Metriocnemus knabi. Larvae of this species are found exclusively within the water-filled leaves of pitcher plants (Sarracenia purpurea) in a system that is naturally patchy at multiple spatial scales . Using generalized linear mixed models and multimodel inference, we estimated effects of the amount of habitat, patch size, interpatch distance, and patch isolation, measured at different spatial scales, on genetic differentiation (FST) among larval samples from leaves within plants, plants within clusters, and clusters within peatlands. Among leaves and plants, genetic differentiation appears to be driven by female oviposition behaviors and is influenced by habitat isolation at a broad (peatland) scale. Among clusters, gene flow is spatially restricted and aspects of both the amount of habitat and configuration at the focal scale are important, as is their interaction. Our results suggest that both habitat amount and configuration can be important determinants of genetic structure and that their relative influence is scale dependent.Despite strong interest in understanding how habitat spatial structure shapes the genetics of populations, the relative importance of habitat amount and configuration for patterns of genetic differentiation remains largely unexplored in empirical systems. In this study, we evaluate the relative influence of, and interactions among, the amount of habitat and aspects of its spatial configuration on genetic differentiation in the pitcher plant midge,  The abundance and distribution of habitat in a landscape  is one of the most influential factors driving species abundance patterns over space and time  may operate at unique scales Wiens . CurrentMetriocnemus knabi larvae are found exclusively within fluid-filled leaves of the purple pitcher plant, Sarracenia purpurea L., throughout patchy peatland habitats across eastern North America. The pitcher plant phytotelma represents an ecological microcosm used to address questions of population regulation, community interactions, and ecosystem processes . The abundance and distribution of leaves within pitcher plants, plants within clusters, and clusters within peatlands vary widely, such that various combinations of habitat amount and configuration occur naturally at each scale.Addicott . MultiplAddicott . Thus, tM. knabi is univoltine and adults emerge in late spring. Little is known about the adult stage, although adults are small (∼3 mm in length) and likely have weak flight abilities  with greater differentiation at the higher spatial scales , from different plants within clusters , and from different clusters within peatlands . Each plant, cluster, and peatland can be considered a replicate “landscape” from which we have sampled multiple habitat patches. At each scale, we estimate genetic differentiation among the sampled patches within each “landscape”, and relate measures of differentiation to select habitat amount and configuration metrics. Specifically, we focus on the size and distance among sampled habitat patches, which are key configuration metrics most commonly measured in genetic studies of habitat fragmentation . Individuals were genotyped at 11 microsatellite loci , thermal cycling, and fragment analysis protocols followed that of Rasic and Keyghobadi  and an infinite allele mutation model. The mean number of alleles (NA), observed (HO), and expected (HE) heterozygosities were calculated across loci and samples for each plant, cluster, and peatland using GenAlEx version 6.4.1 . The large sample size of System 2  exceeded the computational limit of the hierfstat package, so variance components were assessed by removing one peatland at a time and averaging the resulting values.A hierarchical analysis of molecular variance (AMOVA) was conducted to assess the distribution of genetic variation across all spatial scales in both systems. Variance components and hierarchical FST in GenAlEx . Similarly, at the cluster and peatland scales, FST was estimated among the three sampled plants within each cluster, and among the 3–5 sampled clusters within each peatland, respectively. At the cluster scale, only plants containing 9–15 individuals per plant  were included (n = 29 clusters). At the peatland scale, each cluster contained 27–45 individuals (n = 7 peatlands). These FST values were subsequently used as the response variables in models describing effects of habitat amount and configuration on genetic differentiation. Significance of FST was assessed using 9999 permutations and α = 0.05. Note that this response variable (FST) represents a node-based estimate of genetic differentiation. Thus, at the plant scale, a single value of FST was estimated among the three sampled leaves within each plant and the total sample size is the number of plants. This approach is different from most landscape genetic studies where the response variable is typically a pairwise measure of genetic differentiation. Unlike pairwise values, node-based estimates are not inherently interdependent  was measured as the length of the widest part of the pitcher vessel as this metric was a strong predictor of the leaf's potential volume . Patch size of plants (Spl) and clusters (Scl) were measured as the number of leaves per plant, and the number of pitcher plants per cluster, respectively (Table S3). At each scale, the sizes of the three sampled patches  within each study unit  were averaged to give a single patch size metric . As clusters of plants were centered on the point of highest plant density and tend to have indefinite edges, interpatch distance at the peatland scale was measured as the average distance among the centers of sampled clusters .At the plant scale, interpatch distance was measured in the field as the average distance among the three sampled leaves in each plant . We measured isolation of each cluster within the peatland as the number of plants within a 10-m-wide buffer around the cluster (Icl) by analyzing maps of interpolated plant-count data collected in 2009–2010 (Rasic and Keyghobadi Iptld) was measured as the distance from the center of the peatland to the center of the nearest neighboring peatland using Google Earth 6.2. Use of center-to-center peatland distances is justified by the small size of peatlands relative to the distances separating them (edge-to-edge distances are on average three times larger than patch radii) and a high correlation of center-to-center distances with edge-to-edge distances between peatlands (Pearson r = 0.974). Subsequently, models at the plant scale included isolation of each plant within its respective cluster (Ipl), isolation of that cluster within its respective peatland (Icl), and isolation of that peatland (Iptld), as predictor variables. Likewise, cluster scale analyses included Icl and Iptld as predictors, and peatland scale analyses included Iptld  and the average plant density in the sampled peatland . Peatland area was measured in ArcGIS using a combination of high resolution enhanced Forest Resource Inventory aerial imagery  and peatland (Aptld) were included in the model, while at the cluster scale, Aptld was included  in the local “landscape”  was quantified as the number of leaves per plant (FST) and predictor variables were constructed for each spatial scale (Table S3). At the plant scale, eight independent predictors were considered to be important habitat structure metrics: mean patch size of the sampled leaves (Slf), mean pairwise distance among the sampled leaves (Dlf), isolation of the plant (Ipl), cluster (Icl), and peatland (Iptld), and the amount of habitat in the plant, cluster, and peatland . The predictor variables Slf, Dlf, and Apl represent focal scale habitat metrics, whereas Ipl, Acl, Icl, and Aptld represent habitat structure at broader scales. At the cluster scale, the six predictors included: mean patch size of sampled plants (Spl), mean pairwise distance among sampled plants (Dpl), isolation of the cluster (Icl) and peatland (Iptld), and the amount of habitat in the cluster (Acl) and peatland (Aptld). Here, Spl, Dpl, and Acl are focal scale habitat metrics, while Icl, Iptld, and Aptld represent broader scale habitat variables. At the peatland scale, predictor variables included: mean patch size of the sampled clusters (Scl), mean pairwise distance among sampled clusters (Dcl), isolation of the peatland (Iptld), and the amount of habitat in the peatland (Aptld). As the effect of interpatch distance may depend on the amount of habitat in the landscape, interactions between mean pairwise distance among sampled patches and the amount of habitat at the focal scale were also included at each scale .Separate data sets containing the response  at each spatial scale was analyzed using generalized linear mixed models (GLMMs), fit using the lme4 package  were calculated and interpreted as the probability that a model explains genetic differentiation, where a top-ranked model with AICc two units less than the second-ranked model and wi > 0.9 was considered strong evidence in support of the best model (Burnham and Anderson w+[i]) was assessed for each predictor to identify the most important habitat metric at each scale by summing the Akaike weights of the target predictor across the models in which it was present. Model-averaged parameter estimates (R2m)GLMMGLMM. There were 512, 128, and 32 models, respectively, at the plant, cluster, and peatland scales. Models were ranked separately for each scale according to corrected Akaike information criterion values (AICn Bartoń  and AICcn Bartoń  in R (R n Bartoń . Marginahielzeth  when a sNA) at the plant scale ranged from 2.53 to 3.60, while the mean observed (HO) and expected heterozygosities (HE) ranged from 0.51 to 0.66 and 0.34 to 0.57, respectively (Table S8). At the cluster scale, NA = 3.70–4.87, HO = 0.48–0.64, and HE = 0.50–0.55 (Table S9). At the peatland scale, NA = 5.62–6.56, HO = 0.55–0.59, and HE = 0.53–0.56 (Table S10). The average number of alleles across loci and peatlands was 7.77 (SE = 0.99) in System 1 and 9.27 (SE = 0.99) in System 2. Observed and expected heterozygosity were not significantly different between the systems .A total of 1199 individuals were genotyped from 195 leaves, 65 plants, 29 clusters, and seven peatlands. For modeling habitat amount and configuration effects on genetic differentiation, at least three individuals per leaf were required for plant scale analysis, such that the plant scale data set consisted of 921 individuals . The cluster scale data set consisted of 1165 individuals , while all individuals were included in the peatland scale analysis (Table S10). One locus (mk80) was identified as potentially under positive selection and was removed from analyses. Based on 10 remaining microsatellites, the mean number of alleles  and decreased steadily in between-leaf and between-plant comparisons, but then increased slightly in between-cluster comparisons in all peatlands except SB  at all spatial scales, including within individuals. Values at the individual level are equivalent to inbreeding coefficients, and similar to Rasic and Keyghobadi . At the cluster scale, FST measured among the three plants within each cluster ranged from −0.0003 to 0.0871, and 37.9% of values were significant, whereas FST computed among clusters within peatlands ranged from 0.0037 to 0.0169, and 85.7% were significant .At the plant scale, c < 2 and Akaike weight (wi) ranged from 0.063 to 0.025 (Table S14). The cumulative sum of the Akaike model weights (0.339) among the top eight models suggests considerable model uncertainty. Nonetheless, all models within ΔAICc < 2 contained predictors for the isolation of the peatland in the surrounding system (Iptld), and model averaging indicated that Iptld had the highest relative importance with a model-averaged weight (w+[i]) of 0.916 (TableThere were no strong correlations between predictor variables in any of the data sets (Tables S11–S13). Within the plant scale model set, no single model had a high probability of being the “best”, as eight models were within ΔAICc < 2 and had a cumulative wi = 0.501 (Table S14). All models within ΔAICc < 2 contained isolation of the peatland (Iptld), which was assigned high relative importance (0.910) following model averaging (TableIptld) on FST at the cluster scale was positive.At the cluster scale, three models were within ΔAICing Table. The effScl), mean interpatch distance among sampled clusters (Dcl), amount of habitat in the surrounding peatland (Aptld), and an interaction between interpatch distance and amount of habitat in the peatland (Dcl:Aptld) was clearly identified as the best model GLMMGLMM( = 0.999). The second-ranked model had ΔAICc = 11.14. All of the predictors in the top model  had equally high importance (0.996–1.000), and their estimated effects on FST among clusters were all negative. Post hoc examination of the interaction between interpatch distance (Dcl) and amount of habitat in the peatland (Aptld) was conducted by dividing the data into high and low Aptld groups according to positive and negative standardized Aptld values, respectively, and assessing the relationship between FST and interpatch distance (Dcl) for each group. Genetic differentiation increased with interpatch distance when the amount of surrounding habitat was low (Pearson R2 = 0.581), but decreased with interpatch distance when the amount of surrounding habitat was high , should be an important determinant of observed spatial genetic structure among larvae at a fine scale (Anderson and Dunham The transition from fine (leaf) to broad (peatland) spatial scales involves a shift from predominantly individual to population level processes  and second highest hierarchical F-statistic values , samples were highly differentiated with the greatest proportion of significant ues Table. PreviouF-statistic values at the cluster scale in both peatland systems  are scaled up and broader scale processes (gene flow-drift) are scaled down, we might expect the magnitude of the effects of both sets of processes on genetic differentiation to attenuate. Consistent with this expectation, we observed on average the lowest genetic variance components and hierarchical ms Tables,2. OveraIptld), as the only important predictor of FST among leaves  size to be an important predictor of genetic differentiation at this scale. Leaf size is positively correlated with larval abundance  and interpatch distance (Dcl) were important predictors of genetic differentiation among clusters and that higher differentiation, which results from higher levels of drift, was associated with smaller patch size (TableAptld) and its interaction with interpatch distance (Dcl:Aptld) were important , while gene flow should decrease with increased patch distances. We did indeed find that patch size (ize Table. Similarize Table. Howeverant Table. This reant Table.Dcl:Aptld) must be interpreted cautiously given a limited number of data points, and considering that peatlands with low habitat amount were all from System 1 and those with high habitat amount were all from System 2. Further study is needed to determine whether the observed pattern is an artifact of sampling design and to establish more solidly the nature of interpatch distance and habitat amount interactions. Nonetheless, studies examining patch size and distance effects on the genetics of fragmented populations would likely benefit by also considering the amount of habitat in the landscape.For genetic data, we would expect a stronger positive effect of interpatch distance on genetic differentiation when the amount of habitat in the landscape is low; with increasing habitat amount, unsampled intervening patches that can act as stepping-stones for gene flow, and a larger regional effective population size, may dampen such a relationship. Consistent with this expectation, we did see greater genetic differentiation with increasing pairwise patch distance when the amount of habitat in the landscape was low Fig.. HoweverSpl) and interpatch distance (Dpl), which is consistent with the expectations that gene flow within clusters is not limited (Rasic and Keyghobadi Iptld) was the only variable identified as highly important, and the weight assigned to it, as well as the direction of its effect, was similar at the plant and cluster scales (TableAt the intermediate cluster scale, low importance was assigned to focal scale patch size (les Table. This suSimulation modeling experiments to quantify the relative influence of habitat amount and configuration on genetic structure conclude that habitat configuration is more important than habitat amount in predicting genetic differentiation and that patch characteristics are among the strongest individual predictors of genetic structure (Bruggeman et al. Control of habitat amount and configuration in natural systems is extremely challenging, and experimental manipulations are generally impractical for genetic studies because of the time lags required for genetic structure to respond to changes in landscape conditions (Landguth et al.


Calcification in areas of neuronal degeneration is a common finding in several neuropathological disorders including ischemic insults. Here, we performed a detailed examination of the onset and spatiotemporal profile of calcification in the CA1 region of the hippocampus, where neuronal death has been observed after transient forebrain ischemia. Histopathological examinations showed very little alizarin red staining in the CA1 pyramidal cell layer until day 28 after reperfusion, while prominent alizarin red staining was detected in CA1 dendritic subfields, particularly in the stratum radiatum, by 14 days after reperfusion. Electron microscopy using the osmium/potassium dichromate method and electron probe microanalysis revealed selective calcium deposits within the mitochondria of degenerating dendrites at as early as 7 days after reperfusion, with subsequent complete mineralization occurring throughout the dendrites, which then coalesced to form larger mineral conglomerates with the adjacent calcifying neurites by 14 days after reperfusion. Large calcifying deposits were frequently observed at 28 days after reperfusion, when they were closely associated with or completely engulfed by astrocytes. In contrast, no prominent calcification was observed in the somata of CA1 pyramidal neurons showing the characteristic features of necrotic cell death after ischemia, although what appeared to be calcified mitochondria were noted in some degenerated neurons that became dark and condensed. Thus, our data indicate that intrahippocampal calcification after ischemic insults initially occurs within the mitochondria of degenerating dendrites, which leads to the extensive calcification that is associated with ischemic injuries. These findings suggest that in degenerating neurons, the calcified mitochondria in the dendrites, rather than in the somata, may serve as the nidus for further calcium precipitation in the ischemic hippocampus. Brain parenchymal calcification is a well-known finding in various kinds of neuropathological disorders including brain ischemia –9. TheseTransient forebrain ischemia for 10 min causes the selective and delayed neuronal death of hippocampal CA1 neurons in the rat brain –19. CalcTherefore, we performed a detailed ultrastructural study of the onset and spatiotemporal progression of calcification in the CA1 region of the hippocampus during a 4-week survival period after transient forebrain ischemia. We used several different methods including conventional transmission electron microscopy (TEM), field emission (FE)-TEM with electron probe microanalysis, and alizarin red S staining for light microscopy, which is a sensitive and specific method for detecting calcium deposition , 22. To All experimental procedures were conducted in accordance with the Laboratory Animal Welfare Act, Guide for the Care and Use of Laboratory Animals, and Guidelines and Policies for Rodent Survival Surgery, and were approved by the Institutional Animal Care and Use Committee at the College of Medicine of The Catholic University of Korea . All efforts were made to minimize animal suffering and to reduce the number of animals used.ad libitum. They were maintained on a 12-h light/dark cycle. Transient forebrain ischemia was induced by the four-vessel occlusion and reperfusion method described by Pulsinelli and Brierley [Adult male Sprague Dawley rats 250–300 g) were used in this study. Animals were housed in groups of three per cage in a controlled environment at a constant temperature (22 ± 5°C) and humidity (50 ± 10%) with food (gamma ray-sterilized diet) and water (autoclaved tap water) available Brierley , with mi0–300 g wn = 8 per time point for the ischemic group; n = 5 per time point for the sham-operated group) were deeply anesthetized with 16.9% urethane  and sacrificed by transcardial perfusion with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains ware promptly removed, sectioned with a vibratome at 40 μm, and post-fixed with 1% osmium tetroxide (OsO4) and 2.5% potassium-dichromate  for 30 min at room temperature (25°C). After dehydration and embedding in Epon 812, areas of interest were excised and glued onto resin blocks. Ultrathin sections (70–90-nm thick) were lightly stained with uranyl acetate and observed with an electron microscope .Animals were allowed to live for 3, 7, 14, or 28 days after reperfusion. At each of the four time points following reperfusion, animals . The chemical compositions of the selected profiles were analyzed using FE-TEM . The microscope was equipped with Energy Dispersive X-ray Analysis (EDAX), which was installed at the Korea Basic Science Institute . The operative voltage was 200 kV.n = 3 per group). Sequential coronal cryostat sections (25-μm thick) were stained with 1% (w/v) alizarin red S  for 6 min or with 0.0004% FJB  in distilled water containing 0.01% acetic acid for 30 min according to the manufacturer’s protocol. After rinsing in distilled water, the sections were immersed in xylene and cover-slipped with the mounting medium. Sections stained with alizarin red were viewed with a microscope and photographed with a digital camera . Images were converted to the tagged image file format, and the contrast levels were adjusted using Adobe Photoshop v. 10.0 . Since the images were acquired with the same light intensity during microscopy and the same parameters for digital photography, only minor adjustments were made to establish uniform contrast settings for all figures. Slides stained with FJB were viewed with a confocal microscope .To define the relationship between ectopic calcium deposition and cell death, we employed the alizarin red S and Fluoro-Jade B (FJB) histochemical techniques. For this experiment, we used additional groups of rats, including sham controls and experimental rats at 3, 7, 14, or 28 days after reperfusion  male rats, which have been used previously by many researchers in a variety of experimental studies. However, several studies have demonstrated that age and sex have robust effects on stroke outcome and that cortical neurons exhibit sex differences in response to ischemic cell death –50. In tThe present results indicate that intrahippocampal calcification after ischemic insults initially occurs in the dendritic mitochondria of degenerating neurons, with subsequent propagation of the mineralization occurring throughout the dendrites, which then coalesce to form larger mineral conglomerates of adjacent calcifying neurites. In contrast, no prominent calcification was observed in the CA1 pyramidal cell layer, even though what appeared to be calcifying mitochondria were noted in some of the degenerating neurons that became dark and condensed but retained their compact ultrastructure. Collectively, these results suggest that the calcified mitochondria in the dendrites, rather than in the somata, of degenerating neurons, may serve as a nidus for further calcium precipitation in the ischemic hippocampus.


To analyze the efficacies of tumor curettage and resection for treatment of giant cell tumor of the bone (GCTB) around the knee joint (KJ).A total of 126 KJ-GCTB cases were treated at our department from August 2011 to February 2015. These cases were divided into two groups (A and B) according to treatment methods. Group A underwent tumor curettage, while group B underwent tumor resection.P>0.05), while the complication rate in group A was significantly lower than that in group B (P<0.05). In addition, the Enneking score for group A was significantly higher than that for group B (P<0.05); in addition, postoperative local recurrence, histopathological grading according to Jaffe, and radiographic imaging-based Campanacci’s staging positively correlated (P<0.05).The relapse rates did not differ significantly between the groups (Tumor curettage was the preferred surgical approach for patients with KJ-GCTB. Giant cell tumor of the bone (GCTB) was first described by Jaffe in 1940 as one of the common primary bone tumors; while its source remained uncertain, it may originate from the mesenchymal tissues of the bone marrow.3GCTB is typically considered to have low malignant potential. This disease commonly occurs in individuals aged between 20’s to 50’s, and is more common in women than in men.8A variety of clinical treatment methods is used for GCTB, among which tumor curettage and tumor resection are the most common.A total of 126 KJ-GCTB cases were treated at our department from August 2011 to February 2015. The patients included in the present study underwent imaging examinations , and theP>0.05). This study was conducted in accordance with the declaration of Helsinki after approval from the Ethics Committee of Yan’an People’s Hospital. Written informed consent was obtained from all participants.Group B had 28 men and 22 women, with a mean age of 35.9±9.3 years. Among them were 28 cases with tumor on the anterior tibial segment and 22 cases with tumor on the inferior femoral segment. The histopathological examination according to Jaffe revealed nine grade I, 27 grade II, and 14 grade III cases. The general characteristics, including age, sex, and tumor types, did not differ significantly between the two treatment groups  to inactivate the tumor cells. The degree of bone impairment was assessed, and artificial and allogeneic bone transplantation was performed for internal fixation in cases where the impairment might have affected the supporting role of the bone scaffold, compacted the graft, and covered the anterior support. If the affected site was close to the articular surface, the sub-articular surface was also filled simultaneously, and allogeneic bone with the cortical bone was used to cover the affected site in order to ensure cortical bone continuity.Group B underwent tumor resection, which is often used to treat patients with extensive bone destruction; the resection was performed in accordance with the standards of wide excision of bone tumor, and allogeneic bone with large articular surface was used for repair.All patients were followed up for more than six months; the treatment efficacies, relapse rates, and treatment risk factors between the two groups were analyzed. The postoperative functions were scored using the Enneking scoring system.2 test, and factors associated with treatment efficacies of GCTB were analyzed by logistic regression analysis, with P<0.05 considered statistically significant.SPSS Statistics for Windows, version 17.0 was used for statistical analysis of the data, and postoperative Enneking scores were expressed as±s and analyzed with t tests. Postoperative recurrence and complication rates were expressed as % and analyzed by χP>0.05).Group A had 22 cases of postoperative recurrence, corresponding to a recurrence rate of 25.3%. In comparison, while group B had 10 cases of postoperative recurrence and a recurrence rate of 20.0%, the recurrence rates did not differ significantly between the two groups (P<0.05).Group B had four cases of bacterial infection, three cases of graft reaction, and a complication rate of 14.0%. Group A had one case of bacterial infection and one case of graft reaction, corresponding to complication rate of 2.3%. The complication rate in group A was significantly lower than that in group B , P<0.05.P<0.05).The average Enneking score for group A (29.16±8.53), was significantly higher than that for group B (19.22±7.43) (P<0.05).Age, sex, onset time, and pathological grading were included into the statistics for the analysis; the analysis revealed that postoperative local recurrence, Jaffe histopathological grade (r=3.73) and tumor radial Campanacci’s stage (r=4.06) were positively correlated . Previous studiesP<0.05). Therefore, based on these findings, patients without aggressive features should undergo tumor curettage, while those with severe bone destruction, or when reconstruction after tumor curettage cannot meet the patient’s loading requirements, should undergo tumor resection.Tumor curettage and resection are two approaches often used for treatment of KJ-GCTB. Because tumor curettage retains the patient’s own cartilage surface and bone scaffoldP<0.05), and follow-up examinations revealed that the joint function scores of group B were significantly lower than those of group A, suggesting that tumor curettage had more pronounced treatment effects. The method for treatment of KJ-GCTB is generally selected according to patient specific performance and imaging findings.P<0.05). Therefore, patients with Jeffer’s pathological grade I, as well as tumor size and bone diameter less than 1/2 with relatively mild clinical manifestations should undergo tumor curettage. Similarly, patients with Jeffer’s pathological grade III and radiographic Campacci’s stage III, with relatively significant cancer-associated damage and whose cortical bone is easily invaded, should undergo tumor resection.Previous studies on tumor curettage have identified the windowing position to be critical, as the window should be large enough to adequately support the patient’s bone defects and be convenient for surgical procedures. The current methods for filling bone defects include autologous, allogeneic, and artificial bone, etc.; while autologous bone is currently the preferred choice, many patients have large bone defects for which the autologous bone is inadequate. In these situations, an allogeneic or artificial bone could accelerate bone repair.In short, a reasonable surgical approach for treatment of KJ-GCTB should consider the tumor location, the degree of damage, and range of the tumor. Tumor curettage offers effective tumor removal without affecting supporting bone. Therefore, this method should be considered the preferred surgical approach, while tumor curettage should be considered for patients with severe bone destruction, or for whom the loading requirements could not be met after reconstruction.In the treatment of patients with giant cell tumor around the knee joint one should select the right surgical method. Tumors curettage can not affect bone support effective removal of the tumor, surgery is the preferred way. The main defect is that their tumor recurrence rate is higher. After tumor resection of large lesions, the activity of the joints is poor, but the postoperative local recurrence rate is low. Hence keeping in view the actual situation one should choose the right surgical procedure.


Splanchnic venous thrombosis (SVT) includes thrombosis of the hepatic, portal, and mesenteric venous system. Myeloproliferative neoplasms (MPNs) are important factors of SVT in adults. Addition of JAK2V617F mutation in WHO criteria for diagnosis of MPNs has made this test a useful tool for diagnosis. JAK2 is an intracytoplasmic tyrosine kinase that plays a critical role in signal transduction from multiple hematopoietic factor receptors. The mutation is found frequently in patients with SVT; many such patients have no other manifestations of an MPN. Although the correlation of JAK2V617F mutation with thrombotic risk in MPNs has been shown in many studies, the impact of presence of additional thrombophilic factors in these cases is yet not known. As the management of MPNs remains highly dependent on the patient's thrombotic risk, it is important to assess the thrombotic risk factors in detail. Here, we report two cases of JAK2V617F positive MPN who also had other thrombophilic conditions and presented with recurrent thrombosis. Abdominal venous thrombosis represents a manifestation of unusual thrombosis and although rare, these thrombotic events are often severe, accounting for important morbidity and mortality. It may be associated with a wide spectrum of underlying disorders, either local or systemic. Local factors comprise abdominal infections or inflammations, cancer, cirrhosis, abdominal trauma, and recent surgery . The preµL, platelet count of 522 × 109/L, and differential count P 71, L20, M06, and E03. Contrast enhanced CT (CECT) of abdomen revealed thromboses of main and branches of portal vein with massive splenomegaly and normal liver architecture. USG Doppler of both lower limbs did not reveal any abnormality. In view of thrombocytosis with splanchnic thrombosis, sequencing for JAK2 was done which showed homozygous JAK2V617F mutation. Bone marrow examination showed moderately hypercellular marrow with megakaryocytic hyperplasia and predominance of hyperlobulated forms with clustering at places consistent with myeloproliferative neoplasm possibly ET  polymorphism, and prothrombin G20210A were not detected. Workup for lupus anticoagulant was negative. To rule out an acquired cause for protein S deficiency, family screening was done which showed her sister to be protein S deficient but she was asymptomatic. In this case, protein S was the hereditary thrombophilic factor which has amplified the risk of thrombosis.A 40-year-old female presented with dyspepsia, pain with discolouration of finger tips, pain in left leg, and abdominal pain for 20 days. There was no history of fever, jaundice, headache, bleeding from any site, and major surgery. On examination, liver was palpable 3 cm below the right costal margin and spleen was palpable 8 cm below the left costal margin with no significant lymphadenopathy. On investigations, complete hemogram showed Hb of 13 g/dL, WBC count of 10,900/sibly ET . Bcr-AblµL, and platelet count of 500 × 109/L. Her anemia was attributed to coexisting iron deficiency. CECT of abdomen showed thrombus in main, right, and left branches of portal vein with chronic thrombus in splenic and superior mesenteric vein with splenomegaly and grade 1 fatty liver. On further investigations, she was found to be heterozygous for JAK2V617F mutation. Her bone marrow examination showed hypercellular marrow with hyperlobulated megakaryocytes suggestive of MPN possibly ET. Bcr-Abl by RT PCR was negative. She was started on hydroxyurea and anticoagulation. Her INR was stable at 2.26–2.48. After 2 months she came with joint pain, swelling of left side of neck for 2 days which was painful and progressively increasing, and breathlessness. On examination, she had tachycardia and bilateral dullness on chest percussion which on on X ray of the chest was confirmed to be bilateral pleural effusion. Her USG of neck showed complete thrombus of left subclavian vein and partially thrombosed axillary vein. CT pulmonary angiography revealed thromboses of bilateral lower lobe segmental branches of pulmonary artery (right more than left) with a small peripheral wedge shaped infarct seen in right lower lobe. D dimer was 1 µg/mL (positive). She was started on low molecular weight heparin. In this case, also progression of thrombosis to distant sites despite being on adequate anticoagulation was not explainable. Therefore, we did thrombophilic workup after 6 weeks of the event and off anticoagulation, which showed protein C 82%, protein S 75%, and antithrombin 113%. Factor V Leiden, prothrombin G20210A, MTHFR polymorphism, and workup paroxysmal nocturnal hemoglobinuria were negative. Antiphospholipid IgM, anticardiolipin IgM antibodies, and confirmatory test for lupus anticoagulant (LA) were positive. ANA was also positive. Rheumatology opinion was taken and as per the disease activity, patient was planned to start on hydroxychloroquine.A 46-year-old female presented with pain and distension of abdomen for 1 month. She was a known diabetic on treatment. There was no history of fever, headache, jaundice, and bleeding from any site. On examination, mild pallor with spleen was palpable 2 cm below the left costal margin. Her complete hemogram revealed Hb of 10.7 g/dL, WBC count of 8800/The aim of reporting these two cases is to stimulate the large group study for defining the proper role of screening for additional thrombophilic factors in patients of JAK2 positive MPNs. Both patients were <60 years of age, one case was heterozygous, and the other was homozygous for JAK2V617F mutation. Both the cases were started on cytoreductive therapy and anticoagulation owing to high risk MPN. Despite the decrease in platelet count and spleen size with therapeutic INR, these patients had progression of thrombosis which prompted us to work up for other prothrombotic conditions which can exacerbate the thrombotic risk of JAK2V617F positive MPN.JAK2V617F mutation is identified in most patients with polycythemia vera (PV) 95%) and in a significant proportion of patients with ET (50–60%) and primary myelofibrosis (50–60%) . In an I5% and inAlso in a study by Vannucchi et al. the frequency of thrombosis at diagnosis was double in heterozygous ET patients compared to that in WT patients (21.7% versus 10.5%), although the difference did not attain the significance level; however, the difference reached the significance level if all mutant (heterozygous plus homozygous) ET patients were compared with a WT counterpart . WolanskThe impact of JAK2V617F on thrombotic risk likely extends beyond its effect on platelets. The presence of JAK2V617F mutant liver endothelial cells but not hematopoietic cells in patients with Budd-Chiari syndrome suggests that expression of JAK2V617F also directly affects nonhematopoietic cells to augment thrombotic risk . LiteratOne of our cases had protein S deficiency as an additional prothrombotic factor. A decrease in the free protein S is reported in patients with ET due to protein S cleavage by a protease from platelets  and it rThe second case was detected to have ET with antiphospholipid syndrome. This patient also presented with progression of thrombosis despite therapeutic INR which actually can be an overestimation of INR due to LA. Valle et al. have shown that INR determinations obtained with a recombinant PT reagent substantially overestimate the actual degree of anticoagulation of most LA patients and that this is due  assignment) to interference of lupus anticoagulant IgG in PT assays .We suggest that one should look beyond JAK2 in patients of MPNs who present with thrombosis of unusual vascular territory because multiple factors presented together can accelerate thrombus formation and also treatment modalities will differ in case of additional prothrombotic factors.


Pallister-Killian syndrome (PKS) is a sporadic genetic disorder caused by the presence of a tissue-specific mosaicism for isochromosome 12p - i(12) (p10) and is characterized by facial dysmorphism including coarse facies, upslanting palpebral fissures, bitemporal alopecia, pigmentary skin anomalies, developmental delay, hypotonia and seizures. Although typical clinical features of PKS commonly exist, clinicians often do not raise the possibility of this diagnosis.We reviewed the medical records of 10 patients with confirmed PKS followed in our service (since 1990 to 2015). Age at diagnosis varied from prenatal to 3 years and clinical features were consistent with those described in the literature. In all patients, peripheral blood karyotypes were normal and cytogenomic study was performed in order to confirm the diagnosis. Three of these patients had PKS diagnosis confirmed by buccal smear MLPA.An early conclusion from our results demonstrated that MLPA on buccal smears is a good and non-invasive method to detect extra copies of 12p and should be considered as the first exam, before a skin biopsy for a fibroblast karyotype is performed. Pallister-Killian syndrome (PKS) is a sporadic and rare chromosomal disorder caused by the mosaicism for isochromosome 12p – i(12)(p10). There are just over 200 cases described in the literature .Clinical findings of PKS are distinctive, especially the combination of coarse facies, pigmentary skin anomalies, developmental delay, hypotonia and seizures. The facial appearance includes coarse facies (coarseness becomes more pronounced with age), prominent forehead, hypertelorism, upslanting palpebral fissures with epicanthal folds, small nose with upturned nares, high arched palate, macrostomia, long philtrum, micrognathia, bitemporal alopecia and an extension of the philtral skin into the vermilion border of the upper lip, which is termed “Pallister lip”. Other associated findings include the following: congenital heart defects, diaphragmatic hernia, cryptorchidism, renal malformations, imperforate or anteriorly placed anus –4. PatiePKS is caused by a tissue-limited mosaicism for supernumerary i(12)(p10) , 8. AlthA fibroblast karyotype from skin biopsy is commonly used to achieve PKS diagnosis, but other tissues such as lung, tissue from a buccal smear, bone marrow and even amniotic fluid yield higher percentages of mosaicism i(12)(p10), which can help diagnose PKS –11.All ten patients had PKS confirmed by a fibroblast or amniotic fluid karyotype or by buccal smear MLPA. The presence of i12)(p10) in the fibroblast karyotype ranged from 50 % to 100 % and in amniotic fluid from 75 % to 100 % (Table 2(p10) inPeripheral blood karyotypes were normal in nine patients. In one who died at 5 days of age it was not performed but diagnosis was confirmed by amniotic fluid karyotype.Three patients, with clinical features of PKS, had buccal smear MLPA confirming the diagnosis. In two patients fibroblasts culture failed and MLPA was performed in buccal smear sample showing four copies of the short arm of chromosome 12 (p10), whereas analysis of skin and lung tissues that were obtained postmortem exhibited i(12)(p10) in 98.5 % and 97.5 % of the cells, respectively . More stWe used MLPA method for diagnosis with DNA prepared from buccal smear samples in three patients, with the detection of four copies of the short arm of chromosome 12, confirming the diagnosis of PKS. There are few reports of PKS diagnosis using MLPA and DNA from buccal smears in the literature. We recommend this analysis as the first exam, before a skin biopsy.MLPA assay is a recently developed technique that is able to detect variations in the genes copy number and dosage. This test is a high throughput analysis, allowing up to 96 samples to be handled simultaneously allowing the study of several regions of the human genome in a single reaction , 20.Due to this capability, MLPA can be applied in the molecular diagnosis of several syndromes since the duplication of entire genomic regions causes a disease due to the presence of extra copies of the genes, while complete or partial deletion can produce a completely different phenotypic effect .In fact, MLPA assay is one of the most widely used techniques for the molecular investigation of microdeletion/microduplication syndromes. The use of this technique has important advantages such as relative simplicity of approach, low cost, rapid turnaround, ease of multiplexing to permit high confidence in the results, high accuracy of copy number estimation, and the potential for combination of copy number analysis with other applications, such as methylation detection or SNP genotyping , 20.MLPA use target sequences, between 50–80 nucleotides, that applied to DNA extracted from a buccal swab allow identifying genomic aberrations, easier than detected by FISH. Over 300 kits are so far commercially available, dedicated to the study of several diseases .In situ Hybridization) whose have been previously described as alternatives to diagnose PKS, MLPA technique is less expensive and technically uncomplicated method [Finally compared to array CGH (Comparative Genomic Hybridization) and FISH  from 1990 to 2014. The study was approved by the by the ethics committee of the HCFMUSP (University of São Paulo-CAPPesq) and informed consent form was obtained from all families.in situ hybridization (FISH) with chromosome 12p specific subtelomeric probe  according to the technique of Pinkel [Peripheral blood karyotype was performed using a standard phytohemagglutinin-stimulated lymphocyte culture method followed by G-banding. Twenty metaphase cells were analyzed for all patients except one, who died at 5 days of age. Also, we performed a skin biopsy for fibroblast karyotype and the long-term closed flask fibroblast explant cultures were set up according to the protocol adapted from Rooney . Additiof Pinkel , with miIn three of the patients buccal smear was collected with the Oragene Kit  and DNA was extracted using the QIAamp DNA Blood Midi Kit 250  in order to perform Multiplex Ligation-dependent Probe Amplification  assay.RBBP2 and SLC6A12 respectively, determine the DNA copy number variations in 12p region and can provide a definitive diagnosis.MLPA with kits SALSA P070 and SALSA P036 that contain subtelomeric probes, Written informed consent was obtained from the parents of the patients.


Elevated levels of reactive oxygen species (ROS) are detected in 25% to 80% of infertile men. They are involved in the pathology of male infertility. Understanding the effect of increasing levels of ROS on the differential expression of sperm proteins is important to understand the cellular processes and or/pathways that may be implicated in male infertility. The aim of this study was to examine differentially expressed proteins (DEPs) in spermatozoa from patients with low, medium and high ROS levels.6 sperm) (n = 11), Medium (>93-500 RLU/s/106 sperm) (n = 17) and High ROS (>500 RLU/s/106 sperm) group (n = 14). All fertile men had ROS levels between 4-50 RLU/s/106 sperm. 4 subjects from fertile group and 4 each from the Low, Medium and High ROS were pooled. Protein extraction, protein estimation, gel separation of the proteins, in-gel digestion, LTQ-orbitrap elite hybrid mass spectrometry system was conducted. The DEPs, the cellular localization and pathways of DEPs involved were examined utilizing bioinformatics tools.A total of 42 infertile men presenting for infertility and 17 proven fertile men were enrolled in the study. ROS levels were measured by chemiluminescence assay. Infertile men were divided into Low  contains supplementary material, which is available to authorized users. Infertility is defined as the inability to achieve a pregnancy after one year of unprotected intercourse . Male faOxidative stress results from the imbalance between reactive oxygen species (ROS) and the antioxidants that neutralize these excessive free radicals . Oxidati6 sperm) in the ejaculate are considered normal in fertile men (unpublished work). We also found that infertile men and those with unexplained or idiopathic infertility have ROS levels that are significantly higher than 90 RLU/s/106 sperm (unpublished work).Elevated levels of ROS are detected in 25% to 80% of infertile men . InfertiProteomics is developing rapidly as a promising field in the assessment of male infertility. Novel tools developed in the last decade have helped identify more than 6000 different sperm proteins. In addition to identification of unique sperm proteins that may contribute to male infertility, quantitative proteomics can help to determine the physiological and pathological function of these proteins with respect to their cellular localization and biological processes . DiffereThe goal of this study is to determine the effect of increasing presence of ROS in the seminal ejaculate on the human spermatozoa proteome in male infertility patients. ROS levels in the seminal ejaculates of infertile men, categorized as low, medium and high were compared to healthy fertile men with physiological levels of ROS. To our knowledge, this is the first study that is aimed at understanding the impact of ROS levels on sperm protein alterations, biological functions and signaling pathways in infertile men.Following approval from the Institutional Review Board of Cleveland Clinic, semen samples were collected from male infertility patients with different oxidative stress levels (n = 42) as well as healthy men of proven fertility (n =17). All subjects provided written consent to be enrolled in this prospective study.Male infertility patients 20-40 years of age who presented for infertility were enrolled in the study from March 2012 to March 2014. All female partners of the infertile men had undergone gynecologic evaluation and had normal results on fertility assessment. Patients were excluded if they had a recurring fever in the 90-day period prior to semen analysis. Patients with leukocytospermia, azoospermia and oligozoospermia were not included in the study.6 WBC/mL when the round cell concentration was >1 X 106m/mL. This was confirmed by the peroxidase or the Endtz test [All specimens were collected by masturbation at the Andrology Laboratory after 2-3 days of sexual abstinence. Samples were allowed to liquefy completely for 15-30 minutes at 37°C and manual semen analysis was performed using a MicroCell counting chamber  to determine sperm concentration and motility. Smears of the raw semen were stained with a Diff-Quik kit  for assessment of sperm morphology according to WHO criteria . Sample dtz test .6 sperm [6 spermLow ROS group: ROS levels 0- < 93 RLU/sec/X 106 spermMedium ROS: ROS levels >93-500 RLU/sec/X 106High ROS group: ROS concentration > 500 RLU/sec/X 10ROS formation was measured by chemiluminescence assay using luminol  as the probe. Chemiluminescence was measured for 15 min using a Berthold luminometer . Results were expressed as relative light units (RLU)/sec/X106 sperm . ROS con6 sperm are required to give about 1.5 mg/mL of protein. 20 μL of protein are loaded in triplicate for each run. Therefore about 75 -100 μL of protein aliquot with a protein concentration of about 1.5 mg/mL is adequate. To accomplish this, we pooled 4 samples from each group.Pooling of samples is common in proteomic studies. However, it is important to have equal contribution of sperm from each pool. The number is based on the amount of proteins necessary to conduct the proposed measurement of sperm concentration and running of the gel for proteomic analysis. For normalizing the sperm and protein concentration needed for proteomic analysis, a minimum total sperm concentration of about 100 X 10For proteomic analysis, we pooled 4 subjects from the fertile group and 4 subjects each from the low, medium and High ROS group in the infertile men. Equal number of spermatozoa were pooled from patients in each group and washed with PBS three times. Once the supernatant was removed, the spermatozoa were solubilized in radio-immunoprecipitation assay (RIPA) lysis buffer  containing the proteinase inhibitor cocktail . After complete lysis of the spermatozoa, protein concentration was determined using a bicinchoninic acid (BCA) kit  and equal amounts of proteins was fractionated using SDS-Page 1D gel electrophoresis.Twelve bands were cut from a single Coomassie blue stained 1D gel and analyzed in triplicate. The bands were washed, reduced, alkylated, and digested with trypsin. The completely digested extracts were eluted on LC-MS system, and the CID spectra searched against the human reference sequence database. These samples were analyzed using an LC gradient from 2 to 70% acetonitrile in 120 minutes. All of these chromatograms contained several peaks indicating efficient digestion of the samples.Samples were mixed with SDS Page buffer and separated on a 1D gel. For the protein digestion, the bands were cut to minimize excess polyacrylamide, divided into a number of smaller pieces. The gel pieces were washed with water and dehydrated in acetonitrile. The bands were then reduced with dithiothreitol (DTT) and alkylated with iodoacetamide prior to the in-gel digestion. All bands were digested in-gel using trypsin, by adding 5 μL 10 ng/μL trypsin in 50 mM ammonium bicarbonate and incubated overnight at room temperature to achieve complete digestion. The peptides that were formed were extracted from the polyacrylamide in two aliquots of 30 μL 50% acetonitrile with 5% formic acid. These extracts were combined and evaporated to <10 μL in Speedvac and then resuspended in 1% acetic acid to make up a final volume of ~30 μL for LC-MS analysis .The LC-MS system was a Finnigan LTQ-Obitrap Elite hybrid mass spectrometer system. The HPLC column was a Dionex 15 cm × 75 μm internal diameter Acclaim Pepmap C18, 2 μm, 100 Å reverse phase capillary chromatography column. Five μL volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 0.3 μL/min were introduced into the source of the mass spectrometer on-line. The microelectrospray ion source is operated at 1.9 kV. The digest was analyzed using the data dependent multitask capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans.For semen parameters, comparison was made between fertile men and patients as well as fertile men and patients in each ROS group by Wilcoxon–rank sum test.Tandem mass spectra were extracted by Proteome Discoverer version 1.4.1.288. Charge state deconvolution and de-isotoping were not performed. All MS/MS samples were analyzed using Mascot , SEQUEST  and X! Tandem . Mascot, Sequest and X! Tandem were set up to search the human reference with database  assuming trypsin as the digestion enzyme. These searches were performed with a fragment ion mass tolerance of 0.8 Da, and a parent ion tolerance of 10 parts per million (PPM). Carbamidomethylation of cysteine was specified as a fixed modification, and oxidation of methionine was specified as a variable modification.To validate MS/MS-based peptide and protein identifications Scaffold  was used. Peptide identifications were accepted if they could be established at >95.0% probability by the Peptide Prophet algorithm  with ScaFor proteomic analysis, the relative quantity of the proteins was determined by comparing the number of spectra, termed spectral counts (SpCs), used to identify each protein. The total number of mass spectra (SpC) that matched peptides to a particular protein was used to measure the abundance of proteins in the complex mixture. Normalization of spectral counts using the NSAF  approach was applied prior to relative protein quantification. DEPs were obtained by applying different constraints for significance tests and/or fold-change cutoffs based on the average SpC of the protein from multiple runs.Very Low abundance: spectral count range 1.7-7; p ≤ 0.001 and NSAF ratio ≥ 2.5 for Upregulated, ≤ 0.4 for Downregulated proteinsLow abundance: spectral count range 8-19; p ≤ 0.01 and NSAF ratio ≥2.5 for Upregulated, ≤ 0.4 for Downregulated proteinsMedium abundance: spectral count range between 20-79; p ≤ 0.05 and NSAF ratio ≥ 2.0 for Upregulated, ≤ 0.5 for Downregulated proteinsHigh abundance: spectral counts >80; p ≤ 0.05 and NSAF ratio ≥ 1.5 for Upregulated, ≤ 0.67 for Downregulated proteinsAppropriate filters were used to identify DEPs that were dependent on the overall abundance of the proteins. It has been reported  that acchttp://david.niaid.nih.gov), and proprietary software package such as IPA  from Ingenuity® Systems, used to obtain consensus-based, comprehensive functional context for the large list of proteins derived from proteomic study.Functional annotation and enrichment analysis were performed using publicly available bioinformatics annotation tools and databases such as GO Term Finder , GO Term6 sperm, 17 had Medium ROS group levels >93 - 500 RLU/sec/106 sperm and 14 had high ROS levels >500 RLU/sec/106 sperm.Semen analysis results for fertile men, infertile patient group, and the ROS  groups are shown in Tables Among the different ROS groups, sperm concentration, motility, and morphology were significantly reduced in the high ROS group compared to the fertile group. Semen parameters in the other two ROS groups were comparable with the fertile group.The number of proteins identified from the LC-MS/MS analysis of spermatozoa from fertile men was 1337, from patients with Low ROS was 1297, Medium ROS was 1280 and High ROS was 1331 respectively  in fertile men and 1132 (87%), 1094 (85%), 1133 (85%) in the Low, Medium and High ROS groups respectively.two of the three replicates were 72 (5%) in fertile men and 107 (8%), 114 (9%), 125 (9%) in the Low, Medium and High ROS groups respectively.one of three replicates was 60 (4%) in fertile men and 61 (5%), 72 (6%), 73 (5%) in the Low, Medium and High ROS groups respectively.Some of the more abundant proteins found in the fertile group  ROS groups are shown in Figure 33 DEPs were identified as common to all 3  ROS groups when compared to the control group.In the Low ROS group: 132 DEPs were observed. Of these, 51 were unique, 29 were similar to Medium ROS and 19 were similar to High ROS.In the Medium ROS group: 131 DEPs were obtained. Of these, 47 were unique, 29 were similar to Low ROS and 22 were similar to High ROS.In the High ROS group: a higher number of DEPs (178) were expressed compared to the other two groups. Of the 178 DEPs, 104 were unique, 19 were similar to Low ROS, and 22 were similar to Medium ROS.The distribution of overexpressed (OE), underexpressed (UE), and unique proteins in each of the three categories  and the control group is shown in Figure Of the 132 DEPs in the Low ROS group: 37 were OE and 84 were UE respectively compared to the control group. 4 DEP were expressed only in the Low ROS group and 7 DEP were expressed only in the control group.Of the 131 DEPs in the Medium ROS group: 44 were OE and 66 were UE respectively compared to the control group. 9 DEP were expressed only in the Medium ROS group and 12 DEP were expressed only in the control group.Of the 178 DEPs in the High ROS group: 76 were OE and 80 were UE respectively compared to the control group. 15 DEP were expressed only in the High ROS group and 7 DEP were expressed only in the control group.The abundance of DEPs in the 3  ROS groups is shown in Figure High Abundance proteins: 25 proteins were differentially expressed in the Low ROS group, 20 in the Medium ROS group and 36 in the High ROS group.Medium Abundance proteins: 31 proteins were differentially expressed in the Low ROS group, 30 in the Medium ROS group and 52 in the High ROS group.Low Abundance proteins: 38 differentially expressed proteins were identified in the Low ROS group, 32 in the medium ROS group and 45 in the High ROS group.Very Low Abundance proteins: 31 differentially expressed proteins were identified in the Low ROS group, 37 in the Medium ROS group and 38 in the High ROS group.From the Reactome analysis of DEPs, distribution of a select set of pathway categories (either associated with or known to affect reproductive functions) in each of the three groups is shown in Figures A total of 37 proteins were overexpressed in this group  associated with key reproductive related functions. The differential expression of these proteins in the three ROS groups is shown in Table Male infertility is a multifactorial condition emerging from a wide variety of etiologies including gene mutations, malformation of reproductive organs, infectious disease, or environmental exposure to toxicants . In the Some abnormal sperm morphologies are genetically- determined while others are caused as a result of repeated physiological and environmental stresses. These abnormal morphologies are reversible; however, after repeated stress attacks, the testis may fail to fully recuperate and could result in a permanent decrease in number of sperm with normal morphology. These abnormalities can be explained by the effect of elevated level of oxidative stress in high ROS group. We have recently demonstrated the association of teratozoospermia with increased levels of ROS . Basic sROS has been shown to be an independent marker of oxidative stress. ROS levels can predict male factor infertility (MFI) with an accuracy of ≥80% . ROS-indHigh ROS levels are known to significantly affect proteins and lipids, induce apoptosis and cause DNA damage, leading to male infertility . Cells tThe proteome provides a dynamic understanding of post-genomic events and characterizing these events will further our understanding of the underlying functional changes in men diagnosed with various male-infertility-associated etiologies , 48. CurIn the present study, the Reactome software showed that DEPs in Low, Medium or High ROS group were involved in the metabolism of proteins, immune system, disease, transmembrane (TM) transport, extracellular matrix organization, signal transduction, post-translational modifications, and cellular response to stress. Figures  and 5. DThe role of membrane trafficking is well known in spermiogenesis. The sperm proteome is enriched in proteins that mediate membrane fusion and promotes the release of acrosomal contents. The proteins involved in the final sperm-oocyte fusion event are also present in the sperm proteome. Many of the overexpressed proteins are also involved in signal transduction, extracellular matrix organization and cellular response to stress.Many of the DEP that were underexpressed in the 3 ROS groups were also involved in the important pathways such as metabolism of proteins and lipids, signal transduction, biological oxidations, post-translational protein modifications, metabolism of proteins, transmembrane transport of small molecules, signal transduction, apoptosis, and cellular response to stress.Compared to the Low ROS group, there were proteins that were uniquely expressed in the fertile control group. These were involved in disease, apoptosis, biological oxidation, signal transduction and immune system. In the medium ROS group, the proteins that were uniquely expressed in the fertile group were involved in events such as metabolism, metabolism of lipids and lipoproteins, signaling by Wnt and by Robo receptor, hemostasis and signal transduction. Compared to the High ROS group, DEP that were uniquely expressed in the fertile group were involved in metabolism, electron transport from NADPH to ferrodoxin, synthesis of phosphatidylinositol phosphate (PIP) at the Golgi membrane, phospholipid metabolism, mitochondrial iron-sulfur cluster biogenesis, metabolism of lipids and lipoproteins as well as phosphoionositide (PI) metabolism that play a role in lipid signaling, cell signaling and membrane trafficking.The Reactome analysis also identified proteins that were uniquely expressed in each ROS group. Proteins that were unique to the Low ROS group were involved in Toll-like Receptor 9 (TLR9) cascade, toll-like receptor cascades, and innate immune system. These receptors mediate response to unmethylated CpG dinucleotides in bacterial DNA to trigger an immune response. The presence of these receptors may be indicative of leukocyte contamination in ROS generating semen samples. Proteins that were uniquely expressed in the Medium ROS group were involved in disease, gene expression, metabolism of proteins, toll-like receptors cascades, nuclear import of Rev proteins and both innate and adaptive immune system. Innate immunity is an inherent immunity (defense) that is already present at birth.Similarly in the High ROS group, a significantly high percentage of uniquely expressed proteins were involved in metabolism, electron transport from NADPH to ferredoxin, and metabolism of the phospholipids, lipids, lipoprotein synthesis of PIP at the Golgi membrane suggesting that these proteins were playing a key role in optimal spermatozoa function. On the other hand, in the patients within the High ROS group, proteins were unique and involved in cellular response to stress, signal transduction, metabolism of proteins, metabolism of lipids and lipoproteins suggesting that these proteins were operating under high oxidative stress conditions that was affecting the metabolic activity of the sperm and in signal transduction and response to stress.The most significant pathways in the male gamete are those involved in protein metabolism, membrane trafficking, apoptosis, cell cycle, hemostasis and meiosis. Developmental biology and extracellular matrix organization were also detected as putative sperm pathways but with lower probability. Signaling transduction is not a major pathway but some signaling pathways that are seen to be active include signaling by Wnt proteins, a family of secreted lipid-modified signaling glycoproteins . The oveWe also demonstrated that while oxidative phosphorylation is important for sperm function, the major pathway for energy metabolism was the glycolytic pathway. Majority of the proteins were involved in cellular metabolic and regulatory processes. We demonstrated in our earlier work that a number of proteins that were overexpressed or underexpressed in ROS+ group were involved in cellular processes, metabolic process, response to stress and transport. Furthermore, a small number of proteins were involved in post-translational processes and protein folding .DAVID software also identified a number of proteins that were involved in key functions in the 3 ROS groups. In the functional categories related to reproduction and/or spermatogenic events, 12 proteins were identified in the low ROS group, 109 in the medium ROS and 44 in the high ROS group that may be modified and were underexpressed compared to the fertile group. The various functional annotations are shown in Table 2+ ion binding, oxidoreductase, defense response, protein transport, etc. whereas other proteins involved in signal peptide, nucleotide, binding, gamete generation, spermatogenesis, protein folding and spermatid development were underexpressed especially in the high ROS group. This is the category with the largest distribution of proteins that were differentially expressed highlighting the deviation of the various functions from the fertile group.The majority of the overexpressed proteins were associated with various functions such as acetylation, phosphoproteins, CaWe further identified the DEPs in all 3 ROS groups that are known to play a role in reproduction and spermatogenesis that were underexpressed, over or were uniquely expressed to each of the 3 ROS groups compared to the fertile controls Table . These pIn conclusion, we have for the first time demonstrated poor sperm quality that is associated with elevated oxidative stress level by categorizing the patient semen samples into low, medium and high ROS groups. These abnormalities can be explained by the altered expression of specifically DEPs Calmegin, tripeptidyl synthesase, dynein intermediate chain 2, heat shock 70 kDa protein 4L, early endosome antigen 1, plasma serine protease inhibitor in patient groups. These DEPs may serve as potential biomarkers in the identification of the effect of increasing ROS level on protein profile of spermatozoa in infertile men. Further validation of DEPs is necessary to establish the role of these proteins as biomarkers of oxidative stress induced male factor infertility.Additional file 1: Table S2a: Spermatozoa proteins in fertile men. (DOCX 308 KB)Additional file 2: Table S2b: Spermatozoa proteins in Low ROS group. (DOCX 305 KB)Additional file 3: Table S2c: Spermatozoa proteins in Medium ROS group. (DOCX 294 KB)Additional file 4: Table S2d: Spermatozoa proteins in High ROS group. (DOCX 301 KB)


The redundancy hypothesis predicts that the species redundancy in a plant community enhances community stability. However, numerous studies in recent years questioned the positive correlation between redundancy and stability.We explored the relationship between the species redundancy, functional redundancy and community stability in typical steppe grassland in Northern China by sampling grassland vegetation along a gradient of resource availability caused by micro-topography. We aimed to test whether community redundancy enhanced community stability, and to quantify the relative importance of species redundancy and functional redundancy in maintaining community stability.Our results showed that the spatial stability of plant community production increases with increased supply of soil resources, and the functional redundancy instead of species diversity or species redundancy is correlated with the community stability. Our results supported the redundancy hypothesis and have implications for sustainable grassland management. The relationship between species diversity and ecosystem stability and its maintenance mechanism has been one of the hotspots in ecology research –3. HumanMany hypothesized mechanisms for the maintenance of species diversity and ecosystem function have been proposed; including, but not limited to, the diversity-stability hypothesis , rivet-pWhile the large scale relationships between diversity and ecosystem functions in Inner Mongolia grassland has been investigated –29, studLeymus chinensis (Trin.) Tzvel. dominates the community in fertile lowland, while two Stipa species (S. grandis P. Smirn and S. krylovii Roshev.) dominate the communities on the slope and top of the hills where there is a poor resources.The sample area is located in the Ecology Research Base of Inner Mongolia University (within 44.26°N—44.28°N and 116.32°E—116.35°E). The region has a continental climate, with a mean annual temperature of 0.88°C(1983–2005). Mean annual precipitation was 338 mm (coefficient of variance (CV) = 22%), of which 85% occurred from May through to September. The growing season is about 150 days long, with 200–235 frost free days. The land is hilly and the soil is calcic kastanozems and the vegetation is typical steppe. A vegetation gradient occurs along a resource gradient associated with different slope positions; Vegetation was surveyed in plant biomass peak period in this region (early August 2013) using a plot-transect method. Seven line transects perpendicular to the slope were evenly set from the top of hills to the lowland, i.e., the seven line transects were aligned in an ascending order from top to bottom. The length of each line transect was 31 m, and 1 m x 1 m quadrates were set on each line transect with an interval of 1 m, each line transect containing 16 quadrats, totally 112 quadrats. The height of plant vegetative and reproductive layers and the number of plants (abundance) in each quadrat were recorded by species, and aboveground biomass was determined by clipping the plants and obtaining fresh and dry weights . The frequency of each species in each transect was calculated as its presence in the 16 quadrats of one transect. The functional traits of each species that appeared in the 16 quadrats were determined by sampling 6 well-grown plants in the corresponding transect. The measured functional traits include plant height (H), number of leaves, leaf area , and leaf dry weight . The average aboveground weight (W), specific leaf weight (SLW) and specific leaf area (SLA) of individual plants were calculated. The five plant functional traits: W, H, LDW, LA and SLA were examined in this analysis.4+ and NO3-), available P (A-P), and organic C. Total N was determined using selenium-cupric sulfate (CuSO4)-potassium sulfate (K2SO4)-heating digestion method; total P was measured by alkali fusion-Mo-Sb colorimetric method; available N was measured by Kjeldahl nitrogen determination method, and available P was measured by sodium bicarbonate (NaHCO3) leaching-Mo-Sb colorimetric method; and organic C was measured by potassium dichromate (K2Cr2O7) heating oxidation method [Soil samples were collected from three drills to 30 cm in depth within each quadrat. The mixed sample of the 3 drills were brought to laboratory to determine the content of total N, total P, available N  was calculated using Shannon-weaver species diversity index since it represents the complexity of the community .SD=∑i=1S = υ ÷ σ) [Community stability was calculated using reciprocal of variation coefficient of aboveground biomass in 16 quadrats at each line transect (= υ ÷ σ) .Community redundancy was calculated based on two main methods: The first is based on the number of species that were functionally redundant in community. As the keystone or dominant species occupy a large proportion of community function, and the redundant species possess few functional properties , 32, theSD is calculated by FD is represented by Rao coefficient [dij is the functional feature distance between species i and species j, pi and pj are ratios of individual numbers of species i and species j over the individual number of overall species in the community, respectively. S is the overall species number in the community.The second method is according to Bello et al  who belifficient .FD=∑i=1Statistical analyses were performed using SPSS 17.0 software. First, the variation tendencies of the species diversity and community stability along the gradient of resources supply (at different slope positions) were analyzed and their causal relationship was identified using regression analysis. Then, the relationships between the species diversity and community stability, as well as among species redundancy, functional redundancy and community stability were examined using regression analysis to elucidate the role of species diversity, species redundancy and functional redundancy in community stability.Soil fertility indicators total N, total P, available N, and organic C in seven transacts showed an increasing trend with decreasing slope position from the top to the bottom of hills, except the available P which showed no spatial change down the slope . That isp = 0.017, r = 0.947, p = 0.001 respectively; P = 0.056)  .P = 0.283)  .P = 0.001), but not species redundancy   . CommuniThe diversity-stability relationship is a subject of much debate , 35. TheWe have calculated the community redundancy using the first and the third methods mentioned above for the redundancy of communities located in different slope positions in the sample area separately, and found no correlations between the redundancies obtained with these two methods (P > 0.05). The reason for this might be that a small number of keystone species or dominant species account for a large function proportion in the community , while tThe redundancy hypothesis predicts a positive impact of redundancy on stability . HoweverThis study shows that both the species redundancy and functional redundancy are positively correlated with the community stability, indicating a positive effect of redundancy on community stability, and provides some evidence to verify the redundancy hypothesis. In a functional group containing a variety of species, the species often manifest functional redundancy, and a functional group or community containing higher redundancy tends to achieve niche differentiation , 14 duriSpecies redundancy and functional redundancy are two features of community stability. Our results show that both the species redundancy and functional redundancy present a positive correlation with stability, with functional redundancy having a greater effect than the species redundancy. This result is reasonable, and can be explained from the calculation of the redundancy, the relationship between community complexity and stability and the effects of scale. Firstly, the species redundancy calculated here has some shortcomings because it reflects the richness of species with the same function in the community . AlthougS1 Table(XLSX)Click here for additional data file.


Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis.Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results.The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera. Echinococcus granulosus. The life cycle of this cestode involves dogs and other canids as the definitive hosts, while intermediate hosts are usually cattle, sheep, goats and pigs E. granulosus eggs shed with canid feces, man can become an accidental intermediate host and develop CE.Cystic echinococcosis (CE) is caused by the larval form of per 100000 inhabitants The greatest prevalence of CE in human and animal hosts has been recorded in the countries of the temperate zones, including parts of Eurasia, Australia, South America and Africa The hydatid cyst fluid (HCF), collected from infected animals, is the main source of antigens to be used for serological diagnosis. This liquid is a mixture containing a wide range of proteins of both parasite and host origin, the complexity and heterogeneity of which has already been highlighted by the proteomic characterization of HCF collected from sheep, cattle and humans During the last twenty years, many serological tests have been proposed, based mostly on crude HCF, Ag5, and AgB, but their diagnostic values have been proven to be limited in human hydatidosis. This is probably due to a combination of factors, including poor sensitivity and the lack of standardized antigen preparations. Moreover, it is worth noting that many published considerations refer to dated observations, often elaborated without the currently available advanced protein analysis facilities ex novo biochemical characterization of sheep HCF was performed by size exclusion chromatography, western immunoblotting, and mass spectrometry. We report here the set up of a reproducible size exclusion chromatography method that enables to obtain a highly enriched preparation of native Ag5. To evaluate the immunological reactivity of this antigen preparation, limited cohorts of sera from CE patient and healthy controls were evaluated against HCF crude antigen sample and against the Ag5 enriched chromatographic fraction. As preliminary results, we report that the enriched preparation of native Ag5 shows a very high and specific reactivity, both in western immunoblotting and in ELISA platforms, revealing promising perspectives for the development of sensitive immunodiagnostic tools.For all these reasons, an A written informed consent was obtained from patients at the time of sample collection. The study was approved by the ethics committee of the local health authority of Sassari , Prot N. 1123/L.g at 4°C and the supernatant stored at −80°C. A total of 24 blood sera from CE patients and control subjects were obtained from a sample archive at the Istituto Zooprofilattico Sperimentale della Sardegna and were chosen by examining the clinical records available for each patient and control. All samples were leftovers from routine diagnostic procedures and had originally been collected from CE patients (n = 13) at the time of admission at the hospital. Negative sera were from Sardinian healthy blood donors (n = 6) or patients admitted at the hospital for other non-related diseases (n = 5). Diagnosis of CE had been performed by clinical examination and imaging according to standard clinical practice guidelines. Eight patients were surgically treated following admission to hospital and clinical diagnosis. CE was confirmed in all these cases by direct inspection of parasite material.HCF crude samples were collected in two different Sardinian slaughterhouses  and CE/IT2078M, Lula (NU)), after a permission obtained from these slaughterhouses to use the animal parts from animals being processed as part of the normal work of the abattoir. Fluid was aspirated from liver and lung cysts found in infected sheep. The hydatid fluid was centrifuged at 1000×g at 4°C. The supernatants were then five-fold concentrated and desalted by ultrafiltration on centrifugal filter devices with a molecular mass cut-off of 10 kDa . For each sample, total protein concentration, evaluated by the BCA assay , was quantified before and after ultrafiltration. Finally, Laemmli buffer with or without 400 mM dithiothreitol (DTT) as a reducing agent was added to 20 µg of each sample Four different HCF samples (from two lung and two liver cysts) were thawed and centrifuged at 12000×After BCA assay, Laemmli buffer with or without 400 mM DTT was added to four different aliquots  of size exclusion Ag5 fraction, and the samples were subjected to SDS-PAGE on 4–10% acrylamide gel. Gel was stained by silver nitrate procedures Western immunoblotting was performed as described previously g on PVDF 0.22 µm filters . Size exclusion chromatography fractionations were then performed on a Superdex-200 column . Runs were performed at room temperature at a flow rate of 1 mL/min. Column was calibrated, under the same conditions, with standard proteins .Ag5 fraction was obtained by Fast Protein Liquid Chromatography (FPLC) procedures, performed on an AKTA Explorer 10 system . Aliquots of 5 mL of 5 sheep HCF were desalted by HiPrep 26/10 column , lyophilized, solubilized in 500 µL of 50 mM phosphate buffer at pH 7.6, containing 150 mM NaCl (running buffer) and filtered at 12,000×insitu hydrolysis as described previously 4HCO3 buffer with 10 mM DTT at 56°C and then carbamidomethylated in 50 mM NH4HCO3 buffer with 55 mM iodoacetamide at room temperature in the dark. Tryptic digestion was performed at 37°C overnight using variable amount (50–100 ng) of trypsin per gel slice.Gel bands (selected ones or the whole lanes) were cut and subjected to −1 for MS and “ultra scan” mode at 26000 (m/z)sec−1 for MS/MS scans. Peptide analysis was performed scanning from m/z 250 to m/z 2200 in AutoMS (n) precursor selection mode of the three most intense ions (fragmentation mass range from 100 to 2200 m/z). Dynamic exclusion was used to acquire a more complete survey of the peptides by automatic recognition and temporary exclusion (0.15 min) of ions from which definitive mass spectral data had previously acquired. Helium was used as the collision gas. Data Analysis software (6300 Series Ion Trap LCMS), provided by the manufacturer, was used to analyze raw MS and MS/MS spectra and to generate peak lists, as MGF files.Peptide mixtures obtained by selected bands from HCF were analyzed by LC-MS/MS on a XCT Ultra 6340 ion trap equipped with a 1200 HPLC system and a chip cube . After loading, peptides were concentrated and desalted at 4 µl/min on a 40 nL enrichment column, with 0.2% formic acid and then fractionated on a C18 reverse-phase  at a flow rate of 300 nl/min, with a linear gradient of eluent B (0.2% formic acid in 95% acetonitrile) in A (0.2% formic acid in 5% acetonitrile) from 3 to 60% in 20 min. MS method parameters were as follows: Capillary voltage 1730 V. Trap ICC smart target was 300000 units and maximal accumulation time was 100 ms. MS/MS was operated at a fragmentation amplitude of 1.3 V, and threshold ABS was 6000 units. Scan speed was set in “standard-enhanced” mode at 8100 (m/z)secThe whole lane analysis (GeLC-MS/MS) of Ag5 fraction from size exclusion chromatography was performed on a Q-TOF hybrid mass spectrometer equipped with a nano lock Z-spray source and coupled on-line with a nanoAcquity chromatography system (Waters). The peptide mixture was concentrated and washed onto a Trap column  using 0.2% formic acid, and fractionated onto a C18 RP column  at a flow rate of 250 nL/min. The samples were fractionated using a linear gradient of eluent B (0.2% formic acid in 95% ACN) in eluent A (0.2% formic acid) from 0.5 to 50% B in 46 minutes. The mass spectrometer was set up in a data-dependent MS/MS mode where a full-scan spectrum was followed by tandem mass spectra, selecting peptide ions as the three most intense peaks of the previous scan. MS method parameters were as follows: Capillary voltage 2000 V, Scan time 0.5 sec in MS and 1 sec in MSMS. Peptide analysis was performed scanning from m/z 300 to m/z 1600 in MS and from 100 to 2000 m/z in MSMS. Dynamic exclusion was used to acquire a more complete survey of the peptides by automatic recognition and temporary exclusion (10 sec) of ions from which definitive mass spectral data had previously acquired. Argon was used as the collision gas, and collision energy depending on mass and charge of the precursor ion was applied. ProteinLynx software (Version 2.2.5), was used for analysis of raw MS and MS/MS spectra and the production of the pkl lists.Echinococcus granulosusBoth the peak lists from Q-TOF analysis, converted into a MGF file, and the MGF files from Ion Trap mass spectrometer were analyzed by Proteome Discoverer software  using Sequest-HT as search engine for protein identification, according to the following criteria: Database UniprotKB (release 2014_02), taxonomy Mammalia integrated with Protein abundance was expressed by means of the normalized spectral abundance factor (NSAF). NSAF was calculated as follows: For indirect ELISA tests, microplates (Nunc-Maxisorp Immunoplate) were coated with Ag5 fraction by depositing in each well 100 µL of a 100 ng/mL antigen solution in PBS. Plates were then incubated at 37°C until completely dried A total of 4 different HCF samples collected from hydatid cysts, two localized in the lung and two in the liver of infected sheep, were concentrated and desalted by ultrafiltration and then analyzed by SDS-PAGE under non-reducing and reducing conditions . ProteinIn order to characterize the reactivity and specificity of antibodies in sera of CE patients, a pool of crude HCF antigen preparation was subjected to western blotting analysis on a multiscreen apparatus. HCF was run under non-reducing and reducing conditions and resulting membranes were both incubated with a collection of 24 sera, 13 of which were from CE patients and the other 11 were from healthy controls. As an example, To shed light on the identity of the proteins which gave reactivity in CE patients and to gather information on the HCF composition, an SDS-PAGE of HCF was performed and the gel was cut in two parts; one half was subjected to a western blot experiment with serum 1, whilst the second half was Coomassie stained and used for protein identification by tryptic digestion followed by tandem mass spectrometry (LC-MS/MS) analysis . More inThe strongest and immunodominant reactivity observed in patient sera was directed against native, non-denatured Ag5. Hence, a separation protocol by size exclusion chromatography was developed and optimized in order to attain an Ag5 enriched preparation under native conditions . The maiAs clearly evident in According to the standard curve performed on the size exclusion column, the apparent native molecular weight of Ag5 was of at least 600 kDa, corresponding to an oligomeric structure of about ten units, or to a native protein complex involving Ag5. In order to investigate this issue, an SDS-PAGE of fraction 2 was run and silver stained . Both whE. granulosus proteins.By using this data, label free quantitation with the spectral count approach applied to lane 3R allowed also to provide a rough estimate of the relative protein content in fraction 2. As described in To assess its diagnostic value, the chromatographic Ag5 preparation was assayed by immunoblotting on a multiscreen apparatus, with a collection of 24 human sera available in this study . An unamE. granulosus in all clinically positive patients, while patients 4 and 5 tested negative with all the HCF-based assays. In addition, patient 9 tested negative with three out of four commercial assays. Specificity was also high when compared to the most specific commercial assay, indicating that sensitivity can be increased without incurring in detection of false positive results. Because of the unavailability of sera, specificity was not evaluated for patients with different parasitoses.The suitability of the enriched Ag5 preparation to the ELISA format was then evaluated on all patient and control sera (Ag5 ELISA). As a comparison, the same sera were also tested with four ELISA kits currently used for serological evaluation of CE patients . Despite almost fifty years of active investigation, immunodiagnosis of CE E. granulosus. In this work, SDS-PAGE and size exclusion chromatography , is in keeping with a stable complex with Ag5. Accordingly, other authors The Ag5 preparation obtained with the method described in this work is suitable for use as native antigen in immunoassays, as suggested by the sensitive and specific reactivity observed by western blotting against sera collected from CE patients and control subjects . WesternThe ELISA format, given the native conditions used, enables preservation of native Ag5 complex conformation and, therefore, provides better conditions for detection of antibodies with higher efficiency and sensitivity. In a preliminary comparative evaluation with four commercially available ELISA assays , an enriIn conclusion, this work led to the development of a highly reproducible and performing procedure for the rapid, easy and inexpensive preparation of a native Ag5 based antigen. Noteworthy, this procedure can be further simplified and made less expensive by employing a peristaltic pump, instead of an FPLC system, to produce a controlled flow and, once standardized, collecting a time fraction to recover the antigen. Consequently, this method can be easily adopted by most laboratories with average skills in biochemistry, opening the way to its extensive validation. Indeed, while preliminary experiments demonstrated the high performance of the enriched native Ag5 described in this study in indirect ELISA-based immunoassays, its diagnostic value needs to be confirmed by means of future large scale validation studies.Table S1Summary of protein identification data for SDS-PAGE bands.(DOC)Click here for additional data file.Table S2Summary of protein identification data for GeLC-MS/MS analysis of size exclusion chromatography fraction 2.(DOC)Click here for additional data file.Table S3Optical densities of sera in ELISA assays.(DOC)Click here for additional data file.