The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated , and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI  were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects  Phototherapy as a treatment option for neonatal hyperbilirubinemia was first used by Cremer and co-workers in the 1950's –4, as weAlthough phototherapy for neonatal hyperbilirubinemia is accepted as the 'gold standard' of treatment, it may be accompanied with side effects such as impairment of thermoregulation, mineral dysbalance , and dirThe neurotoxicity of bilirubin is directly associated with the concentration of the fraction unbound to albumin (or other solubilizing substances), which is called Bf (bilirubin free) ,14. Bf iin vitro using the neuroblastoma cell line SH-SY5Y.Therefore, the aim of the current study was to isolate and characterize pure forms of bilirubin PI; and then to assess their potential effects on bilirubin-albumin binding, as well as their possible biological effects The bilirubin  was purified before use, according to McDonagh and Assissi . The chlBecause of light-sensitivity of bilirubin and bilirubin PI, all procedures were carried out under dim light in aluminium wrapped flasks. Evaporation was performed under vacuum and stream of nitrogen.3 solution in methanol); the solution underwent 90 minutes of photo-irradiation using a Lilly phototherapeutical device , composed of a field of LEDs emitting light  with a maximal spectral irradiance of 100 μW/cm2/nm corresponding to the total irradiance of 3.1 mW/cm2. The sample was then evaporated under vacuum, dissolved in pure methanol, decanted from the residual bilirubin, and re-evaporated. The residue of bilirubin PI was protected from light, and stored at -20°C until use.The pure bilirubin photoderivatives were prepared as previously described –27 with The residue after photo-irradiation was dissolved in a small amount of methanol:chloroform , and separated by thin layer chromatography . During the first chromatography, the mixture of bilirubin derivatives was separated into 8 major bands, which were extracted using the mobile phase, evaporated to dryness, and then re-chromatographed using the same conditions. The individual separated compounds were re-extracted, the solvent evaporated, and the isolated compounds were stored at -20°C until used. The isolated compounds 1 and 7, corresponding to ZE/EZ-bilirubins and lumirubin, as verified by HPLC ,29 Figs, in a 1:et al. /[HSA] = 1/1, the concentration of the pigment was 1.5 x 10otometry . CD specotometry . The metotometry ,35.p value (oxidation constant of bilirubin) was determined. For enzyme standardization , the solution of bilirubin in PBS without albumin was used (bilirubin concentrations were between 1 and 3 μM). Bilirubin absorbance was measured at 440 nm . Afterwards, 5 μl of H2O2 and 10 μl of HRP were added, the solution was slightly mixed, and the decrease of absorbance at 440 nm in 60 s was measured. The Kp constant was counted according to Kp calculation Kp = constant for oxidation of bilirubin, and V0 = the initial oxidation velocity (expressed as ΔAbs/min).The effect of bilirubin PI on Bf levels was studied by a peroxidase method . BrieflyKp values were similar . We first determined the bilirubin concentration corresponding (under the conditions used) to 140 nM (approximately 24 μM bilirubin). Then, we studied whether Bf is affected by the addition of increasing concentrations of bilirubin PI . A mixtin vitro studies . Authentication of the cell line was confirmed by independent laboratory . The cells were tested for Mycoplasma contamination using the MycoAlert luminescence test . Cells were cultured in a mixture of MEM Eagle and Ham’s F12 media , containing 15% of fetal bovine serum, in 75 cm2 culture flasks, at 37°C, in a 5% CO2 atmosphere. For functional tests, cells were seeded at a concentration of 50,000 cells per 1 cm2.The SH-SY5Y human neuroblastoma cell line was used for the The effect of bilirubin (24 μM), pure bilirubin PI , and the combination of bilirubin with bilirubin PI on cell viability was analyzed by both MTT  as well as by luminescent CellTiter-Glo  tests, using a Sunrise Microplate Reader  and Synergy 2 Multi-mode Microplate Reader , respectively.The effect of bilirubin and bilirubin PI (24 μM bilirubin (corresponding to 140 nM Bf), 15% bilirubin PI, and a mixture of bilirubin and 15% PI) on the expression of genes involved in heme catabolism and in the regulation of the cell cycle  was inveThe activity of heme oxygenase (HMOX) was measured as described previously . In brieCells for flow cytometry analyses were treated with bilirubin (24 μM corresponding to 140 nM Bf), bilirubin PI (15%), and a combination of bilirubin and PI for 1 and 24 hrs. At the end of the treatment the medium was aspired, the cells were washed twice with PBS, then fixed by adding 5 ml of cold (-20°C) 80% ethanol drop-wise under constant gentle vortexing. After centrifugation , the sediments were re-suspended in 1 ml of staining solution in PBS containing 0.1% v/v Triton X-100, 20 μg/ml propidium iodide (PI), and 0.2 mg/ml DNAse free RNaseA. Samples were incubated in the dark for 30 min at RT, and subjected to FACS analysis . Data were collected for 10,000 events per sample.Data are presented as the median and 25–75% range. Differences between variables were evaluated by the Mann-Whitney Rank Sum test. Differences were considered statistically significant when p-values were less than 0.05. Statistical analyses were performed using Prism 6 software .Photo-exposure of unconjugated bilirubin, under the conditions defined above, lead to the generation of 18 different PI . These cOut of these 18 individual substances, several were clearly identified ; the others are likely to represent unstable, oxidized, and as yet undefined intermediates. Pigments 1 and 7 , identifTo investigate whether bilirubin PI might affect Bf levels, we directly analyzed this effect by measuring Bf using a peroxidase method. The addition of 3.6 and 7.2 μM of bilirubin PI  had no effect on the Bf concentration .To a solution with 140 nM Bf concentration (approximately 24 μM bilirubin), bilirubin PI were added in increasing concentrations (15% and 30%); the resultant bar was constructed from the difference between the decrease of absorbance after addition of bilirubin PI or DMSO to the bilirubin solution. n = 6 for each group.Ka) for bilirubin and lumirubin. Lumirubin had a significantly lower Ka compared to bilirubin . In the complexes with HSA, bilirubin shows an absorption maximum at 475 nm, which is shifted closer to the red spectrum compared to the unbound pigment in solution . In contrast, CD spectrometric analysis of the lumirubin-albumin complex revealed that lumirubin has one binding site on HSA with right-helical conformation  of the bound pigment . The obtained lumirubin-HSA complex had also a slight red-shifted absorption band with the maximum at 445 nm, compared to unbound lumirubin that absorbed at 430 nm.The results of the CD analyses further supported this conclusion. Bilirubin was found to be bound to a high-affinity site inducing the CD positive couplet [460(+)/410(-) nm] that is characteristic for the bound P-conformer of bilirubin (see right panel). The HSA-bilirubin system  of bilirubin or its PI did not have any effect on cell viability (data not shown). However, cell viability was significantly reduced after the 24 or 48 hr cell treatments with bilirubin, and this effect further increased after 48 hours of exposure .Bilirubin is the final product of the heme catabolic pathway, and its formation is under the control of both HMOX and biliverdin reductase (BLVRA). Since these enzymes are protective tools of the organism against increased oxidative stress ,42  in sufficient purity and quantity for biological studies.per se. Lumirubin was found to have only one binding site on HSA, and this binding site was the same as for hemin (i.e. in the subdomain IB or close to it). Nevertheless, the bilirubin and lumirubin binding sites on albumin are not totally independent, because the lumirubin binding site is close to the bilirubin high-affinity binding site; to a certain extent it is also able to lower bilirubin's ability to bind to HSA. However, this effect is probably not clinically relevant. In support of this data, we confirmed that lumirubin-HSA's binding constant is much lower, compared to bilirubin. These data collectively demonstrate that although lumirubin binds to albumin, the binding has no biological relevance in terms of any possible influence on bilirubin-albumin binding.We were able to demonstrate that these bilirubin PI do not increase Bf levels, and thus do not increase bilirubin toxicity in vitro model, even after prolonged exposure. This was in striking contrast with the known toxic effects of bilirubin. This indicates that only unconjugated bilirubin is toxic, and that the conformational changes induced by irradiation almost completely abolishes the noxious effects of the pigment on the cell.The experiments, focused on the potential cytotoxicity of major bilirubin PI, revealed no apparent effects on cell viability in our BLVRA mRNA expression was not expected, since biliverdin to some extent is also produced during phototherapy [MRP1 and MDR1 mRNA gene expression, indicating that these transporters are not inducible, at least in our cell system, by either bilirubin or their PI.Neither bilirubin, nor bilirubin PI had any functional effect on two key enzymes (HMOX and BLVRA) important in heme degradation and bilirubin production. The lack of any effect of bilirubin PI on otherapy . Similarin vitro model. However, other brain cells, such as astrocytes, microglia, or even endothelial cells should also be tested; ideally in an organotypic brain slice ex vivo model to bring conclusive evidence.Our study has several limitations. First, we only assessed the biological effects of three major bilirubin PI, ZE/EZ-bilirubins and Z-lumirubin, and thus we cannot exclude that other photoproducts or bilirubin oxidation products formed during phototherapy, especially those short-lived and not detected in our study, might be toxic. Thus, it is still needed to be carefully assessed, whether minor oxidation products produced from bilirubin during phototherapy can exert any biological action. In addition, our studies were only performed on one cell line, representing a neuronal In conclusion, our data indicate that the major bilirubin PI, ZE/EZ-bilirubin and lumirubin, seem to be biologically inert, and do not exert any negative biological effects. The side effects of phototherapy, theoretically attributable to bilirubin PI, are most likely due to other mechanisms.


MLH1 and MSH3, and 5/84 CpGI in the TP, that is, MLH1, MSH3, APC, MSH6, and MGMT. The gene with the highest methylation percentage in the TC and TP was MLH1. Given that MLH1 methylation has been associated with microsatellite instability, we analyzed the status of the microsatellite Bat-26. We found that neither the TC nor the TP presented instability. The methylation of MLH1, MGMT, and APC has been described in GISTs, but to the best of our knowledge this is the first time that the methylation of these genes has been associated with gastric leiomyoma. Further research should be conducted to identify reliable molecular markers that could differentiate between GISTs and gastric leiomyomas. Leiomyomas constitute 2.5% of all resected neoplasms of the stomach. They are usually asymptomatic, but may present mucosal ulceration. Aberrant DNA methylation is a well-defined epigenetic change in human neoplasms; however, gene-acquired methylation may not necessarily be related with a malignant phenotype. In this report we analyzed in a gastric leiomyoma, the methylation status of 84 CpGI in tumor suppressor and DNA repair genes. We analyzed the tumor center (TC) and tumor periphery (TP) separately. We found aberrant methylation in 2/84 CpGI in the TC portion, that is,  Gastric leiomyomas account for 2.5% of gastric neoplasms. Although most of them are asymptomatic, patients may present upper gastrointestinal hemorrhage , 2. EndoTumorigenesis is the result of a multistep process characterized by the accumulation of genetic and epigenetic alterations leading to uncontrolled growth. Among epigenetic modifications, the most studied event in human neoplasms is the deregulation of methylation of DNA, giving rise to widespread changes in the methylome patterns during tumor progression . The epiGene expression profile studies have demonstrated that some genes are hypermethylated in gastric GISTs  –8, but, The objective of this study was to analyze by methyl specific-multiplex ligation probe amplification (MS-MLPA) the methylation status of tumor suppressor and DNA repair genes in a gastric leiomyoma. H. pylori infection 15 years before. No other constitutional symptoms were present. Upper gastrointestinal endoscopy was performed, showing an ulcerated submucosal tumor localized on the cardial region. Upper endoscopic ultrasonography (EUS) evidenced a submucosal lesion, of about 50 mm with decreased echogenicity and homogeneous structure, and no necrotic areas. The lesion belonged to the muscular layer, and EUS suggested GIST . Multisided tomography revealed that the lesion was 5 cm and showed no evidence of other lesions in the abdominal area. The patient was scheduled for laparoscopic surgery and was discharged from the hospital eleven days after the operation.A 63-year-old Hispanic female presented with history of melena for two days. Upon clinical examination the patient mentioned that she was treated forThe histopathological diagnosis for the submucosal lesion was gastric leiomyoma, characterized by a proliferation of bland, spindle-shaped cells with elongated nuclei and eosinophilic fibrillary cytoplasm without necrosis and atypia . The mithttp://www.mlpa.com/) on DNA obtained from the formalin fixed embedded tumor. The methylation status of 84 CpGIs in 41 cancer related genes was assessed . To analyze whether methylation affected gene expression, we performed qRT-PCR assays on 2 cell lines (MDA-MB231 and MCF-7) which presented different percentages of APC promoter methylation ; we confirmed that the methylation of APC on the CpG site −21 nt before TSS reduces gene expression (data not shown). The methylation of MLH1 at 382 nt before TSS has been previously shown to provoke downregulation of gene expression [MS-MLPA assay was performed by kits ME001, ME002, ME003, ME0024, and ME011 according to manufacturer's, MRC-Holland, Amsterdam, The Netherlands, instructions (assessed . We have and APC . As a copression . MLH1 methylation is associated with microsatellite instability (MSI) in sporadic endometrial and colorectal cancers [Given that cancers , 12, we  cancers , 14. IntThe onset and progression of tumorigenesis involves a cascade of genetic and/or epigenetic events. Results from recent investigations have shown that DNA methylation profiles contain tumor type-specific signatures which, in the future, could serve as biomarkers for clinical outcome . Hyperme MLH1 or APC) were the first ones to be epigenetically altered during tumoral progression. Considering that MLH1 presents a high methylation percentage, we hypothesize that its methylation could be an initial epigenetic event during gastric leiomyoma formation.The methylation percentage in the overall sample varied widely (from 9.5% to 77%) for the different genes . This wi MLH1 methylation is associated with MSI in several tumors [Given thatl tumors , 12, we l tumors . Therefol tumors . MLH1, MSH3, MGMT, and MSH6 participate in DNA repair functions whereas APC gene participates in cell proliferation. The methylation of MLH1, MGMT, and APC has been previously described in GISTs, but to the best of our knowledge this is the first time that the methylation of these genes has been associated with gastric leiomyoma, which is considered a benign disease. Interestingly, we also found that there were differentially methylated genes between GISTs and the gastric leiomyoma. For example, in a study performed by House et al., the authors determined that the most frequently methylated genes in GISTs (in decreasing frequency) are MGMT, p16, RASSF1A, E-cadherin, and MLH1  [ MLH1 and MGMT, GISTs also presented methylation in MINT2, p73, p16, E-cadherin, MINT1, p15, and MINT3. Moreover, the methylation of RASSF1A progressively increased from small to malignant GISTs and p16 was specifically methylated in malignant-prone and malignant GIST [ RASSF1, p16, nor p73 is methylated in the studied gastric leiomyoma. These epigenetic differences between malignant GISTs and gastric leiomyoma have not been described before.The genes found methylated in the gastric leiomyoma play different functions in the cell:ant GIST . Note thTo the best of our knowledge this is the first report showing the methylation profile of a gastric leiomyoma. Further research should be conducted to identify reliable and accurate molecular markers that could help in the differentiation between GISTs and gastric leiomyomas.


Japanese diets contain a relatively high amount of carbohydrates, and its high dietary glycemic index and glycemic load may raise the risk of diabetes in the Japanese population. The current study evaluated the associations between the dietary glycemic index, glycemic load, and the risk of type 2 diabetes in a population based cohort in Japan.We observed 27,769 men and 36,864 women (45–75 y) who participated in the second survey of the Japan Public Health Center-based Prospective Study. The dietary glycemic index and glycemic load were estimated using a food-frequency questionnaire. The development of diabetes was reported in a questionnaire administered five years later, and the associations were analyzed using logistic regression after controlling for age, area, total energy intake, smoking status, family history of diabetes, physical activity, hypertension, BMI, alcohol intake, magnesium, calcium, dietary fiber and coffee intake, and occupation.P-trend = 0.01). The association was implied to be stronger among women with BMI < 25 than the women with BMI ≥ 25. The dietary glycemic index was positively associated with the risk of diabetes among men with a high intake of total fat: the multivariable-adjusted odds ratio comparing the highest vs. the lowest quartile was 1.46 . Among women with a high total fat intake, those in the first and second quartiles of the dietary glycemic index had a significant reduced risk of diabetes, compared with those in the first quartile who had a lower total fat level .The dietary glycemic load was positively associated with the risk of diabetes among women: the multivariable-adjusted odds ratio comparing the highest vs. the lowest quartile was 1.52 (95% CI, 1.13-2.04; The population-based cohort study in Japan indicated that diets with a high dietary glycemic load increase the risk of type 2 diabetes among women. Total fat intake may modify the association between the dietary glycemic index and the risk of type 2 diabetes among men and women. As carbohydrates are easily converted into glucose, and produce an insulin response generally , the conThe associations between dietary GI and GL and the risk of diabetes were pronounced in individuals with a low cereal fiber intake ,11,12. PThe Japan Public Health Center-based Prospective Study was launched in 1990 for Cohort I, and Cohort II was added in 1993. The study design has been previously described . BrieflyThere were 140,160 eligible subjects, and 113,403 of them responded to the questionnaire at the time of the baseline survey, yielding a response rate of 80.9%. Among the subjects who responded at the baseline survey, 77,540 (68.4%) responded to both the 5-year and 10-year follow-up surveys. Those who reported that they had been diagnosed as having diabetes or who reported a history of cancer, cerebrovascular disease, myocardial infarction, chronic liver disease, or renal disease at the baseline or 5-year survey were excluded from the analysis . Furthermore, subjects whose dietary information was missing or whose total energy intake lay outside the mean ± 3 standard deviations according to sex were also excluded . After these exclusions, the analysis included 64,633 eligible participants, consisting of 27,769 men and 36,864 women.At the 5-year survey, food frequency questionnaire (FFQ) containing 147 items was administered to assess the habitual diets over the past year . The valSake (Japanese rice wine), which contained 8.8 g of available carbohydrate per serving, was assigned the same GI as beer (GI = 66), as no GI value was available. GI values were also not available for four kinds of vegetables containing more than 3.5 g/serving of total carbohydrate: bitter gourd, chard, and loofah  as well as onion (which contains 3.42 g of available carbohydrate per serving). As the GI values were also not available for similar vegetables in the same family and it is unlikely that they would induce a significant rise in blood glucose [The GI values for single food items on the FFQ were derived based on available publications ,26. When glucose , we assi glucose .The development of newly diagnosed diabetes for 5 years, between the 5-year survey and the 10-year survey, was identified using the questionnaire. The validity of diabetes reported in the questionnaire was previously assessed for study participants in three districts of the study areas. Based on their medical records, 94% of the participants who reported a diagnosis of diabetes were confirmed to have diabetes among those whose medical records were available .All the analyses were performed for men and women separately, as the estimated prevalence of diabetes varies by sex in Japan . The chaFor each dietary variable, we assessed the risk using two models: one adjusted for age and study area, and the other adjusted for age, study area and potential confounding factors including log-transformed total energy intake, smoking status , family history of diabetes , physical activity , hypertension , BMI , intake of alcohol , energy adjusted intake of log-transformed magnesium, calcium, and dietary fiber, coffee consumption , and occupation . Metabolic equivalents per day was obtained to measure the level of physical activity from a questionnaire administered in the 5-year survey, and this measure has been previously validated . To testTable Table Table Table Since we observed a significant association between dietary GI and the risk of diabetes when stratified according to the total fat intake among men as well as a similar, but non-significant, association among women, we conducted an post-hoc analysis to assess the joint effect of dietary GI and total fat intake on the risk of diabetes by cross-classifying participants according to these variables Figures  and 2. AIn this large scale population based cohort study, a higher dietary GL was associated with an increased risk of diabetes among women. The dietary GI was also implied to be associated with the risk of diabetes among women, but the association was attenuated and no longer significant after controlling for potential risk factors of diabetes. This is the prospective study conducted among men and among women in the general Japanese population.A recent study among Japanese male workers reported that the dietary GI was significantly associated with an increased risk of diabetes , but a sThe dietary GL was positively associated with the risk of diabetes among women in the current study. A few studies in Europe have even observed a protective effect of dietary GL on the risk of diabetes in certain models ,34, but 2 had a positive association between dietary GL and the risk of diabetes, whereas the association was weaker among those with a higher BMI. The association with dietary GI was also implied in the same group, albeit non-significantly. Several studies have reported an increased risk of diabetes in individuals with a higher dietary GI or GL among subjects with a high BMI [The stratified analysis shows that women whose BMI was less than 25 kg/mhigh BMI -14. Onlyhigh BMI ,15, and high BMI . As insuhigh BMI ,39. The high BMI -14.A positive association between the dietary GI and the risk of diabetes in both men and women was suggested among individuals with a high level of total fat intake, but not among those with a lower level of total fat intake. Although no previous epidemiological studies have assessed the effect of dietary fat intake on the association between the dietary GI and the risk of diabetes, the findings from human experimental studies have indicated that the consumption of potato, one of the highest GI food items, together with margarine, butter, or corn oil reduced the blood glucose response, compared with the consumption of potato by itself -18. A hiThe current study has several limitations. The development of diabetes was determined using a self-administered questionnaire, the FFQ, which was not specifically designed to estimate the dietary GI or GL. Nevertheless, such FFQ has been utilized in several epidemiological cohort studies -11,14,40The current population-based cohort study in Japan observed an association between the dietary GL and the risk of type 2 diabetes among women. The dietary GI was also associated with the risk of type 2 diabetes among men whose total fat intake was higher than the median level. Further studies are needed to assess the association between dietary GI, GL and the risk of type 2 diabetes in populations with different habitual diets.GI: Glycemic index; GL: Glycemic load; FFQ: Food frequency questionnaire.The authors declare that they have no competing interests.ST, MI, TM and MN were responsible for the study conception and the design of the study. ST and MI acquired data. SO, AN and KK performed the analysis. SO, AN, KK, AG, MK, TM and MN made contributions to interpretation of data. SO drafted the manuscript. All authors revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.


Vacuoles are one of the most prominent organelles in plant cells, and they play various important roles, such as degradation of waste materials, storage of ions and metabolites, and maintaining turgor. During the past two decades, numerous advances have been made in understanding how proteins are specifically delivered to the vacuole. One of the most crucial steps in this process is specific sorting of soluble vacuolar proteins. Vacuolar sorting receptors (VSRs), which are type I membrane proteins, are involved in the sorting and packaging of soluble vacuolar proteins into transport vesicles with the help of various accessory proteins. To date, large amounts of data have led to the development of two different models describing VSR-mediated vacuolar trafficking that are radically different in multiple ways, particularly regarding the location of cargo binding to, and release from, the VSR and the types of carriers utilized. In this review, we summarize current literature aimed at elucidating VSR-mediated vacuolar trafficking and compare the two models with respect to the sorting signals of vacuolar proteins, as well as the molecular machinery involved in VSR-mediated vacuolar trafficking and its action mechanisms. Plant cells contain a variety of endomembrane compartments. Of these, the vacuole is the most prominent organelle, occupying up to 90% of the cellular volume in mesophyll cells. Two different types of vacuoles exist in plant cells: the lytic vacuole in vegetative cells and the protein storage vacuole (PSV) in seed cells ,2. Thesei.e., the Golgi apparatus, trans-Golgi network (TGN), and prevacuolar compartment (PVC) [Of these multiple routes, the most prominent employs multiple intermediate organelles, nt (PVC) ,6,7,8,9.nt (PVC) ,12,13,14nt (PVC) ,15. The nt (PVC) ,18,19,20+-ATPase VHA-a3, and the H+-pyrophosphatase AVP1 are transported via a Golgi-independent route [Alternatively, certain vacuolar proteins are transported directly from the ER to the vacuole via a Golgi-independent route ,22,23. Int route ,25. In pnt route . Tonoplant route .i.e., lytic vacuoles and PSVs, and we discuss the two models describing this process.In this review, we focus on the VSR-mediated trafficking of soluble proteins to the vacuole. For other routes of vacuolar trafficking, we recommend other recent reviews ,29,30,31Ipomoea batatas) and green fluorescent protein (GFP)), AALP:GFP , and Spo:amylase  [atvsr1atvsr4 double-mutant plants, a small amount of AALP is secreted into the apoplast in leaf tissues [atvsr single and double mutants , vacuolar trafficking capacity is reduced to varying degrees depending on the genotype, although these mutants do not show any noticeable defects in vacuolar trafficking in intact leaf tissues [atvsr1atvsr4 double-mutant plants was complemented by transient expression of AtVSR1 or AtVSR4, further confirming that these proteins are involved in lytic vacuolar trafficking. Their role in lytic vacuolar protein sorting has also been directly supported by numerous ways; ER-retained soluble PV72 (PV72-HDEL) caused accumulation of AtALEU in the ER in Arabidopsis transgenic plants [VSRs were originally identified as abundant proteins in the CCV fraction purified from plant extract . Subsequvacuole) ,19,34,35 tissues . In addic plants . Amy-spoc plants . In cultc plants .AtVSR1, significant amounts of 12S globulins and 2S albumins accumulate as precursors and are partially secreted into the extracellular matrix [et al. [atvsr mutants have smaller PSVs than the wild type, which may be due to the reduced levels of PSV proteins in the PSVs resulting from defects in PSV trafficking. However, single and double mutants of atvrs1, atvsr3, and atvsr4 do not show any obvious defective phenotype in their vegetative tissues.In addition, the role of VSRs in PSV trafficking has been confirmed by genetic studies. In a knock-out mutant of r matrix . Zouhar  [et al.  subsequeatvsr mutants, atvsr5atvsr6 double-mutant plants did not exhibit defective trafficking of protein to the two vacuoles (lytic vacuole and PSV) when two lytic vacuolar cargoes, sporamin:GFP and AALP:GFP, and two PSV proteins, 12S globulins and 2S albumins, were examined [While the physiological roles of these AtVSR isoforms have been elucidated, the roles of AtVSR5 and AtVSR6 are not yet known. These two proteins also localize primarily to the PVC in protoplasts, as does AtVSR1 ,38. Howeexamined ,35. The in vitro [When VSRs function as sorting receptors, one of their most important activities is the specific recognition of vacuolar proteins among the numerous organellar proteins that are simultaneously transported through the endomembrane compartments. Vacuolar proteins contain a specific sequence motif, the sorting signal, which is required for specific recognition by VSRs. The sorting signals of various vacuolar proteins are classified into two groups, sequence-specific vacuolar sorting signal (ssVSS) and C-terminal vacuolar sorting signal (ctVSS). The ssVSSs show a consensus sequence while the ctVSSs are poorly defined but generally composed of hydrophobic amino acids . ssVSSs,in vitro ,16. Consin vitro ,16,33. T2+ ion plays a crucial role in cargo binding to VSRs in vitro; cargo binding to VSRs is enhanced by high concentrations of Ca2+ [2+-binding motif, the epidermal growth factor (EGF)-like motif, at the luminal domain. Moreover, Ca2+-binding to the EGF-like motif is crucial for its interaction with other proteins [2+ is involved in the interaction between cargoes and the luminal domains of VSRs [2+ binding to the EGF-like motif contributes to cargo binding to the luminal domain.The mechanism underlying the specific recognition of sorting signals by VSRs has been elucidated at the molecular level. VSRs must bind to cargoes at the donor compartment and release them at the acceptor compartment. Ca of Ca2+ ,43,44,45proteins . Indeed, of VSRs . Howeverin vitro experiments have shown that the optimal pH for cargo binding to VSRs is pH 6–7, while they dissociate at pH 4 [in vitro result showing that cargo release from VSRs occurs at pH 4.0; thus other conditions, including the Ca2+ concentrations in the lumens of the compartments, may contribute to cargo binding to and release from VSRs during vacuolar trafficking.Another important factor for the interaction between cargoes and their receptors is pH. In animal cells, the lysosomal cargo receptor mannose-6-phosphate (M6P) receptor (MPR) binds to M6P  at the TGN and releases these cargoes at the late endosome . In this at pH 4 ,47. Curr at pH 4 ,51. HoweExtensive studies have been carried out investigating the molecular mechanisms of VSR-mediated vacuolar trafficking in various plant species. These studies have resulted in the proposal of two different models describing VSR-mediated vacuolar trafficking: VSR-mediated trafficking from the TGN to the PVC (Model I) and VSR-mediated trafficking from the ER to the TGN (Model II). These models are significantly different, particularly with respect to the locations of cargo binding to and release from the VSRs, as well as the use of carrier vesicles.vps29 mutant. Moreover, in the vps29 mutant plants, VSRs accumulate to the PVC even when anterograde trafficking is inhibited by LatB, an inhibitor of actin filaments [Model I closely resembles the models describing lysosomal and vacuolar trafficking in animal cells and yeast, respectively, whereas Model II appears to be specific to plant cells. One of the key differences in the two models is the localization of VSRs. In Model I, VSRs primarily localize to the PVC, while a significant proportion localize to the TGN. Indeed, immunogold labeling, immunostaining using anti-VSR antibody, and live cell imaging of GFP- or RFP-tagged VSRs have revealed that VSRs localize primarily to the PVC, with a significant proportion localizing to the TGN or trans-face of the Golgi ,53,54,55ilaments .vsr1vsr4 double mutant, atvti11, vps29, epsinr1, and ap1m2 mutant plants exhibit a defect in protein trafficking to the PSV and/or lytic vacuole. Of these proteins, EpsinR1 and AP-1 are monomeric and heterotetrameric adaptors of CCVs, respectively, thus raising the possibility that CCVs function as carriers during VSR-mediated vacuolar trafficking. Indeed, AtVSR1 interacts with EpsinR1. AP-1 may also have a direct interaction with VSRs since VSRs have an AP-1 binding motif, the YXXΦ motif, at their cytosolic tail. In fact, these proteins together with clathrin appear to form an interaction network at the TGN that is crucial for CCV formation [A large number of proteins function either directly or indirectly in VSR-mediated vacuolar trafficking. These protein factors include AtVTI11, EPSIN1 (renamed EpsinR1), AP-1, clathrin, actin filaments, and VPS29. Their physiological roles in VSR-mediated vacuolar trafficking have been confirmed genetically; like the ormation ,52,61. Iormation . Anotherormation , which iormation . Therefoormation ,18,63,64snx1-1 and snx2a-2snx2b-1 mutants. These results raise the possibility that retromer components localize to both the TGN and PVC, and they may not always function together [VSRs require additional molecules for their action. The majority of VSRs localize to the PVC. These VSRs must recycle to the TGN for cargo binding. In animal cells and yeast, retromer  is involved in the recycling of MPRs and Vps10p from the late endosome/PVC to the TGN ,65,66,67together . Indeed,together .vps29 mutant plants show defects in both PSV and lytic vacuolar trafficking [In addition, fficking ,77. A refficking . These rfficking ,18, raisfficking .mag1-1/vps29 mutant plants, which have a defect in retrograde trafficking of VSRs from the PVC to the TGN, vacuolar cargoes accumulate to the TGN or are secreted into the medium [2+ is an important cofactor for cargo binding to VSRs [2+ concentration was reduced from 1 mM to 10 μM, binding of the luminal domains of BP80 and AtVSR4 to a peptide containing the aleurain sorting signal dropped to 20% and 50%, respectively [2+ concentration for cargo binding to VSRs. The ER and Golgi contain 0.05–0.5 mM and 0.7–3 μM Ca2+, respectively [2+ concentration of the TGN is similar to that of its neighboring organelle, the Golgi, it is too low to enable a high level of binding of cargoes to VSRs. However, the exact concentration of Ca2+ at the TGN is unknown. In addition, pH also plays a critical role in the binding of cargoes to VSRs. Currently, the exact pH levels of the TGN and PVC in plants are not well established. Two recent studies reported conflicting data on the pH of these organelles. One study reported that the pH of the TGN and PVC are 6.3 and 6.2, respectively [2+ concentration, determines the binding of cargoes to VSRs. In addition, the homomeric interaction of VSRs may also contribute to the binding of cargoes because the effective concentration of the luminal domain involved in cargo binding is increased by this homomeric interaction [According to Model I, VSRs should bind to their cargoes at the TGN. Indeed, a large number of proteins involved in VSR-mediated trafficking localize to the TGN, including AtVTI11, EpsinR1, AP-1, and clathrin ,79. In ae medium . However to VSRs ,45,47. Wectively . If the ectively , whereaseraction .2+ concentration. However, additional studies are necessary to confirm the dissociation of cargoes from VSRs in the PVC.Another important question is how cargoes are released from VSRs at the PVC in Model I. In fact, there is no direct evidence for vacuolar cargo dissociation from VSRs in the PVC. A peptide containing an N-terminal sorting motif binds to VSRs optimally at pH 6–7 and dissociates at pH 4. However, according to two recent studies showing that the pH level of the PVC is 6.2 or 7 ,51, carget al. [2+ in the ER could be favorable for binding of VSRs to cargo proteins in the ER [et al. [et al. [Recently, a new model has been proposed for VSR-mediated vacuolar trafficking in plant cells. This model is based on several observations that cannot be explained by Model I. Niemes et al.  showed tet al. ,82; thusn the ER ,81. In a [et al.  found th [et al.  also proAnother important difference between the two models is the trafficking of vacuolar proteins from the TGN to the PVC. Model II proposes that the trafficking of vacuolar proteins from the TGN to the PVC occurs in a receptor-independent manner without any carriers. It has been proposed that the TGN is converted to the PVC via organelle maturation . Indeed,2+-dependent manner [2+-ATPase SPCA1 and Ca2+-binding protein Cab45 at the membrane and in the lumen of the TGN, respectively [eca3 mutant plants have a defect in transporting apoplastic peroxidases [2+, and Ca2+-ATPase at the trans-Golgi may lead to a reduction in Ca2+ concentration at the TGN, thereby resulting in the release of vacuolar cargoes from VSRs. Based on these possibilities, Robinson and Pimpl [2+-based vacuolar cargo sorting occurs at the TGN; thus, according to this model, soluble vacuolar proteins interact with VSRs at the ER and the cargo-VSR complexes are transported to the cis-Golgi via COPII vesicles together with secretory cargo proteins. At the trans-Golgi, secretory cargoes bind to the Ca2+-binding protein together with Ca2+-ATPase, while vacuolar cargoes remain in association with VSRs. Secretory cargoes are packaged into secretory vesicles at the “early” TGN, whereas vacuolar cargoes dissociate from VSRs at the “late” TGN due to the low concentration of Ca2+ and low pH achieved by the action of TGN-localized H+-ATPase. At the “late” TGN, ligand-free VSRs recycle back to the ER via retromer, and vacuolar cargoes travel to the PVC via maturation of the TGN into the PVC and, finally, to the vacuole [2+-based model in plants. On the contrary, there were several reports supporting the bulk flow secretion. For example, sporamin lacking the ssVSS and sec:GFP, a chimeric construct consisting of the leader sequence of binding protein (BiP) and GFP, were secreted into the apoplast [2+-binding property which is required by this model. Thus, the Ca2+-based model needs to be further tested in terms of both secretion and vacuolar transport in the future.Model II raises the question of how vacuolar proteins are sorted from secreted cargo proteins at the TGN if the cargoes are dissociated from VSRs at the TGN and the vacuolar cargoes are transported from the TGN to the PVC via maturation of the TGN into the PVC. In animal cells, it has recently been demonstrated that secretory proteins are actively sorted at the TGN in a Cat manner . This acectively ,89,90,91oxidases ,93. In aoxidases , which snd Pimpl  suggeste vacuole . Currentapoplast ,97. sec:2+ concentration at the Golgi, TGN, and PVC may also be crucial for explaining experimental results and designing future experiments.VSR-mediated trafficking of soluble vacuolar proteins through the endomembrane compartments has been studied extensively in plant cells. However, many questions remain unanswered. In fact, there are two different models for VSR-mediated trafficking of soluble vacuolar proteins in plant cells. One key issue is where cargoes are sorted by the receptors, VSRs, and then released from the VSRs. Another key issue is how cargoes are transported from the TGN to the PVC. Currently, it appears that more lines of evidence support Model I than Model II; however, further studies are necessary to exclude either of these two models or to support both of them. More direct evidence for cargo binding to, and release from, VSRs at their respective locations will be important for supporting both models. In addition, for Model I, more direct evidence is necessary for the involvement of clathrin at the TGN. For Model II, little information is currently available about the molecular machinery involved in VSR-mediated trafficking of soluble vacuolar proteins; thus the identification of these molecular factors will provide additional support for this model. Information about the exact pH and CaAnother intriguing question about the role of VSRs in vacuolar trafficking is how they are involved in trafficking proteins to the lytic vacuole and PSV in both leaf and seed cells. Lytic vacuolar proteins and PSV proteins have the sorting signals ssVSSs and ctVSSs, respectively. Thus, further studies are necessary to elucidate how VSRs function in both pathways with two different types of sorting signals.


In group C, the pH, BB, BE, sO2, pO2, hemoglobin (Hb) level, serum Na+ concentration, LDH activity and TP level were significantly reduced, and the pCO2, serum Cl− concentration, alanine transaminase activity, TBIL and urea levels were significantly increased (P<0.05) compared with those in group A. The Hb and ALP levels in group C were significantly lower than those in group B (P<0.05); and the TP, TBIL and urea levels in group C were significantly higher than those in group B (P<0.05). Pathological observation revealed that the alveolar wall was hyperemic, edematous and incrassate, the alveolar epithelium was hyperplastic and infiltrated with neutrophilic granulocytes and the alveolar septum was widened; brain neurons were edematous with enlarged perivascular spaces, and hippocampal neurons were metamorphic and karyopyknotic; and kidney mesangial cells were hyperplastic, both following acute exposure to high altitude and after returning to low altitude. In conclusion, blood gas indices, biochemical indicators and functions of the heart, liver, kidney were significantly changed, and marked pathological changes occurred in the brain, liver and kidney following acute exposure to high altitude and also after returning to low altitude. These changes are likely to seriously affect the pharmacokinetics of drugs.High altitude environments cause the human body to undergo a series of pathological, physiological and biochemical changes, which have a certain effect on drug pharmacokinetics. The objective of the present study was to observe changes in factors affecting pharmacokinetics in rats following acute exposure to high altitude and return to low altitude. A total of 21 male Wistar rats were randomly assigned to three groups. The rats in group A were maintained at low altitude in Shanghai, 55 m above sea level; those in group B were acutely exposed to high altitude in Maqu, Gansu, 4,010 m above sea level; and those in group C were acutely exposed to high altitude and then returned to low altitude. Blood was collected from the orbit for the analysis of significant biochemical indicators and from the abdominal aorta for blood gas analysis. Brain, lung and kidney tissues were removed to observe pathological changes. In group B, the pH, buffer base (BB), base excess (BE), total carbon dioxide content (ctCO Hypoxia is a very important factor affecting the health and life activities of individuals at high altitude, which has serious impacts on physiology and induces pathological changes in the body . There iIn the present study, the physiological and pathological effects in Wistar rats of acute exposure to a high altitude of 4,010 m followed by a return to low altitude were investigated in order to identify the associations between high altitude and drug pharmacokinetics.An automatic blood gas system , automatic biochemistry analyzer , high speed centrifuge , hand-held GPS  and electron microscope  were used.A total of 21 healthy and clean male Wistar rats  were used in the study. The rats weighed 200±20 g and all originated from a low altitude area. They were randomly divided into group A, which was maintained at low altitude ; group B, which was acutely exposed to high altitude  and group C, which was taken to high altitude and then back to low altitude (n=7 per group). This study was approved by the Ethical Committee of Lanzhou General Hospital of Lanzhou Command .The rats in group A were normally fed at low altitude  and had an average weight of 203 g. The rats in group B were acutely exposed to high altitude , where they were fed normally for 72 h, and had an average weight of 198 g. The rats in group C were fed normally during acute exposure to high altitude  for 72 h, and then returned to low altitude where they were normally fed for a further 24 h; these rats had an average weight of 207 g. The rats were transported by aviation in a semi-enclosed polypropylene cage. In each group, examination of the rats began after fasting for 12 h.2), oxygen saturation of arterial blood (sO2), carbon dioxide tension of arterial blood (pCO2), oxygen tension of arterial blood (pO2), hemoglobin (Hb) level, sodium ion concentration (cNa+), potassium ion concentration (cK+) and chloride ion concentration (cCl−).In this study, 1 ml blood was taken from the abdominal aorta following anesthetization by the injection of 1 ml 10% chloral hydrate into the peritoneal cavity. Blood gas analysis was carried out immediately using the automatic blood gas system. The indicators measured were pH, buffer base (BB), base excess (BE), content of total carbon dioxide  and were then stained with hematoxylin and eosin. Pathological changes were observed under the electron microscope.All data are presented as the mean ± standard deviation. Analysis of statistical significance was performed, and P<0.05 was considered to indicate a statistically significant result. An analysis of variance (SNK-q test) was used to perform multiple comparison between the three groups. The analysis was carried out using SPSS software, version 13.0 .2, sO2, pO2 and cNa+ values of group B were significantly decreased by 2.43, 630, 311, 11.48, 91.38, 76.22 and 2.82% respectively, while the pCO2 and cCl− values significantly increased by 47.40 and 6.76%, respectively. In group C, the pH, BB, BE, sO2, pO2, Hb and cNa+ values were significantly decreased by 3.24, 542.00, 296.00, 92.89, 89.46, 32.32 and 4.20%, respectively, while the pCO2 and cCl− values were significantly increased by 75.49 and 4.25%, respectively. The Hb level of group C was decreased by 25.82% compared with that in group B.The results of the blood gas analysis are presented in 2 and pO2 were significantly reduced while ctCO2 was significantly increased following the acute exposure of the rats to high altitude.Statistical analysis demonstrated that the pH, BB, BE, sOBiochemical indicators are main indicators for evaluating the function of major organs. The biochemical indicator results are presented in The results show significant pathological changes in the alveoli among the groups. In Blood gas analysis indicates that the blood pH was significantly decreased when the rats were acutely exposed to high altitude and also when the rats returned to low altitude from high altitude. When the blood pH is low, acidic drugs dissociate less  and the 2 and sO2 levels were similar to those in rats acutely exposed to high altitude. This may due to deadaptation , which seriously affects the metabolism and excretion of drugs. In the present study, there was no significant difference in K+ concentration between the three groups. As for Cl−, in rats that were exposed to high altitude and then returned to low altitude, the Cl− concentration increased compared with that of rats maintained at low altitude. Serum chloride plays a part in the synthesis of gastric acid   . In addi+ levels.The results of the pathological examinations revealed that at high altitude, the alveolar walls were hyperemic, edematous and incrassate while the alveolar epithelium was hyperplastic with infiltrative neutrophilic granulocytes. The alveolar septa were widened. This suggests that oxygen exchange in the lungs becomes difficult, which is consistent with the blood gas analysis results. These pathological changes did not recover after the rats returned to low altitude from high altitude, which explains why there is no significant difference in the results of blood gas analysis, with the exception of K+ was significantly decreased, which suggests that Na+ flowed into cells abundantly under anoxic conditions and led to cellular edema, capillary occlusion, and hypoxia of the microcirculation. As a result, pathological examination revealed pulmonary edema and cerebral edema, which is consistent with the blood gas analysis results. In conclusion, the blood gas index and pathological features of the rats were changed significantly at high altitude, which is likely to seriously affect drug absorption, distribution, metabolism and excretion and ultimately the therapeutic effects and adverse reactions of drugs.At high altitude, the concentration of serum Na− were observed in the present study. The study conducted by Mei et al , clearance (CL) and area under the curve (AUC) associated with the drug metabolism and excretion and are thought to have a significant effect of the therapeutic and side effects of drugs , peak time (Tmax), peak concentration (Cmax), half life (tof drugs . The deg


We also observed an indirect effect of exercise behavior on eating pathology (r = 0.16) through EXD . In females the total variance of eating pathology explained by the SEM model was 9%. In males we observed a direct effect between EXD  on eating pathology. We also observed indirect effect of exercise behavior on eating pathology (r = 0.11) through EXD . In males the total variance of eating pathology explained by the SEM model was 5%.The purpose of our study was to study the prevalence of exercise dependence (EXD) among college students and to investigate the role of EXD and gender on exercise behavior and eating disorders. Excessive exercise can become an addiction known as exercise dependence. In our population of 517 college students, 3.3% were at risk for EXD and 8% were at risk for an eating disorder. We used Path analysis the simplest case of Structural Equation Modeling (SEM) to investigate the role of EXD and exercise behavior on eating disorders. We observed a small direct effect from gender to eating disorders. In females we observed significant direct effect between exercise behavior ( Excessive exercise can become an addiction known as exercise dependence (EXD). EXD occurs when the person is preoccupied with exercise, has withdrawal symptoms upon cessation, continues to exercise when socially or medically contraindicated, and when exercise interferes with relationships and work ,5,6,7,8.American Psychiatric Association in the Diagnostic and Statistical Manual of Mental Health Disorders (DSM-V), EXD as a disorder on its own, has not been officially categorized [Studies have been conducted on the prevalence of EXD and eating disorders. However, there is debate on the methodologies, diagnosis cut off points, and the relationship between the two ,11,12,13egorized . Secondaegorized . Researcegorized .et al. suggests that true vegetarians and vegans appear to be the healthiest in regards to weight and eating and semi-vegetarianism is the most likely to be related to disordered eating [Eating disorders are psychological disorders that involve a persistent disturbance in eating patterns or other behaviors intended to control weight, body size or shape. Eating disorders affect physical and nutritional health, psychological functioning and if untreated, they can be fatal. According to mental illness classification system, there are three categories of eating disorders: anorexia nervosa, bulimia nervosa, and Eating Disorders Not Otherwise Specified (EDNOS), which include binge-eating disorder . Geneticd eating .In summary, EXD is a problematic behavior, which can affect individuals with eating disorders. A review of previous research indicates that EXD could possibly mediate the relationship between exercise behavior and eating pathology ,10, but Therefore, using a nonclinical adolescent ethnically diverse sample, with a wide range of dietary preferences, the goal of this cross-sectional observational study was to (i) report the prevalence of EXD among college students (ii) to investigate the role of EXD and exercise behavior on eating disorders and (iii) to observe the role of gender on eating disorders.With the approval of the University’s Institutional Review Board, students in various undergraduate and graduate classes on the campus of Andrews University were asked to complete a self-reported, anonymous, paper based questionnaire. The questionnaire was composed of four sections: 14 demographic and exercise behavior questions, a 31-item Food Frequency Questionnaire (FFQ), the Exercise Dependency Scale Test (EDS-21), and the Eating Attitudes Test (EAT-26). There were 567 participants who completed the survey; 47 subjects were disqualified because they were not between ages 18 to 25, three subjects were removed because of missing data leaving a study population of 517 , representing a wide range of ethnic backgrounds , class standing  and dietary preferences . Andrews University is a Seventh-day Adventist (SDA) institution of higher learning. SDAs represent a unique population because this religious group actively endorses a healthy lifestyle.The EDS-21 is based on the DSM-IV criteria for substance dependence. It provides the following information: mean overall score of exercise dependence symptoms, and the differentiation between: (a) at-risk for exercise dependence; (b) nondependent-symptomatic; and (c) nondependent-asymptomatic. The items on the scale are based on the following criteria: tolerance, withdrawal, intention effect, lack of control, time, reductions in other activities, and continuance. The scale involves 21 items on a Likert scale ranging from 1 (never) to 6 . The scale has acceptable test-retest and internal consistency reliability, content and concurrent validity ,26.The Eating Attitudes Test (EAT-26) consists of 26 questions on the 6-point Likert scale ranging from 1  to 6 (never). It is useful and valid for determining the prevalence of an eating disorder but not diagnosing the specific disorder. A score of greater than or equal to 20 is considered “at-risk” for an eating disorder. The EAT-26 has been validated and can be regarded as a reliable and valid instrument for screening for eating disorders ,27.Exercise behavior was ascertained using four questions  about the usual exercise habits of the participants. The questionnaire measured the frequency that an individual engages in aerobic, strength and flexibility exercises and the type of aerobic activities done most frequently. Each score was then converted into metabolic equivalents (METS)  and summed up to provide the estimated energy expenditure in METS/week .The FFQ was used to accurately ascertain the vegetarian status of the participants.p Values less than or equal to 0.05 were considered statistically significant.The data was analyzed using SPSS  for descriptive statistics and AMOS 7.0 statistical software for SEM. Structural Equation Modeling (SEM) is a statistical procedure that enables researchers to evaluate specific relationships hypothesized, and Path analysis is the simplest case of SEM where we do not include latent variables . Raw datIn our population, 3.3% of students were characterized as at-risk for EXD, 51.5% as nondependent-symptomatic, and 45.2% as nondependent-asymptomatic. The prevalence of at-risk (EAT-26 score > 20) for an eating disorder was 8%. The EAT-26 test includes four behavioral questions regarding possible eating disorder symptoms; 10% of participants admitted binging, 2.9% admitted vomiting, 3.7% admitted the use of laxatives, diet pills or diuretics and 2.1% admitted being treated for an eating disorder in the last six months. In our preliminary analysis we followed the general analytic consideration suggested by Baron and Kenny  to repliet al. [et al. .2 (Df = 3) 155.238, p = 0.000, CFI = 0.249, TLI = 0.502, RMSEA = 0.314) however significant effects were observed from the independent variables to the dependent.Initial structural model examining the direct effect of the METS, EXD and gender on eating pathology was tested by SEM techniques as it is shown in 2 (Df = 2) 12.499, p = 0.002, CFI = 0.948, TLI = 0.845, RMSEA = 0.101) showing a significant decrease in the chi-square value. r = 0.19, p < 0.001), METS  and EXD  on eating pathology. Finally there is an indirect effect of METS on eating pathology (r = 0.15) through EXD . The total variance of eating pathology explained by this model is 11%.Following an exploratory approach, an alternative model was developed using the same variables with an additional direct effect of METS on EXD, suggesting an indirect effect of METS on eating pathology. In this case, the model improved and was a good fit, (χ2 (Df = 0) 0.000, CFI = 1.000) with coefficients and the direct and indirect effects. Significant direct effect was observed between METS  and EXD  on eating pathology. Finally there is an indirect effect of METS on eating pathology (r = 0.16) through EXD . The total variance of eating pathology explained by this model is 9%.2 (Df = 1) 0.033, p = 0.856) with coefficients and the direct and indirect effects. Significant direct effect was observed between EXD  on eating pathology. There is an indirect effect of METS on eating pathology (r = 0.11) through EXD . The total variance of eating pathology explained by this model is 5%.The first goal of our study was to study the prevalence of EXD among college students, to investigate the relationship between METS, EXD, gender and eating disorders, and to replicate previous research . In our The second goal of our study was to investigate the role of EXD and exercise behavior on eating disorders. A mediator variable is one that explains the relationship between two other variables . The stuIn the next step, we proceeded to use the SEM method to test the effects of METS, EXD, and gender on eating pathology as an initial model . This mor = 0.19, p < 0.001) from gender to eating disorders  populations to clarify the relationship of exercise behavior and eating disorders.In conclusion in our population, 3.3% of students were characterized as at-risk for EXD, 51.5% as nondependent-symptomatic, and 45.2% as nondependent-asymptomatic. The prevalence of at-risk (EAT-26 score > 20) for an eating disorder was 8%; 10% of participants admitted binging, 2.9% admitted vomiting, 3.7% admitted the use of laxatives, diet pills or diuretics and 2.1% admitted being treated for an eating disorder in the last six months.In our population significant direct effect was observed between gender, METS and EXD on eating pathology and an indirect effect of METS on eating pathology through EXD.In our population of females, we observed a significant direct effect between exercise behavior and EXD on eating pathology and an indirect effect of exercise behavior on eating pathology through EXD. In males, we observed a significant direct effect between EXD on eating pathology and an indirect effect of exercise behavior on eating pathology through EXD. Given the limitations of the study, the different pathways observed in the SEM gender specific models between exercise behavior, EXD and eating pathology perhaps could indicate different treatment approaches for different genders.


Ramsay Hunt syndrome (RHS) is characterized by facial paralysis, inner ear dysfunction, periauricular pain, and herpetiform vesicles. The reported incidence in children is 2.7/100,000. The pathogenesis involves the reactivation of latent varicella zoster virus (VZV) in the geniculate ganglion of the facial nerve. The recovery rate is better in children than in adults. This paper discusses a 12-year-old girl with a rare case of peripheral facial paralysis caused by RHS and reviews the literature. In 1907, Hunt first described Ramsay Hunt syndrome (RHS), a disease characterized by facial paralysis, inner ear dysfunction, periauricular pain, and herpetiform vesicles . Only 10Congenital anomalies, trauma, otitis media, Lyme disease, metabolic and genetic factors, and malignancy of the temporal bone or parotid gland can cause peripheral facial paralysis in children. Herpes simplex virus (HSV-1), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus (HHV 6 and 7), and mumps virus can all cause acute peripheral paralysis. However, the etiology is unknown in most cases, resulting in a diagnosis of idiopathic peripheral facial paralysis or Bell's palsy. Bell's palsy is the most common peripheral facial paralysis in children, and it is reported to cause 24–70% of all cases of peripheral facial paralysis in children .Although RHS is the second most common atraumatic cause of facial paralysis, it is very rare in children. Hato et al. reported that RHS caused 16.7% of all peripheral facial palsies in children [This study presents a case of peripheral facial paralysis caused by RHS and discusses the current literature.The patient was a 33 kg, 138 cm-tall, 12-year-old girl. She came to the pediatrics clinic with a chief complaint of facial asymmetry. She first developed a sore throat, cough, and runny nose, and this was followed 2 days later by pain around the right ear and a vesicular rash on the right ear pinna and behind the ear. The facial asymmetry started 3 days after the periauricular rash. She presented to our clinic on the second day of facial paralysis. She had no audiovestibular complaints, such as vertigo, tinnitus, nausea, vomiting, or hearing loss. Her medical history included varicella infection at 5 years of age.The patient had Grade 4 peripheral facial paralysis according to the House-Brackmann (HB) classification . She hadBased on the clinical findings, RHS was diagnosed at this point. The patient was started on methylprednisolone  and oral acyclovir (800 mg/day) simultaneously. The methylprednisolone dose was tapered to 1 mg/kg at the beginning of the second week, tapered during the second week, and stopped on day 14. The acyclovir was continued for 5 days. Artificial tear drops were recommended to protect the cornea. Facial electromyography (EMG) was not required, as the patient's clinical picture improved by day 21. Cranial imaging was also not required, as the patient had no history of peripheral facial paralysis and no neurological manifestations.Herpes zoster oticus or Ramsay Hunt syndrome is a disease that affects cranial nerves 7 and 8. The acute peripheral facial paralysis is characterized by vestibulocochlear dysfunction and a herpetic rash around the ear pinna and outer ear canal. It is reported to be the cause of 2–10% of acute peripheral facial paralysis cases . The repThe primary VZV infection is varicella. The virus moves retrogradely and stays latent in the geniculate ganglion of the facial nerve, and the clinical picture of RHS presents after the reactivation of VZV there.Typically, the clinical picture begins with otalgia that lasts 1~3 days. Then, herpetic vesicles appear in the outer ear canal, tympanic membrane, mouth, and distal 2/3 of the tongue, which can also cause losing taste sensation. If the eighth cranial nerve is also affected, nausea, vomiting, vertigo, nystagmus, tinnitus, and hearing loss can occur . HearingThe disease can also present with only acute peripheral facial paralysis, without the vesicular rash. This condition, termed “zoster sine herpete,” tends to be misdiagnosed as Bell's palsy .To diagnose RHS in children, enzyme-linked immunosorbent assay (ELISA) serum anti-VZV IgG and IgM antibody titers are recommended . In our Starting steroids and antiviral agents in the early phase of the disease is recommended. Antivirals prevent VZV replication and facial nerve involvement, and the steroids prevent inflammation and edema. Murakami et al. examined the recovery of facial nerve function after starting treatment in the first 3 days, at 3–7 days, or later than 7 days and found that the recovery was best when treatment was started within 3 days of disease presentation . We starKim and Bhimani recommended starting oral acyclovir at a dose of 800 mg for 7–10 days and prednisone 1 mg/kg/day for 5 days, followed by weaning over the next 6-7 days . KinishiThe prognosis of the facial paralysis in RHS is worse than that in Bell's palsy, and only 10% of complete facial paralysis in RHS recovers completely . Furuta With this case report, we show that VZV reactivation can be a rare cause of acute peripheral facial paralysis in children aged 6–15 years and that early diagnosis and treatment have a positive effect on disease prognosis.


N-carbamoylputrescine is hydrolyzed to putrescine by N-carbamoylputrescine amidohydrolase . During the hydrolysis, consecutive nucleophilic attacks on the substrate by Cys158 and water lead to formation of putrescine and two by-products, ammonia and carbon dioxide. CPA from the model legume plant, Medicago truncatula (MtCPA), was investigated in this work. Four crystal structures were determined: the wild-type MtCPA in complex with the reaction intermediate, N-(dihydroxymethyl)putrescine as well as with cadaverine, which is a longer analog of putrescine; and also structures of MtCPA-C158S mutant unliganded and with putrescine. MtCPA assembles into octamers, which resemble an incomplete left-handed helical twist. The active site of MtCPA is funnel-like shaped, and its entrance is walled with a contribution of the neighboring protein subunits. Deep inside the catalytic cavity, Glu48, Lys121, and Cys158 form the catalytic triad. In this studies, we have highlighted the key residues, highly conserved among the plant kingdom, responsible for the activity and selectivity of MtCPA toward N-carbamoylputrescine. Moreover, since, according to previous reports, a close MtCPA relative from Arabidopsis thaliana, along with several other nitrilase-like proteins, are subjected to allosteric regulation by substrates, we have used the structural information to indicate a putative secondary binding site. Based on the docking experiment, we postulate that this site is adjacent to the entrance to the catalytic pocket.Putrescine, 1,4-diaminobutane, is an intermediate in the biosynthesis of more complexed polyamines, spermidine and spermine. Unlike other eukaryotes, plants have evolved a multistep pathway for putrescine biosynthesis that utilizes arginine. In the final reaction,  Fabaceae) family and nitrogen-fixing bacteria. Medicago truncatula (Mt), which is the source organism of the enzyme studied in this work, is a model legume plant. Legumes form special organs, root nodules, which are dwelled by nitrogen-fixing bacteria from Rhizobium genus. This extraordinary plant–microbe symbiosis allows legume plants to overcome limited nitrogen availability because rhizobia utilize atmospheric N2 and convert it into ammonia, which plants are able to assimilate. It has been demonstrated that the concentration of PAs affects both number and biomass of root nodules (Polyamines (PAs) are small, aliphatic, polycationic compounds that bear at least two amino groups. Although PAs are found in all domains of life, the most abundant PA in nature is 1,4-diaminobutane, commonly named putrescine (PUT), which, together with 1,5-diaminopentane, cadaverine (CAD), owes its name to the noxious odor of putrefying cadavers. More complexed PAs include, but are not restricted to, triamine spermidine (SPD), tetraamine spermine (SPM) and thermospermine. It has been shown that PAs are some of the key players during plant growth and development, as well as response to both abiotic and biotic stresses . To list nodules . Moreove nodules .For years, the molecular function of PAs was attributed only to their cationic character, that is, affinity to interact with negatively charged biomolecules, such as nucleic acids and proteins, and modulate transcription and translation . MeanwhiS-adenosylmethionine. There are two routes leading to PUT synthesis  (Arabidopsis thanliana (At), rely solely on this method (N-carbamoylputrescine (NCP) to putrescine by N-carbamoylputrescine amidohydrolase , with the release of carbon dioxide and ammonia. The latter enzyme, CPA from Medicago truncatula (MtCPA), is the subject of the studies presented here.In plant cells, the homeostasis of PAs is attained by regulation of their biosynthesis, catabolism, conjugation and transport . The eleigure 1) . One oris method . The argA. thaliana genome  has been examined by Escherichia coli cells, authors reported that AtCPA does not follow Michaelis–Menten kinetics, and its enzymatic activity can be described by Hill equation. The reported Hill coefficient, equal 2.2, indicated positive cooperative binding. The maximal velocity, Vmax was calculated as 86 nanokatals per mg of protein, and half maximal velocity was achieved at 135 μM of NCP. Based on native gel electrophoresis, authors also show that AtCPA in solution forms homooctamers . cDNA was obtained with the use of SuperScript II reverse transcriptase (Life Technologies) and oligo dT (15 and 18) primers. PCR with the primers (Forward: TACTTCCAATCCAATGCCATGGCGGAAGACAAGGGTAGAAAAGTA and Reverse: TTATCCACTTCCAATGTTATCACAGAACAGGATTTTTGCCGTCCAAA) and cDNA as a template was performed to obtain the coding sequence of MtCPA open reading frame. The PCR product was incorporated into pMCSG68 vector  by ligase-independent cloning , transformed with the vector, were used for overexpression of the protein. Culture was carried out to the A600 of 1.0 at 37°C in LB media with the addition of ampicillin (150 μg/ml). The culture was cooled to 18°C before induction. Then, IPTG  was added to the culture. The culture was grown for 20 h and then cooled to 4°C. Cells were pelleted by centrifugation at 3500 × g for 20 min. After the supernatant removal, cells were resuspended in 35 ml of the binding buffer  and frozen at -80°C. Thawed culture was subjected to sonication to disrupt the cells. Bursts of total 4 min duration, with appropriate intervals for cooling, were applied. Disrupted cells were pelleted by centrifugation at 25 000 × g for 30 min at 4°C. The supernatant was used to retrieve the His6-tagged MtCPA protein on the column packed with 5 ml of HisTrap HP resin , which was connected to the Vacuum Manifold (Promega). After application of the supernatant, column was washed five times with 40 ml of the binding buffer. The elution of MtCPA was performed with 20 ml of elution buffer . His6-tagged TEV protease was added to the sample to the final concentration of 0.1 mg/ml to cleave the His6-tag. The sample was dialyzed overnight at 4°C against the buffer: 50 mM Tris-HCl pH 8.0; 500 mM NaCl; 1 mM TCEP. HisTrap HP resin was used to remove the cleaved His6-tag and the His6-tagged TEV protease. Final purification was performed by the size exclusion chromatography on HiLoad Superdex 200 16/60 column , equilibrated with the buffer: 50 mM Tris-HCl pH 8.0, 200 mM NaCl and 1 mM TCEP. The column was connected to the AKTA FPLC system (Amersham Biosciences).BL21 Gold MtCPA-C158S mutant was obtained by introduction of the C158S mutation to the vector used for overexpression of wild-type MtCPA, according to the Polymerase Incomplete Primer Extension method  to 20 mg/ml, as determined by the absorbance measurement at 280 nm, with the extinction coefficient of 38640. Initial crystallization trials were performed by sitting drop vapor diffusion method using the crystallization robot (Mosquito). Conditions were established for MtCPA as the 64th reagent of PEG/Ion Screen HT (Hampton research). Both proteins were then crystallized manually by hanging drop method in the conditions containing 20% PEG3350 and 8% Tacsimate at pH 7.0 (Hampton Research) or 20% PEG3350, 8% Tacsimate at pH 7.0 and 10% glycerol (GOL). Crystals with the best morphology were obtained by streak seeding.Both, MtCPA with N-(dihydroxymethyl)putrescine (DHMP) and MtCPA-C158S with PUT were obtained by incubation of the unliganded proteins with PUT  for 15 min. Samples were then centrifuged at 17 000 × g before crystallization setup. The complex of MtCPA with CAD was formed using the same protocol, with the final 17 mM concentration of CAD. Crystals were cryoprotected by transfer to the solution containing 20% PEG3350, 8% Tacsimate at pH 7.0 and 25% GOL, supplemented with the appropriate ligand.Complexes of XDS  22-ID beamline at the Advanced Photon Source, Argonne National Laboratory, USA. The diffraction images were processed with XDS ; for detMtCPA were processed to the resolution of 2.65 Å. The online version of BALBES, a molecular-replacement pipeline  as the asymmetric unit. In each of the structures presented herein, the octamer-flanking monomers  present higher degree of flexibility than the inner subunits of the octamers. This is most probably due to lack of intermolecular contacts, which allows for higher conformational fluctuations. As a result, the electron density maps for the flanking protein subunits  were not clear enough to model ligand molecules, and the ligands were placed for 12 out of 16 protein chains (B–G and J–O).The polypeptide chain of Figure 2). Therefore, many conclusions, inferred from the presented structures, may be extrapolated to other CPA enzymes. The octameric quaternary structure in the crystal lattice of MtCPA is in agreement with the assembly reported, based on native gel electrophoresis, for the very close ortholog from A. thaliana  . The helical octamer surrounds a central void of 20 Å diameter. MtCPA octamer can be divided into four symmetric dimers that are shifted by about 30 Å along the long axis of adjacent dimer(s). The gap between the first and the last dimer within the octamer is approximately 10 Å. The dimers present αββααββα topology, and are formed by two monomers of αββα sandwich fold, which is very characteristic for the members of nitrilase superfamily  on the inner side of the octamer. The C-terminal swap anchors the monomers to each other in an arm-to-arm manner, sturdily enhancing the monomer-monomer interactions. Furthermore, residues Asp295:Leu301 do not interact with a dimer mate, but instead increase the dimer-dimer interfaces in all but first (A/B and I/J) dimers in each octamer.CPAs show high degree of similarity, especially among plant species (ilarity) . Based oerfamily . To formMtCPA. The length of the swapped fragments varies among proteins with available structures from nitrilase family, from short swap of as little as 10-residue fragment   , through (DmβAS) .DmβAS , whereas the corresponding values for MtCPA are: 110, 20, and 45 Å, with the latter value being only theoretical because the pitch of an incomplete helix is virtual. Authors report that DmβAS can freely form higher helical oligomers by attachment of further dimers to the octamer. In contrast, the helix pitch of MtCPA is too small to accommodate another dimer , and the addition of further dimers would either create severe steric clashes or force significant structural rearrangements (uncoiling) of the octamer. Consistently, the flanking dimers of the MtCPA octamer are separated by only a 10 Å gap, much narrower than in the DmβAS octamer. In DmβAS, the gap between the first and last dimer is almost 40 Å, which is sufficient to accommodate additional dimer, necessary for the formation of the theoretically infinite helical assembly.In the PDB, one protein of the nitrilase family, DmβAS  . More interestingly, DHMP was covalently bound to the catalytic Cys158. There are two conceivable scenarios of the DHMP adduct formation during crystallization. It is possible that PUT reacts with carbon dioxide (or carbonic acid) in solution, outside the catalytic site of the enzyme, for it is known that primary amines react with CO2 in aqueous solution (N-carbamicputrescine (with carboxylic COO- group attached to one of the PUT amines) may enter the active site and, similarly to the NCP, most likely is susceptible to the nucleophilic attack of the Cys158. The DHMP covalent adduct is stabilized by placement of one of its oxygen atoms within the oxyanion hole, created by Nζ of Lys121 and the backbone N of Trp159 (see Active Site and the Reaction Mechanism). The other scenario of events, which might lead to the covalent enzyme-DHMP adduct in the crystallization drop, involves an initial reaction of carbon dioxide (or carbonic acid) with the active Cys158. To our judgment, however, this situation is much less likely for two reasons. Firstly, the covalent C-N bond in that case would need to result from a nucleophilic attack of N atom of PUT on the S-bound carboxyl C, which is very questionable. Secondly, if the second scenario was true, one would expect a similar state in the structure of MtCPA cocrystallized with CAD, because nothing should prevent the Cys158 carboxylation in the first place. In that instance, there would be not enough room to bind entire CAD molecule. This is not the case, and we do indeed observe CAD in the active site . It is, of course, possible that CAD molecules in solution also form CO2 adducts, but due to steric clashes (see Substrate Specificity), they cannot be recruited to the catalytic site, meanwhile non-modified CAD molecules can. CAD in MtCPA complex reaches deeper than PUT into the catalytic pocket, however, due to presence of sulfhydryl group of Cys158 it bends toward Glu132.The structures of wild-type solution . The resMtCPA without addition of any ligands was determined at 2.55 Å resolution (data not shown). Unfortunately, the electron density maps for the content of the catalytic site were unclear, and represented most probably disordered ingredients of the crystallization solution, GOL, and partially oxidized Cys158. Combined with the rather poor data resolution, we were not able to unambiguously interpret the content of the active site of “unliganded” wild-type MtCPA, and we do not report this structure herein. Instead, we decided to construct the MtCPA-C158S mutant. Substitution of sulfhydryl with hydroxyl group preserved binding properties of residue 158 but strongly diminished its reactivity. The structure of unliganded MtCPA-C158S, determined at 2.39 Å resolution, presented significantly lower noise in the electron density maps around the active site when compared to the unliganded wild-type MtCPA. This improvement allowed to identify the ligand bound inside the active site as GOL . However, GOL does not penetrate deep inside the pocket and presents no direct interactions with either of the residues from catalytic triad. Instead, it is hydrogen-bonded with the side chain oxygen of Glu187.The crystal structure of wild-type MtCPA-C158S mutant cocrystallized with PUT clearly shows the ligand without any modifications . In this case, the absence of nucleophilic sulfur prevents from the formation of covalent bond between DHMP and residue 158. In fact, PUT in MtCPA-C158S complex does not interact directly with the residue 158, but creates water-mediated hydrogen bond with the hydroxyl group of Ser158 instead.The crystal structure of MtCPA monomer contains a cavity, which is accessible from the outer surface of MtCPA octamer and, in its depth, comprises the active site. In order to present the active sites in a clear manner, we divide the MtCPA cavities to four parts, each buried deeper in the protein core: (i) the entrance preceding the genuine active site; (ii) the tail amine-binding region, which binds the non-reacting amine group of PA; (iii) the non-polar section, interacting with the ligand aliphatic fragment; and (iv) the catalytic triad, which performs the reaction. The cavities are in a very close proximity to the neighboring protein subunits, with the exception of the outer monomers A, H, I, and P that only have a single neighbor. The entrances to the funnel-like catalytic cavities are actually shaped not only by the amino acids from the protein subunit to which a particular site belongs, but are also fenced by protein chains of its immediate neighbor(s) . Moreover, each dimer-mate subunit surrounds a larger fragment of the active site entrance than the protein chain from the neighboring dimer.Each ∗ (asterisk indicates a residue that belongs to dimer-mate subunit), Cys276∗, Asp126 and Pro128 are responsible for the negative potential localized around the active site . Most probably, they attract the positively charged NCP, when it penetrates its way through the cavity. Walls of the middle section of the active site are composed of the side chains of: Tyr130, Tyr54, Pro125, Trp159, Trp162, Ile184, Ala183, and by Trp277∗ from the swapped C-terminal fragment . In this manner, MtCPA provides the hydrophobic milieu of the central section of the active pocket. This fragment is, therefore, perfectly adapted to bind the aliphatic fragment of the substrate, NCP by hydrophobic interactions.Glu187, Asp190, together with several carbonyl oxygens of Trp277MtCPA of Glu48 (proton acceptor), Lys121 (proton donor) and Cys158 (nucleophile). These three residues are placed deep inside the narrow part of the active site. The nucleophilic Cys, as in any other nitrilase-like protein, is positioned at the apex of the nucleophile elbow  (3) diaminothioalcoholate intermediate . The generated oxyanion is stabilized by the oxyanion hole, formed by Nζ of Lys121 and backbone N of Trp159, similarly to the case reported for Pyrococcus abyssi nitrilase (PaNit) . Then, most probably, the water molecule tethered to O𝜀 of Gln161 and N𝜀 of Trp162 is added to the intermediate, and, in an apparently concerted manner, C-S and C-N bonds are cleaved, which results in the release of PUT and the second by-product, HCO3-.The hydrolysis of NCP by (HpAmiF) , but aft (PaNit) . SubsequAt . All bound amines, CAD, PUT and DHMP, present the same hydrogen bond between terminal amine group and the side chain oxygen of Glu187. Additionally, these amino groups create two water-mediated hydrogen bonds with carbonyl oxygens of Asp126, Trp277∗, and mediated by two waters hydrogen bonds with Pro128 and Cys276∗. Glu187, conserved in CPA enzymes, prevents from substrate mismatch, and stabilizes substrate position within the active site. Moreover, the position of negatively charged Glu187 at the active site entrance determines both the charge and length of the bound substrate. Longer or shorter molecules than NCP, would not be able to create a hydrogen bond with the oxygen of Glu187, and/or would collide with other parts of the active site.We addressed the question of structural features that are responsible for such narrow substrate specificity of CPA enzymes. The comparison of the product (PUT), its longer analog (CAD) and the intermediate (DHMP) bound within the active site of MtCPA/CAD complex, we can assume that longer substrates than NCP, such as N-carbamoylcadaverine, would not obtain stable conformation inside the active site. Although CAD is able to interact via hydrogen bond with Glu187 at the entrance of the pocket, similarly to PUT and DHMP, deep inside the cleft CAD passes by the Sγ of Cys158 and bends toward the pair of glutamates. Thus, this would not leave enough space for the carbamoyl moiety of such longer substrate. In other words, the proper orientation of N-carbamoylcadaverine, with the preservation of the hydrogen bond with the Glu187 side chain is impossible due to steric clashes either with the catalytic triad or with the residues in the middle section of the pocket.The pocket presents very narrow and highly hydrophobic middle section, which excludes bulky substrates form binding. It also limits the spectrum of possible substrates to those which have an unbranched aliphatic fragment of four carbon atoms. From the L-lysine, L-arginine, which would mimic the substrate. Unfortunately, their complexes with neither wild-type MtCPA nor its C158S mutant allowed to unambiguously identify any other ligand of interest within the active site, except for those presented in this paper.The commercial availability of NCP is extremely limited, therefore, during our studies, we attempted to obtain complexes with other ligands, such as urea, sarcosine, A. thaliana does not follow the Michaelis–Menten kinetics. Instead, in the excess of substrate, an accelerating mechanism is triggered. Since PAs are linked with the plants response to stress factors, this phenomenon may actually be significant in vivo. The positive allosteric regulation was found also in other nitrilase-like proteins, β-alanine synthases . The substrate is situated in the niche with the carbamoyl moiety aiming at the entrance to the active site. In the predicted alternative binding site, NCP, would form three hydrogen bonds with Asp194, His198 and Glu248∗. The calculated binding energy of the secondary binding site is -3.6 kcal mol-1, whereas the corresponding value for the genuine catalytic cavity equals -5.8 kcal mol-1. It is, therefore, suggestive that the substrate affinity is significantly lower in the alternative binding site, nonetheless, still within a reasonable range.The architecture of close neighborhood of the catalytic site shows a solvent/cytoplasm accessible niche near the substrate binding pocket. In every structure presented herein, this niche is occupied by a GOL molecule. Considering the fact that in the per se, it would mitigate the delay needed for the substrate diffusion into the active site. By far, it is merely a speculation, and many additional experiments are required to validate this hypothesis, however, it is worth to mention that a standby substrate position was reported, e.g., for tyrosine phosphatase from Selenomonas ruminantium , such as high salinity, drought and/or thermal extrema, could provide a conceivable answer. Assembling into higher oligomers undoubtedly increases enzyme’s tolerance to adverse conditions, for a smaller portion of the protein surface is exposed toward the cytoplasm and, therefore, is less susceptible to changing environment. A similar rationale was anticipated to explain oligomerization of other abiotic-stress-related plant proteins, which form ring-like decamers, δ1-pyrroline-5-carboxylate reductases  and Glu187, placed at the entrance, the key residue responsible for the proper substrate orientation inside the active site. The catalytic triad at the bottom of the cavity is composed of Glu48, Lys121 and Cys158. Glu48, the proton acceptor, in the cooperation with Glu132 enhances the nucleophilic character of Cys158 and maintains the proper charge of Lys121, the proton donor. Lys121, together with backbone N of Trp159 forms the oxyanion hole, which stabilizes the intermediate state during NCP hydrolysis. Cys158, placed at the apex of the nucleophile elbow, formed in MtCPA by β7-turn-η6 motif, is responsible for the nucleophilic attack on the substrate, and creation of the covalent intermediate-enzyme complex. The catalytic water molecule, which performs subsequent nucleophilic attack on the intermediate, is stored within the active site in a small niche, near the catalytic triad. This water is hydrogen-bonded to the side chains of Gln161 and Trp162. The reaction leads to the breakage of two covalent bonds of the substrate, which ends with the release of PUT, ammonia and carbon dioxide. Presented here indication of the key residues, responsible for specificity and activity of MtCPA, sheds new light on CPA enzymes overall. Any future in-depth physiological study of CPAs can be more rational, as the research can benefit, or even directly arise from the structural knowledge presented herein.ards NCP , and theMtCPA and its C158S mutant confirmed high affinity of their active sites for ligands, which were either absorbed from crystallization solution (GOL), or intentionally added to obtain MtCPA complexes (PUT and CAD). Interestingly, in the complex of the wild-type MtCPA, obtained by the cocrystallization with PUT, the reaction intermediate, DHMP, was covalently bound to the active Cys158, inside the active site instead of PUT. DHMP adduct was most probably formed as the result of the reaction of the amine group of PUT with carbon dioxide, absorbed by the crystallization drop, followed by nucleophilic attack of the active Cys158. This showed that the oxyanion hole, next to the MtCPA catalytic triad, is able to stabilize the otherwise labile geminal diol. It would be very interesting to see whether a similar scenario could be relevant in planta, that is, if CO2 to a significant extent reacts with PUT in the cytoplasm and (as carbamic acid) enters the catalytic cavity of MtCPA. If so, the enzyme activity would be modulated by CO2 concentration, which would give another link between PAs and plant stress response.The crystallographic analysis of the complexes of MtCPA, however, is not able to change the oligomerization state, as its octamers are formed in a very coiled manner. We examined MtCPA structure to indicate a secondary substrate-binding site. The analysis led to designation of the conserved (in MtCPA structures) GOL binding location as the potential alternative substrate binding site. This hypothesis was supported by docking experiment, which indicated that the substrate may indeed bind also in a niche, with carbamoyl moiety of the docked NCP partially overlapping the GOL binding site. This venue is very close to the entrance of the active site, but separated from the catalytic triad. However, with present data we are not able to unambiguously determine the activity-accelerating mechanism linked to the alternative binding site. We may suppose that MtCPA might not be allosterically regulated per se, but rather own a site, where the substrate can standby, prior to entering the catalytic site. In that case, the turnover rate of the enzyme would be increased, as the delay needed for the substrate to diffuse in the cavity would be alleviated if it was bound right next to the entrance. This hypothesis, however, needs to be verified using parallel functional and structural approaches, nonetheless, it sketches a perspective for future studies of CPA enzymes. From the physiological perspective, accelerating mechanism is very beneficial, as it triggers increased enzyme activity when it is most needed, bypassing regulatory mechanisms of transcription and translation. During abiotic and biotic stresses as well as in the course of nodulation, when the demand for PAs is aggravated, plant can produce sufficient amount of PUT without engaging the protein synthesis machinery to deliver more molecules of CPA, but instead simply providing more NCP.Several members of nitrilase superfamily have been previously reported to exert enzymatic mechanism suggesting cooperative binding. Due to lack of a candidate secondary substrate-binding position, for most of them this behavior was associated with changes of the oligomerization state. MtCPA/DHMP complex, 5H8I; MtCPA/CAD, 5H8J; MtCPA-C158S mutant, 5H8K; MtCPA-C158S/PUT, 5H8L.Coordinates and structure factors of the related structures were deposited in the PDB: BS produced the protein and solved the crystal structures. BS, MR, and ZD analyzed the data. MM performed docking calculations. MR and BS wrote the manuscript.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.


To the Editor: Zika virus, a member of the Flaviviridae family, is primarily transmitted through Aedes spp. mosquitoes, and evidence of vertical, sexual, and blood transmission of Zika virus has been reported , headache, and dizziness on January 28. He subsequently had rash, chills, retro-orbital pain, and mild diarrhea on February 2, the day on which he departed from Caracas and traveled to Jiangxi Province via Paris and Hong Kong, arriving in Shenzhen, China, on February 5. At the time of hospital admission, the patient had fever (36.7°C), headache, conjunctivae, rash on his back and face, retro-orbital pain, and mild diarrhea. General clinical examination was unremarkable. Results of a complete blood cell count and liver function tests were within reference ranges.4 copies/mL), and Zika virus RNA was detected from urine samples collected on days 10, 11, and 12 .Serum samples were collected at day 9 and day 10 after symptom onset, and urine samples were collected once a day from day 10 through day 14. In the 2 serum samples, no dengue virus (DENV) or chikungunya virus (CHIKV) IgM or IgG were detected by a Panbio IgM and IgG capture ELISA for DENV  or by an indirect immunofluorescence assay slide test kit for CHIKV . Serum and urine samples were negative for DENV nonstructural protein 1 (NS1) antigen on an NS1-ELISA test kit . To detect virus RNA in samples, in-house–designed probe and primers specific to DENV and CHIKV were used  on serum and urine samples. A 1,813-bp of partial genome sequences  was obtained from urine and was used for comparing with selected other strains from GenBank. Phylogenetic analysis showed that the virus was of Asian lineage . PairwisAedes mosquitos, the risk for onward transmission of travel-associated Zika virus should not be overlooked.The clinical findings for the patient were similar to those previously reported among Zika virus–infected patients (