling appearance due to the presence of white colored nodular masses of different sizes in the splenic parenchyma (fig. some of these nodules were coalescent and difficult to distinguish from the splenic parenchyma, while other nodules showed a clear demarcation from the splenic parenchyma in the form of a zone of hemorrhagic and necrotic areas. the findings on the cut surface of the liver were the same as that on the cut surface of the spleen ; however, the nodules in the liver were totally demarcated from the hepatic parenchyma, and the hepatic surface was uneven due to the presence of these nodules (fig. the severity of the gross lesions on the surface and cut surface of the tested organs scored as follows : 17 organs, which included 11 spleens and 6 livers, scored 3 + ; 16 organs, which included 12 spleens and 4 livers, scored 2 + ; 9 organs, which included 6 spleens and 3 livers, scored + ; and the remaining 20 spleens with no significant gross lesions scored as. histopathological findings : the affected spleens showed neoplastic lymphocytic aggregations of various sizes ranging from focal lesions to extensive lymphoma, and these aggregations often included the periarterial lymphoid sheath and the perivenular lymphoid tissue, and appeared as a compressed red pulp. these aggregations were observed in 76% (37/49) of the spleens and 92% (12/13) of the livers. the neoplastic lymphocytes severely infiltrated the hepatic parenchyma with severe dilatation of the hepatic sinusoids. each lesion was composed of small mature lymphocytes, large bizarre lymphocytes, lymphoblasts, tangible body macrophages and pyknotic lymphocytes. the mitotic count ranged from 3/high power field (hpf) in mild lesions to 10/hpf in severe lesions. the large bizarre lymphocytes, which were 23 times larger than the other lymphoid cells with a large nucleus and moderate cytoplasm, were located mainly in the center of the neoplastic lymphoid aggregations ; however, sometimes, these cells were observed at the periphery of the lymphoid mass. on the other hand, multiple and small eosinophilic intranuclear inclusion bodies were observed, occasionally, within the karyomegalic neoplastic cells in 2 livers and 9 spleens. the inclusions were observed in both the small and large bizarre lymphocytes (fig. 2.(a) the spleen shows variable sized tumorous proliferations of large bizarre shaped lymphoid cells with large nuclei and moderate cytoplasm (arrows) located at the center or periphery of the lymphoid mass ; bar=10 m. (b) the large bizarre shaped lymphocytes of the md lymphoma in the spleen show small, multiple and variable sized eosinophilic inclusions in their nuclei (arrows). (a) the spleen shows variable sized tumorous proliferations of large bizarre shaped lymphoid cells with large nuclei and moderate cytoplasm (arrows) located at the center or periphery of the lymphoid mass ; bar=10 m. (b) the large bizarre shaped lymphocytes of the md lymphoma in the spleen show small, multiple and variable sized eosinophilic inclusions in their nuclei (arrows). the distribution pattern of the neoplastic lymphocytic accumulation within the affected spleens and livers was histopathologically divided as follows. 1) multifocal lymphocytic accumulation : this pattern was noticed in 32% (12/37) of the total numbers of spleens with neoplastic lymphocytic accumulation and 69% (9/13) of the total number of livers. in this pattern, the lymphocytic accumulation appeared as multiple, circumscribed and variably sized nodules within the parenchyma. the eosinophilic intranuclear inclusion bodies were noticed in the neoplastic cells of 3 out of 9 of spleens and in 2 out of 2 livers. 2) coalesced lymphocytic accumulation : this pattern was noticed in 43% (16/37) of the total number of spleens with neoplastic lymphocytic accumulation and 23% (3/13) of the total number of livers. in this pattern, the infected spleens showed uncircumscribed accumulations of neoplastic lymphocytes within the splenic parenchyma and these lesions were divided by thin indigenous splenic pulps, whereas the infected livers showed a severe invasion of uncircumscribed and variably sized foci in the hepatic sinusoids and hepatic parenchyma. the eosinophilic intranuclear inclusion bodies were observed in the neoplastic cells of 4 out of 9 infected spleens, while in the liver, no eosinophilic intranuclear inclusion bodies were noted. 3) diffuse lymphocytic infiltration : this pattern represented 24% (9/37) of the total numbers of spleens with neoplastic lymphocyte infiltrations. in this pattern, the normal structure of the spleen was lost, and most of the splenic tissue was replaced by pleomorphic neoplastic lymphocytes. the eosinophilic intranuclear inclusion bodies were observed in the neoplastic cells in 2 out of 9 infected spleens. immunohistochemistry : the cav antigen was detected in the neoplastic cells of 24% (9/37) of the spleens with the neoplastic lymphocyte infiltrations and 15% (2/13) of livers. the eosinophilic intranuclear inclusion bodies in the small and the large bizarre lymphocytes were positive for this antigen (fig. (a) cav antigen was detected within the md lymphoma in the large bizarre and small lymphocytes. (b) cav antigen was detected as intranuclear fine granular inclusions within the large bizarre shaped lymphocytes (arrow) and the small lymphocytes (arrowheads). (a) cav antigen was detected within the md lymphoma in the large bizarre and small lymphocytes. bar=10 m. (b) cav antigen was detected as intranuclear fine granular inclusions within the large bizarre shaped lymphocytes (arrow) and the small lymphocytes (arrowheads). although the presence of coinfection of mdv and cav was proved in several experimental studies [10, 11, 16 ], herein, we evaluated the existence of cav antigen and its inclusions within naturally occurring lymphomas in condemned organs. in this study, we confirmed md lymphoma in 37 spleens and 12 livers based on the cellular morphology and infiltrative nature of the neoplastic lymphocytes.these lymphomas were composed of pleomorphic neoplastic lymphocytes and lymphoblasts with nuclear pleomorphism ; these findings were consistent with previous reports [2, 18 ]. however, cav inclusions were observed in the md lymphomas of 9 spleens and 2 livers. cav inclusions were observed in 2 types of neoplastic lymphocytes, the large bizarre cells and the small sized lymphocytes. these results suggest the possibility of the existence of cav antigen within the mdv lymphomas in > 4-week - old chickens, and this finding supports the experimental results of haridy. and the md lymphomas, according to their pathomorphology, were divided into multifocal, coalesced and diffuse lymphocytic accumulations. cav inclusions were detected in both multifocal and the coalesced patterns in a higher ratio than in the diffuse pattern. because the 3 patterns are considered as sequential infiltrations of the neoplastic lymphocytes, the increase in the infiltration pattern of the md neoplastic lymphocytes appears to have a negative correlation with the existence of cav within the mdv lymphoma. the pathogenesis of the co - infection with both the viruses is still poorly understood. however, we speculate the relationship between cav and mdv during dual infection as symbiotic. cav - neutralizing antibodies were previously believed to clear the virus ; however, recent studies detected chicken anemia viral dna in gonadal tissues and spleens despite the flock seroconversion [4, 5 ]. this may play a role in cav latency in the offspring after reactivation of the virus during the sexual maturity of chickens. this mechanism may result in a subclinical form of cav, which causes an immunosuppressive effect and impairment in the generation of cytotoxic t lymphocytes and in turn, influences the development of other diseases. based on the above - mentioned hypothesis, we assume that the poor vaccine - induced protection against md and the late breakdown of md vaccination might be caused by the reactivation of cav. moreover, the existence of cav antigen and inclusions is closely correlated with the fact that cav needs dividing cells for its replication [1, 20 ]. the neoplastic cells in md lymphomas are considered as uncontrolled dividing cells, which are susceptible to infection with cav. the neoplastic cells of md lymphomas may act as a nest for the replication and multiplication of cav. in conclusion, to our knowledge, this is the first report regarding the persistence of cav antigen and its inclusions in naturally occurring md lymphomas in > 3-week - old chickens, and these md lymphomas may act as a nest for cav, prolonging its dissemination time and increasing the possibility of further infection in chickens.	the chicken anemia virus (cav) and marek s disease virus (mdv) infect chickens worldwide ; a single or dual infection by these viruses has a great impact on poultry production. in the present study, we examined the existence of cav antigen and its inclusions in marek s disease (md) lymphomas in chickens in the slaughterhouses of iwate prefecture, japan. forty - nine spleens and 13 livers with different degrees of nodular lesions were histopathologically examined at our laboratory. grossly, the tested organs showed various sizes and anatomical architectures. based on the cellular morphology and the infiltrative nature of the neoplastic lymphocytes, md was confirmed in 76% (37/49) of the spleens and 92% (12/13) of the livers. the lesions of md, according to the pattern of lymphocytic accumulation in the affected organs, were divided into multifocal, coalesced and diffuse. cav intranuclear inclusion bodies were detected within the small and the large bizarre lymphocytes of the md lymphomas in 2 livers and 9 spleens, and the immunostaining test for cav confirmed the persistence of cav antigens and inclusions in the neoplastic cells. this study demonstrated the persistence of cav infection within the neoplastic cells of naturally occurring md lymphomas in chickens.
color stability of composite resin is an important property influencing its clinical longevity, which continues as a challenge inherent to material. color changes can occur due to various etiologic factors ; extrinsic discoloration can occur due to staining in the superficial layer of resin composite, water absorption, surface roughness, smoking, and diet [2, 3 ]. intrinsic discoloration could occur as a result of physicomechanical reaction within the material (e.g., the filler and the resin matrix properties). in oral conditions, composite resins are exposed to different dietary beverages such as coffee which might result in absorption and adsorption of colorants in coffee into the resin surface and consequently undesirable color change. previous investigations reported the influence of coffee on the color stability of composite resins [610 ]. generally, it is recommended that the resins should be placed in 2 mm increments to obtain sufficient light transmittance and complete curing of composite resins. placing the resin in 2 mm increments has some merits including the reduction of the polymerization shrinkage and the voids incorporation between the layers. however, the application of composite resins in an incremental technique and light curing each increment individually is a time - consuming procedure. there is also an increasing possibility of air bubble inclusion or moisture contamination between individual increments of resin composite restorations. to overcome such fallibility,, these materials can be sufficiently light cured up to 4 mm in a single increment, without influencing the polymerization shrinkage, degree of conversion, or cavity adaptation. in addition, it is claimed that these materials have lower polymerization shrinkage compared to conventional composite resins. thus, some postoperative problems, such as gap formation and the incidence of recurrent carries, may be diminished. such merits are probably due to the increased translucency of the bulk - fill composites, which permits greater light transmission. additionally, the formulation of these materials allows for modulation of the polymerization reaction by applying the stress - relieving monomers, the use of more reactive photoinitiators, and the incorporation of different types of fillers, such as prepolymer particles and fiberglass rod segments. while the manufacturers recommend bulk - filling of these materials up to 4 mm, many clinicians suspect that the depth of cure and mechanical properties might not be suitable for clinical use there are few reports on of the effect of increment thickness and the storage media on the discoloration of these bulk - fill resin composites. since color changes are a concern that affects the population and one of the main reasons for replacing resin - based restorations, this study investigated the effects of the increment thickness and the storage media on the discoloration of one of these bulk - fill resin composites. two a3 shade light cured composite resins, conventional and bulk - fill tetric evoceram, were selected for this study. the specimens of conventional (6 mm diameter and 2 mm thickness, n = 20) and bulk - fill composite resins at two different thicknesses (6 mm diameter and 2 mm and 4 mm thickness, n = 20) were prepared using a polyethylene mold. after applying the composite resin, a mylar strip was placed and pressed with a glass slide to obtain a flat surface. the glass slide was removed and the specimens were cured for 40 s using a halogen light curing unit (1086.67 mw / cm, demetron l.c ; kerr corporation, orange, ca, usa). after removing the specimens from the molds, baseline color was measured using a digital image analysis method as described later. specimens were stored at 37c in distilled water for 28 d. half of the specimens remained in distilled water and the other half were immersed in coffee solution 20 min / d and remained in distilled water in the interval between cycles. to prepare the coffee solution, 25 g of coffee (taster 's choice, nestl usa, inc., glendale, ca, usa) was poured in 250 ml of boiling water, and after 10 min of stirring, the solution was filtered through a filter paper. the color of the specimens was assessed in the commission international de i'eclairege lab (cielab) color space using a digital image analysis method. the cielab system is a chromatic value color space that measures chroma and value in three coordinates : l, the lightness measured from 0 (black) to 100 (white), a, color in the red (a 0) axis, and b, color in the blue (b 0) axis. a setup was designed in a dark room while two 60 w light sources were positioned from the sides (45-degree angle to the samples). at each interval, the specimens were rinsed with distilled water for 5 seconds, blotted dried with tissue paper, and placed on a dark background. the digital images connected with the computer running adobe photoshop cs5 (adobe, san jose, ca, usa) as color assessment software. the results were statistically analyzed using repeated measures two - way anova with factors including materials (conventional, 2 mm and 4 mm bulk - fill resin composite) and immersion media (coffee and distilled water) for each time interval. for multiple comparisons, tukey 's hsd post hoc test was used to compare different materials (p 3.3 are considered appreciable by nonskilled persons and are, hence, not clinically acceptable. therefore, color changes above a value of e = 3.3 were considered clinically unacceptable. in this study coffee staining produced higher color changes in the specimens than those of water storage, which was in line with the previous investigations ; erta. and villalta. immersed composite resin into the different beverages and reported that coffee showed greater color changes compared to water storage [6, 7 ]. the present result seems reasonable because when specimens were submitted to coffee staining, discoloration occurred due to the adsorption and absorption of pigments into the organic phase of resin - based materials. additionally, coffee contains significant amounts of staining agents such as gallic acid, which facilitate staining. the findings of our study present that water storage can also lead to slight discoloration, slightly perceptible, which is in line with the other investigations. the staining susceptibility of specimens after being immersed in distilled water might be due to their degree of water absorption and the hydrophilic / hydrophobic nature of the resin matrix. excessive water absorption can decrease the life of a composite resin by plasticizing and expanding the resin component, causing microcrack formation. as a result, microcracks or interfacial gaps at the interface between the filler and matrix allow stain penetration and discoloration. in addition, discoloration might be due to the differences in the refractive index of filler and matrix which might increase after water absorption. others factors that have been shown to have a significant impact on the color stability of material are the composition of composite resins and the characteristics of filler particle. in this study the color susceptibility of bulk - fill composite resin was significantly higher than that of conventional composite resin after immersion in storage media ; it is claimed that bulk - fill tetric evoceram resins consist of a variety of fillers, prepolymer shrinkage stress reliever, different photoinitiator system, and light sensitivity system. the mechanism of the interaction between the bulk - fill composite resin and the storage media is unknown and requires further investigation. altering the resin thickness is one of the other variables that can affect the final appearance of composite restoration. in the present study we tested the different thicknesses (2 and 34 mm) on the color stability of bulk - fill composite resin. we found that bulk - fill materials showed significantly greater discoloration at increasing increment thickness at 14 d and 28 d. this can be explained by the polymerization process of resin - based composites. depth of cure can be influenced by different factors such as the monomer composition, filler content, and photoinitiator system. light exposure can lead to causing activation of the photoinitiator which is attenuated by composite absorption and scattering. thus, depth of cure relies on the material 's capacity to transfer light into its depths, as well as on the polymerization kinetics. a previous study showed that, in the case of applying composite resin incrementally, no significant difference was observed in values of depth of cure at different depths. but a bulk - fill technique might result in a greater number of particle / resin matrix interfaces and increased light scattering due to the differences in their refractive indices. therefore, lesser amount of photons would reach deeper layers of composite resin and consequently a lower depth of cure value would be obtained at the deepest depths. in accordance with these results, flury. reported lower depths of cure of the bulk - fill specimens with 4 mm thickness compared to the values asserted by the manufacturers. in part, differences in the depth of cure, and even the overall degree of conversion, might affect the uncured monomer released and influence the composite discoloration. as a consequence, mechanical properties are deteriorated leading to greater monomer elution which can result in more water absorption and, hence, discoloration. our findings demonstrated that bulk - fill composite resin had greater color susceptibility after immersion in coffee than conventional composites. considering the increment thickness it can be noted that the discoloration is increased with greater increment thickness. we demonstrated that greater staining susceptibility of thicker specimens might be due to their lower depth of cure when placing bulk - fill materials.	we aimed to evaluate the color stability of bulk - fill and conventional composite resin with respect to thickness and storage media. twenty specimens of a conventional composite resin (6 mm diameter and 2 mm thick) and 40 specimens of the bulk - fill tetric evoceram composite resin at two different thicknesses (6 mm diameter and 2 mm thick or 4 mm thick, n = 20) were prepared. the specimens were stored in distilled water during the study period (28 d). half of the specimens were remained in distilled water and the other half were immersed in coffee solution 20 min / d and kept in distilled water between the cycles. color changes (e) were measured using the cie lab color space and a digital imaging system at 1, 7, 14, and 28 days of storage. data were analyzed using two - way anova and tukey 's hsd post hoc test (p conventional ; p < 0.001). coffee exhibited significantly more staining susceptibility than that of distilled water (p < 0.001). there was greater color changes with increasing the increment thickness, which was significant at 14 (p < 0.001) and 28 d (p < 0.01). color change of bulk - fill composite resin was greater than that of the conventional one after coffee staining and is also a function of increment thicknesses.
an 18-year - old female came to our clinic reporting blurred vision of the left eye for 2 days. she also complained of decreased hearing with tinnitus of the right ear and mild headache. best - corrected visual acuity (bcva) was 20 / 50 in the left eye, and 20 / 20 in the right eye. fundus examination and fluorescein angiography of the left eye showed ischemic retina with signs of branch retinal artery obstruction (fig. 1). we performed brain magnetic resonance imaging (mri) and lumbar puncture to evaluate any brain or central nervous system lesions. the mri scan showed a t2-weighted, high - signal, discrete area around the periventricular region, in the white matter and in the corpus callosum (fig. blood pressure, echocardiography, carotid ultrasonography, full blood count, erythrocyte sedimentation rate, fasting lipids and glucose, autoantibody screen including anti - cardiolipin antibody, protein s and c levels, and antithrombin iii level were all normal. the patient was treated with intravenous prednisolone 1 g / kg for 3 days followed by oral steroid tapering. three months after treatment, bcva recovered to 20 / 25 in the left eye. a mild residual auditory defect remained, but no remaining tinnitus or headache were observed. susac syndrome was first described by susac in 1976 and since then has been called by different names, including red - m (retinopathy, encephalopathy, deafness associated with microangiopathy), sicret (small infarction of cochlear, retina and encephalitic tissue), and retinocochlear vasculopathy. this syndrome is rare and usually shows female predominance with a sex ratio of 3 to 1. approximately 80 cases of susac syndrome have been reported in the literature [1 - 4 ]. the syndrome is often misdiagnosed and should be differentiated from a number of other diseases, including multiple sclerosis, disseminated encephalomyelitis, lupus erythematosus, mnire disease, migraine and thromboembolic strokes. moreover, many patients do not present initially with the clinical triad and show incomplete forms of the disease in which only branch retinal artery obstruction or hearing loss in the absence of encephalopathy is observed. susac and colleagues reported that there was always involvement of the corpus callosum, and callosal lesions typically involve the central fibers with relative sparing of the periphery. yellow retinal arterial wall plaque in association with susac syndrome has been described, but plaques were not identified in our patient. the bilateral or unilateral hearing loss is due to cochlear involvement. hearing loss may be asymptomatic and found only by audimetry. the pathogenesis of this syndrome is believed to be an immunological reaction, leading to small vessel vasculitis causing micro infraction in the retina, brain and apical turn of the cochlea. in a recent autopsy study, the findings of retinas with susac syndrome suggested that the syndrome could be an endothelioapthy. the disease course is known to be monophasic and self - limited, with a duration of 1 to 4 years. different therapeutic protocols such as corticosteroids, immunosuppressive agents, anti - platelet agents, anticoagulant agents and plasmapheresis have been reported. although their efficacy remains difficult to evaluate, early recognition of the disease is important because treatment with immunosuppression seems to reduce permanent cognitive, audiologic, and visual sequelae. to the best of our knowledge, this is the first report of susac syndrome in korea. a high index of suspicion leading to early recognition of this syndrome is important because immunosuppressive treatment may decrease permanent neuropsychological or visual impairment. in cases with retinal arterial occlusion with hearing loss or neuropsychological symptom, early detection of the characteristic brain mri finding, audimetry, and funduscopic examination	the purpose of this article is to report on the first known korean case of susac syndrome. an 18-year - old female came to our clinic reporting blurred vision of the left eye for 2 days. she also complained of decreased hearing with tinnitus of the right ear and mild headache. she was previously healthy and had no remarkable medical history. best - corrected visual acuity was 20 / 50 in the left eye and 20 / 20 in the right eye. an axiomatic triad of ocular, cochlear, and neurologic involvement was observed in the patient. fluorescein angiography showed branched retinal arterial occlusions in the left eye. a sudden right sensorineural hearing loss was observed on audimetry. magnetic resonance images showed a hyperintense lesion in the white matter around the corpus callosum. the patient was treated with high doses of systemic corticosteroids, and no neuropsychological sequelae were observed. this is the first case report of susac syndrome in korea. in cases of retinal arterial occlusion with hearing loss or neuropsychological symptoms, susac syndrome should be suspected.
xanthomas are nonneoplastic, reactive tumor - like processes, usually arising in response to lipid profile disturbances, diseases with secondary hyperlipidemia, like primary biliary cirrhosis and diabetes mellitus and occasionally in normolipidemic states it has been suggested that endothelial, stromal, histiocytic and rarely, epithelial cells may be transformed into xanthoma cells. xanthomata occur more frequently in the skin, tendons, subcutaneous tissue and gastrointestinal tract, but, unlike them, urinary bladder xanthoma appears to be a rare condition, and 12 cases have been reported in english medical literature so far. the patient had no significant medical or surgical history in the past except for tubal ligation done 5 years back as a measure of family planning. ultrasound scan of the abdomen showed 7 4 cm size mass lesion in relation to anterior wall of the bladder. contrast - enhanced computed tomography abdomen and pelvis showed irregular thick wall enhancing area with internal nonenhancing area in the anterosuperior wall of the bladder with perivesical fat stranding and extension of lesion into the right rectus abdominis muscle suggestive of malignant mass of possibly bladder or urachal origin with multiple enlarged enhancing lymphnodes in pre / paraaortic and iliac regions suggestive of metastasis. a flexible cystoscopy examination revealed 6 5 cm mass on the dome of the bladder. blood counts, lipid profile and other hematological and biochemical investigations were within normal limits. transurethral resection of the bladder mass was done, and histological examination lesion showed urothelial papilloma. magnetic resonance imaging abdomen and pelvis of the patient was performed postoperatively which showed approximately 61 23 43 mm sized enhancing polypoidal mass lesion involving anterosuperior wall of bladder on the right side, the mass appeared hypointense on t1wi and t2wi and showed enhancement on post contrast study, there was focal loss of fat plane of the mass anteriorly suggestive of involvement of perivesical fat with invasion of perivesical fat, bilateral recti and subcutaneous fat present, there was peripheral rim enhancement with central nonenhancing necrotic areas, there was loss of fat plane with uterus and right fallopian tube, multiple enlarged pre / paraaortic, bilateral iliac and inguinal lymph nodes were noted [figure 1a ]. contrast - enhanced computed tomography abdomen + pelvis showed irregular thick wall enhancing area with internal nonenhancing area in anterosuperior wall of bladder with perivesical fat stranding and extension of lesion in the right rectus abdominis muscle [figure 1b ]. we performed a partial cystectomy [figure 1c and d ], and histological examination of the mass showed xanthomatous cystitis [figure 2 ]. the patient is kept on follow - up for every 3 monthly ; however, long - term follow - up is not mandatory as it is a benign condition with no malignant potential. 61 23 43 mm sized enhancing polypoidal mass lesion involving anterosuperior wall of bladder on right side with focal loss of fat plane of the mass anteriorly with multiple enlarged pre / paraaortic, bilateral iliac, and inguinal lymph nodes were noted, (b) contrast - enhanced computed tomography abdomen + pelvis - irregular thick wall enhancing area with internal nonenhancing area in anterosuperior wall of bladder with perivesical fat stranding and extension of lesion in the right rectus abdominis muscle (c) intraoperative photograph (d) postoperatively specimen photograph histopathopathological image - showing xanthoma cells with all layers of urinary bladder patients with bladder xanthomas are often asymptomatic or may present with hematuria or lower abdominal pain. in most cases, conservative treatment with antibiotics does not resolve these lesions and complete surgical resection is advised. however, others are of the opinion that long - term follow - up is unnecessary as the lesion remains static and is not premalignant. the differential diagnosis on gross appearance includes malakoplakia, which can only be excluded by histological examination. histologically, foamy cells in the bladder mucosa can occur in xanthogranulomatous cystitis, malakoplakia, atypical mycobacterial infection, and signet ring cell carcinoma. the histiocytes in malakoplakia are associated with small basophilic extracellular or intracytoplasmic calcospherules called michaelis - gutmann bodies which stain positive for von kossa stain, iron, and periodic acid - schiff. the differences between xanthoma and xanthogranulomatous cystitis are that the latter consists of xanthoma cells with multinucleated giant cells and chronic inflammatory cells, while the former comprises neither chronic inflammatory cells nor giant cells. from the literature review, it seems that urinary bladder xanthoma is slightly more common in women than in men. the coexistence of xanthoma and carcinoma in a bladder diverticulum has been documented, as bladder xanthoma can develop adjacent to urothelial cell carcinoma. treatment in the presence of symptoms and no other lesion to account for them may require complete transurethral resection. on the other hand, urinary bladder xanthoma does not cause symptoms, and after checking the lipid profile, long - term follow - up is not mandatory, as urinary bladder xanthoma has no malignant potential. in summary, diagnosis of xanthomatous cystitis is difficult preoperatively, and the condition may be mistaken for malignancy. surgical resection is curative with no postoperative recurrence reported. simple excision may suffice for a localized xanthoma, but if the disease is combined with an urachal remnant or adenoma, partial cystectomy is preferred.	xanthoma cystitis of urinary bladder is a rare entity and may present as an intravesical mass. a 38-year - old female presented with abdominal pain and imaging was done which was suggestive of a malignant mass with surrounding tissue infiltration. partial cystectomy was performed, and histological examination of the mass showed xanthomatous cystitis.
patients with nephrotic syndrome often require immunosuppression to achieve remission, yet many patients either relapse after remission or are resistant to therapy. for example, while up to 90% of adults with minimal change disease (mcd) will respond to initial therapy with prednisone, approximately one - third of these same patients will relapse within 6 months and require further immunosuppression.1,2 with diseases such as idiopathic membranous nephropathy (imn) and focal segmental glomerulosclerosis (fsgs), for which first - line therapies produce substantially lower response rates than for mcd, physicians are often compelled to use second-, third-, and even fourth - line therapies to achieve remission.38 in several european studies, tetracosactide, a synthetic adrenocorticotropic hormone (acth) analog, has shown efficacy as primary and secondary therapy for nephrotic syndrome. the initial reports came in a case series of patients with various etiologies of nephrotic range proteinuria, including mcd, imn, fsgs, and membranoproliferative glomerulonephritis (mpgn).9 subsequently, a randomized, controlled study by ponticelli reported similar remission rates in patients with imn randomized to synthetic acth or to therapy with alternating months of steroids and cyclophosphamide.10 these reports have generated renewed interest in using acth as treatment for nephrotic syndrome, particularly in patients who are resistant to conventional therapies. synthetic acth is not currently available for use in the united states, but a natural, highly purified acth gel formulation (h.p. acthar gel [repository corticotropin injection ], questcor pharmaceuticals, inc, union city, ca, usa ; abbreviated acth gel) is both available and approved for use in nephrotic syndrome. to date, however, there is no modern published experience on using acth gel in nephrotic patients. we therefore explored the initial use of acth gel for nephrotic syndrome in nonresearch settings (ie, by prescription), collecting data from treating nephrologists of all known patients in the united states whose treatment with this agent was initiated by the end of 2009. in this retrospective case series, we evaluated all known cases of idiopathic, nondiabetic nephrotic syndrome treated with acth gel outside of research settings (ie, by prescription) with initiation of therapy by december 31, 2009, allowing a minimum of 6 months follow - up. questcor pharmaceuticals, the drug s manufacturer, provided contact information for nephrologists whose patients had acth gel prescriptions filled during this time period. treating nephrologists were asked to provide data on patients demographics, diagnoses, previous immunosuppressive regimens, indications for acth gel therapy, dosing of acth gel, clinical response to therapy (creatinine, proteinuria, serum albumin, cholesterol), and adverse events from initiation of therapy to most recent clinical encounter. we defined complete remission as stable or improved renal function (estimated glomerular filtration rate, gfr [egfr ], based on serum creatinine) with final proteinuria falling to 3500 mg / day. failure to meet the above criteria was classified as no response. given the small number of patients and the observational nature of this study, in the united states, 25 patients with nondiabetic nephrotic syndrome began treatment with acth gel before december 31, 2009. full data were available for 21 patients with the following diagnoses (table 1) : imn (n = 11), mpgn (n = 4), fsgs (n = 1), mcd (n = 1), immunoglobulin a (iga) nephropathy (n = 1), class v systemic lupus erythematosus (sle) glomerulonephritis (n = 1), monoclonal diffuse proliferative glomerulonephritis (n = 1), and unbiopsied nephrotic syndrome (n = 1). acth gel was used as primary therapy for only 3 patients, one each with diagnoses of imn, iga nephropathy, and nonbiopsied nephrotic syndrome. the remaining 18 patients had failed a mean 2.3 immunosuppressive regimens prior to acth gel therapy, of which 9 patients had failed at least 3 prior therapies. nine of 21 patients were female, all but 3 patients were white, and there was a wide range of ages (24 to 81 years). most patients had impaired baseline renal function (egfr range from 0.2 to > 1.0 ml / s/1.73 m [11 to > 60 ml / min/1.73 m ]) with 12 patients demonstrating stage 4 or 5 chronic kidney disease (egfr 0.5 ml / s/1.73 m [30 ml / min/1.73 m ]). pretreatment proteinuria ranged from 1340 mg / day to 18,553 mg / day ; only 3 patients had proteinuria 8000 mg / day (table 2). the 3 patients whose baseline proteinuria was in subnephrotic range exhibited other signs of nephrotic syndrome (hypoalbuminemia, hyperlipidemia, and edema). g / l (2.9 g / dl), and mean pretreatment total cholesterol was 6.10 mmol / l (236 mg / dl) (data available for 12 patients). the most common treatment regimen was 80 units subcutaneously (sc) twice weekly for 6 months, which was used in 13 of 21 patients (table 2). five patients treated for fewer than 6 months were taken off therapy as a result of clear lack of response ; 1 patient discontinued therapy due to weight gain but had achieved a temporary response while on therapy. the longest treatment duration was 14 months, of which the last 2 months included a taper of the dose from 40 units twice weekly to 40 units once weekly. most patients stopped treatment without a taper, however, and without obvious rebound in proteinuria. overall, 11 of 21 patients (52%) achieved a complete or partial remission, with 4 (19%) in complete remission (table 2). one patient achieved a limited response, while 9 patients had no overall response to therapy. the patient with class v sle glomerulonephritis transiently responded to therapy (proteinuria declining to 1.0 ml / s/1.73 m [11 to > 60 ml / min/1.73 m ]) with 12 patients demonstrating stage 4 or 5 chronic kidney disease (egfr 0.5 ml / s/1.73 m [30 ml / min/1.73 m ]). pretreatment proteinuria ranged from 1340 mg / day to 18,553 mg / day ; only 3 patients had proteinuria 8000 mg / day (table 2). the 3 patients whose baseline proteinuria was in subnephrotic range exhibited other signs of nephrotic syndrome (hypoalbuminemia, hyperlipidemia, and edema). g / l (2.9 g / dl), and mean pretreatment total cholesterol was 6.10 mmol / l (236 mg / dl) (data available for 12 patients). the most common treatment regimen was 80 units subcutaneously (sc) twice weekly for 6 months, which was used in 13 of 21 patients (table 2). five patients treated for fewer than 6 months were taken off therapy as a result of clear lack of response ; 1 patient discontinued therapy due to weight gain but had achieved a temporary response while on therapy. the longest treatment duration was 14 months, of which the last 2 months included a taper of the dose from 40 units twice weekly to 40 units once weekly. most patients stopped treatment without a taper, however, and without obvious rebound in proteinuria. overall, 11 of 21 patients (52%) achieved a complete or partial remission, with 4 (19%) in complete remission (table 2). one patient achieved a limited response, while 9 patients had no overall response to therapy. the patient with class v sle glomerulonephritis transiently responded to therapy (proteinuria declining to < 500 mg / day) but relapsed as soon as therapy was stopped, thus not meeting criteria for remission. of the 11 patients who achieved complete or partial remission, 9 had imn, 1 had fsgs, and 1 had iga nephropathy. of the 11 patients with imn (figure 1), 3 achieved complete remission and 6 achieved partial remission despite having previously failed a mean 2.4 therapies. of the 10 patients with nephrotic syndrome diagnoses other than imn (figure 2), 1 patient with iga nephropathy achieved complete remission, 1 patient with fsgs achieved partial remission, and 1 patient with mpgn had a limited response to therapy. follow - up time for patients ranged from 6 to 14 months. five patients reported steroidlike adverse effects with therapy : 2 patients had impaired blood glucose control, 2 patients had significant weight gain, and 1 patient showed evidence of accelerated bone loss on bone densitometry not observed prior to acth gel therapy (table 3). in addition, 1 patient with monoclonal diffuse proliferative glomerulonephritis developed acute renal failure within 1 month of starting therapy, with creatinine rising from 327.1 mol / l (3.7 mg / dl) to 751.4 mol / l (8.5 mg / dl). the drug was stopped along with the patient s diuretics and angiotensin - converting enzyme inhibitor ; after intravenous fluid hydration, the creatinine returned to 353.6 six months after starting acth gel, the patient s creatinine ranged between 442.0 and 530.4 mol / l (5.0 and 6.0 mg / dl), with transplant evaluation underway. we present here a retrospective case series evaluating the initial experience of 21 patients in the united states treated with acth gel for nephrotic syndrome. the majority of these patients received acth as second-, third-, or fourth - line therapy for resistant nephrotic syndrome. idiopathic membranous nephropathy was the leading diagnosis among these patients and also showed the greatest benefit of therapy, with 9 of 11 patients (82%) achieving either complete or partial remission. although 3 patients with other etiologies of nephrotic syndromedemonstrated response to therapy, the early clinical data suggest that the drug s greatest potential benefit may be in treating refractory imn. our observations are concordant with previously published reports from europe using a synthetic acth analog for nephrotic syndrome due to imn. berg and arnadottir, in a seminal paper from 2004, reported the results of acth treatment in 23 cases of nephrotic syndrome of various etiologies, including 10 cases of imn.9 half of these imn patients had previously been treated with at least 1 prior immunosuppressive therapy, and all 10 achieved sustained remission of proteinuria. a more recent series from germany reported the results of 8 months of synthetic acth therapy for 4 patients with imn refractory to prior therapies of steroids, cyclosporine, cyclophosphamide, mycophenolate mofetil, or azathioprine.11 within the first year, 2 had achieved complete remission, and 2 had achieved partial remission. the most convincing data, however, come from the randomized trial by ponticelli, in which 16 patients with imn received steroids alternating with a cytotoxic drug for 6 months versus 16 patients with imn treated with synthetic acth for 1 year.10 most of these patients were on renin angiotensin system blocking drugs, but none had previously received immunosuppression. after a median follow - up of 24 months, there were 4 complete and 8 partial remissions in the steroid / cytotoxic therapy group versus 8 complete and 6 partial remissions in the acth group. the results presented here, on the other hand, do not wholly agree with the successful experiences of berg and arnadottir in treating nephrotic syndromes other than imn.9 thirteen of the 23 cases presented in their series were nephrotic syndrome diagnoses other than imn. the remaining 12 patients with diagnoses of mpgn, mcd, fsgs, diabetic nephropathy, and hereditary nephritis all experienced at least a 50% reduction in proteinuria during synthetic acth therapy, and 8 patients achieved sustained complete remission (proteinuria < 500 mg / day). in this case series, us patients treated with acth gel for diagnoses other than imn did not fare as well : for example, only 1 of 4 mpgn patients demonstrated a limited response. in part, this may be due to these patients having worse baseline renal function and having failed more therapies than those reported by berg and arnadottir. notably, 11 of the 21 patients reported here had advanced renal insufficiency (gfr < 0.5 ml / s/1.73 m [30 ml / min/1.73 m ]) when prescribed acth. eight of these 11 patients were diagnosed with diseases other than mn, and their response rate to acth was generally poor. the lack of response exhibited by these patients may reflect the degree of renal failure more than the specific diagnosis itself. theoretically, the difference in response could also be explained by inherent differences between the synthetic and natural formulations of acth. although encouraging, the initial results of acth gel therapy in the united states must be interpreted cautiously in light of the limitations of an observational series such as this. these patients were not randomized to therapy, and there is no comparison or control group against which to interpret these results. in some respects, however, the patients may serve as their own controls, having failed on average between 2 and 3 prior immunosuppressive regimens. we did not have detailed data on duration of time between prior immunosuppressive therapies and initiation of acth gel ; conceivably, if this duration was short, some of the response could be attributed to a delay in response to prior agents. in addition, the data presented here reflect only short - term follow - up, most patients having less than 1 year of follow - up at the time of this report. given that 1 of the patients presented here has relapsed off therapy, the follow - up period is likely too short to label the remissions as sustained. in previous reports of synthetic acth therapy, some patients have relapsed but responded to second courses of acth with good results.9,11 the short duration of follow - up may also understate adverse events in this cohort, which thus far have been mild and mostly steroidlike in presentation. finally, our report does not address cost analysis of the various agents available to treat the nephrotic syndrome. these data also do not provide any further understanding of the mechanism of action by which acth ameliorates proteinuria in the nephrotic syndrome. speculatively, its better performance in imn than other causes of nephrotic syndrome might point to a target of action ; eg, antibodies against the phospholipase a2 receptor.12 however, a recent study using rats with passive heymann nephritis, an animal model of membranous nephropathy, proposes that acth may work at the melanocortin receptor mc1r in podocytes to reduce proteinuria, improve glomerular morphology, and reduce oxidative stress.13 this finding suggests that acth should be equally, if not more, effective in podocytopathies such as mcd and fsgs than in imn. as only 2 of our patients had mcd or fsgs, further study of acth gel in these conditions is clearly warranted. in conclusion, we present a case series on the initial experience in the united states of using acth gel as treatment of nephrotic syndrome. overall, 11 of 21 patients including 10 of 18 patients who could be classified as treatment resistant achieved either complete or partial remission with at least 6 months of follow - up. further, 9 of 11 patients with refractory nephrotic syndrome due to imn achieved remission despite having previously failed at least 2 prior immunosuppressive regimens. this retrospective data analysis suggests that further studies are warranted to evaluate acth gel in the treatment of nephrotic syndrome.	purpose : a synthetic adrenocorticotropin (acth) analog has shown efficacy in europe as primary and secondary therapy for nephrotic syndrome, but there is no published experience using the natural, highly purified acth gel formulation, available in the united states, for nephrotic syndrome. we therefore investigated the use of acth gel for nephrotic syndrome in the united states.patients and methods : twenty - one patients with nephrotic syndrome treated with acth gel outside of research settings in the united states, with initiation of therapy by december 31, 2009, allowing a minimum 6 months follow - up. we defined complete remission as stable renal function with proteinuria falling to 50% reduction in proteinuria from 500 to 3500 mg / day.results : twenty - one patients with nephrotic syndrome were treated : 11 with idiopathic membranous nephropathy (imn), 4 with membranoproliferative glomerulonephritis (mpgn), 1 with focal segmental glomerulosclerosis (fsgs), 1 with minimal change disease (mcd), 1 with immunoglobulin a (iga) nephropathy, 1 with class v systemic lupus erythematosus (sle) glomerulonephritis, 1 with monoclonal diffuse proliferative glomerulonephritis, and 1 with unbiopsied nephrotic syndrome. acth was used as primary therapy for 3 patients ; the remaining patients had previously failed a mean 2.3 immunosuppressive regimens. eleven patients achieved a complete or partial remission, with 4 (19%) in complete remission. of the 11 patients who achieved remission, 9 had imn, 1 had fsgs, and 1 had iga nephropathy. of the 11 patients with imn, 3 (27%) achieved complete remission and 6 (55%) achieved partial remission despite having previously failed a mean 2.4 therapies. five patients reported steroid - like adverse effects, but there were no severe infections. the limitations were retrospective data analysis with short - term follow-up.conclusion:acth gel may be a viable treatment option for resistant nephrotic syndrome due to membranous nephropathy. short - term data suggest that remission rates may approach 80%.
micrornas (mirnas) are small non - coding rnas (ncrnas) of approximately 22 nucleotides responsible for specific regulation of gene expression in a post - transcriptional manner, and, thereby, have an important role in several biological processes, such as development, cell proliferation, and apoptosis, among others [1 - 3 ]. their genes correspond to 1 - 3% of all genes of the genome, and may be responsible for the regulation of approximately 60% of the coding genes. the specificity of mirnas is given by the seed region (nucleotides 2 to 8) that requires full complementarity to the mrna - target [6 - 8 ]. the rnase iii members, drosha and dicer, together with their specific partners, dgcr-8 and trbp, are crucial for mirnas biogenesis, since they slice, respectively, primary and precursor mirnas to yield the mature ones [9 - 12 ]. in vertebrates, approximately 70% of the mirna genes forty percent of all mirnas are organized in clusters and transcribed in a polycistronic fashion. usually, these clustered mirnas are members of a family, showing overlapping functions [13, 14 ]. each cell type has a combination of either isolated or clustered expressing mirnas, which regulate coding genes in a tissue - specific manner and, therefore, they are essential to the maintenance of cell identity and functional phenotype. in this article, we review mirnas involved in pluripotency maintenance, cell fate decision, differentiation - state safeguarding, focusing on how this knowledge has been used in tissue engineering. embryonic stem cells (escs) are derived from pre - implantation blastocysts, and have the ability of self - renewal and the latent capacity to differentiate in cells of all three embryonic germ layers, therefore being pluripotent [16, reviewed in 17 ]. because of these remarkable characteristics, escs have been extensively used as model in developmental and therapeutic studies, including tissue regeneration, transplantation, and drug screenings [18 - 21 ]. to sustain the undifferentiated state, they have a unique gene expression profile [22 - 26 ] regulated by a highly expressed set of transcriptional factors, including oct4, nanog, and sox2 that, in this scenario, have overlapping functions [27 - 29 ]. in addition to oct4 and nanog, the most commonly used markers of escs are the cell surface antigens (ssea-3, ssea-4, tra-1 - 60, tra-1 - 81) and tissue - nonspecific alkaline phosphatase. mouse pluripotent stem cells can also be derived from the epiblast of peri - implantation blastocysts, and are called episc [31, 32 ]. these are epigenetically more similar to human escs (hescs), being dependent on bfgf for self maintenance. the human equivalent of mouse escs (mescs), which is dependent on lif, small molecule inhibitors of erk1/erk2 and gsk3b signaling, and express ssea-1, has only recently been derived from human blastocysts and appear to be in an earlier epigenetic developmental state therefore, they are called nave hescs, whereas the fgf - dependent hescs are called primed hescs [34, 35 ]. the early steps of embryonic development are marked by global dna demethylation, and this permissive epigenetic state at pre - implantational phase is pivotal for the expression of the above mentioned transcription factors. by the time of implantation, methylation of cpg dinucleotides is reestablished throughout the genome as result of the de novo activation of highly expressed dna methyltransferases dnmt3a and dnmt3b, which triggers the differentiation process [37, 38 ]. when most of the dna methylation marks have already been established, the expression of many genes belonging to the epigenetic machinery is decreased, concomitantly with the differentiation process [39, reviewed in 40 ]. more recently, the maintenance of pluripotency has also been associated to mirna expression. since the first discoveries of the regulatory effect of small ncrnas lin-4 and let-7 in caenorhabditis elegans, it is well established that mirnas are involved in development [1, 41 ]. indeed, disruption of dicer gene in mice compromises the entire mirna biogenesis and is lethal early in development. although mouse dicer - null escs are viable, they have a slow proliferation rate and fail to differentiate [43, 44 ]. mouse dgcr8-deficient escs also exhibit delayed proliferation rates with prolonged g0 and g1 phases, and when induced to differentiation, show aberrant expression of specific differentiation markers, such as a delayed expression of primitive ectoderm marker (fgf5), a weak or absent expression of endoderm (hnf4a and afp), mesoderm (brachyury, bmp4, gata) and ectoderm (sox1) markers, and incomplete repression of pluripotency [43, 46 ]. additionally, mouse dicer - null escs showed significant hypomethylation of the genome, including the promoter of oct4 gene (also known as pou5f1 gene) [47, 48 ], which impairs differentiation. three mirnas clusters hsa - mir-371 - 373 (ortholog of the mouse cluster mmu - mir-290 - 295), hsa / mmu - mir-302 - 367 and hsa / mmu - mir-17 - 92 are highly expressed in escs [49 - 54 ], therefore known as esc - enriched mirnas. they share similar seed sequences, suggesting an overlapping regulation of their targets [45, 55 ]. the hsa - mir-371 - 373 cluster transcribes four mirnas, whereas its murine counterpart, mmu - mir-290 - 295 transcribes seven, and accounts for up to 70% of the total mirnas expressed in mescs. at the stage of four to eight cells, mouse embryos already show mir-290 - 295 expression, which decreases after embryonic day 6.5, and during in vitro differentiation of escs to embryoid bodies. these mirnas are involved in (a) the regulation of transition from g1 to s phase of the cell cycle through targeting of cell - cycle inhibitors ; (b) repression of mesoderm and primordial germ cell differentiation pathways ; (c) repression of epithelial - mesenchymal transition. transfection of mirnas belonging to the mir-290 - 295 cluster restores many of the defects exhibited by mouse dicer - null escs. this phenomenon was seen by two independent studies showing that mir-290 - 295 members inhibit the expression of the retinoblastoma - like 2 protein (rbl2), which is a transcriptional repressor of dna methyltransferases dnmt3a and dnmt3b [47, 48 ]. thus, the authors of these two studies [47, 48 ] proposed that this is the major mechanism by which cells regulate dna de novo methylation during early development. accordingly, introduction of mir-290, mir-302 and mir-17 - 92 mimics was able to re - establish the proliferation rates of dgcr8-deficient escs. however, mir-290 - 295 function is still controversial, since recently it was shown that, although rbl2 mrna levels are increased in mouse dicer - null escs, its protein remains at low levels. the other highly expressed mirna cluster in escs, mir-302 - 367, transcribes eight and five mirnas, in human and mice, respectively [49, 50 ]. this cluster confers stemness proprieties to hescs by controlling lefty1 and lefty2 expression, two inhibitors of tgf/ nodal pathway that have an essential role in signaling early cell fate determination. this cluster is also important to control cell cycle, since its inhibition leads to arrest of hescs in g1 phase by targeting cyclin d1. finally, mir-17 - 92 cluster comprises six mirnas in human and mice, is overexpressed in mesc and hesc [50, 51 ], and has an important role in cell cycle regulation [63, 64 ]. despite being associated to maintenance of esc pluripotency, this cluster is also widely expressed in many cell types [65 - 67 ]. in humans, its overexpression may lead to several malignancies, since it is located at the genome region 13q31-q32 that is frequently found amplified in lymphomas and other cancer types [68, 69 ]. interestingly, expression profiles for mescs and episcs reveal that they have differences in the expression of several mirnas, including these important esc - enriched mirna clusters, mir-17 - 92, mir-290 - 295 and mir-302 - 267. the former two are more highly expressed in mesc, whereas the latter is in episc. additionally, although barely expressed, members of let-7 mirna family, which are differentiation markers, are enriched in episc in comparison to mesc, and that may reflect a degree of commitment to differentiation. thus, mirnas may have redundant and specific roles in regulation of pluripotency. in humans, hsa - mir-302b expression is indicative of pluripotency in nave and primed hesc, while expression of hsa - mir-371 - 373, the human ortholog of mmu - mir-290 - 295, is increased in nave when compared to primed hescs [33, 55 ]. nevertheless, oct4, sox2 and nanog regulate mir-290 - 295 and mir-302 - 367 gene clusters in mice and humans, reinforcing mirnas role in pluripotency control and in the early steps of differentiation [56, 62, 70 ]. adult stem cells, such as mesenchymal, bone marrow and hematopoietic stem cells are also capable of self - renewal and have the plasticity to differentiate into one or multiple cell types, functioning as a quiescent reservatory for tissue maintenance and repair throughout the life span [reviewed in 71 ]. as its embryonic counterparts, they have mirnas that may participate in the maintenance of cell identity [72, 73 ], such as mir-489 that is highly expressed in mouse muscle stem cells (satellite cells), but is downregulated during cell activation. since the reversion of differentiated fibroblasts into pluripontent cells by the introduction of the defined reprogramming factors oct4, sox2, klf4, and c - myc (oskm) in 2006, induced pluripotent stem cells (ipscs) have been the center of many studies in cell therapy [75, 76 ]. ipscs raise great interest in regenerative medicine because of their potential to overcome the issue of histocompatibility between cells and patient. different methods have been used to deliver the reprogramming factors, including (a) integrative retrovirus vectors ; (b) non - integrative vectors, such as adenovirus, sendai virus, and plasmids ; (c) dna - free transfections, such as mrnas and fusion proteins ; and (d) excision after integration, such as piggybac transposon and retrovirus with loxp construction followed by cre recombinase mrna transfection [reviewed in 77 ]. accordingly, new approaches to promote a better understanding of the mechanisms involved in maintenance of pluripotency, to achieve higher reprogramming efficiency, and to guarantee the safe use of ipscs to therapy are of great importance. in this context, the introduction of esc - enriched mirnas has been used to improve cell reprogramming. transient transfection of mirnas mir-291 - 3p, mir-294, and mir-295 enhances the efficiency of the reprogramming of mouse embryonic fibroblasts achieved by retroviral deliver of oct4, sox2 and klf4. among them, mir-294 showed the best results, increasing tenfold the efficiency rates. similarly, the expression of mirnas from the mir-302 - 367 cluster also enhances retroviral reprogramming of human fibroblasts with oct4, sox2 and klf4, either with or without c - myc. conversely, inhibition of let-7 family members, which are known to be robust maintainers of the differentiated state in mouse embryonic fibroblasts, enhanced in over fourfold the efficiency of reprogramming carried out by oct4, sox2 and klf4. other examples of mirnas expression manipulation to improve the efficiency of reprogramming are the transfection of mir-93 and mir-106b, the knockout of mir-34 and mir-199a-3p, among others [84 - 86 ]. strikingly, the reprogramming of human skin cancer cells and human fibroblasts into a pluripotent state by the introduction of mir-302 - 367 cluster per se was reported. however, few studies have used this methodology recently, and reprogramming of somatic cells through transfection of members of the cluster mir-302 - 367 alone or combined with mir-200c or mir-369 yield no clones or resulted in a low efficient rate. nevertheless, the use of mirnas as oskm adjuvant to produce ipsc might be a good strategy to improve the efficiency of somatic cells reprogramming. as any other cell - therapy approach, mirna use must be in consonance with the applications intended for ipscs, since some differentiated cells retain more plasticity than others, and some cell types present a more robust epigenetic memory after reprogramming [reviewed in 91 ]. cell differentiation is a complex pathway that depends on both activation of lineage - specific genes and repression of pluripotency - related ones. however, a coordinated modulation of oct4, sox2 and nanog expression in early steps of differentiation process contributes to specific germ layer induction of mescs, despite combined expression of oct4 and sox2 suppresses germ layer differentiation. therefore, high oct4 or sox2 levels promote mesendodermal or neural ectoderm differentiation, respectively, while nanog downregulation is decisive for lineage commitment. similar phenomenon was seen in a study using hescs, whereas each factor is per se involved in a specific cell fate. likewise, mir-302 - 367 cluster that, as seen before, has an important role in the maintenance of pluripotent cells, is also expressed in the human endodermal lineage, providing evidence that this cluster has a role in organogenesis. indeed, hsa - mir-302 - 367 cluster promoter is targeted by gata6 transcription factor in early stages of lung epithelial development, promoting the proliferation of lung endoderm progenitor cells, proper apical - basal polarity and preventing its complete differentiation. therefore, this cluster seems to be essential for the correct development of a single - layered lung epithelium. additionally, mir-17 - 92 that is also enriched in hesc, has an important role in the early stages of lung morphogenesis, regulating the proliferation - differentiation balance of lung epithelial progenitor cells. as the cells commit to differentiation, oct4, sox2 and nanog are downregulated, and consequently the clusters regulated by them, mir-290 - 295 and mir-302 - 367 in mice and human, respectively, are also silenced [56, 62 ]. prior to being silenced, oct4, sox2 and nanog also upregulate the expression of some mirnas specifically associated with differentiation in mescs, such as mir-9, mir124a, mir-155 and mir-708, which at least are in part responsible for proper cell fate determination. indeed, mirnas are essential for escs specific - differentiation and maintenance of the differentiated status. accordingly, mirnas expression is frequently globally downregulated in tumors, which are less differentiated cells [15, 96 ]. similarly, adult stem cells also have mirnas involved in the commitment of their differentiation. for instance, expression of mir-590 and mir-199a in adult cardiomyocyte promotes re - entrance in cell cycle, resulting in cardiac repair in an ex - vivo mouse model. one of the first mirnas recognized by its role in differentiated tissues was let-7. with its orthologs organized in large families along the vertebrate genomes, let-7 is up - regulated in differentiating and differentiated mouse cells [98 - 100 ]. the processing from pri - let-7 to let-7 mature duplex is inhibited by the rna binding protein lin28, which prevents differentiation and stabilizes mesc status. since then, many other mirnas have been reported as having an important role in early steps of differentiation and maintenance of the differentiated status.. indeed, nanog and sox2 are direct targets of mir-21, and this mirna may have an important role promoting adipocyte differentiation as well as in bone formation, since it is overexpressed during the initial steps of osteogenic differentiation. mir-22, by its turn, has been reported as a maintainer of progenitor cells in murine mammary epithelium, promoter of osteogenic differentiation and inhibitor of adypogenic differentiation. moreover, a set of mirnas was found to be up - regulated (mir-297, mir-96, mir-214, mir-125a, mir-424, mir-21, mir-29c, mir-7) or down - regulated (mir-376a) in mouse blastocysts when compared to the morula stage, indicating that they are involved in trophectoderm determination. furthermore, the different mirna profiles characterizing the three germ layers in gastrulating embryo implicate the involvement of mirnas in the differentiation of mesoderm, endoderm, and ectoderm [107 - 109 ]. in the supplementary table s1 we show a comprehensive list of mirnas involved in tissue regulation, organogenesis and development. once differentiation is established, each cell type will express its own set of mirnas. accordingly, tissues with the same ontogenetic origin have similar expression profiles, which are different from those of tissues originating from different embryonic layers. since mirnas have been widely implicated in the control of stem cells fate, a better understanding of the relationships among mirnas, transcription factors, signaling pathways, chromatin remodeling factors, and extracellular clues have a pivotal importance in developing new strategies to tissue engineering. tissue engineering (te) is an interdisciplinary field that combines cells, engineered materials, and biomedical technology towards the development of bio - artificial tissue - like structures to restore, replace, maintain or improve the function of tissues or organs. the obstacles in te are to attain specific cell types, to develop appropriated scaffolds, and to promote the release of growth factors and other molecules from scaffolds in order to resemble organogenesis [115, 116 ]. the success of implant engineered tissues is also challenged by the difficult formation of blood vessel network, tissue innervation, and by inflammatory and immunological responses [reviewed in 117 ], making the transition from research stage to clinical trials limited to avascular or thin tissues, such as cartilage and skin [118, 119 ]. given the role of mirnas in many biological processes, including cell differentiation and maintenance of cell identity, modulation of these small molecules in combination with stem cells and/or bioartificial scaffolds has been providing encouraging results. indeed, strategies for vascularization of bioartificial tissues, which are insofar mainly based on the delivery of angiogenic growth factors [120 - 122 ], have recently advanced with the use of mir-132 that indirectly induces ras overexpression, enhancing neovascularization rate. when this mirna is encapsulated in biodegradable polymer nanoparticles, it improves vessel formation in human endothelial cells transplanted in immunodefficient mice. this approach allows the release of these small rnas for weeks, longer than conventional lipid - based transfection. similarly, localized and sustained expression of mir-26a in vivo positively regulates osteogenesis - angiogenesis coupling, therefore, providing an enhanced efficiency in bone regeneration. zhang and colleagues have also reported that the inhibition of mir-29 in bioengineered vessels may increase the expression of its target gene, eln, which has a major function in maintaining the integrity of the extracellular matrix of arteries. other studies also showed that te could be potentially improved by the transfection of cells with mir-21, since it promoted high proliferation rates and high matrix synthesis of rat chondrocytes cultured in atelocollagen gel, and by implanting scaffold embedded with mir-29b in cutaneous injury, which was able to improve extracellular matrix remodeling of treated excisional wounds. examples of this are (a) the inhibition of mir-133, which enhances skeletal murine myoblast differentiation and the response to electrical stimulation in three - dimensional (3d) bioartificial muscle ; (b) the transfection of mir-206 in satellite cells, which increases their differentiation in a bioartificial muscle construct ; (c) the introduction of mir-148b mimic and mir-489 inhibitor, which improves osteogenesis from human mesenchymal stem cells and the expression of osteogenic markers, also in a 3d scaffold, (d) the usage of nano - bioglass ceramic particles (nbgc) that stimulates mir-30 expression in osteoblastic cells, inducing their differentiation, (e) the transfection of mir-31 inhibitor in bone marrow stromal cells, which increases osteogenic differentiation, bone mineral density, biocompatibility and regeneration rate. finally, mirnas seem to have an important role in the development of engineered tissues with a refined architecture. for instance, articular cartilage is subdivided in specific zones that seem to be frequently lost in monolayer expanding cultures of chondrocytes. superficial zone - specific mirnas expression is also lost in this process, and the tgf--directed differentiation of chondrocytes in a 3d agarose culture was able to reestablish their expression. therefore, manipulation of mirna expression might be useful to the correct assembly of a complex engineered tissue. a complete list containing all mirnas used so far in te is reported in table 1. thus, we expect that mirnas will become an increasingly important tool for controlling cell fate for te, and the prominent candidates to this purpose are listed in supplementary table s1. finally, mirnas might potentially be used to monitor the graft status, since in a mouse model of heart transplantation, allograft rejection seems to be associated with specific mirna signatures. moreover, mir-182 was found overexpressed in peripheral blood mononuclear cells and plasma in mice with allograft rejection. micrornas have an essential role in maintenance of cell pluripotent and differentiated states, as well as in cell fate decisions, working as modulators of cell identity. accordingly, these small regulators might (a) assist the reprogramming of ipsc, an important source of cells for te ; (b) direct and maintain tissue - specific differentiation ; (c) guarantee proper vascularization of engineered tissue. a better understanding of mirnas involvement in tissue formation, regeneration and function will provide more efficient engineered tissues. thus, on the whole, despite few studies have been performed so far, the results are very promising and warrant remarkable advances in the next future.	micrornas post - transcriptionally regulate the expression of approximately 60% of the mammalian genes, and have an important role in maintaining the differentiated state of somatic cells through the expression of unique tissue - specific microrna sets. likewise, the stemness of pluripotent cells is also sustained by embryonic stem cell - enriched micrornas, which regulate genes involved in cell cycle, cell signaling and epigenetics, among others. thus, micrornas work as modulator molecules that ensure the appropriate expression profile of each cell type. manipulation of microrna expression might determine the cell fate. indeed, microrna - mediated reprogramming can change the differentiated status of somatic cells towards stemness or, conversely, micrornas can also transform stem- into differentiated - cells both in vitro and in vivo. in this review, we outline what is currently known in this field, focusing on the applications of microrna in tissue engineering.
in egypt, childhood acute lymphoblastic leukemia (all) is the most common cancer in children, accounting for almost one - third of newly diagnosed pediatric cancer cases. the annual incidence of pediatric all is approximately four cases per 100,000 children per year in the national cancer institute nci, cairo university, egypt. the 210 years age group constitutes 68.5%.1,2 fortunately, improvements in treatment, including multimodal therapy and hospital care, have improved survival such that over 80% of children diagnosed with all survive at least five years.2,3 however, childhood cancer survivors are at increased risk of developing chronic health conditions, some of which manifest during or soon after treatment whereas others emerge years after therapy.4 obesity is a particularly significant problem among all survivors, which can intensify cardiovascular outcomes and place these individuals at greater risk for other chronic health conditions.5 evidence from the childhood cancer survivor study (ccss) suggests that survivors of all (who lived 5ys after treatment) experience a higher rate of obesity than their same - sex siblings, especially for female survivors (all : 31.7% vs. siblings : 22.2%).6 obesity may further compound the risk of other late effects, such as increased rate of cardiovascular diseases observed in childhood cancer survivors.7 previous studies have attributed obesity to the cranial irradiation (crt) patients received to prevent central nervous system (cns) relapse, however, since the 1990s, cns prophylaxis with crt protocols has gradually been replaced with intrathecal and systemic chemotherapy by several consortia.8 a study of the children s oncology group (cog) found excessive weight gain also occurred in children receiving chemotherapy alone.9 treatment with glucocorticoids has been implicated in the physiology of adiposity, and there is data that dexamethasone may act more potently than prednisone.10 prolonged use of corticosteroids has shown effects on body composition associated with increases in the percentage of body fat in pediatric all survivors.11 also, hepatic abnormalities are well documented in survivors of childhood malignancies. a spectrum of liver diseases has been described including hepatitis b and c, iron overload, hepatic fibrosis, cirrhosis and hepatocellular carcinoma.12 less commonly reported hepatobiliary complications include cholelithiasis, focal nodular hyperplasia (fnh), nodular regenerative hyperplasia, hepatic microvesicular fatty change and siderosis.13 the childhood cancer survivor study (ccss) noted an almost two - fold excess risk of gallbladder disease among childhood cancer survivors compared to sibs (1.9 95 % 1.72.2).13 acute or sub - acute hepatobiliary injury is recognized with varying incidence following radiation, multiple chemotherapies, or hematopoietic stem cell transplantation (hsct).14 additionally, hepatobiliary toxicity is associated with supportive care measures, such as transfusion - acquired hepatitis, transfusion - associated iron overload or cholestatic disease from total parenteral nutrition (tpn). these conditions may predispose to clinically significant liver disease in aging childhood cancer survivors.15 in this study, we aimed to estimate the prevalence of overweight, obesity, and hepatic late adverse effects in pediatric all survivors who lived 5 years after treatment. a comparative cross - sectional case - control study was performed on thirty - five all survivors, treated at hematology and oncology unit, pediatric department, menoufia university hospital. all of them completed treatment with st jude total xv chemotherapy protocol 5 years before the time of examination. in that protocol, on hematopoietic recovery, consolidation therapy with high - dose methotrexate, mercaptopurine, triple intrathecal treatment began, and the dose of methotrexate was based on risk classification. during initial continuation therapy, patients with low - risk disease received daily mercaptopurine and weekly methotrexate with pulses of mercaptopurine, dexamethasone, and vincristine. two re - induction treatments were given between weeks 7 to 9 and weeks 17 to 19. patients with standard - risk disease received weekly asparaginase and daily mercaptopurine with pulses of doxorubicin plus vincristine plus dexamethasone. they also received two re - induction treatments between weeks 7 to 9 and weeks 17 to 20. for the remaining part of continuation therapy, patients with low - risk disease received mercaptopurine and methotrexate, with pulses of dexamethasone, vincristine, and mercaptopurine, and patients with standard - risk disease received three rotating drug pairs continuation treatment lasted 120 weeks for girls and 146 weeks for boys.16 none of those survivors received radiotherapy. a control group of 35 healthy children with matched age and sex was selected from volunteers from a local school. they were apparently healthy with no history of chronic illnesses or previous history of steroid intake. a written informed consent was obtained from the parents of all children, and oral assent was obtained from children of both groups. this study was approved by the ethical committee of the faculty of medicine, menoufia university. the survivors and controls were subjected to anthropometric measurements and laboratory investigations : anthropometric measurements (weight, height, and body mass index), bmi was assessed by z - score. body mass index = weight in kilograms/(height in meters)2 was plotted on age and gender - specific percentile charts (for 2 to 20-year - olds). bmi over the 95 percentile indicates obesity, between 85 and 95 indicates risk of overweight.17 we evaluated longitudinal changes in obesity rate and bmi z scores in survivors of pediatric all as for survivors of childhood cancer aged 16% values were regarded as correct ones.25 serum ferritin levels were measured to assess the iron status of our patients by enzyme linked immune sorbent assay (elisa) technique using (ramco laboratories kit, inc., usa), and hcv antibodies were detected by elisa using (autobio diagnostics, china) kit on microplate reader (bio - rad 680 hercules, california, usa). the statistical presentation and analysis of the present study were conducted using spss v.20 (spss inc., data were expressed in two phases : continuous parametric variables were presented as means sd while for categorical variables numbers (%) were used. chi - square (test) and fisher s exact test were used for qualitative variables, student s t - test for parametric continuous variables and man whitney (u) test for non - parametric variables the mean age of the survivors at the time of the study was (11.014.6) years. fourteen of them (40%) are females, and 21 (60%) are males. sixty % were leukemia of low risk, and 40 % were of standard risk the mean age of them at diagnosis was (5.861.5) years. the mean age of controls was (9.63.3). the weight and bmi were significantly higher in the survivors controls (p value = 0.002 and 0.039 respectively) while no significant difference was found between the two groups regarding the height (p - value = 0.351) (table 1). (p - value = 0.003) and highly significant correlation with weight at diagnosis and after chemotherapy (p value = < 0.001). also, a highly significant value was detected between obese survivors and positive family history of obesity (p value = < alt, total & direct bilirubin, serum ferritin and transferrin saturation were significantly higher in the survivors than controls (p value = 0.03, 0.036, 0.044, 0.006 and 0.03 respectively) (table 3). while no significant difference was found between the two groups regarding ast, albumin, creatinine, bun, serum iron and tibc (table 3). ten (28.6%) of survivors had hepatitis c antibodies with none (0%) of controls (p value = 0.02) (table 4). a correlation was calculated between cumulative doses of asparaginase and alt, ast, total bilirubin, direct bilirubin. a positive highly significant correlation was found between cumulative dose of asparaginase and liver enzymes (alt, ast) (p value = < 0.001) and with total bilirubin (p - value = 0.003) but not with direct bilirubin (p - value = 0.052) (table 5). no significant correlation was found between liver function (transaminases and hyperbilirubinemia) and ferritin levels in survivors (table 6). the first aim of this study was to assess the prevalence of overweight and obesity in pediatric all survivors. our results revealed that survivors of pediatric all were at risk becoming overweight or obese with long - term follow - up. centers for disease control and prevention (cdc) definition growth charts, salazar - martinez.26 said that the prevalence of overweight and obesity was 12.1% and 6.2%, respectively, among the healthy egyptian adolescents. this study revealed that overweight and obesity were more prevalent in all survivors compared to general egyptian population, suggesting an impact of chemotherapy on weight gain in all survivors. this datum is in agreement with asner.27 who examined the prevalence and the risk factors for overweight and obesity in a cohort of all survivors treated and living in the french speaking part of switzerland reported that there is a significant prevalence of obesity in young all survivors. fang.28 indicated a significantly higher bmi in pediatric all survivors than the reference population. however, a study by murphy.29 found that on - treatment and survivor groups had a significantly lower body cell mass index than matched controls, and 53% of the survivors were considered undernourished. in a study done on 56 adolescent, all survivors in saudi arabia, with a mean age of 13.4 years an average of 9.1 years post - diagnosis who did not receive crt, the prevalence of bmi for age defined overweight, and obesity (combined 28.5%) were lower than in the general population in saudi arabia. the authors supposed that overweight and obesity observed were probably not an all specific problem.30 our results demonstrated that the survivors who had high bmi z - score at diagnosis also had increased risk of being overweight /obese after treatment completion. study which is a retrospective cohort of 83 pediatric patients with all ; they examined bmi status at several key time points : diagnosis ; end of induction ; end of consolidation ; every 6 months during maintenance ; and yearly for up to 5 years post - treatment. at diagnosis, 21% were overweight (bmi = 8594.9th percentile) or obese (bmi 95th percentile). at the end of treatment and weight gain during treatment was associated with being overweight / obese 5 years post - treatment (or = 3.8, 95% ci : 1.112.5).31 all of the involved survivors had received dexamethasone with the mean cumulative dose of 927 135 mg / m which may be the cause of weight gain. one theory is that, during glucocorticoid treatment, all patients have an increased energy intake32 and reduced energy expenditure on habitual physical activity33 and that this effect continues after treatment ceases. other theories are that glucocorticoid treatment causes increased adiposity by suppressing growth hormone secretion or that it causes resistance to leptin.34 the second aim of this study was to assess the hepatic late adverse effects in pediatric all survivors. at our study, there was a significant increase in d. bilirubin, t. bilirubin, alt, serum ferritin and soluble transferrin saturation in the survivors group more than the control group. ten of our survivors (28.6 %) have hcv positive antibodies detected by elisa. these results go with the previous findings of mulder.35 who concluded that abnormal high alt level was detected in survivors of childhood cancer. also, schempp.36 found elevated levels of serum ferritin and soluble transferrin (iron overload) in survivors of childhood cancer and attributed this to transfusion volume. this iron overload causes tissue damage through the chronic formation of free radicals leading to liver dysfunction.37 in a study of 118 children (with standard - risk leukemia) receiving native e. coli asparaginase or peg - asparaginase, abnormal liver function (grade 3/4), including elevated transaminases and hyperbilirubinemia, was found in 8% of patients receiving native e. coli asparaginase and in 5% of patients receiving peg - asparaginase.38 there are no clear pediatric guidelines for the management of asparaginase in patients with hepatic toxicity, and treatment recommendations vary across protocols. in the dcog all-11 pediatric protocol, patients are required to display aspartate aminotransferase / alanine aminotransferase < 10uln and no signs of jaundice with bilirubin < 3 uln before starting asparaginase treatment.39 patients with hematologic malignancies were at a very high risk of hcv infection due to the large transfusional support they often needed.40 the previously immunocompromised status of the leukemia survivors may have promoted more rapid viral replication or impaired host viral clearance and led to rapidly progressive liver disease.41 moreover, it is known that chemotherapeutic drugs (methotrexate and 6-mercaptopurine) increase the risk of liver toxicity during or soon after cancer treatment.13 pediatric all survivors are at increased risk of overweight / obesity, iron overload, hcv infection and delayed hepatic complications.	backgroundchildhood acute lymphoblastic leukemia (all) with current cure rates reaching 80% emphasizes the necessity to determine treatment - related long - term effects. the aim of this study is to estimate the prevalence of overweight, obesity, and hepatic late adverse effects in a cohort of all survivors treated at the hematology and oncology unit, pediatrics department, menoufia university, egypt.methodsin this case - control study, height, weight, and body mass index (bmi) were assessed for 35 pediatric all survivors and 35 healthy children. these parameters were plotted on the growth and who standard deviation charts for both males and females. overweight and obesity were defined by bmi > 85th and 95th percentile respectively. laboratory investigations were done in the form of iron profile, liver enzymes, total and direct bilirubin levels, serum urea & creatinine and detection of hepatitis c virus antibodies by elisa.resultsthe weight and bmi were significantly greater in the survivors than controls (p value = 0.002 and 0.039 respectively). alt, total & direct bilirubin, serum ferritin and transferrin saturation were considerably higher in the survivors than the controls (p value = 0.03, 0.036, 0.044, 0.006 and 0.03 respectively). ten (28.6%) of survivors had hepatitis c antibodies with none (0%) of controls (p value = 0.02)conclusionspediatric all survivors are at increased risk of overweight / obesity, hepatic dysfunction in the form of elevated liver enzymes, bilirubin levels, and c viral hepatitis. screening of those survivors for such complications should be considered.
we report the case of a previously healthy woman who presented with signs and symptoms of sepsis and disseminated intravascular coagulation (dic) secondary to ia. post mortem findings confirmed non - hodgkin 's lymphoma as the cause of her immunosuppression. a 62 year old woman presented to the accident & emergency department (day 0) with acute abdominal pain, diarrhoea and confusion., she was found to be profoundly hypotensive (blood pressure 60/25 mm hg), tachycardic (heart rate 135 per minute) and tachypnoeic (respiratory rate 33 per minute). her abdomen was tender particularly in the right upper and lower quadrants. she was resuscitated aggressively with intravenous fluids and inotropic support, started on intravenous (iv) antibiotics (amoxicillin clavulanic acid, gentamicin, and metronidazole) and transferred to the intensive care unit. initial blood results showed high bilirubin (30 mol / l), low albumin (27 g / l) and modestly elevated c - reactive protein (35 mg / l). the white cell count was 8.810/l and the neutrophil count was 5.210/l with toxic granulations, reactive lymphocytes, and occasional blast forms on blood film. the clotting screen was grossly abnormal (platelets 1710/l, prothrombin time 16 s, fibrinogen 67 mg / dl) consistent with dic. she was administered four units of fresh frozen plasma, four cryoprecipitates and two pools of platelets. abdominal computed tomography (ct) scan showed thickened right colon suggestive of pseudomembranous colitis, for which she was given oral vancomycin empirically. chest x - ray showed extensive alveolar shadowing throughout the whole of the visualised right lung and the left upper zone in keeping with active infection. aspergillus fumigatus was isolated from her sputum and endotracheal aspirate on day 1 and although the significance of this finding was uncertain, she was commenced on iv voriconazole for a presumed active infection. despite aggressive therapy, the most significant findings at post - mortem examination were pleural effusion, pulmonary oedema, and thrombosis of small pulmonary vein branches without infarction. the gastric mucosa was thickened and haemorrhagic with multiple ulcers, the largest being 20 mm in diameter with features of recent perforation. the liver was slightly enlarged (1821 g) and showed patchy pallor and congestion. histologically, small foci of intravascular thrombus were confirmed in lung and stomach, some of which were associated with septate fungal hyphae at both sites. intra - bronchial fungal elements were also identified and were associated with underlying necrosis and destruction of the bronchial wall with invasion of hyphae into the adjacent lung parenchyma (fig. there was a neoplastic lymphoid infiltrate of medium and large sized cells with marked nuclear pleomorphism in the lymph nodes, the liver mainly in portal areas and widely in the stomach including the site of perforation. there was probable involvement of spleen but this showed marked post - mortem autolysis. on immunohistochemical staining, the neoplastic cells were positive for common leucocyte antigen, cd3, cd43, cd7, and focally for cd57, cd30 and granzyme (fig. the cells were negative for cd4, cd8, cd20, cd79a, alk-1 and epstein barr virus. peripheral t cell non hodgkin 's lymphoma, not otherwise specified. a. fumigatus dna was detected in the tissue samples by polymerase chain reaction. ia is common in immunocompromised patients, particularly in those who suffer from prolonged neutropenia. aspergillus is ubiquitous in the environment where it sporulates releasing conidia. due to their small size (2.53 m) the conidia remain airborne and when inhaled they reach the alveoli where they germinate and transform into hyphae. disseminated disease results from direct invasion of the intra - thoracic structures (heart and great vessels) or by haematological spread to distant organs. the most common site of metastatic disease is the central nervous system, but invasion of many solid organs has been reported including the thyroid, liver, kidneys and heart. however ia as a presenting feature of lymphoma is very rare with only two cases published cases in literature. garcia - gonzalez and colleagues reported a case of diffuse small and large cell multi - centric lymphoma that was diagnosed at autopsy in a previously healthy man who presented with a sudden onset of a respiratory illness that progressed to respiratory failure leading to death. histopathology sections of both lungs revealed septate hyphae with branching at 45 angles with vascular invasion consistent with ia. definitive diagnosis of ia requires one of the following : microscopic documentation of infection namely presence of hyphae in histopathologic, cytopathologic or direct examination of a specimen obtained by needle aspirate or biopsy accompanied by evidence of tissue damage or a positive culture from a normally sterile material obtained by a sterile procedure from a clinically or radiologically abnormal site consistent with an infectious disease process (but excluding specimens such as bronchoalevolar lavage fluid, urine, and cranial sinus cavity specimens), or a positive blood culture consistent with infection. the diagnostic finding of a. fumigatus in sputum and tracheal aspirate soon after admission is unusual in our experience and although it did not satisfy the criteria for definitive illness at the time of admission, it prompted us to start treatment with voriconazole. timely initiation of treatment for ia is a crucial determinant of survival and treatment should not be delayed while awaiting definitive diagnosis. in scenarios where ia is suspected, the need for thorough investigation including high - resolution ct scan and serum galactomannan testing can not be overemphasised. the post - mortem evidence of hyphal invasion with necrosis in our patient established the diagnosis beyond doubt. isolation of aspergillus from acutely ill patients presenting with community - acquired infection should trigger a search for an underlying cause including lymphoma. this patient died on day 2 following admission in the intensive care unit so it was not possible to obtain consent.	invasive aspergillosis (ia) is a life - threatening infection. ia is usually seen in severely immunocompromised patients. however, ia as a presenting feature of non - hodgkin 's lymphoma is rare. the patient we describe had no signs or symptoms of lymphoma prior to hospital admission. a. fumigatus was isolated from respiratory tract specimens on the day of admission and fungal elements were detected on autopsy. isolation of aspergillus in patients with severe sepsis should trigger a search haematological malignancy.
a blowout fracture is characterized by outward fracture of the orbital wall with ocular symptoms such as diplopia, eyeball movement restriction and enophthalmos of the invaded orbit., is described as a trapdoor orbital floor fracture, linear and hinged in form, which allows herniation of the orbital contents through the fracture. the incidence of trapdoor - type medial blowout fracture is extremely low compared with that of floor one, and is easily missed due to the lack of other prominent soft tissue injury signs. here we describe a case of trapdoor - type medial blowout fracture with horizontal eye ball movement limitation that was treated via an endoscopic endonasal approach. a 38-year - old woman presented diplopia with left lateral gaze after sustaining trauma to the left orbit. ophthalmologic examination results were within normal except of ocular motility revealed an abduction deficit in the left eye (fig. computed tomography (ct) demonstrated a trapdoor - type left medial orbital wall fracture with entrapment of the medial rectus muscle (fig. endoscopic endonasal reduction surgery for medial blowout fracture, first introduced by yamaguchi was used to expose the fractured medial orbital wall. the entrapped medial rectus muscle and herniated orbital tissue were gently released from the fracture after ethmoidectomy. the herniated orbital tissue and fractured bone were reduced to the original position, and then supported with a suitably sized silastic sheet placed in the ethmoid sinus in an inverse u shape. a piece of merocel was packed between the silastic sheets to provide support and prevent orbital tissue herniation, which were removed 4 weeks later at out - patients clinic (fig. computed tomography scans demonstrated that the medial rectus muscle was reduced after surgery (fig. 2b). figure 1:(a) left ocular movements showed prominent limitation of abduction preoperatively. figure 2:(a) ct showed a trapdoor fracture of the left medial orbital wall with the medial rectus muscle entrapped (arrow) within the fracture. (b) the entrapped medial rectus muscle and fractured medial wall were resolved on post - operative ct. figure 3:(a) a suitably sized silastic sheet implant (black arrow) was inserted in an inverse u shape after assessment of ethmoid volume. (b) a piece of merocel (white arrow) was packed between the silastic sheets. (a) ct showed a trapdoor fracture of the left medial orbital wall with the medial rectus muscle entrapped (arrow) within the fracture. (b) the entrapped medial rectus muscle and fractured medial wall were resolved on post - operative ct. (a) a suitably sized silastic sheet implant (black arrow) was inserted in an inverse u shape after assessment of ethmoid volume. (b) a piece of merocel (white arrow) was packed between the silastic sheets. a blowout fracture is a common injury of the orbit and typically involves the thin bones of the medial wall and/or floor. although the medial orbital wall is thinner than the floor, the floor is more susceptible to fracture because the floor has no central support. conversely, the medial wall has many bony septa within the ethmoid sinus that support the wall and make deformation less likely [4, 5 ]. some reported that most blowout fractures involved the orbital floor with only 412% involving the medial wall [1, 4 ]. indeed, there is no real medial rim to transmit energy to medial wall, thus medial wall fractures are most likely purely hydraulic in nature, which may be an additional reason of low incidence trapdoor - type medial blowout fracture. jordan. first described the white - eyed blowout as an orbital floor fracture which is more commonly seen in children than adolescents. the bone in children is thought to be relatively thicker and more elastic, and when pressure increases within the orbit from a blow, the bones may crack and form a hinged trapdoor that is transiently displaced to the sinus. on release of orbital pressure, the trapdoor snaps back in position, entrapping the orbital tissue in the sinus. however, the medial wall has many supporting bony septa and it is uncommon to sustain a trapdoor - type fracture. thus, to date, the majority of reports have been concerning trapdoor - type fractures of the orbital floor, whereas medial wall trapdoor - type fractures have been discussed relatively infrequently. additionally, an adult case of trapdoor - type medial blowout fracture the clinical presentation of medial blowout fractures includes horizontal diplopia, restricted abduction and limited adduction due to entrapment of the medial rectus muscle. treatment guidelines for medial wall fractures are variable ; classic indications for surgical intervention include severe oculocardiac reflex, entrapment of extra - ocular muscle, early enophthalmos and persistent dipolpia. many authors have demonstrated better clinical outcomes in trapdoor fractures of the floor when surgery is performed within days of the injury [7, 8 ]. yamaguchi. reported the first application of endoscopic intranasal reconstruction of the medial orbital wall. high - resolution endoscopes and the advent of endoscopic instruments for sinus surgery now provide the surgeon with excellent endonasal visualization and access to the orbital walls without major complications. malhotra. insist that endoscope - assisted orbital surgery provided magnified view with good visualization of the fractured structures of the medial orbital wall, good local illumination and allowing the trainee to safely perform selected complex orbital procedures. in this case, we surgically repaired the orbital medial wall fractures using an endoscopic endonasal approach with successful l results. in conclusion, as with white - eyed blowout fracture, we recommend early surgical intervention and prompt surgical release of entrapped soft tissues, which can result in a complete resolution of symptoms for trapdoor medial wall fracture with rectus muscle entrapment in adults.	orbital blowout fracture frequently occurs along the floor or medial aspect of the orbital wall, which are the two thinnest areas of the bony orbit. true trapdoor injury of the orbit is less common and is rare as an isolated medial wall injury, because the medial orbital wall has several bony septa within the ethmoid sinus that provide support and decrease the risk of a trapdoor fracture. additionally, the incidence of trapdoor - type blowout fracture in adults is lower than in children. in a trapdoor - type blowout fracture with restricted ocular movement, prompt diagnosis and early intervention are associated with better clinical outcomes. we encountered a case of trap door - type medial blowout fracture with horizontal eye ball movement limitation in an adult. she underwent endonasal endoscopic reduction surgery for the medial blowout fractures. here we report this case, and suggest early diagnosis and prompt surgical exploration.
a schematic illustration of our nzmw single - molecule positioners is shown in figure 1a. the device consists of a silicon chip that has been processed using established methods to contain an array of zmws on a 100 100 m freestanding sinx membrane (see supporting information for details). the zmw arrays were passivated from reaction with piranha solution by depositing an 11 nm - thick sio2 layer using atomic - layer deposition. nanopores were then drilled in predetermined locations in the zmw arrays using transmission electron microscopy (tem), followed by treatment with piranha solution in order to hydrate the pores prior to experiments (see supporting information). the device was assembled in a custom cell that allows fluidic access to both sides of the membrane, as well as optical access to the bottom side of the chip as shown in figure 1a (see supporting information). an array of zmws is positioned on a 35 nm silicon nitride membrane with nanopores at the bases of waveguides (inset). a voltage bias actively draws complexes of biotinylated dna and fluorescently labeled streptavidin to the pore, which places the fluorophore in the zmw excitation volume. (b) an afm scan of the zmw membrane illustrates the topography of the surface. line scans of each zmw demonstrate uniformity with an average top diameter of 86.2 6.4 nm (n = 21). the scans have a pointed bottom profile because the afm tip can not penetrate the full depth of the waveguide. (c) dark - field scanning transmission electron micrograph (inverted contrast) of four zmws in the array (scale bar = 1 m). (d) tem images of zmws with 3 to 3.5 nm nanopores drilled in their centers (scale bars = 20 nm). zmws have a measured base diameter of 64.9 3.7 nm (n = 57). figure 1b shows an afm scan of a 2 4 zmw array on a sinx membrane (4 m 1.3 m spacing). the zmws are seen as dark uniform circles in the image for which height profiles through the zmw centers are shown as insets to the figure. although the base of the zmws can not be accessed in afm due to the tip geometry, the height profiles reveal uniform top diameters measured to be 86.2 6.4 nm (n = 21). the base diameters were measured by dark - field scanning tem (figure 1c) and bright - field tem (figure 1d) to be 64.9 3.7 nm (n = 57). the dark - field images of figure 1c in which the contrast was inverted for clarity, display a polycrystalline structure with grains in the range of 50150 nm, characteristic of a thermally evaporated metal film. the images in figure 1d show four typical nzmws that contain 33.5 nm diameter pores drilled at their center. we note that the tem images shown in figure 1 represent the first noncross - sectional view (e.g., top - view) of zmws using the tem, because prior zmw devices have all been fabricated on 100 m thick glass substrates that are too thick for tem imaging. on the basis of these afm and tem measurements of the top and base diameters, respectively, we arrive at a funnel - like zmw shape, as previously obtained with other zmw devices fabricated on fused silica substrates. capture of charged molecules into the nzmws can be greatly impacted by an electric field gradient present near the nzmw volume. it is established that dna capture into a nanopore is strongly assisted by the residual electric field near the pore mouth, which generates a localized electromotive force that migrates the molecule and focuses it to the pore. to examine the impact of zmw presence on the electric field profile near the pore, we used finite - element simulations to numerically compute the voltage profile in the vicinity of a 3 nm diameter nanopore in the absence (figure 2a) and presence (figure 2b) of a 60 nm diameter zmw above it. as the simulations show, the addition of the zmw constriction results in an electric field gradient with significant presence beyond the zmw top. the dotted contour lines, which indicate the positions at which the voltage drop is one percent of the total trans - membrane bias, highlight a 4-fold extension of the field away from the pore. numerical solution for the voltage profile induced by applying 800 mv to (a) a 3 nm pore without a zmw and (b) a 60 nm diameter zmw with a 3 nm pore ([kcl ] = 400 mm, t = 25 c). dotted lines i and i indicate the equipotential contour line where the voltage drop is 1% of the total transmembrane voltage. (c) i v curve for an array of three nzmws in 400 mm kcl (blue curve) compared to that for a sinx pore under the same conditions (red curve). (d) power spectral density of electrical noise for a nzmw membrane under different experimental conditions as indicated in the legend (= 488 nm, p = 20 mw, 40 w / cm sample intensity). figure 2c plots an i v curve measured on an array of three nzmws with 3 nm diameter pores in 400 mm kcl (blue) as well as an i v curve of a single 3 nm diameter pore (red). in both cases, the i v curves are linear, indicating open pores are present in the devices. the minor hysteresis in the i v curve of the nzmw array is an artifact of the additional capacitance of the zmw structure, which does not adversely interfere with our ability to capture and observe molecules inside nzmws. power spectral densities (psd) of the current noise for an nzmw device under various experimental conditions are shown in figure 2d. the psd plots show a typical shape for nanopore measurements, characterized by 1/f noise at low frequencies, thermal (johnson) noise at intermediate frequencies, and capacitive - dominated noise at high frequencies. laser illumination at zero bias (red curve) affects the thermal noise, while having little impact on the 1/f and capacitive regimes. in contrast, upon application of voltage (black) the 1/f noise dominates the psd, as previously observed in nanopore experiments. despite the presence of zmws on the membrane, using a device that contains a single nzmw with a 2.5 nm diameter nanopore, we demonstrate the ability to capture a dna / protein complex and dissociate its biotin streptavidin bond in a zmw under high bias. a solution that contains 1003 bp 5-biotinyated dna complexed to alexa fluor 647-labeled streptavidin (see materials and methods) was added to the cis chamber, which resulted in voltage - driven electrophoretic focusing of the complexes into the zmw volume. when the dna threads into the pore, the force on the dna against the streptavidin that is anchored to the zmw base causes the eventual dissociation of the complex. mounting our custom cell on an inverted microscope equipped with 640 nm laser illumination (coherent cube, coherent, inc.) and emccd detection (see supporting information) we simultaneously recorded nanopore current and nzmw fluorescence. upon application of 850 mv, a stable open pore baseline current was observed followed by a stochastic series of spikes that correspond to dna and/or dna / streptavidin interactions with the nanopore in the nzmw. in addition, we observed occasional long - lived events (> 1 s) that correspond to long - lived presence of the complex within the nanopore. these long - lived events were coincident with discrete increases in fluorescence from the nzmw (figure 3, points 15). notably, in events 1 and 2 of figure 3 we observed relatively shallow current blockades, which may represent a complex present in the nzmw without one of its dna molecules being fully threaded. this explanation is supported by events 1 and 2, which respectively show a complex temporarily adhering before diffusing away, and a complex remaining near the pore during which we observed other dna translocation events. for events 35, we observed deeper blockade levels accompanied by increases in fluorescence, which indicate full dna threading and streptavidin presence at the nzmw base. streptavidin bond at high voltage has previously been observed in a solid - state nanopore under similar applied voltage values. in each of these events, the simultaneous reduction of the nanopore current and increase in fluorescence indicates the capture of individual dna / protein complexes in the pore. finally, reversal of the voltage results in immediate ejection of the complex from the nzmw (e.g., events 2, 4, and 5), as observed by a coincident decrease in fluorescence intensity. simultaneous current (250 khz sampling, 10 khz filtering) and fluorescence (1 pixel region of interest, 10.02 ms exposure time, signal - averaged to 400 ms) traces from a single nzmw containing a 2.5 nm pore for a sample of biotinylated 1003-bp dna conjugated to alexa fluor 647-labeled streptavidin. protein complex entered the zmw illumination volume and occluded the pore, resulting in simultaneous fluorescence from the nzmw and blockage of the nanopore current. dna is pulled into the pore but prevented from translocating by the streptavidin, giving long - lasting current blockage. while immobilized in the pore, the labeled streptavidin sits in the zmw excitation volume, resulting in fluorescence. zmw devices are ideal for high - throughput fluorescence - based biomolecular analysis, which requires immobilization of the molecule inside the zmw excitation volume for extended periods of time. we have tested the principle of voltage - driven capture of multiple complexes in a 2 4 array of nzmws that contain 34 nm diameter nanopores, as shown in figure 4. we imaged the membrane while applying alternating biases of + 500 and 500 mv to trap and eject the same dna / streptavidin complex as used for the experiment in figure 3. figure 4a shows a fluorescence image of the nzmw array (left), as well as a series of three images during different time periods of the experiment. the bright spots in the images represent fluorescence that is due to occupied nzmws. we note that five of the eight nzmws in the array were active during the experiment, with the remaining nzmws not displaying optical signal. this yield of 60% is a reasonable yield of active nanopores in this diameter range. in figure 4b we plot time traces of fluorescence from five nzmws, identified as 15 in figure 4a, as well as from a zmw that does not contain a nanopore, labeled as n. at the beginning of the trace a (+) voltage was applied, during which molecules are clearly observed in the zmw volume. with the exception of pore 1 (indicated by), application of () voltage resulted in ejection of complexes from the nzmws, as indicated by a return of the fluorescence signal to the baseline level. upon restoring the (+) voltage we observed fluorescence activity in all five nzmws, indicating molecular loading. this infrequent occurrence of noncorrelated signals in nzmw 1 is a possible result of protein sticking to the surface of the device. (a) fluorescence images of a 2 4 nzmw array (enclosed by dotted line) with immobilized complexes of 1003 bp biotinylated dna and alexa fluor 647-labeled streptavidin. five nzmws that captured molecules are identified in the leftmost image, which is a projection of all frames from the experiment. the next three images from different points in the experiment illustrate molecules entering and leaving nzmws. n (1 pixel region of interest for each nzmw, 42.55 ms exposure time, signal - averaged to 500 ms) superimposed with membrane bias. in regions with green background, the transmembrane voltage is 500 mv. in regions with red background, it is 500 mv. identifies points where a protein adheres to the membrane, resulting in fluorescence persisting through negative voltage pulses. we note that while activity was seen in many of the nzmw devices, no activity was observed in the remaining zmws that contain no nanopores (e.g., zmw we suggest two main reasons for this observation : first, the radius of gyration of a 1003 bp dna molecule is 40 nm, which is slightly larger than the zmw radius (35 nm). this mismatch presents an energy barrier for diffusion of the dna streptavidin complex into the zmw. second, because we have not applied chemistry to covalently link the diffusing dna to the zmw surface, there is no mechanism to immobilize the complex in the zmws. finally, we investigate the efficiency of dna capture into nzmw devices. a solution of 230 pm 6000 bp dna labeled with yoyo-1 intercalating dye (10:1 bp / dye ratio, 488 nm excitation, see materials and methods) was placed on the cis side of the membrane. to monitor dna entry, we imaged a zmw array that contained a single nzmw while the applied voltage was toggled between + 850 and 850 mv. figure 5 shows fluorescence traces from the nzmw, as well as traces from three representative zmws. the inset shows three fluorescence images of the device that correspond to a time - averaged stack of frames from the whole experiment (i), as well as time - integrated images at negative (ii) and positive (iii) voltages. the nzmw (red arrow) was clearly visible based on its notable fluorescence at positive voltage values, while the remaining three zmws did not exhibit a voltage - induced fluorescence enhancement (see supporting information). similarly, the traces in figure 5 clearly show distinct entry of individual dna molecules into the nzmw volume, as indicated by a stochastic set of fluorescence enhancement spikes. we find dna capture to be highly efficient ; the on - time of dna within the nzmw was 51% when the voltage was (+), whereas the off - time was > 99% for negative voltages additionally, we find a prolonged 6.0 5.5 s mean duration of fluorescence spikes, during which we observe a very dynamic fluorescence signal that points to stochastic dna fluctuations within the zmw that occur on a slow time scale. fluorescence time traces from a single nzmw that contains a 3 nm diameter pore in an array of zmws is monitored for the fluorescence from 6000 bp dna labeled with yoyo-1 (9 pixel region of interest for each nzmw, 10.8 ms exposure time, signal - averaged to 100 ms). inset illustrates dna entering the illumination volume of a nzmw as it migrates toward the pore, resulting in increased zmw fluorescence. zmw arrays are shown in fluorescence images (i)(iii) with (i) being an averaged image of all frames in the experiment, and (ii) and (iii) being the membrane under respective 850 and 850 mv. green and red backgrounds in the fluorescence traces correspond to periods of positive and negative voltage, respectively (see supporting information for electrical trace). to quantify the dna loading we compared the on - time of the nzmw with on - times of other neighboring zmws in our experiment for times in which positive voltage was applied. for the random sample of 13 zmws, we have analyzed the resulting ratio of on - times tnzmw / tzmw is 580, highlighting the utility of nanopores as biomolecular focusing elements for zmw - based studies (see supporting information). from a smrt - sequencing perspective, we also compare the input dna requirements for nzmws to those of ordinary zmws. a typical protocol for diffusive loading of a 2000 bp template uses a 150 pm dna concentration and 60 min of reaction time, yielding a concentration - normalized loading rate of 1.9 10 pm s. magnetic bead loading results in improvements on the concentration requirement (330 pm) but still requires long incubation times (60 min) for optimal poisson loading, translating to a concentration - normalized loading rate of 1.7 10 pm s. in contrast, based on the mean dna arrival time in our nzmw experiment (3.5 s), the loading rate in nzmws is 1.3 10 pm s, orders - of - magnitude more efficient than in the case of diffusive or magnetic bead loading. we have demonstrated a novel device that consists of nanopores at the base of zmws for efficient and versatile positioning of single molecules. the fabrication process for these devices involved a combination of electron - beam and photolithography methods and resulted in the first demonstration of zmws on freestanding sinx membranes that contain nanopores at their bases. using synchronous optical and electrical recordings, we have demonstrated the reversible, voltage - driven positioning and ejection of individual dna protein complexes, as well as a mechanism for greatly enhancing the entry of long dna molecules into zmws. we note that loading of long dna molecules into zmws for smrt sequencing is inefficient because of the large dna coil size with respect to the zmw dimensions. this need to package dna into zmws results in a conformationally restricted dna that is unlikely to encounter a dna polymerase at the zmw base on short time scales. current protocols for activating zmws for sequencing involve incubation of the zmws with a preformed complex of dna and a streptavidin - polymerase fusion protein, which still results in a slow binding to the zmw surface due to the imposed conformational restriction. the need to prereact dna and polymerase in solution, as well as the need for higher dna concentrations, has limited certain studies involving precious dna samples using this method. finally, we have demonstrated the first zmw platform in which the zmw can be reused by releasing a molecular complex from the zmw volume at the click of a button. the combination of zmws and nanopores greatly increases the efficiency of dna loading, which can aid in the development of future smrt sequencing applications in genetics and epigenetics. in addition, this ability to focus, hold, and release biomolecules from the illumination volume of the zmw should allow many biophysical studies at the molecular level. dna protein complexes were prepared from pcr - synthesized biotinylated dna and alexa fluor 647-labeled streptavidin (life technologies, carlsbad, ca). biotinylated dna was incubated with labeled streptavidin at a 4:1 dna / streptavidin ratio for 15 min (see gel image in supporting information). yoyo - labeled dna was prepared from 6000 bp dna (thermo scientific, tewksbury, ma) and yoyo-1 intercalating dye (life technologies, carlsbad, ca). dna and dye were incubated for 20 min at 50 c with a 10:1 base pair / dye molar ratio. voltage distributions near pores (figure 2a, b) were computed with comsol multiphysics (comsol, burlington, ma). planck equations were numerically solved for a geometry consisting of two micron - scale cylindrical compartments (i.e., cis and trans) connected by a nanopore embedded in a perfectly insulating membrane. an element size as fine as 0.1 nm and additional boundary meshing layers inside the pore were used to ensure no edge effects skew the physical results. a positive bias voltage of 800 mv was enforced at the bottom surface of the trans chamber and ground to the top surface of the cis chamber. tem imaging and pore fabrication were performed with a jeol 2010feg (northeastern university). nzmw chips were cleaned for 5 min in heated piranha solution, rinsed thoroughly in deionized water, dried under vacuum, and immediately assembled for experiment in a peek flow cell (see supporting information). the cell was mounted in a faraday cage on the stage of an olympus ix81 inverted microscope with a 60, 1.2 na water immersion objective. the membrane was illuminated with a coherent cube 640 nm laser and a coherent sapphire 488 nm laser. an axopatch 200b amplifier was used for current monitoring off ag / agcl electrodes. electrical data was recorded using custom - made labview software (national instruments, woburn, ma). images were taken with a hamammatsu imagem emccd, recorded with hcimage live software (hamamatsu, sewickley, pa), and analyzed with imagej.	we have developed a hybrid nanopore / zero - mode waveguide device for single - molecule fluorescence and dna sequencing applications. the device is a freestanding solid - state membrane with sub-5 nm nanopores that reversibly delivers individual biomolecules to the base of 70 nm diameter waveguides for interrogation. rapid and reversible molecular loading is achieved by controlling the voltage across the device. using this device we demonstrate protein and dna loading with efficiency that is orders of magnitude higher than diffusion - based molecular loading.
as an aftereffect of the evolution of man and his eternal quest for time - efficient, effective and practical instruments and technology, dental practice has had its share of profit as well.. the classic systematic procedures of cavity preparation have resulted in a more tissue - saving way of treatment determined by one underlying theme adhesive dentistry. the possibility of bonding dental materials to enamel and dentin has brought forward significant developments of dental equipment together with improved sources of energy and means for holding and controlling the cutting instruments. in the 1870 s the introduction of the electric motor as a power source was one of the most significant advances. after that, cutting techniques were revolutionized when diamond burs capable of cutting enamel were produced.1 conventional diamond burs are the most common rotary instruments used in the dental office and dental laboratory. conventional diamond burs are made by plating small industrial or mineral diamond particles on stainless steel shanks using a galvanic process. these conventional diamond burs show several limitations such as the heterogeneity of grain shapes, the difficulty of automation during fabrication, the decrease of cutting effectiveness due to repeated sterilization, and short functional lifetime. an additional shortcoming may be represented by the potential release of nickel (ni) ions from the metallic binder into the body fluids.2 according to the literature, clinical performance of abrasive instruments depends on the size, spacing, uniformity, exposure and bonding of the diamond particles, so, the significant loss of diamond particles during cutting procedure is an undesirable factor.2 at present, there are some alternative methods of cutting enamel and dentin. air abrasive instruments for example, developed in the mid 1950 s, and approved by the fda in 1992, have not reached widespread clinical acceptance because of the difficulty of eliminating residual aluminum oxide dust. leo goldman s research in 1964 and subsequent research has identified many laser wavelengths for possible use in dentistry. presently, the er : yag laser s ability to ablate dental tissue efficiently, owing to transmission of specific wavelengths absorbed by water and hydroxyapatite, is regarded as the most efficient laser.3 the general lack of knowledge of many dental professionals regarding this technique and the cost of the equipment has restricted its use. this technology is available to the clinicians, but can only be used effectively provided that dentists have the scientific information necessary to use them appropriately, and are aware of their advantages as well as their limitations. although the use of the ultrasonic devices for dental preparation is a fairly recent and updated technology, ultrasonics have been part of the clinician s everyday routine for some time and are easily utilized and accepted.4,5 recently, a special diamond coated bur coupled to an ultrasonic handpiece for dental applications was developed at the brazil, by coating a molybdenum substrate through a chemical vapor deposition (cvd) process. hereafter we will refer to that device as a cvd bur, as labelled by the manufacturer (cvdentus brazil). developed from a continuous diamond film, this bur is characterized by a pure diamond cutting surface without a metallic binder between crystals (figure 1). these burs can be adapted by an appropriate mandrel to almost any ultrasonic handpiece system commercially available, producing a cutting speed about half that of a conventional rotary instrument.5,6 the aim of this study is to report two clinical cases in which cvd burs are used for cavity preparation. in the clinical case number 1 (figure 3), esthetic deficiencies and secondary caries, detected by a previous bite - wing radiographic exam were the reasons for replacement of the amalgam restorations with resin composite. the removal of the amalgam restorations in a maxillary pre - molar and first molar was carried out using a cvd bur (cylindrical shaped point - figure 4). the adjacent tooth and soft tissue were protected by a plastic matrix, and not harmed by the cvd bur (figure 5). the patient did not request or require anesthetic during the procedure, which is a major advantage of this approach. an inverted cone shaped cvd bur was used to remove the amalgam from the retention areas. ultrasonic cvd burs were used under constant water cooling, but the amount of water released by this equipment is much less than by conventional rotary instruments, as it produces less heat. this offers minimal risks of pulp damage, and makes the visibility of the operation field better requiring less frequent interruption during the procedure (figure 6). based on the amplitude of the handpiece movement, a different type of result is obtained, and for each bur, the manufacturer indicates the best amplitude to work with, which must be adjusted in the ultrasonic equipment by the professional. the authors present sem images obtained in an in vitro previous study that demonstrated a typical cutting pattern that is expected to be seen in the prepared surfaces when working with cvd burs4 (figure 7). to confirm this pattern, the authors also investigated, in an in vitro research, dentin surfaces prepared with the cvd burs, by using an atomic force microscope (afm) (nanoscope iiia - veeco instruments - santa barbara california), operated in contact mode.7,13 the afm obtained a three - dimensional image that illustrates the typical dentin surface that should be expected when working with this particular instrument (figure 8). the qualitative results of the prepared dentin surface investigated in sem and afm, corroborates with other authors13 who have stated that cvd burs produce a characteristic dentin surface, where a thin smear layer is detected (figure 9). for this reason, the authors selected a self - etch dentin adhesive (adhese - ivoclar vivadent) that was applied according to the manufacturers instructions. as a clinical aspect of a bright wet layer was observed, light curing was conducted.8 the curing light used was a led (light emitting diode - radii sdi) with 1400 mw / cm for 40 seconds (figure 10), and the composite resin (4 seasons - ivoclar vivadent - shade a2) was inserted in oblique increments.9 the superficial resin film was covered by a glycerine gel to prevent the formation of an oxygen - inhibition layer during the polymerization of the composite material.10,11 an afm image of the surface of a polymerized 4 seasons dental composite, when a glycerin gel is used, is presented in figure 11. after light curing, the rubber dam was removed and occlusion checked. the restoration was polished with enhance silicone abrasive points followed by a felt disk, to achieve an excellent final result (figure 12). the authors present case number 2 to demonstrate the important capability of the cvd burs of working at high inclination angles. moreover, because of the pendular vibratory movements with nanometric variations of amplitude, these burs do n't cut if they touch soft issues, allowing cavity preparations at gingival level without damaging this tissue.17,18 ultrasonic cvd burs are produced in a reactor in which a mixture of methane and hydrogen gases results in the formation of a single artificial diamond layer without space between the grains on the substrate (a molybdenum rod). conventional facturing methods weld the diamond layer using a galvanic process to the substrate which results in a relatively large area between the diamond grains. this new technique allows the diamond to have sufficient adherence to the metal rod to bear the vibration effect of ultrasound. according to the manufacter, these tips have to be used under constant water cooling, but the amount of water released by this equipment is much less than by conventional rotary instruments which produces better visibility of the operation field requiring less frequent interruption during the procedure and less heat.16 researches that assessed intrapulpal temperature variation with different equipments presented controversial results. higher intrapulpal temperature with ultrasound has been reported than with er : yag laser and high - speed rotation, while on the other hand other studies demonstrated no statistically significant difference in the temperature increase between high - speed rotation and ultrasound. however, other authors agree that the use of ultrasound can be considered safe, since this increase is lower than the critical value of 5.5c, thus offering no risk of pulpal damage.12,14,15 the main disadvantage related to this technique seems to be the time required for a complete ultrasonic cavity preparation that is significantly higher than that required for preparation using a high - speed hand - piece, approximately 4 times.2,16 the ultrasound - coupled cvd bur was found to be an efficient method for tooth preparation in this instance. it was effective and safe for working close to the gingival margin, and improved the operating field visibility, because of access inclination angles that are not available when using a conventional handpiece. the ultrasonic preparation procedure also potentially eliminates undesirable psychological effects associated with conventional rotary instrument noise. consequently, the ultrasonic cavity preparation using cvd coated diamond bur offers promising clinical utility for certain dental procedures.	before any restorative procedure can be undertaken a proper cavity preparation is required. this clinical step is the mechanical alteration of the tooth to receive a restorative material with which a satisfactory form, function and the esthetics of the tooth will be established. in recent years improvements in materials and techniques have been devised and new technologies are now available for this purpose. the aim of the present study is to report two clinical cases in which a cvd coated diamond bur coupled to an ultrasonic handpiece is used in dental preparation. this technique provides an accurate and conservative tooth preparation with ideal access and visibility and because of enhanced efficiency can also play a role in eliminating some of the patient discomfort of the dental treatment.
the human papillomavirus (hpv) belongs to the papillomaviridae family and persistent infection of high - risk hpv is the direct cause of cervical carcinoma, which is the second most common malignancy among women worldwide. hpv detection and genotyping is the most effective and accurate approach in screening of the early cervical lesions and cervical cancer. with hpv genotyping becoming more prevalent, over 120 types of hpv have been identified, of which at least 40 types are indicated to infect the genital epithelium. its genotypes are generally classified into high - risk (hr-) and low - risk (lr-) groups based on their carcinogenic potential. hr - hpvs include hpv 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, and so on, and the lr - hpvs include 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, cp6108, and so on. it should be noted that it is of particular interest to know the difference in the clinical properties of cervical neoplasia according to hpv types, which will help us in estimating the gravity of the disease and evaluating the prognosis after therapy according to different hpv types. although there are geographical differences in the distribution of hpv types among populations, globally, it has been shown that hpv 16, 18, 45, 33, and 31 are the most prevalent hpv types associated with cervical cancer. it is of indicated that a vaccine that included the 7 most common hpv types can prevent 87.4% of cervical cancer worldwide. therefore, it is of considerable clinical value to establish a reliable and convenient method to detect and genotype hpv. food and drug administration) commercially available method for the detection of hpv dna, the well - established hybrid capture ii system (hc - ii) has been proven to be a sensitive and reliable assay, which can detect 13 types of carcinogenic - hpv types or 5 types of low - risk hpv in a single test [6, 7 ]. however, its main undeniable limitation is that hc - ii can not distinguish between different hpv genotypes definitely [5, 8 ]. recently, a new hpv - genotyping method combining two advanced techniques, the flow - through hybridization and gene chip (hybrimax) has been used to detect and genotype hpv, which can distinguish 21 different types of hpv dna in a single test and diagnose multiple infections [911 ]. in this study, we have evaluated the efficacy of hybrimax on cervical hpv genotyping through comparison of the results with hybrid capture ii (hc - ii) and in situ hybridization (ish). we showed that the most common hpv types tested by hybrimax in different grades of cervical disease could be determined, suggesting that hybrimax is an efficient method for hpv genotyping and is more suitable for clinical use. 591 out of 7520 women who accepted liquid - based cytology examination in china - japan friendship hospital from august 2004 to may 2005 were randomly selected for detection of the 21 hpv genotypes by hybrimax, and their mean age was 35.4 7.7 (ranging from 20 to 64). among them, 138 women (mean age was 35.8 7.8), who diagnosed within normal limits with cervical cytology for at least two years without any cervical disease or operation, were described as a total of 453 women were diagnosed with abnormal cytology, and the mean age was 34.1 6.9. cytological diagnosis (according to the 2001 bethesda system) of those 453 patients were as follows : 161 cases with atypical squamous cells (asc), 187 cases with low - grade squamous intraepithelial lesion (lsil), 105 cases with high - grade squamous intraepithelial lesion (hsil), or squamous cell carcinoma (scc). patients were classified into 6 groups according to their histopathology diagnosis from specimens of olcposcopic biopsy, loop electrosurgical excision procedure (leep), or cold - knife conization. these 6 groups include 152 cases of chronic cervicitis, 101 cases of cervical intraepithelial neoplasia (cin, which indicates that dysplasia is seen on a biopsy of the cervix) i (mild dysplasia), 77 cases of cin ii (moderate to marked dysplasia), 76 cases of cin iii (severe dysplasia to carcinoma in situ), 27 cases of scc, and 20 cases of condyloma acuminata. with liquid - based cytology, samples were taken with the cervical brush at gynecological examination for hpv dna testing. it is prohibitive to apply vaginal douching three days prior to the collection of samples or to have sexual intercourse within one day. hpv genotyping by hybrimax was performed using an hpv genoarray test kit (hybribio ltd., this assay can determine 21 hpv types, including 14 high - risk hpv types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), five low - risk hpv types (6, 11, 42, 43, and 44), and two unknown - risk types (53 and cp8304), by the flow - through hybridization technique using hpv dna amplified by pcr. in brief, 0.5 ml specimen was centrifuged at 13,000 g for 15 min with kubota 6930. then, the supernatant was removed and the pellet was resuspended in 200 l pbs buffer. high - quality dna was yielded from lysis of cells by isolation of dna, precipitation, and purification. the instrument used for pcr amplification was pe 9600 thermal cycler (applied biosystems, usa). we prepared the pcr master mix by mixing 19.25 l pcr - mix solution and 0.75 l dna taq polymerase for each reaction, adding 5 l dna template in each tube, and then running the amplification program. the amplification program was denaturing at 95c for 9 min, 40 cycles at 95c for 20 seconds, 55c for 30 seconds, and 72c for 30 seconds, and finally extension at 72c for 5 min. the flow - through hybridization was made on a prewarmed instrument at 45c, and the hybrimem hpv-21 dna microarray membrane was placed, which is marked with 21 hpv - genotype probes including hpv 6, 11, 42, 43, 44, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and cp8304. the number of samples tested in a batch could be adjusted from 1 to 15 as required. the pcr products were denatured at 95c for 5 min just before hybridization and then was chilled on ice for at least 2 min. we mixed the pcr products with hybridization solutions and then added the mixture into sample wells to proceed with flow - through hybridization for about 510 min. the membrane was washed with hybridization solution, and the empty space was blocked without reaction. adding nbt / bcip solution to display the results, a positive result was indicated by a clearly visible indigo dot. the hpv - genotype result was determined according to the position of the hpv - genotype probes on the microarray chip. 413 samples were detected by the commercially available hc - ii assay (digene co., gaitherburg, md, usa). the probes used were designed to detect 13 types of high - risk hpv, including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. this enzyme - linked immunosorbent assay is based on a sandwich hybridization followed by a nonradioactive alkaline phosphatase reaction with chemiluminescence in the microplate. samples were classified as positive for hpv dna if their chemiluminescence was more than 1.0 pg / ml of control. it was performed with rembrandt universal dish and ap detection kit (panpath, the netherlands). after being dewaxed and hydrated, paraffin sections (5 - 6 m) specimens were denatured at 95c for 5 min by applying the probe solution to them and hybridized at 37c for 2 hours. then, they were incubated with pan wash at 37c for 15 min except the positive control. we dropped the conjugate by heating for 30 min and the nbt / bcip substrate by heating for 10 min at 37c in the dark and finally counterstaining and mounting. the statistical analysis was performed using the spss software (version 10.0) for chi - square test and kappa index was calculated to evaluate the accordance of the results. the positive rate of hybrimax for 13 types of hr - hpv (detected by hc - ii) was 73.7% (303/413) and the positive rate of hc - ii was 70.7% (292/413) (table 1). in general, hpv - detection rates by hybrimax agreed well with those of hc - ii with a total accordance rate of 92.5%. it is shown that the accordance rates were excellent in general (kappa index (ki) = 0.814) in the group with normal cytological diagnosis (ki = 0.750), chronic cervicitis (ki = 0.781), condyloma acuminata (ki = 0.80), group cini (ki = 0.755), cin ii (ki = 0.723), and good at group cin iii (ki = 0.547) (0.75 > ki > 0.4, good ; ki 0.75, excellent). in addition, we found that there were 21 cases with hc - ii - negative and hybrimax - positive diagnoses, including 8 cases of hpv18 (or multiple infections that include hpv18), 5 cases of hpv 68 (or that include hpv 68), 4 cases of hpv 31 (or that include hpv 31), 2 cases of hpv 33 (or that include hpv 33), and 2 cases of hpv 51 (or that include hpv 51). another two cases with genotyping results of hpv 66, 2 cases with hpv 6, one case with hpv 6/11, and one case with hpv 44 by hybrimax were hc - ii positive, the results of which may be out of the hc - ii testing limits. it is shown that the accordance rate of the two methods was 89.1%, and their kappa index was 0.776. the total hpv - positive rate of patients with abnormal cytological diagnosis was 89.6% (406/453), with 80.9% in group a, 90.1% in group b, 92.2% in group c, 97.4% in group d, and 100% in groups e and f. in the group of patients with normal cytological diagnosis, the positive rate was 30.4%. the 10 most common genotypes and their infection rates with abnormal cytological diagnosis in turn (descending) were hpv 16 (28.9%), 52/58 (19.0%), 18 (16.8%), 33 (9.9%), 31 (9.7%), 81 (8.4%), 53 (8.6%), 68 (8.4%), 66 (5.1%), and 43 (0%). the 10 most common genotypes in normal groups were hpv 16 (8.0%), 68 (7.2%), 18 (6.5%), 52/58 (3.6%), 11 (2.9%), 53 (2.2%), 31/39 (1.4%), and 33 (0.7%), while hpv 35, 45, 59, 66, 42, 43, and 44 were not detected. the 10 most common genotypes in different groups (in descending order) were as follows : hpv 16, 18/58, 52, 31, 53, 68, 81, 33, and 39 in the group of chronic cervicitis ; hpv 58, 16/52, 18, 33, 68, 53, 56/81, and 31 in the group of cin ; hpv 16, 52, 58, 18, 33/81, 31/51, 53, and 68 in cin ; hpv 16, 58, 52, 18, 31, 33, 81, 53/68, and 66 in cin iii ; hpv 16, 18, 52, 58, 33, 66, 68, and 31/51/53 in group of scc. we come to the conclusion that hpv 16, 18, 52, 58, 33, and 31 were the 6 most common hpv types that can infect the patients with cervical lesions. the most common hpv types causing condyloma acuminata were hpv 11 (with a total positive rate of 55.0%) and hpv 6 (with a total positive rate of 30.0%). the positive rates of the 6 most common genotypes in different groups were shown in figure 1. hpv 16 was the most frequent type in almost all the groups (except for cin i, less than hpv 58). hpv 18 was the second most frequent type in scc, but the fourth most frequent type in all groups of cin. similarly, the positive rates of hpv 18 increase with the development of disease. hpv 52 and hpv 58 were the third and fourth most common types in the group of scc, but the second and third most frequent types in all groups of cin. hpv 33 was the fifth most frequent type in the group of scc, and the positive rates increase with the progress of disease in general. previous studies have documented that hpv plays a central role in the etiology of cervical cancer [12, 13 ]. it is indicated that women positive for hpv dna have a risk of developing cervical cancer 1550 times higher than those without hpv dna. therefore, it is a preferred approach to combine the liquid - based cytology diagnosis with hpv dna testing in cervical - cancer screening. traditional hpv - genotyping methods, such as southern - blot hybridization, direct sequencing, and restriction fragment length polymorphisms (rflp) based on polymerase chain reaction (pcr) are unsuitable for clinical use due to various reasons, such as low sensitivity, difficultly of handling, and time consumption. ish is an easy to handle, reliable method for hpv detection and typing, working on pap smears and paraffin - embedded sections [17, 18 ] ; however, its sensitivity and genotype detection are limited. hc - ii, as the most wildly accepted hpv - dna - testing method for clinical use, is considered to be reliable, sensitive and easy to handle [6, 7 ]. however it has a limitation in discriminating hpv genotype and multiple infections, for hpv infection can only be attributed to a low - risk or high - risk group [5, 8 ]. flow - through hybridization is the most efficient method for molecular hybridization [911 ]. a newly developed biotechnology named hybrimax, combining two advanced techniques, the flow - through hybridization and gene chip, is developeded to be used in clinical practice for the detection and genotyping of 21 different types of hpvs at one test. the hpv genotypes detected by hybrimax include not only the 5 low - risk hpvs and 13 high - risk hpvs that hc - ii can detect, but also hpv 66, 53, and cp8304 that hc - ii can not detect. in addition, hybrimax provides much more information than that afforded by hc - ii. in this study, our results indicated that hybrimax was highly comparable to hc - ii in the detection of 13 types of hr - hpv, and has good accordance with ish in the detection of hpv 16/18. therefore, it is suggested that hybrimax can serve as an ideal method for hpv genotyping. among the 21 cases which hc - ii diagnosed negative while hybrimax revealed positive, there were 8 cases of hpv 18 and 5 cases of hpv 68, which probably suggested that hc - ii was less sensitive to those hpv types. there were some samples detected hpv 6, 11, 44, and 66 positive by hybrimax, present also positive by hc - ii, which should be negative (not included in the range of genotype which can detected by hc - ii). it was indicated that there was cross - reaction between the probes of the hc - ii hpv types. it had been reported that the probes of hc - ii can have a cross - reaction with less than 22 types of hpv dna other than 13 types of hc - ii. a potential disadvantage of hybrimax comes from the procedure of pcr, which generally was confronted with the problem of contamination. in addition, this study revealed that, in china, the 6 most common genotypes in cervical lesions were hpv 16, 18, 52, 58, 33, and 31 included in cervical cancer. the recent international prevalence surveys by the international agency of research on cancer (iarc) reported that the most common hpv types of invasive cancer were 16 (57.4%), 18 (16.6%), 45 (6.8%), 31 (4.3%), 33 (3.7%), 52 (2.5%), 58 (2.3%), 35 (2.2%), 59 (1.5%), and 56 (1.3%), but the study did not include the chinese population. a meta - analysis made by clifford in 2003 revealed that in cases from asia, hpv 58 (5.8%) and 52 (4.4%) were more common than hpv 45, 31, and 33, which supports our results. a large - scale survey on the hpv types of 809 cervical cancer cases in china showed that hpv 16 and hpv 18 were the first and second most common hpv types, and hpv 58 and 52 were the third and fourth most common genotypes, followed by 31 and 33, which also supported our results. this study also found that the positive rates of hr - hpvs in groups of abnormal cytological diagnoses were prominently higher than that of normal groups. therefore, it is suggested that hpv detection is especially important for women with abnormal cytological findings.	persistent infection of high - risk human papillomavirus (hpv) has been recognized as the direct cause of cervical carcinoma. therefore, detection and genotyping of hpv are important to cervical - cancer screening. in this study, we have evaluated the efficacy of flow - through hybridization and gene chip (hybrimax) on hpv genotyping through comparison of the results with hybrid capture ii (hc - ii) and in situ hybridization (ish). 591 women were classified into 6 groups according to their histological diagnoses. the overall accordance rate on 13 types of hpv genotypes between hybrimax and hc - ii were 92.5% and 100% in the cancer group. the overall accordance was excellent with the kappa index (ki) of 0.814. the value of ki in each group was 0.750 (normal cytological diagnosis), 0.781 (chronic cervicitis), 0.80 (condyloma acuminatum), 0.755 (cervical intraepithelial neoplasia (cin) i), 0.723 (cin ii), and 0.547 (cin iii) (0.75 > ki > 0.4, good ; ki 0.75, excellent). the 10 most common hpv subtype detected by hybrimax were 16, 52/58, 18, 33, 31, 81, 53, 68, and 66 in patients, and 16, 68, 18, 52, 58, 11, 53, 31/39, and 33 in normal controls. in conclusion, hybrimax is an efficient method for hpv genotyping and more suitable for clinical use.

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