Whether IgA nephropathy is attributable to mesangial IgA is unclear as there is no correlation between intensity of deposits and extent of glomerular injury and no clear mechanism explaining how these mesangial deposits induce hematuria and subsequent proteinuria. This hinders the development of a specific therapy. Thus, precise events during deposition still remain clinical challenge to clarify. Since no study assessed induction of IgA nephropathy by nephritogenic IgA, we analyzed sequential events involving nephritogenic IgA from IgA nephropathy-prone mice by real-time imaging systems. Immunofluorescence and electron microscopy showed that serum IgA from susceptible mice had strong affinity to mesangial, subepithelial, and subendothelial lesions, with effacement/actin aggregation in podocytes and arcade formation in endothelial cells. The deposits disappeared 24-h after single IgA injection. The data were supported by a fluorescence molecular tomography system and real-time and 3D in vivo imaging. In vivo imaging showed that IgA from the susceptible mice began depositing along the glomerular capillary from 1 min and accumulated until 2-h on the first stick in a focal and segmental manner. The findings indicate that glomerular IgA depositions in IgAN may be expressed under the balance between deposition and clearance. Since nephritogenic IgA showed mesangial as well as focal and segmental deposition along the capillary with acute cellular activation, all glomerular cellular elements are a plausible target for injury such as hematuria. Immunohistological analysis is the crux of the diagnosis of IgA nephropathy (IgAN) for which dominant or co-dominant mesangial deposition of IgA is essential. Significant number of patients is reported to have elevated levels of circulating IgA immune complexes (IC), presumably due to aberrantly glycosylated IgA and endogenous antiglycan antibodies In contrast, we also know patients who exhibit minor clinical symptoms, such as very low levels of hematuria and proteinuria but have massive glomerular IgA depositions, suggesting that not all IgA immune deposits possess equivalent pathogenic potential. These conflicting clinical findings make it difficult to identify the true therapeutic target of this disease, thereby hindering the development of a specific therapy O-linked glycans in the hinge region of human IgA1 is linked to human IgAN O-glycans are absent in the hinge region of murine IgA. However, our recent paper We recently established a strain of spontaneous IgAN-prone gddY mice in which the disease phenotype and genetic regulation largely overlap with human IgAN ad libitum in a specific pathogen-free (SPF) room at the animal facility of Juntendo University, Tokyo, Japan.The gddY mice were established by the selective mating of early-onset ddY mice for more than 20 generations and have a 100 % incidence of severe disease, even at a young age Same-age female Balb/c  mice were used as controls. Female Balb/c AJcl-nu/nu (nude) mice were used for the injection of serum or IgA from the gddY mice with early-stage disease and from the Balb/c mice. Balb/c and nude mice were purchased from CLEA Japan Inc., Tokyo, Japan. The experimental protocol of the present study was approved by the Ethics Review Committee for Animal Experimentation of Juntendo University Faculty of Medicine.Serum samples were obtained from gddY mice aged between 20 and 25 weeks who showed severe renal injury with glomerular IgA deposits. Serum from age-matched healthy Balb/c mice was also collected as the control serum. Serum levels of IgA were measured using a single radioimmunodiffusion assay . The obtained serum samples, including 2 mg IgA of gddY or Balb/c mice  were injected once into the tail vein of nude mice aged between 10 and 12 weeks. The nude mice were euthanized either 2 or 24 h after the injection of serum. Kidneys were collected from the nude mice after perfusion with normal saline solution. The serum samples and kidneys were stored at −80°C until use. Renal histological analysis was performed using electron and confocal microscopy.Snap-frozen 3-µm-thick renal sections were used for immunofluorescence with a fluorescein isothiocyanate-conjugated goat antimouse IgA antibody . For electron microscopy, the renal specimens were fixed in 2 % glutaraldehyde, washed in phosphate buffer, and postfixed in 1 % osmic acid. The specimens were washed, dehydrated, and embedded in Epon 812. The ultrathin sections were contrasted with uranyl acetate and lead citrate and then examined under an electron microscope .Serum samples were obtained from the gddY and Balb/c mice aged between 20 and 25 weeks. Each serum sample was incubated with a rat antimouse IgA antibody with CNBr Sepharose overnight at 4°C. After washing with phosphate-buffered saline (PBS), IgA was eluted with 0.1 M glycine (pH 2.5) using a fraction collector. Molecular size of purified IgA obtained from ddY and Balb/c mice were analyzed by Western blot analysis. The purified IgA samples were used for fluorescence molecular tomography and confocal laser microscopy analyses.These studies were conducted on female nude mice (n = 3). The purified IgA samples obtained from gddY or Balb/c mice were labeled with Vivo Tag-S 750 . Purified IgA samples were dissolved at 2 mg/mL in PBS, and 0.5 mL of the solution was alkalized with 50 µL of 1 M sodium bicarbonate solution. The mixture was incubated with Vivo Tag-S 750 for 1 h at room temperature, with stirring. The labeled IgA was purified using resin column chromatography. Fluorescein-labeled IgA (0.5 mg/mL) was dissolved in 200 µL PBS and injected into nude mice aged between 10 and 12 weeks. After 2, 4, and 24 h, IgA signals were measured using a fluorescence molecular tomography system .Kinetic studies by confocal laser microscopy were performed in female nude mice (n = 3). Purified IgA obtained from gddY or Balb/c mice was labeled using an Alexa Fluor 633 protein labeling kit . Purified IgA samples were dissolved at 2 mg/mL in PBS, and 0.5 mL of the solution was alkalized with 50 µL of a 1 M sodium bicarbonate solution. The mixture was incubated with Alexa Fluor 633 dye at room temperature for 1 h, with stirring. The labeled IgA was purified using resin column chromatography. Before analysis, the nude mice were anesthetized with sodium pentobarbital. An incision of approximately 10 mm was made on the lower back, and a kidney was removed using small forceps. The surface of the lower pole of the kidney was sliced and placed on a microscope stage. Dextran (500 kDa) is not filtered and is a good marker for blood vessel wall integrity. First, fluorescein-labeled 500-kDa dextran  was injected into nude mice to confirm the staining of glomeruli. Following this, fluorescein-labeled IgA (0.5 mg/mL) was dissolved in 200 µL PBS and injected into mice. After 1 and 120 min, the kinetics of labeled IgA in glomeruli were analyzed using confocal laser microscopy . Single or time-series images of glomeruli were recorded in fluorescence mode.7 BMCs were injected into the tail vein of irradiated recipient mice (n = 3) at 700 rad. The recipient mice were housed under SPF conditions. Young and old gddY transplant recipients were euthanized 12 weeks after BMT. IgA deposits were assessed by immunofluorescence and electron microscopy, and urinary protein was measured by immunoassay .The procedure for murine BMT was previously described in detail We first tested whether a serum from gddY mice induced glomerular IgA deposition in nude mice. Serum from Balb/c mice was used as a control. Renal tissue was obtained from nude mice euthanized 2 and 24 h after a single injection of the serum. Serum from gddY mice, but not Balb/c mice, induced mesangial IgA deposition 2 h after the injection . The elein vivo kinetics of IgA using a fluorescence molecular tomography system after a single injection in nude mice. After the injection of IgA from gddY or Balb/c mice, fluorescence signals in the kidneys, liver, and bladder were evaluated after 10 min and 2, 4, and 24 h. There was no difference in the signals in the liver and bladder between the mouse groups that received labeled IgA from either gddY or Balb/c mice .To further clarify the sequential manner of the deposition process, we injected Alexa Fluor 633 protein-labeled IgA (red) from gddY and Balb/c mice and fluorescein-labeled 500-kDa Dextran (green) to visualize the glomerular capillary in the nude mice. We then analyzed the glomerular capillaries using confocal laser microscopy from 1 min to 2 h after the single injection . Yellow ly 1 min . IgA accly 1 min ; howeverTo clear IgAN in gddY mice sine qua non. This notion may lead us to believe that mesangial IgA deposition directly induces hematuria. However, there are no studies proving a direct relationship between the amount mesangial IgA immune deposits and the extent of glomerular injury leading to hematuria. Our earlier comprehensive observations emphasized the variable characteristics of the IgA/IgA IC, particularly the nature of antigens and the molecular form of IgA, as major determinants of glomerular injury The primary abnormal clinical manifestation of IgAN in patients is recurring bouts of hematuria with or without associated proteinuria The present analysis using tomography showed a similar time course of liver signals peaking at 2 h in both the groups of mice, indicating that most of the injected IgA from the gddY and control mice was trapped in the liver in a similar fashion. The parts of injected IgA from both the groups of mice passed through into the bladder in a similar manner. Nevertheless, renal signals were conspicuously stronger in the group injected with IgA from gddY mice than in those injected with IgA from healthy mice. This finding indicates that some clones of IgA in gddY mice have a strong affinity for kidney tissues and may be nephritogenic. Our recent studies have shown that IgA in gddY mice is polymeric and that it has a high capacity for activating complement cascades, including the lectin pathway The disappearance of the glomerular deposits in this single-injection model is one of most important findings of the present study. This finding indicates that a continuous clearance mechanism of IgA deposition may be at work in the glomerulus. Thus, glomerular IgA deposits in IgAN may be explained as an imbalance between deposition and clearance. Although most IgAN patients have undoubtedly had glomerulonephritis for years before a renal biopsy, it seems that a certain percentage of IgAN patients may go through a clinical period just after the onset, when neither IgA nor C3 is detectable in the renal biopsy but when there are histological lesions and aberrant urinary test results Increasing evidences suggest that mucosal type polymeric IgA1 is produced in BM of IgAN patients In vivo serial imaging showed that such subendothelial/subepithelial IgA deposits seemed to be formed as a result of an accumulation on the focal/segmental initial aggregates of IgA, suggesting that the initial aggregates (microdeposits) may change the local physiological conditions, thereby leading to the increased glomerular affinity of IgA. These physiological changes may include the local deceleration of the glomerular capillary flow or increased permeability of the glomerular basement membrane interposed between the endothelial and epithelial layers Although most glomeruli in mice injected with gddY IgA showed mesangial IgA deposition, deposits along capillaries were detected in a focal and segmental manner. This study revealed the kinetics of glomerular deposition over the course of IgA-induced IgAN. These IgA molecules have a strong affinity for focal and segmental subendothelial, subepithelial, and glomerular mesangial lesions. Rapid cellular activation of endothelial cells and podocytes by IgA deposition may precede the events of hematuria in IgAN. The significant differences between human and murine IgAN limit the translation of these data to the human disease. Further, similar study using human GdIgA1 is needed. However, the present findings regarding the kinetics of IgA deposition may indicate that not only glomerular mesangial cells but also endothelial cells and podocytes are plausible targets of glomerular injury in IgAN.Video S1In vivo 3D imaging of glomerulus after injection of nephritogenic IgA. 3D images of glomeruli at 2 hours after single injection of purified IgA from gddY mice were evaluated by a confocal laser microscopy. Alexa Fluor 633-labeled IgA from gddY (red) and 500-kDa fluorescein-labeled dextran (green) were injected for analyzing kinetics of IgA deposition and visualizing blood vessel wall integrity, respectively. IgA signals were detectable after 2 h in a focal and segmental manner in mice with IgA from gddY mice.(MOV)Click here for additional data file.Video S2In vivo 3D imaging of glomerulus after injection of IgA from Balb/c mice. 3D images of glomeruli at 2 hours after single injection of purified IgA from Balb/c mice were evaluated by a confocal laser microscopy. Alexa Fluor 633-labeled IgA from and Balb/c mice (red) and 500-kDa fluorescein-labeled dextran (green) were injected for analyzing kinetics of IgA deposition and visualizing blood vessel wall integrity, respectively. IgA signals were detectable after 2 h in a focal and segmental manner in mice with IgA from gddY mice (Video images S1 and S3). In contrast, mice who received Balb/c IgA did not show clear signals after 2 h.(MOV)Click here for additional data file.Video S3In vivo real-time imaging of glomerulus after injection of nephritogenic IgA. Real-time images of glomeruli at 2 hours after single injection of purified IgA from gddY mice were evaluated by a confocal laser microscopy. Alexa Fluor 633-labeled IgA from gddY mice (red) and 500-kDa fluorescein-labeled dextran (green) were injected for analyzing kinetics of IgA deposition and visualizing blood vessel wall integrity, respectively. IgA signals were detectable after 2 h in a focal and segmental manner in mice with IgA from gddY mice.(MOV)Click here for additional data file.Video S4In vivo real-time imaging of glomerulus after injection of IgA from Balb/c mice. Real-time images of glomeruli at 2 hours after single injection of purified IgA from Balb/c mice were evaluated by a confocal laser microscopy. Alexa Fluor 633-labeled IgA from Balb/c mice (red) and 500-kDa fluorescein-labeled dextran (green) were injected for analyzing kinetics of IgA deposition and visualizing blood vessel wall integrity, respectively. IgA signals were detectable after 2 h in a focal and segmental manner in mice with IgA from gddY mice (Video images S1 and S3). In contrast, mice who received Balb/c IgA did not show clear signals after 2 h.(MOV)Click here for additional data file.


APPL1, RAB5A, EEA1, PDCD6IP, NOX4 and SORT1 were altered in malignant patient tissue, when compared to indolent and normal prostate tissue. These findings support the initiation of a case-control study using larger cohorts of prostate tissue, with documented patient outcomes, to determine if different combinations of these new biomarkers can accurately predict disease status and clinical progression in prostate cancer patients.Prostate cancer continues to be a major cause of morbidity and mortality in men, but a method for accurate prognosis in these patients is yet to be developed. The recent discovery of altered endosomal biogenesis in prostate cancer has identified a fundamental change in the cell biology of this cancer, which holds great promise for the identification of novel biomarkers that can predict disease outcomes. Here we have identified significantly altered expression of endosomal genes in prostate cancer compared to non-malignant tissue in mRNA microarrays and confirmed these findings by qRT-PCR on fresh-frozen tissue. Importantly, we identified endosomal gene expression patterns that were predictive of patient outcomes. Two endosomal tri-gene signatures were identified from a previously published microarray cohort and had a significant capacity to stratify patient outcomes. The expression of  More than 1.1 million new cases of prostate cancer are diagnosed each year and two thirds of these patients are from the Western world. The marked increase in the age adjusted incidence rate for prostate cancer has been partly attributed to the prostate specific antigen (PSA) test identifying men without clinical symptoms of the disease. Unfortunately, the PSA biomarker neither discriminates between patients who are at a higher risk of progressive disease/mortality and those who have a more favorable prognosis, nor can it adequately distinguish between prostate cancer and benign pathologies [Prostate cancer is the second most commonly diagnosed cancer in males , and thehologies . TherefoAMACR), which was highly expressed in prostate cancer and may have value as a prognostic marker for the disease [AMACR is an androgen-regulated gene and exhibits variable expression upon androgen-deprivation therapies or androgen-independent disease progression [The investigation of biomarker expression by microarray analysis in patient cohorts, in relation to known clinical parameters, can be used to develop methods for determining patient prognosis -6. Conse disease . Howevergression . Therefo® test measures the expression of 46 genes involved in cell cycle progression [® Prostate Cancer Test measures the expression of genes involved in stromal response, cellular organization, proliferation, basal epithelial function, androgen signaling and stress response [Commercial tests have recently been developed in an attempt to distinguish between aggressive prostate cancer and indolent disease (reviewed by Sartori & Chan 2014 ). The Prgression , whilst response . While tresponse . ProstatWe recently reported that the biology of endosomes is markedly altered in prostate cancer cells , 14 and Here, we have investigated endosomal gene expression in multiple independent prostate cancer cohorts and developed two endosomal gene signatures that were predictive of patient outcome. We have also evaluated endosomal gene expression in fresh-frozen tissue sections from radical prostatectomies and demonstrated a capacity to distinguish indolent from aggressive tumors. This study provides evidence that endosomal genes can distinguish prostate cancer patient outcomes and predict disease progression, warranting further investigation of these findings in larger case-control studies.APPL1 and EEA1 was significantly increased in primary prostate cancer when compared to non-malignant controls  and PIN tissue . The expression of NOX4 was significantly increased in metastatic prostate tissue when compared with PIN (P ≤ 0.05). Acid ceramidase (ASAH1) expression was significantly increased in PIN when compared to non-malignant and primary prostate cancer tissue (P ≤ 0.05), and was significantly reduced in metastatic tissue when compared to primary prostate cancer (P ≤ 0.01), PIN (P ≤ 0.0001), and non-malignant tissue (P ≤ 0.01). Cathepsin B (CTSB) expression was significantly reduced in both primary cancer tissue and metastatic cancer tissue when compared to non-malignant prostate tissue  in patients who had a greater risk of relapse  expression revealed patients with lower CTSB expression had significantly increased risk of biochemical recurrence (P = 0.0306). There was also a significant stratification of patients with Sortilin (SORT1) expression, with those patients who expressed greater amounts of SORT1 at an increased risk of relapse (P = 0.004). Myosin 1B (MYO1B) stratified patients at risk of recurrence (P = 0.03), with patients having increased expression being associated with a poorer prognosis. There was a trend for lower expression of ALIX (PDCD6IP) to stratify patients with increased prostate cancer recurrence (P = 0.059), and reduced expression of Syntaxin 12 (STX12) was also indicative of at-risk patients, with significant stratification . We postulated that the altered endosome gene expression in this patient group may indicate changes in cell biology that promote a more aggressive disease, and that this change could stratify patients at risk of recurrence, where the expression of PSA was low or borderline in blood samples.RAB5A, APPL1 and EEA1 tri-gene signature, using K-means clustering methodology, robustly separated patients into two groups with low or high expression of each of the three genes . The expression of NOX4 and SORT1 were also significantly increased in aggressive cancer tissue when compared with non-malignant (P ≤ 0.01) and indolent cancer tissue (P ≤ 0.05). There was a significant reduction of PDCD6IP mRNA in indolent cancer tissue when compared to both non-malignant and aggressive prostate cancer tissue (P ≤ 0.05 respectively).qPCR analysis of endosome associated mRNA in fresh-frozen prostate cancer tissue demonstrated significantly increased expression of Prostate cancer is one of the most frequently diagnosed cancers in men, and a leading cause of cancer related deaths worldwide, particularly in the Western world , 21. ThePCA3 and PCA expression in prostate cells extracted from urine, can indicate the likelihood of prostate cancer, even if biopsies return a negative result [PCA3, which is associated with prostate cancer-cell survival and androgen receptor signaling [Prognostic nomograms that incorporate continuous as well as categorical variables can potentially be improved by the integration of gene expression analysis and prognostic molecular signatures . Our rece result ; providiignaling . This suWe recently reported that the biogenesis of endosomes is altered in prostate cancer, providing significant data on gene and protein expression in cultured prostate cells , 14. We ASAH1) has been associated with improved outcomes [ASAH1 maps to chromosome 8p22, which is frequently deleted in prostate cancer patients [CTSB (cathepsin B) is also found on chromosome 8p22 and the down regulation of CTSB in metastatic tissue may also relate to these chromosomal break points. The reductions in both ASAH1 and CTSB expression in metastatic tissue may therefore be related to this deletion and furthermore is linked to the altered biogenesis of endosomes as these organelles are used to transport both of the latter enzymes.The genes employed for these prognostic signatures are associated with separate populations of early-endosome compartments, thus reflecting that the early endosome population as a whole is disturbed in prostate cancer, rather than one specific compartment, complementing the prior discovery of altered endosome biogenesis in prostate cancer . Alteredoutcomes . Interespatients , 29. EndAPPL1 (3p21), RAB5A (3p24) and EEA1 (12q22) and RAB4A (1q42) in PIN or primary cancer tissue compared to their expression in non-malignant tissue may discriminate metastatic tissue and provide an avenue for monitoring disease progression. The changes in the expression of endosomal genes observed in the Tomlins cohort suggested that they may be involved in the initiation or promotion of pre-malignant cell survival, and that sustained expression is not essential for the continued function of the malignant cell. Indeed, the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of prostate cancer displays elevated gene expression in PIN tissue, which is later reduced in tumors [The changes in n tumors . The widMicroarrays provide only a ‘snapshot’ of a cell population and there is potential for mRNA to be secreted prior to, or post protein-synthesis -33, compWe have noted the limitations of microarray gene expression analysis and consequently performed qPCR analysis of fresh-frozen prostate tissue to confirm our findings on endosomal gene expression. It should be recognized that the Glinsky cohort does not provide information on the gene expression in normal tissue from the cancer patients, or healthy individuals that have increased PSA independent of cancer occurrence, thus the current gene signatures are indicative of prognostic potential rather than necessarily stratifying healthy individuals from those with prostate cancer. Furthermore, incorporating the Gleason scores from the Tomlins and Glinsky cohorts have been precluded from this study, due to the variability in the prognosis from this scoring system . HoweverAPPL1, RAB5A and EEA1 gene expression in prostate tissue from aggressive disease compared to normal tissue or indolent-disease tissue indicates that these biomarkers have significant potential to monitor disease progression and to stratifying patients at risk of progressive and or metastatic disease. They also have important biological relevance that could provide a therapeutic target, since alterations in endosome biogenesis can result in significantly altered intracellular cell signaling, result in increased cell proliferation and in combination with NOX4 the expression promote inflammation and angiogenesis [The significant changes to ogenesis .APPL1, EEA1 and RAB5A, and analysis of the endosomal genes MYO1B, PDCD6IP and STX12 in prostate cancer patients expressing PSA ≤ 10 ng/mL, led to patient stratification into high and low-risk recurrence groups. These findings justify further clinical investigation of these biomarkers and gene signatures in a dedicated case-control study. Recognizing that prostate cancer patients have altered endosomal gene expression is a significant step towards the development of new, biology-driven biomarkers that could provide accurate prognosis for this important disease.In summary, endosomal gene expression and specific gene signatures have the potential to prediction disease progression in prostate cancer patients and this may lead to improved treatment outcomes. Multivariate risk analysis and stratification of patients into low- and high-risk groups may provide a more individualized approach to prostate cancer patient management, reducing the amount of over-treatment . Active-The Tomlins  cohort w® gene expression assays (Single Tube TaqMan® Gene Expression Assays Life Technologies); APPL1 (Hs00179382_m1), RAB5A (Hs00991290_m1), EEA1 (Hs00929215_m1), PDCD6IP (Hs00994346_m1), NOX4 (Hs00418356_m1), SORT1 (Hs00361760_m1) and TaqMan® Gene Expression Master Mix in a total volume of 10 μL. Three biological replicates were performed consisting of three technical replicates. Expression of each gene was normalized to an average of ALAS1 (Hs00963534_m1) and HPRT1 (Hs02800695_m1) reference genes and relative quantities calculated by ΔCt.Quantitative RT-PCR was performed on patient samples from the Irish PCRC cohort. RNA was extracted from non-malignant (n = 11), indolent (n = 5) and aggressive cancer tissues (n = 6). Reverse transcription of 500 ng of total RNA was performed using a high-capacity cDNA Reverse Transcription Kit . Real-time PCR was performed using 2 μL cDNA for each of the following TaqManK-means clustering by Cluster 3.0 [K-means clustering methodology to cluster the expression values of the three genes from each tissue, resulting in two clusters representing high or low expression for each of the three genes. One-way ANOVA was performed to evaluate expression differences between non-malignant, indolent and aggressive prostate cancer tissues from the PCRC bioresource. All statistical tests were performed using GraphPad Prism 6.05 . Significant results showed greater than 95% confidence (P < 0.05).A Kruskal-Wallis test with Dunn's multiple comparison was performed to evaluate differences in gene expression between multiple groups in the Tomlins cohort. Data from the Glinsky cohort was analyzed using ster 3.0  to deter


Grain yield in wheat is a polygenic trait that is influenced by environmental and genetic interactions at all stages of the plant’s growth. Yield is usually broken down into three components; number of spikes per area, grain number per spike, and grain weight (TGW). In polyploid wheat, studies have identified quantitative trait loci (QTL) which affect TGW, yet few have been validated and fine-mapped using independent germplasm, thereby having limited impact in breeding.2 and BC4 near isogenic lines (NILs), we validated the three QTL effects across environments. In four of the five experiments the Rialto 6A introgression gave significant improvements in yield (5.5%) and TGW (5.1%), with morphometric measurements showing that the increased grain weight was a result of wider grains. The extended green canopy duration associated with the high yielding/TGW Rialto allele was comprised of two independent effects; earlier flowering and delayed final maturity, and was expressed stably across the five environments. The wheat homologue (TaGW2) of a rice gene associated with increased TGW and grain width was mapped within the QTL interval. However, no polymorphisms were identified in the coding sequence between the parents.In this study we identified a major QTL for TGW, yield and green canopy duration on wheat chromosome 6A of the Spark x Rialto population, across 12 North European environments. Using independent germplasm in the form of BCThe discovery and validation through near-isogenic lines of robust QTL which affect yield, green canopy duration, thousand grain weight, and grain width on chromosome 6A of hexaploid wheat provide an important first step to advance our understanding of the genetic mechanisms regulating the complex processes governing grain size and yield in polyploid wheat. Triticum aestivum L.) is one the world’s major staple crops, supplying approximately twenty percent of the global total calorie intake [[Wheat . ]). Oryzaewed in [–15]], b], bOryzaOsGW2 encodes a previously unknown RING-type E3 ubiquitin ligase and functions as a negative regulator of grain width and weight ]. H]. HOsGW2 et al.  identif allele , suggesn wheat , suggesThe objective of this study was to evaluate a doubled haploid mapping population across North European environments for thousand grain weight, yield and additional morphological and developmental traits. We identified a meta-QTL for TGW, yield, and green canopy duration on wheat chromosome 6A. We developed near isogenic lines (NILs) to validate the 6A QTL effects across environments, and morphometric grain analyses were conducted to determine the specific grain size components being affected by the QTL.Rht-D1b perfect marker, and a morphological GA test. The total length of the map was 1,471 cM across 30 linkage groups which were assigned to specific chromosomes using published consensus maps ]. W]. W27]].25 days , yet thQyld-jic.6A. In four of the five NIL validation experiments the Rialto introgression gave significant improvements in yield (5.5 ± 1.5%) and TGW (5.1 ± 1.6%), with morphometric measurements showing that the increased grain weight was predominantly a result of wider grains. The final weight of individual grains is the last of the yield components to be established once the number of spikes per surface area and the grain number per spike have already been fixed ]. P]. PTaGW2 et al.  and Bed et al.  who shoIn rice, the grain size QTLs identified from bi-parental mapping populations have relatively large effects on TGW and yield, whereas in wheat the majority of studies have identified QTL with relatively small additive effects on grain size (and yield). These differences could be due to several reasons. First, the biological mechanisms determining grain size and yield might be different across species. For example, several rice QTL affect grain size indirectly through the modulation of spikelet hull and glume size –[32–[3230]–], e], eTriti assays ], a], aTriti assays ]. H]. HTritiTaGW2 candidate gene within the QTL interval, although it is still premature to assign a specific contribution of this gene to the QTL effect. The validation of this QTL provides an important first step to advance our understanding of the genetic mechanisms regulating the complex processes governing grain size and yield in polyploid wheat.In this study we identified major and stable QTL for yield, grain weight, and green canopy duration in polyploid wheat across North European environments. These effects were validated using a series of NILs which showed, on average, over 5% improvement in yield and TGW in four of the five environments tested. Our results suggest that the yield effect is driven primarily by increased grain weight due to wider grains. The lack of correlation between prolonged green canopy duration and yield/TGW argue against a direct/causal relationship between these traits within the germplasm studied. Using a promoter polymorphism, we mapped the Triticum aestivum L.), Spark and Rialto. The cross was chosen with the aim of discovering QTL controlling yield variation in a bread making background and from independent breeding programmes. Spark, released in 1993 by Nickersons Seeds (now Limagrain), is a Group 1 premium bread making variety whereas Rialto, released in 1991 by PBIC (now RAGT), is a Group 2 bread making variety, as classified in the nabim wheat guide ], I], Ipsp ], W], Wpsp [m [[wmc , Beltsv [[barc  and INRcfa/cfd  collectcfa/cfd  aiming markers  and altmarkers  for Divmarkers   markers (Hap-6A-P1 and Hap-6A-P2) as previously described [[Xgwm570 [[To increase marker resolution across the 6A QTL interval, an additional 9 single nucleotide polymorphism (SNP) markers derived from expressed sequence tags (EST ) were mscribed . An addXgwm570  were moth edition . The most suitable variance–covariance structure was detected  and genetic predictors were computed for a step size of 4 cM. The significance levels and QTL effects were determined by a final backward selection step at a significance level of 0.05.QTL Cartographer v2.5  was use−2.The DH population was grown in a randomised complete block design with three replications at five sites ; Sandringham, England ; Balmonth, Scotland ; Bohnshausen, Germany ; and Froissy, France ). The experiments were grown over three years (2001–2003) at Norwich and Sandringham, and for two years (2002–2003) at the other three locations. Trials were sown in large-scale yield plots (1.1 × 6 m) and standard farm pesticide and fertiliser applications were made to reproduce commercial practise. Trials were sown by grain number to ensure comparable plant densities for each plot aiming for a target population of 275 seeds*mFinal plot yield, after correction for plot size, was recorded at all sites and additional phenotypic assessments of developmental traits and yield components were made at Norwich and Sandringham. These included heading date at Zadoks growth stage 57  and phy2 NILs) and 2013 (8 BC4 NILs) and at Wohlde, Germany  in 2012 (10 BC2 NILs). Trials were sown with the same experimental practices as the DH population, with the exception of the number of replicates (5–10 at Norwich and 3 at Wohlde). Field phenotyping was conducted in the same manner as for the DH population (described in preceding paragraph) with the following exceptions; biomass and harvest index were only measured in 2011 and tiller number, seeds/spike and seeds/spikelet were measured only in 2011 to 2013 in Norwich. Morphometric measurements  were recorded from 300–400 grains per sample using the MARVIN grain analyser . In addition, field grown NILs were checked with DNA markers to confirm their identity both in the ground and after harvest.The NILs were grown at Norwich in 2010–2012 . SPAD measurements were taken at several time-points after anthesis, aiming for an initial reading when there was no visible difference between the NILs, and then at suitable time points thereafter as senescence progressed. Ten main tillers per plot were tagged at anthesis and used for measurements throughout grain filling. Three measurements were taken across the flag leaf  and a mean value obtained for each tagged tiller.Xgdm36 to Xgwm570 genetic interval . Based on this classification, general one-way analyses of variance (ANOVA) were performed for the multiple traits and Pearson’s correlation coefficients were calculated between selected traits. NILs were evaluated using two-way ANOVA in which the interaction between environment and the 6A interval was included in the model. ANOVA and correlations were performed using GenStat 15th edition .DH lines were classified according to their genotype across the TaGW2 from Spark and Rialto were designed based on the International Wheat Genome Sequencing Consortium (IWGSC) Chinese Spring chromosome arm survey sequence available at URGI [[TaGW2 cDNA sequence [[Primers to amplify the genomic DNA of the A-genome homoeologue of at URGI . Contigequence . Four hTaGW2. Primers were designed to amplify the three homoeologous genomes. Using these primers and the cDNAs of Spark and Rialto, a RT-PCR was used to amplify the complete TaGW2 CDS of the two varieties . Purified RT-PCR products were modified with A-overhangs by incubating with 10 mM dATP and 1u of Taq polymerase  in 1× manufacturer’s buffer for 10 mins at 72°C. They were then cloned directly using a pGEMT-Easy Kit (Promega) following the manufacturers’ instructions. Miniprep DNA of clones was insert-sequenced using M13 primers and sequenced by TGAC. Sequence reads were aligned and assigned to the A genome using the sequence of the flow sorted chromosome arm DNA [[Total RNA was extracted from leaf sections of one 4-leaf seedling each of Spark and Rialto using Tri-Reagent  and cDNA was synthesized using MMLV-RT  using the manufacturer’s protocols. Wheat ESTs and bespoke assemblies from the 454 5× raw data of hexaploid wheat Chinese Spring  were usarm DNA  and thearm DNA .The data sets supporting the results of this article are included within the article and its additional files.The authors declare that they have no competing interests.TaGW2. VK coordinated field trials in Germany. JSn coordinated and conceived the DH population field trials. CU conceived the NIL validation experiments, analysed the data, and wrote the manuscript. All authors read and approved the final manuscript.JSi developed the germplasm used in this study, carried out the mapping, coordinated all field evaluations and statistical analysis and wrote the manuscript. PS provided assistance with phenotypic assessments, field trial preparation and glasshouse husbandry. MLW led the mapping of the original Spark x Rialto DH population and assisted with phenotypic assessments of the population. AT and JB carried out the sequencing of Yield and TGW QTL effects in DH population across individual environments. Genome-wide QTL analyses for yield (a) and thousand grain weight (b) in the Spark x Rialto DH population. Environments are determined by colour  and years by line style . The threshold value for significance is set at 2.5 LOD.Click here for fileEffects of the Rialto 6A region on yield, TGW and GCD in DH lines across environments. DH lines were classified according to their genotype across the Xgdm36 to Xgwm570 genetic interval  and the phenotypic values averaged for each location. Differences between the Rialto and Spark NIL phenotypes are presented as a percentage of the Spark NIL (yield and TGW) or as the absolute value (GCD).Click here for fileMTME output for agronomic and yield related traits. Green canopy duration across 4 environments (a), thousand grain weight across 12 environments (b), tiller number across five environments (c), and yield across 12 environments (d). Significant markers are represented with a square, the colour represents the increasing parental allele  and the intensity of the colour demonstrates the significance level (dark blue and red indicates a higher significance).Click here for fileEffects of the Rialto 6A segment on additional agronomic and yield related traits. Plant biomass, harvest index, spike length, spikelet number, spike yield, seeds/spike, and seeds/spikelet in BC2 and BC4 NILs. Significant differences are represented by *(P <0.05), **(P <0.01) and ***(P <0.001).Click here for fileGenotype of the Spark x Rialto DH population. Genotype of 129 DH lines with 263 genetic markers scored for the Spark (A) or Rialto (B) allele.Click here for filePhenotypic scores of the Spark x Rialto DH population across 12 environments. Yield (kg/plot) and thousand grain weight (g) of the 129 DH lines grown across different environments.Click here for filePrimer sequences used to amplifyTaGW2-Agenomic DNA and cDNA.Click here for file


Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin.Leptospirosis, a re-emerging disease of global importance caused by pathogenic L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32.Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although definitive determination of the role of these PTMs must await further investigations, the reduced immune recognition of a modified LipL32 epitope suggests the intriguing possibility that LipL32 modification represents a novel mechanism of immune evasion within Leptospira.The exclusive detection of PTMs on lysine residues within LipL32 from  Leptospira spp., constitutes an increasing global public health threat. Humans are accidental hosts, and acquire the disease primarily from contact with water sources that have been contaminated with urine from infected animals. Rats are asymptomatic carriers of infection and are critical for disease transmission to humans, particularly in urban slum environments. In this study, investigation of Leptospira directly isolated from the urine of infected rats showed acetylation or tri-methylation of the highly abundant leptospiral lipoprotein, LipL32. In comparison, Leptospira grown in culture did not result in any LipL32 lysine modifications. A synthetic peptide derived from LipL32 that incorporated a tri-methylated lysine modification exhibited less reactivity with serum from leptospirosis patients compared to an unmodified version of the peptide, suggesting LipL32 modifications may alter protein recognition by the immune response. This study reports, for the first time, modification of a Leptospira protein during infection, and suggests these modifications may have a functional consequence that contributes to bacterial persistence during infection.Leptospirosis, caused by pathogenic  Leptospira spp. are the causative agents of leptospirosis, which is considered to be the world's most widespread zoonotic disease Pathogenic Leptospira contain the spirochetes within the renal tubules of the kidney. Dissemination of pathogenic Leptospira occurs largely via urine excreted from these infected hosts, with bacterial acquisition by new hosts occurring through cuts/abrasions or mucous membranes as a consequence of direct exposure to infectious urine or urine-contaminated water Leptospira is the brown rat, Rattus norvegicusLeptospira in urine at high densities (104 to 107 cells/ml) over a prolonged period of time due to persistent renal colonization, and without apparent detrimental effects on the health of the rat Leptospira within the urine of patients for weeks and, in rare instances, months have been reported Mammalian hosts that are chronically infected with Leptospira spp. are able to persist within mammalian hosts are incompletely understood, we have put forward the hypothesis that post-translational incorporation of methyl groups in leptospiral outer membrane proteins (OMPs) may enhance bacterial survival by modifying proteins and their respective functional interactions within the host. In support of this we have previously reported the addition of O-methyl esters to selected glutamic acid residues within the leptospiral protein OmpL32 et al. have detected mono-, di- and tri-methylations on proteins from L. interrogans serovar Lai, including the major leptospiral lipoprotein LipL32 Rickettsia prowazekii that methylation of OmpB alters the immunogenicity of this outer membrane protein Rickettsia Breinl and Evir strains have been shown to be more extensively methylated, and more protective, than OmpB from the avirulent Madrid E strain Although the underlying mechanisms by which pathogenic Leptospira serovars and constitutes the most abundant protein within the cell  in vitro-cultured Leptospira. Through these analyses we show that PTMs, composed of either tri-methylations or acetylations, occur solely on lysine residues from in vivo-derived LipL32. Four PTMs occurred in regions of LipL32 previously identified as B cell epitopes, and one PTM decreased epitope recognition by human leptospirosis serum. This study provides the first in vivo proteomic investigation of post-translational modifications of a major leptospiral lipoprotein and provides insight into the potential functional consequences of such modifications.In the current study we investigate the presence of PTMs, including methylations, occurring within the major leptospiral lipoprotein LipL32. Since LipL32 is found only within pathogenic Samples were obtained from patients with confirmed leptospirosis Leptospira interrogans serovar Copenhageni strain RJ16441, a human clinical isolate In vitro cultivated Leptospira (IVCL) samples were cultured at 30°C in Ellinghausen-McCullough-Johnson-Harris liquid medium  g for 10 min at 4°C when cultures reached a density of 1×108 leptospires/ml, and bacterial pellets were washed three times with 10 mM Tris pH 7.4/1 mM EDTA and stored at −20°C. Rat urine isolated Leptospira (RUIL) samples were prepared as previously described Rattus norvegicus Wistar strain animals  were infected by intraperitoneal injection with 5×107 low passage in vitro cultivated Leptospira. Upon detection of infection, rats were placed into metabolic cages and urine was collected twice weekly for six weeks. Urine samples were centrifuged at 12,000× g for 10 min at 4°C and pellets were washed and stored at −20°C as outlined above.8 cells) and RUIL pellets  were solubilized by overnight incubation at room temperature in a total volume of either 80 µL (IVCL) or 240 µL (RUIL) rehydration buffer . The protein concentration of the IVCL and RUIL samples was determined using the RC/DC protein assay kit . A total of 100 µg of protein from the IVCL samples and 400 µg of protein from the RUIL samples was used to rehydrate 18 cm 4–7pH IPG strips overnight, and proteins were separated by two dimensional gel electrophoresis (2DGE) and stained with SYPRO Ruby as previously described IVCL pellets  as previously described L. interrogans proteome in UniProtKB annotation (http://www.uniprot.org/uniprot/?query=leptospira+interrogans+serovar+copenhageni&sort=score&format=*) that was maintained in-house at the University of Victoria-Genome BC Proteomics Centre. Search parameters used were as follows: fixed modification was set to carbamidomethyl (C) and variable modifications were set to methyl (D/E/K), tri-methyl (K), acetyl (K), peptide mass tolerance +0.3 Da, fragment mass tolerance +0.15 Da and trypsin enzyme specificity with 2 missed cleavages. Peptides with ion scores at or above the significance threshold (set at 28) were included in the analysis. Post-translational modifications comprising tri-methylations and acetylations were confirmed via manual de novo sequencing using the user input sequence for a given peptide spectrum. To be classified as a confirmed PTM and to identify the precise location of the PTM within the peptide, at least one of the following requirements needed to be achieved: (1) the presence of a neutral loss ion (MH+-59) unique for a tri-methylated lysine Δ m/z of 42 daltons.MS/MS data were searched by centroiding with Analyst QS 1.1 Mascot script 1.6 b24 (MDS Sciex) to create the Mascot generic format file (.mgf) for data base searching. The.mgf files were searched against the Treponema pallidum Tp0751 protein sequence 2 fragment-peroxidase . Wells were washed six times with PBST and developed at room temperature with the TMB peroxidase substrate system . Plates were read at 600 nm with a Synergy HT plate reader , and statistical analyses were performed using the Student's two-tailed t-test.Two LipL32 peptides corresponding to previously identified LipL32 immunodominant epitopes 2+-bound LipL32 structure http://www.pymol.org/pymol.Annotation of the identified lysine modifications within the solved CaL. interrogans serovar Copenhageni cells grown under in vivo and in vitro growth conditions. Total protein was prepared from leptospires shed in the urine of rats housed in metabolic cages  and from leptospires cultured in EMJH medium , and each of the whole cell proteomes was independently subjected to two-dimensional gel electrophoresis  or acetylation (Δ m/z 42.0106). Due to the highly similar m/z ratios obtained for tri-methylation and acetylation, differentiation of these two modifications via mass spectrometry relies upon detection of the neutral loss ion (MH+-59) which is unique for a tri-methylated lysine +-59 neutral loss ions  we detected tri-methylation of residue K152. For the LipL32 epitope encompassing residues 162–185 of the mature protein  we detected modifications on K166, K172 and K178, with the latter constituting a confirmed tri-methylation. In order to investigate if the modifications altered immune recognition of these epitopes, synthetic peptides corresponding to the two previously identified epitopes were prepared; specifically, unmodified and K152 tri-methylated versions of P1 were synthesized, while for P2 an unmodified and a representative modified peptide containing a tri-methylated K178 residue were synthesized (152 tri-methylated peptide version was significantly decreased (p<0.0001). As a control, polyclonal antiserum raised against recombinant LipL32 was tested for reactivity against the unmodified and modified versions of P1; interestingly, no difference in reactivity was observed.Analysis of the modified LipL32 peptides detected within the RUIL samples highlighted four peptides that overlap with regions previously identified through epitope mapping studies as antigenic regions of LipL32 thesized . When tethesized , pooled et al. against P2 using hyperimmune rabbit serum In our study minimal reactivity of both patient sera and rabbit serum was observed against the unmodified and modified versions of P2, a result that differs from the reactivity observed by Lottersberger 2+-bound LipL32 crystal structure 1-K253 which corresponds to the mature LipL32 protein  set forward by Vivian et al.et al.29), the loop between the β8 and β9 strands (K152), the β9 strand (K166), the loop between the β9 strand and the α3 helix (K172), the α3 helix (K178), the loop between the β10 and β11 strands (K199) and the α4 helix (K245 and K246). All eight modified lysines localized to surface accessible regions of the structure  and the Ca2+ ion-coordinating residues in the α1β7 loop (D113 and T114) and the β8β9 loop  53, V54, Y62, W115, R117, Y151 and Y198199 is positioned on a flexible loop between strands 10 and 11 that bridges the Ca2+ and reported collagen-binding sites of LipL32, suggesting that modification of K199 may play a role in regulating protein architecture and function.The structural position of the eight modified lysines detected within the LipL32 sample originating from the rat urine-isolated leptospires was determined through examination of the Catructure . Of noteL. interrogans. The leptospiral protein OmpL32 (corresponding to LIC11848) was shown to be differentially methylated on glutamic acid residues from L. interrogans grown under in vitro conditions in vitro-cultured L. interrogans serovar Lai detected multiple PTMs on a broad range of proteins, including 46 proteins with 54 lysine acetylation sites, 104 proteins with 135 glutamic acid/glutamine methylation sites, and 58 proteins with 64 lysine/arginine methylation sites. One of the lysine-modified proteins detected within the latter study was LipL32, which was shown to be devoid of lysine acetylations but to contain mono-, di-, and tri-methyl groups on residue K152152, as well as seven other lysine residues, within LipL32 from in vivo-grown leptospires. Of the modified lysines that were detected an unambiguous assignment of tri-methylation could be made for only residues K152, K178 and K246. By extrapolation from these results it is plausible that the remaining five detected PTMs constitute similar lysine tri-methylations, however definitive assignment could not be made from the spectra obtained in this study.Two previous proteomic studies have established the presence of post-translationally modified proteins within in vitro-cultured L. interrogans. Our study extends this observation and establishes that, at least for modification of lysine residues within the major leptospiral protein LipL32, this phenomenon also occurs under biologically relevant in vivo infection conditions. Of particular interest is the fact that in our study modification of LipL32 lysine residues was restricted to in vivo-grown organisms, while in the study performed by Cao and colleagues LipL32 lysine modification was observed at K152 within in vitro-cultured Leptospirain vitro studies . Alternatively, the difference may have arisen due to the higher in vitro culture density attained for L. interrogans in the Cao et al. study, which reached approximately 6.6×108 bacteria/ml compared to 1×108 bacteria/ml in our study. The higher density may be more representative of that achieved during kidney colonization by Leptospira and thus may indicate the process of protein modification is dependent, at least partially, upon elevated bacterial density. Such a phenomenon has recently been described for Pseudomonas aeruginosa with respect to the PrmC methyltransferase, a protein that mediates methylation of key virulence factors and whose activity is controlled by quorum sensing The prior proteomic studies outlined above establish that post-translational protein modification is a phenomenon which occurs within et al. to be most strongly reactive with acute phase sera 152 within one of these epitopes significantly decreased the reactivity of leptospirosis patient serum compared to the unmodified epitope version suggests the possibility that lysine tri-methylation within LipL32 may decrease immune recognition of this protein during infection. Supporting this theory is our finding that decreased reactivity was solely observed using patient sera and not rabbit serum raised against recombinant LipL32, suggesting the decreased immune recognition of the modified LipL32 epitope is a phenomenon observed only under conditions of infection. Additionally, the modifications present within Leptospira isolated from rats may differ from those arising during infection in humans, a possibility that would further explain the decreased immune recognition of patient sera against a rat-derived modified LipL32 peptide.In this study we observed a correlation between lysine modifications occurring within LipL32 and regions of the protein that have been previously identified as immunogenic. Seven out of eight of the identified lysine modifications occurred in regions identified by Hauk and colleagues as being reactive with patient sera 113, T114, D145, D146, and Y159, with the latter three residues being localized to the β8β9 loop within the LipL32 structure 152 similarly lies within the β8β9 loop. The relatively large size and charge modification of a tri-methyl group at K152 and either an acetyl or tri-methyl modification at K199, combined with the close proximity to the Ca2+-coordinating residues D145, D146 and Y159, has the potential to affect Ca2+ binding. This may affect the stability of LipL32 and/or impact LipL32 functions that are linked to calcium binding, such as the putative immune system modulatory function that has been ascribed to the calcium-bound state of LipL32 (promotion of interaction with the Toll-like receptor 2 to induce an inflammatory response) et al. to be independent of calcium binding An alternative, or additional, proposed function for LipL32 lysine modification comes from determination of the location of the modified residues within the solved LipL32 structure. LipL32 has been shown to possess a calcium-binding cluster that comprises residues Din vivo-isolated organisms, indicating that subpopulations of modified/unmodified LipL32 proteins are present during the course of an infection. These could correspond to distinct subpopulations comprised of surface-exposed/modified and sub-surface/unmodified LipL32 molecules. Additionally, since the RUIL samples analyzed in this study constitute a pool collected over a six week time frame, PTMs could have arisen over time in response to immune pressure, with a higher proportion of modified LipL32 being present at later time points. Both scenarios suggest LipL32 modification could play a role in establishment of persistent infection. However, a study by Murray and co-workers showed no reduction in the ability of an L. interrogans lipL32 transposon mutant to colonize rats lipL32 mutant to persist beyond 15 days post-infection was not assessed, thus leaving open the possibility that wild-type and LipL32-deficient Leptospira may exhibit differences in the capacity to establish long-term infection within rats.Although originally identified as a surface-exposed outer membrane lipoprotein, a recent study using multiple, independent methods has suggested that LipL32 has limited surface exposure 29, K152, K166, K172, K178). The possibility exists that LipL32 modification decreases both humoral and cell-mediated immune recognition of the protein, thus preventing effective bacterial clearance and promoting leptospiral persistence. The importance of prevention of renal tubule colonization with respect to protection from leptospiral infection is exemplified by the study performed by Seixas et al. In this study, hamsters immunized with Mycobacterium bovis bacillus Calmette-Guérin (BCG)-expressing LipL32 were more likely to survive Leptospira challenge than hamsters immunized with BCG alone, and enhanced survival corresponded with an absence of renal tubule colonization The discovery of LipL32 lysine modification in organisms shed in the urine has potential implications for understanding how leptospires persist for extended periods of time in the renal tubules. Such persistence is responsible for the ability of leptospires to maintain high rates of shedding, transmission, and infection among reservoir host populations. An unanswered paradox in leptospiral biology is why leptospires exhibit high levels of LipL32 expression in the renal tubules in vivo, but not in vitro, conditions and provides insight into the potential functional and immunological consequences of these modifications. In addition this study provides a potential molecular mechanism for, and link between, establishment of chronic Leptospira infection in rats and acute Leptospira infection in humans. Of importance to assess through future studies are the temporal occurrence, prevalence, and biological relevance of LipL32 lysine modifications at both the cellular and community levels, as well as whether PTMs are detected on other leptospiral OMPs during the course of infection. These investigations may uncover the elusive function of this highly abundant leptospiral protein and may reveal a novel mechanism of bacterial immune evasion.In summary, this study reports the detection of multiple modified lysine residues within the major leptospiral protein LipL32 isolated from leptospires grown under Figure S1MS/MS spectra for peptides containing a modified lysine. Modified lysines are underlined in the peptide sequence. MH+-59 ions, which are neutral loss ions indicative of peptides containing a tri-methylated lysine (PDF)Click here for additional data file.Table S1MS/MS data for unmodified and modified peptides detected in IVCL and RUIL LipL32 protein spots. Peptides containing a confirmed modified lysine residue are shaded grey, and the modified residue is bolded and underlined in the peptide sequence.(XLSX)Click here for additional data file.


Acute liver injury is a clinically important pathology and results in the release of Danger Associated Molecular Patterns, which initiate an immune response. Withdrawal of the injurious agent and curtailing any pathogenic secondary immune response may allow spontaneous resolution of injury. The role B cells and Immunoglobulin M (IgM) play in acute liver injury is largely unknown and it was proposed that B cells and/or IgM would play a significant role in its pathogenesis.Tissue from 3 models of experimental liver injury  and patients transplanted following paracetamol overdose were stained for evidence of IgM deposition. Mice deficient in B cells (and IgM) were used to dissect out the role B cells and/or IgM played in the development or resolution of injury. Serum transfer into mice lacking IgM was used to establish the role IgM plays in injury.Significant deposition of IgM was seen in the explanted livers of patients transplanted following paracetamol overdose as well as in 3 experimental models of acute liver injury . Serum transfer into IgM-deficient mice failed to reconstitute injury (p = 0.66), despite successful engraftment of IgM. Mice deficient in both T and B cells (RAG1-/-) mice (p<0.001), but not B cell deficient (μMT) mice (p = 0.93), were significantly protected from injury. Further interrogation with T cell deficient (CD3εKO) mice confirmed that the T cell component is a key mediator of sterile liver injury. Mice deficient in B cells and IgM mice did not have a significant delay in resolution following acute liver injury.IgM deposition appears to be common feature of both human and murine sterile liver injury. However, neither IgM nor B cells, play a significant role in the development of or resolution from acute liver injury. T cells appear to be key mediators of injury. In conclusion, the therapeutic targeting of IgM or B cells (e.g. with Rituximab) would have limited benefit in protecting patients from acute liver injury. The term acute liver injury (ALI) encompasses a spectrum of sterile or infective hepatocellular insults characterised by acute inflammation within the liver. Injury results in the release of Danger Associated Molecular Patterns (DAMPs), which initiate an immune response. Withdrawal of the injurious agent and curtailing any pathogenic secondary immune response may allow spontaneous resolution of injury , 2.ALI may progress to acute liver failure, which is associated with a mortality of up to 50% , 4. In tIRI results from the interruption then reinstatement of an organ’s blood supply. It limits access to donor organs and has been linked to early graft failure, as well as both acute and chronic rejection , 8. IRI B cells are capable of shaping the nature of an immune response through their ability to present antigen and via their ability to produce both cytokines and antibodies. This may have a pro-inflammatory or regulatory influence on the resulting immune response . B cellsImmunoglobulin M (IgM) is produced by B cells and is a normal component of serum. Natural IgM is thought to be produced as a response to self-antigen; it is polyreactive and has a role in the clearance of apoptotic cells . IgM wasThe role IgM and B cells play in acute liver injury is largely unknown and it was proposed that IgM and/or B cells would play a significant role in its pathogenesis.Following local ethical approval at the University of Edinburgh, animal work was carried out according to UK Home Office regulations  under licenses 60/4045 and 60/2605. Mice were housed under specific pathogen-free conditions at the University of Edinburgh. General anaesthesia (GA) was induced via inhaled isoflurane and post-operatively subcutaneous opioid analgesia (buprenorphine) was administered. Animals were sacrificed under GA by terminal exsanguination by way of cardiac puncture.The Tissue Governance Unit (South East Scotland SAHSC BioResource) provided ethical approval for the human tissue used in this study.All wild type (WT), knockout and transgenic mice were on a C57BL/6J background and matched for age and sex. RAG1-/- lack IgM and both mature B and T cells . μMT micIn this model of warm partial hepatic IRI, an atraumatic clamp was applied to the vascular pedicle supplying the left lobe for 40 minutes; the liver was then allowed to reperfuse for between 1 hour and 7 days. Care was taken to maintain intraoperative core body temperature at 36°C to minimise the masking effects of hypothermia on liver IRI . In seruParacetamol-induced liver injury and concanavalin A (con A) hepatitis models were used as alternative models of sterile liver inflammation. Briefly, following a period of 18–24 hours of fasting, mice were given a 200mg/kg intraperitoneal injection of paracetamol . Con A hepatitis is a model of immune-mediated hepatitis  and was The extent of any liver injury was assessed in terms of the serum level of alanine aminotransfersase (ALT), a biochemical marker of liver injury . ALT wasThe Tissue Governance Unit (South East Scotland SAHSC BioResource) kindly provided formalin-fixed sections from the explanted livers of patients requiring transplantation following paracetamol overdose; tissue blocks were specifically chosen by a consultant pathologist with a special interest in transplantation to include areas of both injured and uninjured liver. Antigen retrieval on these sections was performed by heating samples with Borg Decloaker . Tissue from the models of murine liver injury were fixed in methacarn and did not require a specific stage of antigen retrieval.Sections were deparaffinised and rehydrated before endogenous peroxidase and avidin/biotin activity were quenched, prior to incubating with a goat anti-mouse IgM antibody  at a dilution of 1 in 5000 or goat anti-human IgM antibody  at a dilution of 1 in 1000. Slides were subsequently incubated at room temperature with a polyclonal rabbit anti-goat biotinylated secondary antibody  at 1 in 400 dilution for 40 minutes. Sections were then developed with VectaStain RTU Elite (Vector Laboratories) followed by diaminobenzidine, before being counterstained.IgM deposition was quantified from 6 randomly taken images at 200x magnification from each sample using ImageJ software ; it is expressed as percentage of total area.Single cell preparations from the liver were generated by a combination of mechanical  and enzymatic  digestion. This was then passed through a 70μm filter and centrifuged at 50g for 5 minutes to remove hepatocytes, clumps of cells and debris. Red cells were lysed . Immune cells were isolated by positive selection using a CD45+ MicroBead AutoMACS separation (Miltenyi Biotec) and then stained with a fixable Live-Dead marker  and a multi-colour panel of antibodies, including CD3, CD4, CD8, CD19 and NK1.1 . Samples were then run on a BD LSR II Fortessa  and analysed with FlowJo software . B cells were defined as CD3-CD19+ cells, within a forward-side scatter defined lymphocyte gate.Groups were analysed with the aid of Prism 5 for Mac OSX ; statistical methods are referred to specifically in the results section.To investigate whether IgM was deposited in areas of acute liver injury, sections from the explanted livers of patients transplanted for fulminant liver failure following paracetamol overdose were stained for IgM. Significant staining for IgM was seen in areas of injury . This deRAG1-/- are deficient in mature T cells, B cells and IgM . RAG1-/-Given the protection seen in RAG1-/- mice was not attributable to deficiency in immunoglobulins normally present in serum , it was Following reperfusion, there was a rapid accumulation of immune cells within the injured liver ; these wB cell deficient (μMT) mice were subjected to hepatic IRI and no significant protection was seen compared to wild type controls . This isDeposition of IgM occurred early after injury and disappeared with injury resolution . To testThis implies that neither B cells nor IgM were important in the resolution of tissue injury in the post-ischemic liver.Paracetomol toxicity and hypoxic liver injury typically cause zone 3 necrosis. In this study, the pattern of IgM staining in experimental models of these injuries, as well as in sections of liver from patients transplanted as a result of liver failure induced by paracetomol toxicity, mirrored this zone 3 distribution of injury . DepositGiven the time-frame in which this occurs (hours), the IgM “painting” injured cells is likely to represent polyreactive natural antibody (IgM) produced by B cells and found naturally in serum . This “pWe also found that RAG1-/- mice, which also lack both B and T cells, were significantly protected from injury ; this coThis is the first study, to look directly at the role T cells play in hepatic IRI with the use of a specific T cell knockout mice (CD3εKO). Work using TCR-/- mice has been performed in models of renal IRI, where T cells were also found to be key mediators of injury , 39. TheBased on our data, therapeutic intervention targeting IgM or B cells (such as with Rituximab) would have no significant benefit to patients or impact on their recovery following liver injury.


Painful Diabetic Neuropathy (PDN) is a debilitating syndrome present in a quarter of diabetic patients that has a substantial impact on their quality of life. Despite this significant prevalence and impact, current therapies for PDN are only partially effective. Moreover, the cellular mechanisms underlying PDN are not well understood. Neuropathic pain is caused by a variety of phenomena including sustained excitability in sensory neurons that reduces the pain threshold so that pain is produced in the absence of appropriate stimuli. Chemokine signaling has been implicated in the pathogenesis of neuropathic pain in a variety of animal models. We therefore tested the hypothesis that chemokine signaling mediates DRG neuronal hyperexcitability in association with PDN.We demonstrated that intraperitoneal administration of the specific CXCR4 antagonist AMD3100 reversed PDN in two animal models of type II diabetes. Furthermore DRG sensory neurons acutely isolated from diabetic mice displayed enhanced SDF-1 induced calcium responses. Moreover, we demonstrated that CXCR4 receptors are expressed by a subset of DRG sensory neurons. Finally, we observed numerous CXCR4 expressing inflammatory cells infiltrating into the DRG of diabetic mice.These data suggest that CXCR4/SDF-1 signaling mediates enhanced calcium influx and excitability in DRG neurons responsible for PDN. Simultaneously, CXCR4/SDF-1 signaling may coordinate inflammation in diabetic DRG that could contribute to the development of pain in diabetes. Therefore, targeting CXCR4 chemokine receptors may represent a novel intervention for treating PDN. Neuropathic pain in diabetes, Painful Diabetic Neuropathy (PDN), is a debilitating affliction present in 26% of diabetic patients -3 with sThis lack of understanding of the pathogenesis and the molecular mechanisms underlying neuropathic pain represents a barrier to further progress in this field as emphasized by the failure of numerous potential therapeutic approaches to successfully treat PDN. Opiates are not particularly effective in treating neuropathic pain and given the chronic nature of this syndrome their use is problematic . Other dPain is a physiological response to potentially dangerous noxious stimuli. However, pathological or “neuropathic pain” is associated with sustained excitability of sensory neurons within pain pathways leading to reduced nociceptive thresholds and the development of pathological spontaneous activity, so that pain is produced in the absence of appropriate stimuli -9. The mChemokines, or chemotactic cytokines, are a large group of proteins important for the regulation of leukocyte migration ,16. In tBased on this evidence, we hypothesized that excitatory CXCR4/SDF-1 signaling in DRG neurons may play a critical role in the pathogenesis of PDN.We now demonstrate that administration of a selective CXCR4 antagonist, AMD3100, substantially reverses neuropathic pain in animal models of type II diabetes. These results indicate that CXCR4 mediated signaling is necessary for PDN in mice. This hypothesis is consistent with the expression of CXCR4 by a subpopulation of DRG neurons and increased excitatory effects of the chemokine SDF-1 on diabetic DRG neurons in culture. Additionally, we observed numerous CXCR4 expressing inflammatory cells infiltrating the DRG of diabetic mice.Overall, these data suggest that CXCR4/SDF-1 signaling mediates enhanced calcium influx and excitability in DRG neurons responsible for PDN. CXCR4/SDF-1 signaling may simultaneously coordinate inflammation in diabetic DRG that could contribute to the development of pain in diabetes. Therefore, CXCR4 signaling may constitute a novel target for therapeutic intervention to PDN.In our first series of experiments we set up a model of type II diabetes by feeding mice with a High Fat Diet (HFD) for 10 weeks. These mice exhibited a gain in weight and became glucose intolerant indicating the development of diabetes  injection. We elected to use this concentration of AMD3100 as we have previously demonstrated it to be effective in other animal models of neuropathic pain ,20. Von These experiments demonstrated that AMD3100 substantially reversed neuropathic pain in HFD diabetic mice. Indeed, 2 hours after antagonist injection in HFD diabetic mice the paw withdrawal threshold increased significantly compared with HFD induced diabetic mouse injected with saline. Statistical analysis revealed that the 50% threshold was 1.001 g with SE 0.07 in HFD diabetic mice injected with ADM3100 (n = 8) compared to 0.157 g with SE 0.013 in HFD diabetic mice injected with saline (n = 8). Values are expressed as means +/− SE (p <0.001)  mouse line. Db/db mice are known to display mechanical allodynia after 6 weeks of age . In keepOverall this first series of experiments demonstrated that SDF-1/CXCR4 signaling is necessary for the expression of pain hypersensitivity in two separate animal models of type II diabetes. As a result of these observations we next wanted to investigate the status of excitatory CXCR4/SDF-1 signaling in DRG neurons taken from diabetic animals.One way that chemokine signaling could contribute to chronic pain is by directly exciting DRG neurons, something that is associated with increases in neuronal Ca2+]i . Our lab+i . Our These results indicate that a increased number DRG neurons express functional CXCR4 receptors in diabetic mice and suggest that increased excitatory CXCR4/SDF-1 signaling in DRG neurons could contribute to PDN.Extensive characterization of DRG neurons has revealed the existence of molecularly and functionally defined subpopulations of cells. According to one type of classification following sensory neurogenesis prospective DRG nociceptive neurons undergo two distinct differentiation pathways leading to the formation two major classes of molecularly defined DRG neurons: 1) TrKA positive sensory neurons, also known as “peptidergic” neurons which express Calcitonin Gene Related Peptide (CGRP) or Substance P and 2) Ret positive sensory neurons also known as “non-peptidergic” neurons, first identified for their ability to bind isolectin IB4 . These tin situ localization of CXCR4 mRNA. These experiments showed that some DRG neurons normally exhibited very high levels of CXCR4 mRNA , 18 weeks old heterozygous mice , 8 weeks old CXCR-4 eGFP mice and SDF-1-mRFP mice, generated in our laboratory i imaging assays. The reason for this overall shift in the pattern of DRG neurons expressing CXCR4 receptors in diabetic mice is elusive at this point. However, we did note that DRG neurons from diabetic mice exhibited greatly increased levels of SDF-1.[Ca2+]i imaging experiments clearly demonstrated increased calcium mobilization in response to SDF-1 in cultured diabetic DRG neurons compared non diabetic control animals. Our laboratory and others have previously shown that activation of CXCR4 receptors by its ligand SDF-1 excites DRG neurons. Increased [Ca2+] i can be used as a readout of DRG neuron hyperexcitability in animal models of neuropathic pain ,25,36. TWe have demonstrated that SDF-1 can be released from DRG neurons ,21. HencOverall, these results are particularly interesting in light of recent publications using gene arrays to profile sural nerve samples from PDN patients, which reported that CXCR4 chemokine receptor expression was up-regulated in these samples . Hence, in situ hybridization studies also revealed the existence of a second population of DRG neurons, which express lower levels of CXCR4 receptors although we did not identify the properties of this neuronal subtype. Nevertheless, excitatory effects of CXCR4 activation in these neurons may also contribute to pain in diabetic mice.During embryonic development DRG sensory neurons differentiate into three primary subtypes: nociceptors, mechanoreceptors and proprioceptors. In the DRG, these are characterized by the expression of the neurotrophin receptors TrkA, TrkB and TrkC, respectively . During In addition to their pronociceptive effects on DRG neurons chemokines and their receptors are important in the recruitment of leukocytes. The observed recruitment of numerous inflammatory cells into diabetic DRG suggests a critical role for inflammation in the pathogenesis of PDN. Immune cells can certainly secrete numerous algogenic factors, which may also have direct excitatory effects on DRG neurons or may sensitize DRG neurons to the excitatory effects of SDF-1 or other mediators ,14. SDF-The role of inflammation in the pathogenesis of PDN has been proposed elsewhere . SpecifiIn conclusion, these studies establish CXCR4/SDF-1 chemokine signaling as a new candidate responsible for the manifestation of PDN. The data suggest a mechanism as to how ongoing sensory neuron excitability could be increased in order to induce and maintain neuropathic pain in diabetes. This concept is that chemokine signaling has a fundamental role in in producing increased excitability of sensory neurons during the transition from physiological to neuropathic pain in diabetes, as well as its subsequent maintenance. This is something that has not been previously appreciated with respect to PDN. Furthermore, SDF-1 could potentially play a crucial role in both recruiting inflammatory cells and producing direct excitatory effects on DRG neurons thereby coordinating the interaction between peripheral neurons and immune cells. Hence, targeting CXCR4/SDF-1 signaling may lead to advanced therapeutic approaches for the treatment of PDN. This novel and more targeted therapeutic intervention has advantages over existing and currently available therapeutic approaches which aim only to reduce symptoms generally by suppressing neuronal activity and are not particularly effective. In contrast, modulation of proalgesic chemokine signaling provides an opportunity for disease modification by inhibiting inflammatory process and neurobiological alterations that support the development of persistent pain.The authors declare that they have no competing interests.DMM prepared the manuscript, performed Von Frey behavioral studies, calcium imaging studies, immunohistochemical labeling, confocal analysis and statistical analysis, AB performed calcium imaging studies, AS performed immunohistochemical labeling, CF helped with Von Frey behavioral studies, DR cultured DRG neurons for calcium imaging studies and in situ hybridization, RJM supervised the project and help writing the manuscript. All authors read and approved the manuscript.


Currently there is little effective treatment available for castration resistant prostate cancer, which is responsible for the majority of prostate cancer related deaths. Emerging evidence suggested that cancer stem cells might play an important role in resistance to traditional cancer therapies, and the studies of cancer stem cells (including specific isolation and targeting on those cells) might benefit the discovery of novel treatment of prostate cancer, especially castration resistant disease. In this review, we summarized major biomarkers for prostate cancer stem cells, as well as their functional mechanisms and potential application in clinical diagnosis and treatment of patients. Prostate cancer (PCa) is the most common non-skin cancer in American men ,2. StandCancer stem cells (CSCs) were defined as cells with capacity of self-renewal and proliferation in cancer tissue . Over yeIntegrins are a family of transmembrane receptors known to participate in cell-cell adhesion and cell-surface mediated signaling, serving as bridges for cell-cell and cell-extracellular matrix (ECM) interactions . IntegriMicroarray experiments performed by several independent groups found that Integrin-α6  is consistently overexpressed in hematopoietic, neural, and embryonic stem cells, and it is suggested as an effective cell stemness marker . It has + PCa cells retain certain intrinsic properties of progenitor cells [+ cells express high levels of stemness genes including Oct-3/4, Bmi, β-catenin and Smoothened (SMO) [+ and CD44− Cells and tested the gene expression of stem cells by RT-PCR. Low expression of luminal cell markers  and AR were observed in CD44+ cells, whereas the genes highly related to stem cell proliferation and differentiation were overexpressed [+ cells had positive correlation with genes related to cancer proliferation and metastasis [+ prostatic adenocarcinoma cells [CD44 is a single-pass type I transmembrane protein and an important cellular adhesion molecule related to signaling to extracellular matrix. CD44 was considered as a marker of cancer stem cells from many organs including prostate ,22,23. Ior cells . CD44+ ced (SMO) ,27. Kasped (SMO) . Van et xpressed . Recentlxpressed , and protastasis . Howevertastasis . Their rma cells .Recently, quite a few thorough analyses have been done on CD44 isoforms that are generated through alternative splicing of CD44 precursor mRNA. Those CD44 variants function distinctly in PCa and might serve as independent markers comparing to total CD44 expression level. For example, CD44v2 correlated with a better recurrence-free survival rate in PCa patients and is underexpressed in metastatic PCa cell lines . AnotherTaken together, those results demonstrated CD44 RNA isoforms, but not total CD44 protein, might serve as specific marker for prostate cancer stem cells, though total CD44 protein level might still serve as a stem cell marker for other types of cancers .+/α2β1hi/CD133+ cells and tumor grade [CD133 is a glycoprotein with five transmembrane domains, generally expressed in various stem cells and endothelial progenitor cells but not in mature endothelial cells . CD133 hor grade . In normor grade . Althougor grade .+ stem cells but was consistently observed in the CD133− population [− basal cells are more susceptible to tumorigenesis compared to the CD133+-enriched stem cells. These findings challenged the current dogma that normal stem cells and cells of origin of cancer are the same cell type(s) [Prostatic basal cells could be enriched based on α2β1 integrin (hi) expression and further enriched for stem cells using CD133 in non-tumorigenic BPH-1 cells . It is dpulation . These d type(s) . Intensi+ cells also displayed high colonization in vitro and highly invasive tumorigenesis and aggressive metastasis characteristics in vivo [ALDH1 was suggested as a stem cell marker for both normal and tumor tissues . As a cy in vivo . p63 cyt in vivo . These c in vivo .The aldehyde dehydrogenase enzymes are likely to protect stem cells by detoxification of cell toxic compounds, which indicates that ALDH1 might prevent prostate cancer stem cells from conventional chemotherapy attack, while effective inhibition of ALDH1 could enhance the chemotherapy efficiency. Thus, ALDH1 could not only be used as a prostate cancer stem cell marker for prognosis, but also as a potential drug target in cancer treatment.Studies have shown that prostate cancer contains side population cells (SP cells), which could be isolated by flow cytometry techniques based on behavioral characteristics of stem cells. SP cells have stem cell properties that are exclusively mediated by ABCG2. As a result, ABCG2 is considered as a marker of SP cells, as well as a cancer stem cell marker. ABCG2 is ATP binding membrane transporters, and is related to prostate cancer multi-drug resistance . After c+. On the other hand, CD44 was found in 97% of benign prostate and HGPIN cases, and in 72% of prostate cancer cases. CD133 was found in only a small portion of PCa tissues . Oct4 expression was found to be closely correlated with benign and HGPIN, but not with PCa. It is believed that CD44 and Oct4 were expressed in most of benign and malignant prostate cells, which is not likely to be representative for a very small proportion of cancer cells (such as cancer stem cells).SOX2 and EZH2 are essential for the development of human embryonic stem cells. SOX2 is a transcription factor and plays a key role in maintaining undifferentiated status and keeping self-renewal ability of embryonic stem cells . EZH2 isCD166 is a newly discovered molecular surface marker of prostate cancer stem cells . CD166 bcPAcP is a prostate specific differentiation antigen. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas . It was p = 0.001). In multivariate Cox regression, preoperative PSA and HGF positivity had the potential to be independent predictors of biochemical recurrence following prostatectomy [It was found that prostate cancer stem-like cells (CSCs)/cancer initiating cells (CICs) express hepatocyte growth factor (HGF) and that the HGF/c-MET proto-oncogene product (c-MET) signal has a role in the maintenance of prostate CSCs/CICs in an autocrine fashion. Immunohistochemical staining of HGF was compared to biochemical recurrence after radical prostatectomy, and patients with PCa tumors exhibiting HGF positivity of 5% or more had a significantly shorter biochemical recurrence-free period than that of patients whose tumor HGF positivity was less than 5%  is a type I membrane glycoprotein which transduces intracellular calcium signal and acts as a cell surface receptor ,67. TropCD117  is a receptor tyrosine kinase protein, and has been used as an important cell surface marker to identify hematopoietic progenitors in bone marrow ,86,87. CAR splice variants were found to promote EMT as well as induce the expression of stem cell signature genes . Over 10Transglutaminases are enzymes that catalyze the crosslinking of proteins by epsilon-γ glutamyl lysine isopeptide bonds. While the primary structure of transglutaminases is not conserved, they all have the same amino acid sequence at their active sites and their activity is calcium-dependent. The protein encoded by this gene acts as a monomer, is induced by retinoic acid, and appears to be involved in apoptosis. TGM2 expression is shown to negatively regulate AR expression and to attenuate androgen sensitivity of prostate cancer cells . TGM2 acStudies of prostate cancer stem cells have gained much progress in the past few years and numerous potential approaches were discussed for novel PCa treatment ,110. Thi


Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, with a prevalence of 5–8%. Type 2 diabetes and cardiovascular disease (CVD) are its long-term complications. Targeted therapies addressing both these complications together are lacking. Glucagon like peptide-1 (GLP-1) agonists that are used to treat type 2 diabetes mellitus have beneficial effects on the cardiovascular system. Hence we hypothesized that a GLP-1 agonist would improve both cardiovascular and metabolic outcomes in PCOS. To test this hypothesis, we used an established rat model of PCOS. Prepubertal female Sprague Dawley rats were sham-implanted or implanted s.c. with dihydrotestosterone (DHT) pellets . At 12 wks of age, sham implanted rats received saline injections and the DHT treated animals were administered either saline or liraglutide (0.2mg/kg s.c twice daily) for 4 weeks. Subgroups of rats were implanted with telemeters between 12-13 weeks of age to monitor blood pressure. DHT implanted rats had irregular estrus cycles and were significantly heavier than the control females at 12 weeks  and 4 weeks of treatment with liraglutide in DHT treated rats significantly decreased body weight . Liraglutide treatment in the DHT implanted rats significantly improved glucose excursion during oral glucose tolerance test . DHT rats were hypertensive and liraglutide treatment significantly improved mean arterial pressure. These results suggest that GLP-1 treatment could improve DHT–induced metabolic and blood pressure deficits associated with PCOS. Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder in women of reproductive age , 2. The Type 2 diabetes that is commonly associated with PCOS most likely occurs due to obesity and insulin resistance. Several conditions involving insulin resistance have been linked to an altered incretin effect. Incretin effect is a phenomenon whereby oral ingestion of glucose elicits a greater increase in insulin secretion than glucose administered via intravenous route  due to tThe GLP-1 receptor agonist liraglutide, is currently approved for use in type 2 diabetes to improve glycemic control. It also has beneficial effects on the cardiovascular system , 17. TheSprague Dawley rats were obtained  at 4 weeks of age and housed in temperature controlled animal quarters with constant light/dark cycles (12 hours light/12 hours dark) with free access to standard chow and water. Between 4–5 weeks of age the rats were implanted s.c. with a non-aromatizable dihydrotestosterone pellet  at the back of the neck (n = 31) or had sham surgery with no pellet implant (n = 13). Starting at 8 weeks of age they had daily vaginal cytology and body weight measurements. At 12 weeks of age, a subgroup of the DHT-implanted female rats (n = 15) received twice daily s.c. injections of liraglutide 0.2mg/kg  and the rest received saline injections twice daily. Between 12–13 weeks of age, telemeters were implanted in a subgroup of the rats in the three groups, DHT+ liraglutide, DHT and controls, and telemetry data were recorded hourly and averaged every 12 hours. At around 16 weeks of age, a subgroup of rats had an oral glucose tolerance test in all three groups. Rats were euthanized by decapitation under isoflurane anesthesia between 88–90 days from DHT implantation during diestrus phase of the estrus cycles and serum was separated from trunk blood. Abdominal adipose tissue was collected and weighed. Serum was stored at -80°C until analysis. All protocols were reviewed and approved by the Institutional Animal Care and Use committee at Michigan State University .Rats were weighed at 4 weeks of age and then weekly from 9–13 weeks and again at 16 weeks of age.Animals were subjected to vaginal cytology on a daily basis starting from 8 weeks of age. A drop of the vaginal lavage was placed on a glass slide and air-dried. The smears were stained with Methylene blue. The cell composition of the smear was used to determine the stage of the estrous cycle as described previously .DHT levels were measured in duplicate using an ELISA kit obtained from My Biosource  at 88 days from DHT implantation using serum samples from all three groups. The assay was performed according to the manufacturer’s instructions.At 16 weeks of age, OGTT was conducted in a subgroup of rats  that were fasted for 10–12 hours overnight and received a 2 g/kg dose of dextrose by oral gavage. Blood glucose levels were measured using an Accu-check Aviva glucometer by tail cut bleed at 0, 30, 60, 90, 120 and 150 min post gavage.Fasting serum insulin levels were measured at 16 weeks of age in all three groups of rats using rat insulin elisa kit . The assay was performed per manufacturer’s guidelines.Total cholesterol was measured in the serum between 88–90 days from DHT implant under fasted condition (fasted for 10 hours) using the Amplex kit  .Telemeters were implanted in the rats at 12–13 weeks of age (n = 9–10 per group) under isoflurane anesthesia using aseptic technique as described previously . BrieflyAll data in the study are expressed as mean ± Standard error of the mean (SEM). Two way repeated measures ANOVA with post hoc Fisher’s LSD test was used for analysis of telemeter data, oral glucose tolerance test and body weights. One way ANOVA was used to determine differences in rate of body weight gain, serum cholesterol, DHT and abdominal fat. P value less than 0.05 was considered to be statistically significant.As shown in As shown in Changes in adipose tissue mass in the abdomen in the three groups are shown in Fasting glucose levels were not different between the groups and neither were the fasting insulin levels . However blood glucose levels in DHT-treated animals were significantly higher than that in control animals post glucose load at 30, 60 and 90 minutes (p<0.01). On the other hand, treatment with liraglutide significantly reduced blood glucose levels at 30, 60 and 90 min post glucose load in the DHT-treated rats (p<0.01) . Area unChanges in blood pressure in the three groups are shown in Average SBP during the entire observation period with DHT treatment was significantly elevated compared to control rats. However, treatment with liraglutide, significantly reduced SBP (p<0.05) (Panel B).Mean arterial pressure (MAP) remained consistently high in DHT-treated rats compared to controls. Average MAP in DHT-treated animals was significantly higher compared to control rats (p<0.01). Liraglutide treatment produced a marked reduction in MAP in the DHT-treated rats (p<0.05) (Panel C). In contrast to blood pressure, there were no significant differences in heart rate between the 3 groups.Blood pressure measurements during the night time were markedly higher in general compared to day time blood pressure . AverageIn this study, we demonstrate for the first time that a GLP-1agonist has beneficial effects on metabolic perturbations, including weight gain, abdominal adiposity and systemic glucose homeostasis, and improves hypertension in a well-established hyperandrogenic rat model of PCOS.PCOS is a condition that is characterized by the presence of polycystic ovaries, elevated circulating androgens with increased prevalence of obesity, insulin resistance and alterations in lipid profile, 26, 27.Liraglutide is a long acting Glucagon-like peptide 1 (GLP-1) receptor agonist. It is FDA approved for treating type 2 diabetes and improves glycemic control by augmenting insulin secretion from the pancreas . LiragluPCOS is also frequently associated with type 2 diabetes and impaired glucose intolerance , 38. It In the present study, we also observed increases in abdominal adipose tissue deposition and higher levels of serum cholesterol in DHT-treated rats supporting findings from previous studies, 23. OnePCOS patients have higher prevalence of hypertension and this is being reported in much younger patients in recent years, 43. MorHyperandrogenism is also an independent risk factor for metabolic syndrome in adolescents. One study reported that 37% of adolescents with PCOS had metabolic syndrome after adjusting for body mass index and insulin resistance. In the same study prevalence of hypertension was greater in girls with PCOS compared to controls. The mecDiet and exercise have been shown to have a positive impact on menstrual irregularity, hirsuitism and insulin resistance in PCOS  but thisWith the increasing prevalence of polycystic ovarian syndrome in adolescent females and higher incidence of hypertension in this syndrome, it is vital to study and target this aspect of the syndrome to prevent long term cardiovascular morbidity. Hence, development and or discovery of therapeutic agents that address not only the hyperandrogenism and insulin resistance observed in PCOS but also the cardiovascular impairment seen in this condition are crucial. Our study provides early evidence of the beneficial effects of a GLP-1 agonist in improving glucose sensitivity, abdominal obesity, serum cholesterol and hypertension. Further studies are required to specifically understand the role of GLP-1 in cardiovascular impairment in PCOS to develop new therapeutic strategies.