Type 1 Diabetes (T1D) is a chronic multifactorial disease with a strong genetic component, which, through interactions with specific environmental factors, triggers disease onset. T1D typically manifests in early to mid childhood through the autoimmune destruction of pancreatic β cells resulting in a lack of insulin production. Historically, prior to genome-wide association studies (GWAS), six loci in the genome were fully established to be associated with T1D. With the advent of high-throughput single nucleotide polymorphism (SNP) genotyping array technologies, enabling investigators to perform high-density GWAS, many additional T1D susceptibility genes have been discovered. Indeed, recent meta-analyses of multiple datasets from independent investigators have brought the tally of well-validated T1D disease genes to almost 60. In this mini-review, we address recent advances in the genetics of T1D and provide an update on the latest susceptibility loci added to the list of genes involved in the pathogenesis of T1D. Type 1 Diabetes (T1D) is a chronic multifactorial disease with a strong genetic component. It arises as a consequence of autoimmune destruction of pancreatic β-cells, resulting in insufficient insulin production. The prevalence of diabetes is increasing worldwide . T1D rep+ and CD8+ T cells, natural killer (NK) cells, B lymphocytes, macrophages, dendritic cells (DC), and antigen-presenting cells (APCs). Studies in human and animal models have shown that both innate and adaptive immune responses participate in disease pathogenesis, possibly reflecting the multifactorial nature of this autoimmune disorder.T1D develops at all ages and occurs through the autoimmune destruction of pancreatic β-cells with resulting lack of insulin production. The immune system participates in β-cell destruction through several of its components including CD4In this review, we will provide an updated summary of genome-wide association studies (GWAS) including recent meta-analyses and discuss the latest associated regions added to the growing repertoire of gene networks predisposing to T1D.The risk of developing T1D is determined by a complex interaction between multiple genes and environmental factors. The discovery of T1D susceptibility genes started as early as 1974 with five genes discovered, using family and candidate gene approaches. The advent of GWAS led to flurry of novel genes associated with T1D reaching the excess of 40 by 2006, and 60 by 2012 as depicted in versus non self-immune recognition. The class I molecules are ubiquitously expressed and present intracellular antigen to CD8+ T cells. Class II molecules are expressed mainly on professional APCs: DCs, macrophages, B lymphocytes and thymus epithelium. Class II molecules are composed of A and B chains, and present antigens to CD4+ T cells, which promote inflammation by secreting cytokines upon recognition of their specific targets. Approximately half of the genetic risk for T1D is conferred by the genomic region harboring the HLA class II genes . In 1984, insulin (INS) gene encoded on chromosome 11p15 was identified as the second loci linked with T1D [et al., 2005 reported interleukin 2 receptor alpha (IL2RA) gene as the fifth T1D loci on chromosome 10p15 [et al. identified the interferon-induced with helicase C domain 1 (IFIH1) gene on chromosome 2q24.3 as the sixth candidate to be strongly associated with T1D through genotyping of only 6,500 non-synonymous SNPs genome wide [Historically, prior to GWAS, only six loci were fully established to be associated with T1D. The human leukocyte antigen (HLA) region on chromosome 6p21 was the first known candidate to be strongly associated with T1D in the 1970s ,9,10. Thwith T1D . Anotherwith T1D . Vella eme 10p15 . In 2006The advent of GWAS in the mid-2000s has changed the situation dramatically, increasing the pace and efficiency of discovery for the T1D associated loci by a factor of ten. The critical platform for this work was laid by the HapMap project ,17. The et al., 2007 replication study confirmed these findings in 4,000 cases, 5,000 controls, and 3,000 T1D families [p-value of 9.13 × 10−10 [et al. [et al. reported an association between SNP at the UBASH3A locus on 21q22.3 and T1D by using SNP genotyping data from a linkage study of affected sib pairs in nearly 2,500 multiplex families [The first full-scale GWAS for T1D were published simultaneously by our group  and by t × 10−10 , previou × 10−10  and Toddfamilies . UBASH3Afamilies . Follow-families . A recenfamilies .p-values were taken forward for further genotyping in an additional British cohort of approximately 6,000 cases, 7,000 controls, and 2,800 families. As a result, the IL2-IL21 association strengthened further and they found strong evidence for four additional loci: a 6q15 region harboring BACH2; a 10p15 region harboring the protein kinase C, theta gene (PRKCQ); a 15q24 region harboring nine genes including cathepsin H (CTSH) and 22q13 harboring the C1q and tumor necrosis factor-related protein 6 (C1QTNF6) and somatostatin receptor 3 (SSTR3) genes [In order to get the most from GWAS and to increase the statistical power researchers carried out meta-analyses using datasets from different investigative groups. First meta-analysis was performed by combining the T1D datasets from the Wellcome Trust Case Control Consortium  and the 3) genes . Study o3) genes . Additiop < 10−6) by using datasets from WTCCC [p < 5 × 10−8) and four additional regions provided nominal evidence of replication (p < 0.05). The meta-analysis observed association to 1q32.1 , 9p24.2 contains only Glis family zinc finger protein 3 (GLIS3), which was first suggested by us in [in silico replication efforts [et al. study was later replicated in 2012 by T1DGC to exclude the possibility that any of the 18 loci were false-positives due to population stratification. Seventeen of the 18 susceptibility loci reached nominal levels of significance (p < 0.05) in the expanded family collection, with 14q24.1 just falling short (p = 0.055) [Meta-analysis by T1DGC  providedom WTCCC , the GoKom WTCCC , and conom WTCCC ,33,34 anby us in , 12p13.3 efforts  further  efforts . The ent= 0.055) . All susp = 5.66 × 10−11) resided in an intronic region of the LMO7 (LIM domain only 7) gene on 13q22. The second most significantly associated SNP  resided in an intronic region of the EFR3B (protein EFR3 homolog B) gene on 2p23; however the region of linkage disequilibrium is approximately 800 kb and harbors additional multiple genes, including NCOA1, C2orf79, CENPO, ADCY3, DNAJC27, POMC, and DNMT3A. The third most significantly associated SNP  was in an intergenic region on 6q27, where the region of association is approximately 900 kb and harbors additional genes including WDR27, C6orf120, PHF10, TCTE3, C6orf208, LOC154449, DLL1, FAM120B, PSMB1, TBP, and PCD2. These latest associations add to the growing repertoire of gene networks predisposing to T1D. To identify additional genetic loci for T1D susceptibility, we examined associations in the largest meta-analysis to date between the disease and ~2.54 million SNPs in a combined cohort of 9,934 T1D cases and 16,956 controls . TargeteThe immune system is well organized and well regulated with a basic function of protecting the host against pathogens. This places the immune system in a vital position between healthy and diseased states of the host. Its protective task is regulated by a complex regulatory mechanism involving a diverse army of cells and molecules of humoral and cellular factors working in concert to protect the body against invaders. Our immune system has two components: innate and adaptive. Innate immunity is comprised of physical, chemical, and microbiological barriers to the entry of antigen, and the elements of immune system , which provide immediate host defense. Adaptive immunity is the hallmark of the immune system of higher animals with T and B cells as the key cellular players that provide more specific life-long immunity .vs. immune (45). However, recent studies indicate that many T1D candidate genes are also expressed in human islets suggesting that functions are not restricted to immune cells, but also play roles in the islets and β cells [In T1D this system breaks down: insulin-producing β-cells are subjected to specific attack by the host immune system. To better understand the etiology of T1D for prevention and cure, a plethora of research has been done to link the systematic destruction of β-cells and the role of the immune system, however the exact mechanism of T1D pathogenesis is not completely elucidated. Linkage studies in the 1970s revealed MHC as the first key contributor to T1D susceptibility ,9,10. Fu β cells . The funThe complex crosstalk between innate and adaptive immune cells is broadly categorized in three phases, which results in the development or the prevention of T1D and is illustrated in Phase I (the initiation phase of T1D) involves β-cell death and APC activation. It takes place in the pancreas where conventional dendritic cells (cDCs) capture and process β-cell antigens. Natural cell death (apoptosis) or viral infection can lead to β-cell death. Antiviral responses are mediated by invariant natural killer T (iNKT) cells; crossplay between iNKT, and plasmacytoid DCs (pDCs) controls viral replication thus prevents subsequent inflammation, tissue damage, and downregulation of T1D pathogenesis .Phase II (the expansion phase) involves expansion of self-antigens and specific T cells. Migration of activated cDCs to the draining lymph node primes pathogenic islet antigen-specific T cells. This activation is promoted by macrophages through IL12 secretion. B cells present β-cell antigen to diabetogenic T cells and secrete autoantibodies in response. The activation of islet antigen-specific T cells can be inhibited by cDCs through engagement of programmed cell death ligand 1 (PDL1). iNKT cells can further promote the recruitment of tolerogenic cDCs and pDCs. These DCs promote expansion of regulatory T (TReg) cells through the production of indoleamine 2,3-dioxygenase (IDO), IL10, transforming growth factor-β (TGFβ) and inducible T cell co-stimulator ligand (ICOSL) .Phase III  occurs in the pancreas where β-cell can be killed by diabetogenic T cells (adaptive component) and NK cells (innate component) through the release of interferon-γ (IFNγ), granzymes, and perforin, as well as by macrophages through the production of tumor necrosis factor (TNF), IL-1β and nitric oxide (NO). IL12 produced by cDCs sustains the effector functions of activated diabetogenic T cells and NK cells. TReg cells that inhibit diabetogenic T cells and innate immune cells through IL10 and TGFβ can prevent β-cell damage. Tolerogenic pDCs stimulated by iNKT cells could also control diabetogenic T cells through IDO production. Lastly, β-cells can inhibit diabetogenic T cells by expressing PDL1 and escape the cell death ,51.There is increasing evidence that innate cells play critical roles in T1D onset. In our 2007 GWAS we identified CLEC16A as a novel T1D susceptibility gene . CLEC16AFour decades of intensive studies have discovered nearly 60 T1D susceptibility loci; however the exact mechanisms by which associated loci confer T1D susceptibility remain elusive and require in depth characterization. Several novel T1D susceptibility genes are discussed below.Our 2007 GWAS in a large pediatric cohort of European descent identified CLEC16A as a novel T1D susceptibility gene within a 233-kb linkage disequilibrium block on chromosome 16p13. Three common non-coding variants of the CLEC16A gene  reached genome-wide significance for association with T1D . ImportaThe 2007 WTCCC study independently discovered CLEC16A  as a T1D susceptibility locus associated with the non-coding variant rs12708716. This finding was confirmed immediately for T1D in populations of European descent . To dateSeveral GWAS in different autoimmune diseases such as multiple sclerosis ,58,59, pet al. reported intron 19 of the CLEC16A gene behaves as a regulatory sequence, which affects the expression of a neighboring gene dexamethasone-induced (DEXI) [Recently, Davison d (DEXI) . While iet al., 2010 characterized an endosomal membrane protein “ema” to be required for endosomal trafficking and promotes endosomal maturation in fruit flies [In vitro studies indicated that the up-regulation of CLEC16A may be involved in astrocyte activation following LPS challenge [Little is yet proven about CLEC16A functions. Kim it flies . Expressit flies . Expresshallenge .CLEC16A is well-established T1D susceptibility gene, which probably contributes to the disease by modulating immunity and thus the encoded protein, is of high interest for further functional studies.In our latest effort to identify additional genetic loci for T1D, we examined associations in the largest meta-analysis to date between T1D and ~2.54 million SNPs in a combined cohort of 9,934 cases and 16,956 controls. Targeted follow-up of 53 SNPs in 1,120 affected trios uncovered three novel loci associated with T1D that reached genome-wide significance .p = 5.66 × 10−11) resides in an intronic region of the LMO7 (LIM domain only 7) gene on 13q22 [The most significantly associated SNP  resides in an intronic region of the EFR3B (protein EFR3 homolog B) gene on 2p23; however, the region of linkage disequilibrium is approximately 800 kb and harbors additional multiple genes, including NCOA1, C2orf79, CENPO, ADCY3, DNAJC27, POMC, and DNMT3A. Protein EFR3B is an 817 amino acid and exists as three alternatively spliced isoforms. The gene encoding EFR3B maps to human chromosome 2p23.3. A number of genetic diseases have been linked to genes on chromosome 2 including Harlequin icthyosis [The second most significantly associated SNP  is a member of the p160/steroid receptor co-activator (SRC) family. The product of this gene binds to a variety of nuclear hormone receptors in a ligand-dependent manner suggesting that NCOA1 may play a role as a bridging molecule between nuclear hormone receptors and general transcription factors ,86.C2orf79 is peptidyl-tRNA hydrolase domain containing 1 (PTRHD1) predicted protein with unknown function.Centromere protein O gene (CENPO) encodes a component of the interphase centromere complex. The protein is localized to the centromere throughout cell division and is required for bipolar spindle assembly, chromosome segregation and checkpoint signaling during mitosis .Adenylate cyclase 3 gene (ADCY3) encodes a membrane-associated enzyme. This protein catalyzes the formation of the secondary messenger cyclic adenosine monophosphate (cAMP) and is highly expressed in human placenta, testis, ovary, and colon . ExpressDnaJ/Hsp40 homolog, subfamily C, member 27 gene (DNAJC27) encodes 273 amino acid protein with RAB-like GTPase and DNAJ domains. EST database reports high expression in nervous and reproductive systems .Pro-opiomelanocortin gene (POMC) encodes a polypeptide hormone precursor protein synthesized mainly in corticotroph cells of the anterior pituitary. POMC is essential for normal steroidogenesis and maintenance of adrenal weight. Mutations in this gene have been associated with early onset of obesity, adrenal insufficiency, and red hair pigmentation ,92. The de novo methyltransferase that can methylate unmethylated and hemimethylated DNA with equal efficiencies [DNA (cytosine-5)-methyltransferase 3 alpha gene (DNMT3A) encodes a protein that functions as a ciencies .Additional fine gene mapping and functional studies are needed for above-mentioned genes to determine causal variants for 2q23 region and their role in T1D.p = 8.06 × 10−9) in our recent study [Intergenic region on 6q27 contained the third most significantly associated SNP  finger protein 10 gene (PHF10) encodes a subunit of an ATP-dependent chromatin-remodeling complex that functions in neural precursor cells .Delta-like 1-Drosophila gene (DLL1) is a human homolog of the Notch Delta ligand and a member of the delta/serrate/jagged family. It plays a role in mediating cell fate decisions during hematopoiesis and cell communication ,97. The The family with sequence similarity 120B gene (FAM120) encodes protein belonging to the constitutive coactivator of peroxisome proliferator-activated receptor gamma (PPARG) family. FAM120B functions in adipogenesis through PPARG activation in a ligand-independent manner .Proteasome  subunit, beta type, 1 gene (PSMB1) encodes a member of the proteasome B-type family, also known as the T1B family, that is a 20S core beta subunit . This geProgrammed cell death 2 gene (PDCD2) encodes a nuclear protein highly expressed in placenta, heart, pancreas, lung, and liver, and lowly expressed in spleen, lymph nodes, and thymus. Expression of this gene is shown to be repressed by B-cell CLL/lymphoma 6 (BCL6), a transcriptional repressor .In addition, despite not reaching the genome wide significance, our study observed evidence for association at three additional loci containing the candidate genes LOC100128081, TNFRSF11B, and FOSL2 . Of theset al., 2012 re-analyzed the original 2007 WTCCC study by using the 1,000 Genomes imputation and reported refined variant rs1265564 in Cut-like homeobox 2 (CUX2) region for association with T1D [CUX2 (12q24): Huang with T1D . CUX2 iswith T1D . CUX2 gewith T1D . NeuroD/with T1D ,107. Thuet al. confirmed [HTR1A (5p13-q13): Asad onfirmed  the prevonfirmed . None ofonfirmed . The stuonfirmed . The HTRonfirmed . Howeveronfirmed . Studiesonfirmed . Sumatrionfirmed . Previouonfirmed . HTR1A aonfirmed . Hence, This review provides a summary of recent advances in the identification of risk variants associated with T1D. Genome wide association studies have revolutionized the discovery approach to autoimmune mediated disorders. In T1D only six genetic factors were well known before GWAS. GWAS has contributed greatly by expanding the number of established genetic variants to 59 loci. Most of these genes are novel and were not in any investigator’s favorite list. For the first time there is real consensus on the role of specific genetic factors underpinning T1D pathogenesis.The discoveries of genetic factors involved in T1D through GWAS present the first step in a long process leading to cure. Genes uncovered using this approach are indeed fundamental to disease biology and will define the key molecular pathways leading to cure of T1D. However, such genome wide scans can lack coverage in certain regions where it is difficult to genotype, thus, it is possible that other loci with reasonable effect sizes remain to be uncovered through whole genome sequencing approaches.To date most of T1D associated variants have been discovered utilizing cohorts of European ancestry because the SNP arrays were designed to optimally capture the haplotype diversity in this ethnicity. Novel SNP arrays are needed with the same degree of capture in diverse populations to elucidate the full role of each locus in a worldwide context.In addition to identifying genes influencing disease susceptibility GWAS can be utilized to facilitate implementation of personalized medicine based on genetic make-up of the individuals. Our pilot study showed a proof-of-principle that use of whole-genome data, rather than a few ‘‘validated’’ susceptibility loci, could improve predictive accuracy . This apThe next challenge is to resolve the specific causal variants and determine how they affect the expression and function of these gene products. The Next-Generation Sequencing (NGS) technology has opened new avenues to elucidate the role of coding and noncoding RNAs in health and disease and is speeding up the identification of causative gene variants in T1D. in vitro and in vivo biology of these genes will be fascinating areas of exploration for many scientists. Only after scientists have fully uncovered the functional context of T1D associated genes, is the promise of new therapies and preventive strategies likely to materialize.No doubt, the


During surveillance for pneumonia of unknown etiology and sentinel hospital–based surveillance in Beijing, China, we detected avian influenza A(H7N9) virus infection in 4 persons who had pneumonia, influenza-like illness, or asymptomatic infections. Samples from poultry workers, associated poultry environments, and wild birds suggest that this virus might not be present in Beijing. During April 1–June 30, samples were collected during surveillance and sampling programs in Beijing. Patients with pneumonia of unknown etiology in all hospitals were reported to the correspondent district CDC. Respiratory specimens were collected and sent to the district CDC for testing for avian influenza A(H7N9) virus by real-time PCR. Suspected positive specimens were verified by the Beijing CDC.,Surveillance was conducted in 23 sentinel hospitals and 17 collaborating laboratories during April 22–June 30. Surveillance participants were defined as patients with influenza-like illness (ILI) ≤3 days after illness onset (Persons in close contact with patients infected with influenza A(H7N9) virus or infected animals were medically observed for 7 days after the most recent exposure during April 1–June 30. Pharyngeal swab specimens were collected from close contacts on each day during medical observation and tested for influenza A(H7N9) virus by real-time PCR.Poultry workers from 5 districts in Beijing were sampled during April 19–28. Pharyngeal swab specimens were collected from poultry workers and sent to the correspondent district CDC for detection of influenza A(H7N9) virus by real-time PCR.At the same time that poultry workers were sampled, environmental samples were collected from associated poultry environments. These environmental samples were sent to district CDC laboratories for detection of influenza A(H7N9) virus.Samples were collected from wild birds in 317 parks and 10 wetland natural reserve regions in Beijing  during MThirty-nine patients with pneumonia of unknown etiology were reported, and 1 of these patients was infected with influenza A(H7N9) virus. This case was in a 7-year-old girl who had pneumonia, which was confirmed on April 12. The girl’s mother and a 4-year-old acquaintance (boy) were positive for influenza A(H7N9) virus but were asymptomatic. This cluster involved 2 families of chicken dealers who lived in Beijing. On April 4, both families had purchased live chickens from the same batch, which had been transported from Tianjin, China. Samples from environments of places of residence of both families were positive for influenza A(H7N9) virus.In sentinel hospital and laboratory-based surveillance, 3,526 pharyngeal swab specimens were subjected to PCR. One specimen was positive for influenza A(H7N9) virus and 73 specimens were positive for seasonal influenza viruses. The patient infected with influenza A(H7N9) virus was a 6-year-old boy who resided in Beijing. On May 21, fever, sore throat, and headache developed in this patient. He visited a sentinel hospital during May 21–24 and recovered on May 23. He did not have pneumonia or any history of exposure to birds or other animals. He had visited the hometown of his family in Shandong Province during May 14–15.A total of 1,422 poultry workers were recruited into this surveillance study, and 14 (0.98%) had ILI symptoms ≤2 weeks before recruitment. All workers were sampled but none were positive for influenza A(H7N9) virus. From the environments of poultry workers, 679 samples were collected; all were negative for influenza A(H7N9) virus. However, 3 samples from 2 other locations were positive for influenza A(H9N2) virus. In addition, 3,401 fecal samples of wild birds were collected; all were negative for influenza A virus.,Clinical manifestations of human infections with influenza A(H7N9) virus found in Beijing included pneumonia, ILI, and asymptomatic infection. Although patients infected with this virus in China have so far had lower respiratory tract infections (The first patient infected with influenza A(H7N9) virus in Beijing was found by surveillance for pneumonia of unknown etiology, which was initially designed for finding patients with severe acute respiratory syndrome or influenza A(H5N1) virus infection. Although hospital-based surveillance is less efficient in finding mild and asymptomatic infections, it may be the most feasible approach for identifying severe cases of infection with influenza A(H7N9) virus.The second patient infected with influenza A(H7N9) virus, the 6-year-old boy, was found through sentinel hospital and laboratory-based surveillance. Before emergence of influenza A(H7N9) virus, surveillance was conducted by using cell culture–based virus isolation techniques. To increase assay sensitivity and rapidity, real-time PCR preceding virus isolation was adopted to replace the strategy of only using virus isolation. In addition, because only specimens positive for seasonal influenza viruses were subjected to virus isolation, this procedure could help avoid the risk for propagating influenza A(H7N9) virus from unknown specimens, as is caused by conducting virus isolation directly in biosafety level 2 laboratories. Our findings and those of another report  .


Interestingly, H2O2-induced GSTT1 was only inhibited by a p38 inhibitor. An inhibitor of MK2, a downstream regulator of p38, also diminished H2O2-induced GSTT1 upregulation. Notably, both p38 and MK2 were significantly inactivated in cells carrying an shRNA construct of GSTT1 (ΔGSTT1 cells), suggesting that the p38-MK2 pathway is essential for age-associated upregulation of GSTT1. The relevance of GSTT1 in mitochondrial activity was then determined. ΔGSTT1 cells displayed enhanced polarization of mitochondrial membrane potential without increasing the apoptosis, suggesting that the age-associated upregulation of GSTT1 may influence the mitochondrial activity of granulosa cells.We previously reported that GSTT1 was upregulated in human granulosa cells during aging and that activation and localization of p38 MAPK was changed in parallel. Although oxidative stress is responsible for these changes, the age-associated expression of GSTT1 regulated by MAPKs and the role of GSTT1 in aged granulosa cells remain unclear. Therefore, we examined the relationship between the expression of GSTT1 and MAPK signaling pathways using human granulosa-like KGN cells stimulated with HCollectively, it appears that the age-associated expression of GSTT1 is induced through the p38 signaling pathway and GSTT1 influences homeostatic activities in granulosa cells. Caenorhabditis elegans in which CeGSTP2-2 belonging to the pi-class of GSTs was reported to conjugate 4-HNE and its overexpression was shown to elongate lifespan [Glutathione S-transferases (GSTs) are well known for removing environmental pollutants and endogenous toxic compounds as part of the phase II detoxification process through glutathionylation of diverse electrophilic substrates. This self-defense system is highly conserved among all organisms including prokaryotes and eukaryotes . Becauselifespan , 9. In clifespan .The expression level of GSTs is decreased in various tissues and organs during aging , indicatin vitro [The p38 MAPK signaling pathway has been involved in a number of important biological activities, such as proliferation, inflammation, cell death, and aging . The actin vitro , 30. Thein vitro . Since pin vitro , 33, thein vitro , 34, musSome GSTs have been shown to be upregulated through the MAPK pathways as self-defense responses to toxins and growth factors , 36. How2O2 is able to induce these changes in vitro, it remains uncertain whether GSTT1 induction by H2O2 is controlled through the p38 MAPK signaling pathway. Therefore, we examined the expression of GSTT1 induced by H2O2 in the presence or absence of distinct MAPK inhibitors. The immunofluorescence study revealed that GSTT1 was highly upregulated in response to H2O2, as has been reported previously  cells, whereas both were severely suppressed in ΔGSTT1 and Δp38α cells , and the expression of GSTT1, as well as the phosphorylation of p38 and MK2 was examined for each cell line  was purchased from Wako Pure Chemical Co. Ltd . Hoechst 33342, protease inhibitor cocktail, phosphatase inhibitors 1 and 2, and a rabbit polyclonal antibody against phosphorylated p38 (Thr180/Tyr182) for the immunofluorescence analysis were purchased from Sigma-Aldrich Inc . SB203580 (p38 inhibitor) and rabbit polyclonal antibodies against GSTT1 and phosphorylated MK2 (Thr222) were purchased from Santa Cruz Biotechnology Inc . SP600125 (JNK inhibitor) was purchased from Enzo Life Sciences, Inc. . PD98059 (ERK inhibitor) and CMPD1 (4-(2'-fluorobiphenyl-4-yl) - N -(4-hydroxyphenyl) - buty-ramide, MK2 inhibitor) were purchased from Merck Ltd. . A rabbit antibody against phosphorylated p38 (Thr180/Tyr182) for the immunoblot analysis was purchased from Novus Biologicals Inc. . A mouse monoclonal antibody against β-actin, a control rabbit IgG, a control mouse IgG, and a donkey anti–mouse IgG conjugated with HRP were purchased from Chemicon International Co. Ltd. . A goat anti–rabbit IgG conjugated with HRP was purchased from Thermo Fisher Scientific Inc. . A rabbit polyclonal antibody against lamin was purchased from Abcam Inc. . A goat anti–rabbit IgG conjugated with Alexa Flour 488 and MitoTracker CMXRos were purchased from Molecular Probes, Inc. .Hydrogen peroxide (H4 cells / ml (200 ml / well) in 8-well culture slides  for the subsequent immunofluorescence studies or in culture dishes  for immunoblot analysis. The medium was replaced by DMEM/F12 containing 0.1% BSA (serum-free medium) before stimulation. The cells were then treated with 200 μM H2O2 for 24 h with or without SB203580 (10 μM), SP600125 (10 μM), PD98059 (10 μM) or CMPD1 (330 nM). The final concentrations of inhibitors are determined according to the previous reports in which these inhibitors were used in various cell types [The human granulosa-like cell line, KGN, was maintained as described previously , 51. Brill types -54. Aftell types .To construct shRNA knockdown vectors, the target sequences of the desired genes were synthesized and obtained from Invitrogen Corp. . GSTT1: Top CACCGCAGGAATGGCTTGCTTAAGACGAATCTTAAGCAAGCCATTCCTGC, Bottom AAAAGCAGG AATGGCTTGCTTAAGATTCGTCTTAAGCAAGCCATTCCTGC, p38α: Top CACCGCCGAGCTGTTGA CTGGAAGACGAATCTTCCAGTCAACAGCTCGGC, Bottom AAAAGCCGAGCTGTTGACTGAAGATTC GTCTTCCAGTCAACA GCTCGGC, Lamin: Top CACCGCTGGACTTCCAGAAGAACACGAATGTTCTTCTGGAAGTCCAG, Bottom AAAACTGGACTCC AGAAGAACATTCGTGTTCTTCTGGAAGTCCAGC, These oligonucleotides were annealed and cloned into a BLOCK–iT RNAi entry vector  according to the manufacturer's instruction. After verification by sequencing of the DNA sequence inserted into the vector, the target sequences were transferred into the destination vector (pLenti6/BLOCK–iT-DEST) using the Gateway system (Invitrogen Corp.). Lentiviruses were then produced in 293FT cells using ViraPower Lentiviral Expression Systems, and KGN cells were subjected to lentiviral infection. Cells stably carrying each shRNA construct were maintained in culture medium containing blasticidin at 5 μg/ml. The efficiency of gene knock-down by shRNA was verified by either immunofluorescence or immunoblot analyses.Immunofluorescence staining was performed as described previously . Briefly2O2 After treatment, the cells were washed twice with Tris-buffered saline  containing protease inhibitor and phosphatase inhibitor cocktails and placed on ice as quickly as possible. They were then lysed by direct application of a lysis buffer to the dish.KGN cells were passaged and cultured in a culture medium for 3 days  and then maintained for 6 h in a serum-free medium before treatment with HThe nuclear/cytosol fractionation kit  was used to separate nuclear and cytoplasmic proteins, according to the manufacturer's protocol. After isolation of the proteins, the concentration of the samples was determined using a micro BCA assay kit (Thermo Fisher Scientific Inc.). Fifteen micrograms of sample per lane were electrophoresed on a 12% reducing SDS-polyacrylamide gel and transferred onto a PVDF membrane . After blocking with 10% goat serum and 90% Block Ace at room temperature for 1 h, the membranes were treated with a primary antibody at 1:1000 dilution in TBST and subsequently with a secondary antibody at 1:10,000 dilution. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific Inc.) was then used for visualization, and the signal was developed on an X-ray film . The band intensity of phosphorylated p38 was measured using Adobe Photoshop Elements 2.0 software and the background was subtracted. It was then divided by the band intensity of total p38 for normalization. Data were collected from three independent experiments and are shown as the mean changes in band intensity ± SEM.2O2 for 24 h and labeled with the MitoTracker CMXRos probe at 100 nM for 30 min. They were washed twice with TBS and fixed with 4% formaldehyde for 1 h at room temperature. Fluorescence images were taken as described above and the fluorescence intensity of each image was measured with Adobe Photoshop Elements 2.0. It was then divided by the number of cells in each field. The fluorescence intensity per cell was defined as 1.0 for the wild-type KGN cells to obtain fold changes in fluorescence. Data were collected from three independent experiments and are shown as the mean fold increase in fluorescence ± SEM.To assess the changes in ΔΨm, the cells were treated with or without 200 μM H2O2 for 24 h as described above. They were then washed with PBS three times and fixed with 2% paraformaldehyde in PBS for 1 h. Next, the cells were permeabilized and labeled with flurescein-dUTP. During the TUNEL labeling Hoechst 33342 at 10 μM was included. The number of flurescein-labeled cells and the total number of cells stained with Hoechst 33342 was counted simultaneously in each field of view under an epi-fluorescence microscope. The incidence of apoptosis was calculated by dividing the number of TUNEL-positive cells by the total number of cells per field. The experiments were repeated three times, and the data are shown as the mean percentage of apoptosis ± SEM.Cell apoptosis was detected using TUNEL enzyme and labeling mix according to the manufacturer's instructions . In brief, cells were plated in 8-well culture slides for a couple of days and stimulated with 200 μM H2O2 were analyzed by Student's t-test or modified Student's t-test (Welch's correction) following an F test. For the analysis of apoptosis, a one-way ANOVA followed by Student's t-test was used. Differences were considered statistically significant if P < 0.05. All statistical analyses were performed using Microsoft Excel software.For all immunostaining experiments, statistical analysis was conducted by one-way ANOVA. For the immunoblot analysis, differences in the phosphorylation of p38 in cells treated with or without H


Anas acuta) ducks wintering in Japan. This study describes characteristics of pintail-originated H1N1, H1N2, H1N3, H5N2, H5N3, H5N9, and H7N7 viruses. Most of the viruses were revealed to be avian strains and not related to pandemic and seasonal flu strains. Nevertheless, the NP genes of 62.5% (5/8) viruses were found closely related to a A/swine/Korea/C12/08, indicating exchange of genetic material and ongoing mammalian-linked evolution of AIVs. Besides, all the viruses, except Aomori/422/07 H1N1, contain PSIQSR∗GLF motif usually found in avian, porcine, and human H1 strains. The Aomori/422/07 H1N1 has a PSVQSR∗GLF motif identical to a North American strain. This findings linked to an important intercontinental, Asian-American biogeographical interface. Phylogenetically all the viruses were clustered in Eurasian lineage. Cocirculation of allele A and B (NS gene) viruses was evident in the study implying the existence of a wide reservoir of influenza A viruses in pintail wintering in Japan.The pandemic influenza virus strains of 1918 (H1N1), 1957 (H2N2), 1968 (H3N2), and 2009 (H1N1) have genes related to avian influenza viruses (AIVs). The nonstructural (NS) gene of AIVs plays a significant role in host-viral interaction. However, little is known about the degree of diversity of this gene in Northern pintail ( Influenza A virus infections in birds account for important inputs into the evolutionary porcine-human complex of this prominent anthropozoonotic pathogen. Among influenza A viruses, which broadly exhibit 17 HA and 9 NA antigenic subtypes, only three haemagglutinin (HA) subtypes H1, H2, and H3) and two neuraminidase (NA) subtypes (N1 and N2) have circulated widely in swine and human populations since the 20th century , and H3 . Avian-oPigs have been postulated, hence, to be the ultimate “mixing vessels” for mammalian influenza viruses and AIVs and can play an important role in the genetic reassortment of influenza viruses. Therefore, detection and characterization of AIVs, especially those subtypes having the potential to transmit to mammals, including pigs and humans, are significant. Avian H7N7 and H3N8 strains are contracted and circulated by horses, as well, and an equine H3N8 virus apparently was involved in the formation of the pandemic H3N2 virus, and perhaps the H3N8 pandemic virus of 1889 [The genome of influenza A viruses consists of eight different segments of single-stranded negative-sense RNA. Most gene segments encode for one protein; whereas the gene segments 2 (polymerase basic1—PB1), 7 (matrix—M), and 8 , each encodes one additional protein from alternatively spliced mRNA. Among the proteins, 9 are structural, while the rest two, namely PB1-F2 and NS1, are nonstructural. Thirteen genes have already been identified all over. Aquatic birds are the primary reservoir of type A influenza viruses and are classified into various antigenic subtypes, based on their two surface glycoproteins, the HA, and the NA. So far, seventeen HA (H1–H17) and nine NA subtypes (N1–N9) have been identified from avian species , represeAnas acuta) (thereafter referred to as pintail (s)) ducks, a globally major influenza host wintering in Japan, is poorly studied. In this paper, we focused on genetic analysis of H1N1, H1N2, and H1N3 strains of AIVs that we isolated from apparently healthy migratory pintails wintering in Japan [It has been demonstrated that the amino acids at positions 627 and 701 of the polymerase basic 2 (PB2) protein influence the outcome of infection in mice , 13. Sevin Japan , 18. Alsin Japan , were fuViruses used in this study  were of Total viral RNA was extracted from infected allantoic fluid using the Isogen-LS , in accordance with the manufacturer's instructions. Reverse transcription was carried out with the Uni12 primer (5′-AGC AAA AGC AAG G-3′) and MMLV reverse transcriptase , followed by full length amplification of each gene segment, as described earlier . The PCRThe PCR product was sequenced by dideoxy chain terminating method, using Dye Terminator Cycle Sequencing FS Ready Reaction Kit . Sequencing was carried out with the same primers used to amplify the gene and continued with subsequently designed sequence-specific primers. Nucleotide sequences were determined using an automated DNA sequencer , edited and assembled with GENETYX-Mac .Multiple sequence alignments and processing were performed with the Molecular Evolutionary Genetics Analysis (MEGA) version 4.1.0 software, with an engine based on the ClustalW algorithm . Blast sThe nucleotide sequences generated in this study have been deposited in GenBank and are available under accession number AB546149–AB546193.As a part of longitudinal virological studies, we have isolated various subtypes of AIVs from fecal materials of migratory, apparently healthy pintails wintering in Tohoku district, Japan , 18. Of n = 6) of H1 subtype with different NA subtype combinations  isolated from pintails during 2007-2008 were closely related to each other  and N2 (n = 3), respectively, for each subtype. On the other hand, the amino acid sequence identities were found to range from 97.4 to 99.8% (N1) and 99.4–99.8% (N2). A duck-originated Japanese strain obtained 2 years earlier, A/duck/Tsukuba/67/05 H1N1 (Tsukuba/67/05 H1N1) was found most closely related to one of the isolates—A/northern pintail/Aomori/422/07 H1N1 (Aomori/422/07 H1N1) (>98% homology at both nucleotide and amino acid sequences)—of the present study, while two other viruses have maximum homology to a Mongolian strain, A/duck/Mongolia/116/02 H1N1 (Mongolia/116/02 H1N1), isolated in 2002 (>98% homology at both nucleotide and amino acid sequences). For N2 NAs, a Chinese strain, namely A/goose/Gui Yang/3799/05 H5N2 (Gui Yang/3799/05 H5N2) isolated from goose in 2005 was found rather closely related (>98% nucleotide and amino acid identity). The N3 NA gene of Aomori/1130/08 H1N3 has >99% nucleotide and amino acid homology to a duck strain, namely A/duck/Niigata/514/06 H5N3 (Niigata/514/06 H5N3) isolated in 2006 in Japan  was found among the NS genes of the sequenced viruses . When anSignificantly, the NS1 genes of influenza viruses in general have been divided into two alleles: A and B . Regardlhttp://alaska.usgs.gov/science/biology/avian_influenza/pintail_movements_virus.php. Thus, inter- and intra continental exchange of genes and genomes of AIV could occur through congregation of North American and Asian migrants at shared summer habitats in eastern Russia, or by means of pintails that migrate between North American nesting grounds and Japanese wintering grounds (assessment of virus movement across continents: using Northern Pintails (Anas acuta) as a test. http://alaska.usgs.gov/science/biology/avian_influenza/migration_ecology.php. Also the number of samples may possibly have effect on the results found in this study.Prevalence of allele A and B viruses in their natural hosts has been reported to vary spatially, as well. Generally, allele B viruses were reported to be less common in their natural hosts than allele A. Out of 11 NS genes sequenced in this study, 72.7% (8/11) were classified as allele A, and 27.3% (3/11) as allele B. In Asia, the prevalence of allele B viruses in all avian species has been reported to be only 15% , which iThe NP genes of the sequenced viruses have 90.4–99.9% nucleotide homologies among each other, regardless of their temporal and spatial differences in isolation . BroadlyThe HA amino acid sequences deduced from nucleotide sequences analysis revealed that all the viruses contain seven potential N-linked glycosylation sites (5 in HA1 and 2 in HA2) throughout the HA molecule. Except for Aomori/422/07 H1N1, the amino acid motif of the HA0 cleavage site of all the sequenced H1 viruses was PSIQSR*GLF (*cleavage point), which is common in all avian, porcine, and human viruses. Aomori/422/07 H1N1 contains a rare amino acid motif, PSVQSR*GLF. Among the H1 avian strains isolated globally and sequences reported in GenBank during 1980–2009, only one strain, namely A/mallard/New York/170/82 H1N2 has identical (PSVQSR*GLF) amino acids. However, Aomori/422/07 H1N1 solely clustered with the Eurasian viruses , implyinOn the other hand, the amino acid sequence analysis of NAs revealed that all the viruses contain full length NA protein with seven potential N-linked glycosylation sites in N1 and N2 known to be conserved in wild ducks . In all PSD-95; discs large, Dlg; zonula occludens-1, ZO-1) domain ligand at the C terminus of NS1 (ESEV-COOH), which plays an important role in many key signaling pathways of viral replication [Deletion of several amino acids in the NS1 gene has been observed more frequently in AIVs in recent years, a feature of possible adaptation of these viruses to poultry , 43. Thelication .When compared to pandemic and seasonal influenza viruses, the HA genes' nucleotide identity of viruses of the present study was found to range from 67.1 to 76.4%, while amino acid sequence homology was found 81.8–92.1% . In diffIn conclusion, our genetic analysis suggests that the sequenced viruses were to an appreciable degree characteristic of the pintail populations we sampled, which typically winter in Japan. Except in one strain, the HA0 cleavage site of H1 was found as usually found in all avian, porcine, and human viruses. The residues that compose the catalytic and framework sites of the NA enzyme were completely conserved in the studied viruses. The NP gene of the majority of the strains sequenced in this study (5/8) was related to that of the porcine strain Korea/C12/08 H5N2. Thus, it may be presumed that our isolates evolved through reassortment process during the cocirculation of these strains. Our findings clearly demonstrate that two distinct gene pools, corresponding to both NS allele A and B, were present within the pintail populations wintering in Japan. It is noteworthy that some strains contain NS gene highly related to a duck isolate obtained in Russian in 1972, about 35 years prior to our isolate, indicating significant gene conservation. It is also concluded that although bearing the same antigenic subtype (H1N1) and obtained during 2007-2008, our isolates were unrelated to 2009 pandemic and seasonal flu strains.


Cyclin D1 is a key regulator of the cell cycle that is over expressed in more than half of breast cancer patients. The levels of cyclin D1 are controlled primarily through post-translational mechanisms and phosphorylation of cyclin D1 at T286 induces its proteasomal degradation. To date, no studies have explored the involvement of phosphatases in this process. Here we treated human breast cancer cells with the structurally distinct toxins calyculin A, okadaic acid, and cantharidin, which are known to inhibit Ser/Thr phosphatases of the PPP family. At low nanomolar concentrations calyculin A induced T286 phosphorylation and degradation of cyclin D1 via the proteosome in MDA-MB-468 and MDA-MB-231 cells. Cyclin D1 degradation also was dose-dependently induced by okadaic acid and catharidin, implicating a negative regulatory role for type-2A phosphatases. These effects occurred without increasing phosphorylation of p70S6K, cyclin D3, or myosin light chain that were used as endogenous reporters of cellular PP2A and PP1 activity. A reverse phase phosphoprotein array analysis revealed increased phosphorylation of only 6 out of 33 Ser/Thr phosphosites, indicating selective inhibition of phosphatases by calyculin A. Calyculin A treatment induced cell cycle arrest in MDA-MB-468 and MCF-7 breast cancer cells. These findings suggest that a specific pool of type-2A phosphatase is inhibited by calyculin A leading to the degradation of cyclin D1 in human breast cancer cells. The results highlight the utility of toxins as pharmacological probes and points to the T286 cyclin D1 phosphatase inhibited by calyculin A as a possible target for chemotherapy to treat triple negative breast cancer.  This decrease has been attributed to cyclin D1 phosphorylation at Thr286, causing export from the nucleus via CRM-1 [Cyclin D1 regulates the G protein . The cyc protein ,4. E2F fia CRM-1 . GSK3β hia CRM-1 ,10. Contin vitro screen of 76 deubiquitinating enzymes showed that one particular enzyme, USP2, reacted with monoubiquitinated cyclin D1. Over expression of USP2 in 293 cells stabilized cyclin D1 protein levels by preventing ubiquitin-mediated proteasomal degradation. Knockdown of USP2 in HCT116 colorectal cancer, MCF-7 breast cancer, and PC-3 prostate cancer cells destabilized cyclin D1 and reduced cell proliferation [There is evidence that defects in cyclin D1 degradation might account for the cyclin D1 protein accumulation found in cancers. An 1 cyclins  that correspond to mammalian cyclin D1 are regulated in part by the Ser/Thr phosphatase Sit4 [1 cell cycle arrest with a corresponding reduction in cyclin D1 levels [Phosphorylation of cyclin D1 induces its proteasomal degradation, therefore protein phosphatases have a putative role in dephosphorylation cyclin D1 to prevent its degradation. To date there is little information about the identity of the protein phosphatase responsible for dephosphorylation of cyclin D1. In yeast, levels of the Gase Sit4 . In PC-31 levels . This reSeveral toxins in nature have been found to potently inhibit the PPP family of Ser/Thr phosphatases that includes the type-2A phosphatases  and the type-1 phosphatase, PP1. These toxins include the polyketals okadaic acid and calyculin A, purified from marine sponges, cyclic peptides of the microcystin and nodularin groups produced by blue green algae, and the compound cantharidin, an epoxycyclohexane dicarboxylic anhydride produced by blister beetles. The toxins calyculin A, okadaic acid, and cantharidin were chosen for this study based on their cell permeability and chemical stability. These three toxins are reported to show preferential inhibition of type-2A phosphatases compared to type-1 phosphatases ,15. The 15 media (Gibco 11415) containing 10% fetal bovine serum at 37° in non-CO2 conditions. MCF-7 cells were maintained in MEM media (Gibco 11095) containing 10% fetal bovine serum, 1 mM non-essential amino acids, 10 μg/mL bovine insulin, and 1 mM sodium pyruvate at 37 °C in 5% CO2. Cells were passaged every three days. MDA-MB-468 and MDA-MB-231 cells were maintained in LMDA-MB-468, MCF-7, or MDA-MB-231 cells were plated at 300,000 cells per well of a six-well dish. The next day they were treated with 20 μM MG132 (Sigma C2211) for one hr, then calyculin A (Sigma C5552), okadaic acid , or cantharidin (Sigma C7632) for the times indicated. Cells were collected in media by scraping and centrifugation at 100 ×g for 5 min. The pellet was resuspended in 100 μL 10% trichloroacetic acid and incubated on ice for 15 min. The precipitated proteins were pelleted by centrifugation at 16,000 ×g for 15 min, washed twice with 500 μL ice cold acetone and dried at room temperature for 15 min. The pellets were resuspended in 2X SDS loading buffer, allowed to dissolve overnight, and boiled for 5 min. Protein was quantified using the BioRad DC colorimetric assay and samples were resolved on a 4-15% precast gradient gel (Biorad 345-0028). Proteins were transferred from gels to nitrocellulose membranes by electrophoresis for 1 h using a semi-dry transfer apparatus. Membranes were blocked with 3% bovine serum albumin (phospho antibodies) or 3% dry milk  dissolved in Tris-buffered saline with 0.1% Triton X-100. Membranes were probed overnight at 4 °C with antibodies against cyclin D1 , pT286 cyclin D1 , actin (Sigma A2103), cyclin D3 , phospho myosin light chain (Rockland 600-401-416), p70S6K  were treated with calyculin A for 24 hr before they were fixed with 70% ethanol overnight at 4 °C, and then washed with PBS. The cells were then incubated in 1 mL PI solution (0.1% (v/v) Triton X-100, 0.2 mg/mL DNase-free RNase A and 20 μg/mL propidium iodide in PBS) at 37 °C for 30 min. The cells were sorted on a Calibur flow cytometer for DNA content and profiles analyzed by system software. MDA-MB-468 or MCF-7 cells  by Aushon 2470 Arrayer (Aushon BioSystems). Total 5,808 array spots were arranged on each slide including the spots corresponding to positive and negative controls prepared from mixed cell lysates or dilution buffer, respectively. Each slide was probed with a validated primary antibody plus a biotin-conjugated secondary antibody. Only antibodies with a Pearson correlation coefficient between RPPA and western blotting of greater than 0.7 were used in RPPA. i.e., dilution series) on a slide with the signal intensity as the response variable and the dilution steps as independent variable. The fitted curve is plotted with the signal intensities-both observed and fitted-on the y-axis and the log2-concentration of proteins on the x-axis for diagnostic purposes. The protein concentrations of each set of slides were then normalized by median polish, which was corrected across samples by the linear expression values using the median expression levels of all antibody experiments to calculate a loading correction factor for each sample.The signal obtained was amplified using a Dako Cytomation-catalyzed system (Dako) and visualized by DAB colorimetric reaction. The slides were scanned, analyzed, and quantified using a customized software Microvigene (VigeneTech Inc.) to generate spot intensity. Each dilution curve was fitted with a logistic model . This fits a single curve using all the samples  human breast cancer cell line MDA-MB-468 with the cell permeable yculin A A and B. yculin A A. Even ayculin A A. At con50 ~3 nM B. A simi50 ~3 nM C. TreatmIn vitro experiments showed that PP1 but not PP2A dephosphorylated cyclin D3, and PP1 associated with His-cyclin D3 in Reh, a B-cell precursor cell line [Calyculin A is known to inhibit both type-1 and type-2A members of the PPP phosphatase family . We examined three different endogenous phosphoproteins in MDA-MB-468 breast cancer cells as reporters for phosphatase inhibition by low nanomolar doses of calyculin A . Cells well line . Increasell line ,18. Likeell line . These dBecause calyculin A treatment of MDA-MB-468 cells did not affect known substrates of PP1 and PP2A, we were prompted to carry out a broader screen of changes in Ser/Thr phosphorylation. We used a reverse phase protein array, which was interrogated with 40 different phosphosite-specific antibodies . Duplicate samples of vehicle vs. calyculin A treated cells were analyzed and the results calculated as the -fold increase in phosphorylation and displayed as rank order, left to right . CalyculIf calyculin A caused depletion of cyclin D1 in human breast cancer cells by PPP phosphatase inhibition, one would predict that other structurally distinct toxins would elicit a similar response because active sites of type-1 and type-2A Ser/Thr PPP phosphatases are the intracellular targets of these toxins. Therefore, we examined the effects of okadaic acid and cantharidin, two other toxin phosphatase inhibitors on cyclin D1 and other phospho proteins. Cells were treated for 1 h with various doses of the toxins and the levels of cyclin D1, and electrophoretic migration of p70S6K and cyclin D3 as reporters of PP2A and PP1 activity, respectively were compared by Western blotting . Cyclin 1 (p = 0.016) phase of the cell cycle and a statistically significant decrease in the fraction of cells in the G2/M (p = 0.018) phase of the cell cycle in response to 10 nM calyculin A (vs. 5B). There was not a change in the fraction of cells in S phase. These results show that 10 nM calyculin A arrested MDA-MB-468 cells in the G1 and S phases of the cell cycle.Cell cycle status of MDA-MB-468 cells treated with calyculin A was evaluated by fluorescence activated cell sorting (FACS) analysis A and 5C.yculin A D vs. 5B.1 phase  with almost no change in the G2/M population. These results show that 10 nM calyculin A induced proteosome degradation of cyclin D1 and cell cycle arrest in MCF-7 cells.Human breast cancer MCF-7 cells were treated with calyculin A to determine effects on cyclin D1 levels and cell cycle progression . Cells w1/S phase of the cell cycle. The results from this study demonstrate that three structurally distinct toxins, calyculin A, okadaic acid, and cantharidin, induce proteosome degradation of cyclin D1 in human breast cancer cell lines. These toxins are known to be inhibitors of PPP protein Ser/Thr phosphatases of the type-1 and type-2A classes, with some reputed preference for the type-2A enzymes (see below). Despite no similarity in structure between these toxins, the fact that they produce the same effect on cyclin D1 argues for a common Ser/Thr phosphatase as their intracellular target. The simplest explanation for our results is that the three toxins inhibit a type-2A phosphatase that dephosphorylates Thr286 in cyclin D1. An increase in cyclin D1 phosphorylation would promote SCF-mediated ubiquitination and proteasomal degradation . The hal50 of 0.25 nM, compared to PP1 and PP4 with an IC50 of 0.4 nM. Phosphatases PP2B, PP5 and PP7 were inhibited by calyculin A at much higher, micromolar concentrations [50 of ~200 nM for PP2A and >1,000 nM for PP1 [i.e., PP2A, PP4, PP6) which serves to limit phosphodegron-mediated ubiquitination and degradation by the proteosome. The PPP family of protein Ser/Thr phosphatases is comprised of multisubunit enzymes where catalytic subunits associate with regulatory subunits to control their activity, substrate specificity, and cellular localization . The toxtrations . In a sttrations . Okadaictrations ,27,28. C for PP1 . Taken tIn vitro phosphatase assays using purified recombinant PP6c and PP2Ac catalytic subunits showed that PP6 was more sensitive than PP2A to each of these toxins using myelin basic protein as a substrate [Degradation of cyclin D1 in MDA-MB-468 cells, however, was especially sensitive to calyculin A, compared to the dephosphorylation of other endogenous proteins we used as reporters of PP1 and PP2A activity. In a screen of 33 Ser/Thr phosphoproteins only 6 showed a pronounced increase in phosphorylation in response to 10 nM calyculin A. These results suggest that in the MDA-MB-468 cell line calyculin A targets a specific pool of Ser/Thr phosphatases, one of which dephosphorylates T286 in cyclin D1. One candidate is PP6 that might be inhibited by lower concentrations of calyculin A, compared to PP2A or PP1. ubstrate . Furtherubstrate . On the vs. okadaic acid and cantharidin. This separation contrasts dramatically from the relative in vitro potency of these inhibitors with type-2A phosphatases. The difference in potency for promoting cyclin D1 degradation in live cells might be attributed in part to cell permeability. The toxins are hydrophobic molecules that are cell permeable, despite having negatively charged groups. We have been unable to find comparative biophysical properties, such as logP, for these toxins in the literature. Regardless, cell permeability is unlikely to fully account for the 2 to 4 orders of magnitude difference in effectiveness of calyculin A vs. okadaic acid or cantharidin on cyclin D1 degradation. The toxin dose-dependent induction of cyclin D1 degradation in MDA-MB-468 cells shows a rather surprising separation between calyculin A vs. cantharidin involved different genes, indicating that binding to a common phosphatase does not fully account for cellular actions of these toxins [Is some other phosphoprotein affected by calyculin A involved in regulating cyclin D1 levels? Although we are not able to answer this question at this moment, it might be a direction for future studies. Maybe calyculin A at low doses increases phosphorylation of heat shock protein αB-crystallin to promote association with Fbx4 . This ase toxins . There ae toxins , and it The toxins calyculin A, okadaic acid, and cantharidin are cell permeable inhibitors of protein Ser/Thr phosphatases of the PPP family. Calyculin A induces T286 phosphorylation and proteosome-mediated degradation of cyclin D1 in human breast cancer MDA-MB-468 and MDA-MB-231 cells. Degradation of cyclin D1 occurs at toxin concentrations below those required to alter the phosphorylation of most endogenous proteins used as reporters of phosphatase inhibition. This indicates selective inhibition of T286 cyclin D1 phosphatase that may be an effective target for pharmaceutical treatment of triple negative breast cancers.


Unlike CMS, the 2sh had significant positive cytoplasmic effects on the seed set rate, weight per panicle, yield, and general combining ability (GCA) for plant height, seed set rate, weight per panicle, and yield. These effects indicated that 2sh cytoplasm may show promise for the improvement of rice yield. Genetic analysis suggested that the phenotype of 2sh was controlled by a single recessive locus. We mapped 2sh to a 152 kb region on chromosome 6, where 22 candidate genes were predicted. None of the 22 genes had previously been reported to be responsible for the phenotypes of 2sh. Sequencing analysis showed a 12 bp deletion in the sixth exon of Loc_Os06g40550 in 2sh in comparison to wild type, suggesting that Loc_Os06g40550 is the best candidate gene. These results lay a strong foundation for cloning of the 2SH gene to elucidate the molecular mechanism of male reproduction.Cytoplasm has substantial genetic effects on progeny and is important for yield improvement in rice breeding. Studies on the cytoplasmic effects of cytoplasmic male sterility (CMS) show that most types of CMS have negative effects on yield-related traits and that these negative effects vary among CMS. Some types of genic male sterility (GMS), including photo-thermo sensitive male sterility (PTMS), have been widely used in rice breeding, but the cytoplasmic effects of GMS remain unknown. Here, we identified a GMS mutant line,  IR19661-283-3-2 has a significant negative effect on both yield and days to flowering but does not affect plant height. The male sterile cytoplasms of IR46828, IR46831, IR48483, IR54752, IR17492-18-10-2-2-3, IR54753, IR19661-283-1-3-2, IR54758, and IR54756 have been found to have different effects on grain yield Cytoplasmic male sterility (CMS) is essential to the exploitation of heterosis in three-line hybrid rice breeding programs. CMS cytoplasm has been reported to have substantial genetic effects on various traits of hybrid rice 1 hybrid. Genetic interaction limits the flexibility of germplasm selection and increases production costs 2sh) identified in our breeding program to evaluate cytoplasmic effects on plant height, yield-related traits, and general combining ability (GCA) for these traits.In the three-line system of hybrid rice, male sterility (MS) and its restoration are governed by genetic interaction between male sterile cytoplasm and the nucleus. The interaction maintains the MS, allows the regeneration of the MS line, and restores MS for enjoyment of yield advantage in the FPMS3  P/TMS12-1  japonica background and TMS in indica background. Recently, a number of non-photo-thermo-sensitive genic MS genes have been identified in rice, including WDA1 (wax-deficient anther1) CYP704B2TDR (tapetum degeneration retardation) GAMYBDPW  PTC1  2SH gene and for elucidating the molecular mechanism of anther and pollen development.GMS includes photoperiod-sensitive male sterility (PMS), temperature-sensitive male sterility (TMS), photo-thermo-sensitive male sterility (PTMS), and non-photo-thermo-sensitive genic male sterility. The MS in PMS, TMS, and PTMS can be maintained and reproduced under certain light and temperature conditions. PMS and PTMS have been successfully applied to the production of hybrid seeds, such as Nongken58s, Peiai64s and Y58s 2sh mutant (a spontaneous mutant identified from an indica maintainer 2SChuannong H) and F2 mapping populations generated by respectively crossing 2sh as female with Zhenshan97B and Nipponbare were planted in a paddy field at Sichuan Agricultural University  and at the Hainan rice station . Five isonuclear alloplasmic lines were generated by crossing heterozygote (2sh+/−) as male with Zhenshan97A, D702A, G46A, K18A and XieqingzaoA and then backcrossing 8 times using 2sh+/− as the recurrent parent to substitute the nuclei of Zhenshan97A, D702A, G46A, K18A, and XieqingzaoA with the 2sh nucleus. The 2sh mutant was named A1, and the resultant isonuclear alloplasmic lines were A2, A3, A4, A5, and A6, respectively  and R1–R5 as parent 2 (P2).The ectively . The nuc2sh mutant were cytologically examined for anthers at the meiosis, post-meiosis, young microspore, and vacuolated pollen stages. The preparation for chromosome spreads followed the method described in Chen et al. The wild type and the 1s and their parents were sown on April 4 and transplanted on May 20, 2006 in the Sichuan Agricultural University paddy  to evaluate cytoplasmic effects. A randomized complete block design with three replications was used. For each replication, three rows with 12 plants in each row were planted in plots 2.12 m×0.8 m in size. Rows were 26.6 cm apart and plants within rows were 17.7 cm apart. Ten randomly selected plants from each plot were measured for plant height (cm), effective panicle number, 1,000-grain weight (g), grain number per panicle, seed set rate (%), and weight per panicle (g). Mean values were used for analysis of variance (ANOVA). Most of the plants from each plot were bulk harvested for grain yield, but one plant from each end of each row was omitted to exclude any border effect. This experiment was repeated in the summer of 2008, with the same design. A fixed model with type III sum of squares in SPSS software  was adopted and used to perform an ANOVA. Least significant difference (LSD) was estimated for mean comparison of general combining ability (GCA) effects. The A1–A6 GCA was calculated by the difference between a mean over all combinations for a line A such as A1 to be a parent with all restores (R1–R5) and the overall mean for a trait. The effects of 2sh cytoplasm on plant height and yield-related traits were estimated as follows: [2sh (A1)/R (R1–R5)] F1– [isonuclear alloplasmic line (A2–A6)/R (R1–R5)] F1.Thirty F2sh gene was mapped using a procedure described previously by Chen et al. 2 populations were generated by crossing 2sh as a female with Zhenshan97B and Nipponbare, respectively. We used 342 SSR markers to examine polymorphism between 2sh and Zhenshan97B. Preliminary mapping was based on 1532 male sterile F2 individuals from the cross between 2sh and Zhenshan97B. For fine mapping, 35 SSRs and 74 indel markers were used to examine polymorphism for the target region between 2sh and Nipponbare, and 2400 male sterile F2 individuals produced from this cross were analyzed. Among the 74 indel markers, the closest marker to the mutation locus was W11 . Genetic distance was estimated using Mapmaker 3.0 software http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi) and the rice anther expression database The 2O was used as a template for PCR. The RNA used for RT-PCR was from wild type and mutant anthers at the post-meiosis stage, and the primer pair for Loc_Os06g40550 was W70  and OsActin1 .Total RNA was isolated using a Qiagen RNA plant mini kit with on-column DNAse digestion (Qiagen). Two micrograms of RNA was used for reverse transcription using M-MLVRT (Promega) with oligo (dT18) primer, and 1 µL RT product diluted with 20 µL ddH2sh mutant showed shorter spikelets with smaller anthers than the wild type , indicating a single recessive mutation in the spontaneous 2sh mutant. 2sh also showed stable MS in both Sichuan  and Hainan , suggesting that 2sh was not a PTMS.All F2sh nucleus. This allowed reliable comparison of the cytoplasmic effects among A1–A6.No differences in genetic background among the six isonuclear alloplasmic lines (A1–A6) were detected , indicat2sh on plant height and yield-related traits were estimated by comparing two hybrids with the same male parent. Out of 25 paired comparisons, all but two showed that 2sh cytoplasm had a positive effect on yield. These positive effects were significant in 12 (48%) and 10 (40%) paired comparisons in 2006 and 2008, respectively (2sh cytoplasm had positive effects on the seed set rate. Thirteen (52%) and fourteen (56%) of such comparisons were found to be significant in 2006 and 2008, respectively. Six (24%) and seven (28%) paired comparisons showed significant positive cytoplasmic effect on weight per panicle in 2006 and 2008, respectively. For plant height, effective panicle number, and 1000-grain weight, there were fewer comparisons with significant positive cytoplasmic effects, even though the high proportion of paired comparisons showed positive cytoplasmic effects    and pland yield)  in both her year . The sig2sh had considerable potential for the improvement of rice yield, the male sterility of 2sh severely restricts its utility in the production of hybrid seeds. This is because 2sh is a non-photoperiod/thermo-sensitive genic male sterile mutant. However, one previously described approach provides a possibility to maintain the MS of recessive GMS, so that 2sh could be used to produce hybrid seed in breeding practices 2SH gene using a mapping-based approach.Although cytoplasmic analysis of agronomic traits and GCA indicated that 2 population was generated from a cross between 2sh and Zhensan97B to map the 2sh gene. A bulk segregant analysis was conducted using 52 polymorphic SSR markers between 2sh and Zhensan97B with two DNA bulks, each with ten sterile F2 plants and ten fertile F2 plants. Of the 52 SSRs, only RM3 on chromosome 6 was polymorphic between the two DNA bulks, indicating that the putative 2sh gene was located on chromosome 6. We then examined 23 SSRs flanking RM3 for polymorphisms between 2sh and Zhensan97B, and the polymorphic SSRs were used to analyze 612 male sterile F2 individuals. Both RM454 (located at 27832 kb) and RM528 (located at 26544 kb) were found to be linked with the 2sh locus, with 7.47 and 9.19 cM genetic distance, respectively  and RM20366 (located at 24196 kb) with 0.2 and 0.3 cM genetic distance, respectively  transporter domain. Its homolog in Arabidopsis, ABCG26/WBC27, has been reported to be involved in pollen exine formation, and the abcg26/wbc27 mutant exhibits impaired pollen wall formation that causes MS in ArabidopsisSNF7 (sucrose non-fermenting 7) protein encoded by Loc_Os06g40620 in Arabidopsis is a subunit of the ESCRT III complex. It may be involved in vesicle-mediated transport Loc_Os06g40630 encodes a protein containing a SFT2 domain, its homolog in yeast is involved in trafficking to the Golgi complex Loc_Os06g40650 encodes copine-1, whose homolog in Arabidopsis (ring domain ligase 2) negatively regulates drought stress response LOC_Os06g40540 and LOC_Os06g40660) are predicted to be retro-transposons, and the genetic functions of three other genes  are unknown at present. Among the ten candidate genes, Loc_Os06g40550 is the only one to be expressed in anthers around the tetrad stage Loc_Os06g40550 homolog in Arabidopsis, abcg26/wbc27, also exhibits MS, and the spatial and temporal expression of Loc_Os06g40550 was consistent with 2sh phenotype, we chose Loc_Os06g40550 as a top candidate gene for further study.Analysis of expression patterns for the 22 candidate genes showed that ten are expressed in the inflorescence GCCACCCTCCTC) deletion in the predicted sixth exon of Loc_Os06g40550 was detected in the 2sh genomic DNA and RNA by sequencing and RT-PCR analysis  of 30 combinations of 6 isonuclear alloplasmic lines (A1–A6) with 5 restorers (R1–R5) during both years.PPTXClick here for additional data file.Figure S3Mean grain number per panicle and CV (coefficient of variation) of 30 combinations of 6 isonuclear alloplasmic lines (A1–A6) with 5 restorers (R1–R5) during both years.PPTXClick here for additional data file.Figure S4Mean 1000-grain weight and CV (coefficient of variation) of 30 combinations of 6 isonuclear alloplasmic lines (A1–A6) with 5 restorers (R1–R5) during both years.PPTXClick here for additional data file.Figure S5Mean effective panicle number and CV (coefficient of variation) of 30 combinations of 6 isonuclear alloplasmic lines (A1–A6) with 5 restorers (R1–R5) during both years.PPTXClick here for additional data file.Figure S6Mean plant height and CV (coefficient of variation) of 30 combinations of 6 isonuclear alloplasmic lines (A1–A6) with 5 restorers (R1–R5) during both years.PPTXClick here for additional data file.Figure S7Mean yield and CV (coefficient of variation) of 30 combinations of 6 isonuclear alloplasmic lines (A1–A6) with 5 restorers (R1–R5) during both years.PPTXClick here for additional data file.Figure S8Mean weight per panicle and CV (coefficient of variation) of 30 combinations of 6 isonuclear alloplasmic lines (A1–A6) with 5 restorers (R1–R5) during both years.PPTXClick here for additional data file.Table S1Isonuclear alloplasmic lines used in cytoplasm effects analysis.(DOCX)Click here for additional data file.Table S22sh cytoplasmic effects on seed set rate, weight/panicle and yield in comparison with Zhenshan97A (A2), D702A (A3), G46A (A4), K18A (A5) and XieqingzaoA (A6).(DOCX)Click here for additional data file.Table S3GCA effects on seed set rate, weight per panicle and yield among cytoplasms of 2sh (A1), Zhenshan97A (A2), D702A (A3), G46A (A4), K18A (A5) and XieqingzaoA (A6).(DOCX)Click here for additional data file.


Trypanosoma brucei relies on Spliced leader trans splicing to generate functional messenger RNAs. Trans splicing joins the specialized SL exon from the SL RNA to pre-mRNAs and is mediated by the trans-spliceosome, which is made up of small nuclear ribonucleoprotein particles and non-snRNP factors. Although the trans spliceosome is essential for trypanosomatid gene expression, not all spliceosomal protein factors are known and of these, only a few are completely characterized. In this study, we have characterized the trypanosome Splicing Factor, SF3a60, the only currently annotated SF3a component. As expected, epitope-tagged SF3a60 localizes in the trypanosome nucleus. SF3a60 is essential for cell viability but its depletion seem to have no detectable effect on trans-splicing. In addition, we used SF3a60 as bait in a Yeast-2-hybrid system screen and identified its interacting protein factors. The interactions with SF3a120, SF3a66 and SAP130 were confirmed by tandem affinity purification and mass spectrometry. Trypanosoma brucei is responsible for Human African Trypanosomiasis (HAT) and Animal African trypanosomiasis (AAT) in sub-Saharan Africa. This parasite relies on trans splicing where a common spliced leader (SL) is added to the 5′end of all immature mRNA transcripts to generate functional messenger RNAs. Trans splicing is mediated by the trans-spliceosome, which consists of small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP factors  and TAP-tagging analyses.We focus here on trypanosome SF3a. In humans and yeast, SF3a contains three subunits: SF3a60 (yeast Prp9), SF3a66 (Prp11) and SF3a120 (Prp21), and each is essential for pre-splicing complex A formation Trypanosoma brucei bloodstream and procyclic 427–1313 cells containing plasmid pHD 514 T. brucei 927 strain using the primers SF3a forward, GGATCCAATGTTTGGTGGAGTTTTAGAAAAGAT bearing BamHI and reverse, TCAACCTGTTTTCCTCGACATCTG. The PCR product was cloned into pGEM-T (Promega), subsequently digested with BamHI and NotI and subcloned in frame into the ProQuest Yeast Two-Hybrid Gateway compatible System (Invitrogen). Bait cloning and Y2H screening were performed by Hybrigenics, S. A., Paris, France (http://www.hybrigenics.com/services.html). DNA from Trypanosoma brucei 927 strain, prepared by Dr. M. Turner, Glasgow Biomedical Research Centre, University of Glasgow, Scotland, UK, was randomly sheared and used to construct genomic library into the Y187 yeast strain. The library contained 7.5 million independent fragments, and was used for screening The coding sequence TbSF3a60 (TriTrypDB accession number Tb927.6.3160) was PCR amplified from 100 ng of total genomic DNA of tet) repressor from plasmid pHD1313 SF3a60 (1629 bp) was amplified using primers CZ3371 ) and CZ3372 ) and genomic DNA of T. brucei strain Lister 427 as template. To express C-terminally TAP-tagged SF3a60, the open reading frame (ORF) was cloned in the ApaI and HpaI sites of pHD 918 Procyclic trypanosomes expressing the tetracycline , incubated with a 1∶50000 dilution of rabbit anti-Protein A antibody in transfer buffer and probed for expression of TbSF3a60-TAP using the peroxidase-anti-peroxidase complex . Subsequently, clones expressing C-terminally TAP-tagged SF3a60 were harvested and the subcellular localization of the tagged SF3a60 determined by indirect immunofluorescence 6 trypanosomes were sedimented, re-suspended, then fixed in 4% paraformaldehyde (w/v) in 1x PBS for 18 min, sedimented again for 2 min, re-suspended in phosphate buffered saline (PBS), and allowed to settle down on a poly-lysine-coated glass surface for 25 min. Cells were blocked with 0.5% gelatin/PBS and thereafter incubated with a 1∶50000 dilution of rabbit anti-Protein A antibody (Sigma) for 1 h and with a 1∶500 dilution of the second antibody Alexa Fluor 594 goat a- rabbit IgG (Molecular 25 probes) for 40 min. The kinetoplast and the nuclear DNA were then stained with 100 ng/ml DAPI/1x PBS for 10 min.Western blotting was performed as previously described 5′-GAGAAGATCTGCATGCGAGAAGCCACCTTCACTCGA–3′, 5′- CGGAATTCGTCGACGTTCCACCACATACCTCGCA–3′ for the 1629 bp SF3a60. In this vector, the RNA interference (RNAi) fragments are flanked by opposing T7 promoters with downstream tet operators. The resulting plasmids were linearized with Not I and transfected into bloodstream-form trypanosomes expressing the tet repressor (pHD 1313) and T7 RNA polymerase (pHD514) For RNAi in bloodstream forms, gene-specific fragments of ∼500 nt T. brucei cell lines using Trifast (PeqLab Biotechnology GmbH). Total RNA (10 μg each) were separated on a denaturing formaldehyde agarose gel and blotted onto a Hybond N nylon membrane (Amersham Pharmacia). The TbSF3a60, Tubulin (TUB) and SRP DNA probes were labeled with [α32P]dCTP by random priming (Stratagene). After overnight hybridization, blots were subsequently washed in 1× SSC (0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate (SDS) for 30 min at room temperature, in pre-warmed 1× SSC and 0.5% SDS for 45 min at 42°C, and in 0.1× SSC and 0.2% SDS for 30 min at 42°C before exposure to film phosphor imager.Total RNA was isolated from bloodstream TbSF3a60 RNAi depletion on splicing, the amounts of SLRNA and Y-structure splicing intermediates were assessed by primer extension 5′-GCAGGAACCAACAGCACAATGCG-3′) or a U3 snRNA (primer CZ2712 5′-TGCCGTTCATCGAAC-3′) before incubation with reverse transcriptase and dNTPs, including radioactive dCTP. The products were run on a 6% polyacrylamide/urea gel with 32P-labelled DNA markers and detected by phosphorimaging.To assess the effect of Trypanosome SF3a60 (Tb927.6.3160) is already annotated as such in the genome database, although without experimental evidence. To determine its location, we expressed SF3a60 with a C-terminal TAP-tag in procyclic trypanosomes . Cells eSLRNA, and the migration of the full-length primer extension product was very slightly slower than normal, due to a decrease in cap methylation SLRNA or the relative abundance of the Y structure. It is evident that although SF3a60 is essential, RNAi does not cause a detectable decrease in splicing, at least under conditions when growth inhibition is not yet detected.The down regulation of SF3a60 expression using RNA interference resulted in growth inhibition within 48 hours  and effiT. brucei SF3a60, like protein homologues from human S. cerevisiaeS. cerevisiae SF3a60 contains a second C2H2 zinc finger domain in its central region. This second zinc finger has also been noted in S. pombe, N. crassaT. brucei (accession no. XP951536), human S. cerevisiae2H2 zinc finger domain. The C-terminal sequence of T. brucei and yeast are relatively shorter and lack the proline-rich heptapeptide repeats S. cerevisiaeTo identify the components of SF3a we purified it using C-terminally TAP-tagged SF3a60. Purification of an empty TAP-tagged construct served as a control. In addition, we identified Tb927.7.6980. Although this protein is not yet annotated, it is clearly SF3b130 as judged both by BLAST searches . Using a predicted biological score (PBS), calculated according to www.ebi.ac.uk/Tools/psa/) revealed that Tb927.1.3560 is 18% identical and 29.9% similar to the yeast U1 snRNP component Prp40; it contains WW domains, two highly conserved tryptophans that bind proline-rich peptide motifs found in the splicing factor Prp40 Tandem affinity purification normally results only in the detection of stable interactions. To see if we could trace any other interactions, which might be sub-stoichiometric or transient, we screened a Yeast 2 hybrid We have here presented experimental evidence identifying potential trypanosome homologues to yeast Prp9 (human SF3a60), Prp21 (human SF3a120) and Prp11 (human SF3a66). Compared to the human and yeast homologues, the trypanosome SF3a proteins show N-terminal sequence divergence but as shown in Although the Northern blots of SF3a60-depleted cells showed reduced amounts of mature SF3a60 mRNA as expected, we detected no effect on trans-splicing . In addiS. cerevisiae Prp9p and Rse 1p, the yeast homologue of SF3b130 In the TAP purification, the excised SF3a60 band was actually a doublet , with a T. brucei SF360 is a positive step towards its complete annotation. Six SF3a60 associated proteins have TPR motifs, which predisposes them to various aspects of protein-protein interactions, including during splicing To detect sub-stoichiometric or less stable interactions, we conducted a yeast two-hybrid screen using SF3a60 as bait. The differences with respect to number of hits identified between TAP pull-down and two hybrid screens is striking, but expected. Although likely to be transient, these hits were nonetheless biologically significant and are likely to occur in vivo and could be validated further via experimental functional analyses. While we identified hits across all the Global PBS confidence intervals, several of these proteins have domains that suggest involvement in aspects of gene regulation. An interaction between human SF3a120 and SF3a60 was previously documented trans splicing, but is required for the cis splicing of at least two trypanosome mRNAs One of the high-confidence interaction partners, Tb927.1.3560, could be the putative trypanosome homologue of yeast Prp40, a component of the U1 snRNP - although it was not found to associate with tagged TbU1–70K cis splicing discuss mainly an association between the spliceosome and the phosphorylated C-terminal heptad repeats of RNA polymerase II (reviewed in trans splicing acceptor site - in which SF3 is involved - are independent of the transcribing polymerase The most interesting 2-hybrid interaction was with the largest subunit of RNA polymerase II (RPB1). There is a voluminous literature describing the strong link between polymerase II transcription and splicing in metazoans and yeast  are shown.(PDF)Click here for additional data file.Figure S2Domain organization of high confidence SF3a60 associated proteins. The hits, their corresponding domains and the amino acid portion involved in the interaction with SF3a60 (SID) are shown.(PDF)Click here for additional data file.Figure S3Domain organization of good confidence SF3a60 associated proteins. The hits, their corresponding domains and the amino acid portion involved in the interaction with SF3a60 (SID) are shown.(PDF)Click here for additional data file.Figure S4Domain organization of moderate confidence SF3a60 associated proteins. The hits, their corresponding domains and the amino acid portion involved in the interaction with SF3a60 (SID) are shown.(PDF)Click here for additional data file.Figure S5T. brucei FBP11 homologue where they are absent.The FF domains of PRP40 proteins. The corresponding FF domains of human FBP11 (O75400.2), yeast PRP40 (NP_012913.3) are shown here in bold and starting points marked with an asterisk (*) within the alignment, except for (PDF)Click here for additional data file.


Using NOD.CD1d−/− mice, we show that this delay of T1D is CD1d-dependent. Interestingly, the latter delay or protection from T1D is associated with the enhanced secretion of IL-10 rather than IFN-g by C24:0-treated CD4+ T cells and the deviation of the islet-reactive diabetogenic T cell response. Both C16:0 and C24:0 sulfatide isoforms are unable to activate and expand type I iNKT cells. Collectively, these data suggest that C24:0 stimulated type II NKT cells may regulate protection from T1D by activating DCs to secrete IL-10 and suppress the activation and expansion of type I iNKT cells and diabetogenic T cells. Our results raise the possibility that C24:0 may be used therapeutically to delay the onset and protect from T1D in humans.Sulfatide-reactive type II NKT cells have been shown to regulate autoimmunity and anti-tumor immunity. Although, two major isoforms of sulfatide, C16:0 and C24:0, are enriched in the pancreas, their relative role in autoimmune diabetes is not known. Here, we report that sulfatide/CD1d-tetramer Sulfatide , a glycosphingolipid expressed primarily in ceramide and negatively charged sulfate moieties. The ceramide galactosyl sulfotransferase enzyme adds a sulfate group to galactosylceramide  to produce sulfatide in the trans-golgi apparatus. Arylsulfatase A degrades sulfatide to GalCer, and in pancreatic islet beta cells GalCer is recycled back to the cis-Golgi apparatus Sulfatide binds to MHC class I-like CD1 group 1  and group 2 (CD1d) glycoproteins and presented to T cells The conformation of the A’ and F’ channels of CD1d differs between their antigen-bound and antigen-unbound states Type 1 diabetes (T1D) is an autoimmune disease that results from the selective T cell-mediated destruction of insulin-producing pancreatic islet beta cells. T1D is commonly studied in nonobese diabetic (NOD) mice, which spontaneously develop T1D due in part to functional and numerical deficiencies in Treg cells and iNKT cells –CD49high NKT cells that proliferate in response to sulfatide can protect against T1D in NOD mice A rapidly increasing role for the regulation of various states of inflammation and disease by type II NKT cells is emerging, as is evident from recent experimental studies of the amelioration of autoimmune disorders such as EAE + effector T cells mediating autoimmunity. Sulfatide-mediated regulation of type I NKT cells not only plays an important role in the control of autoimmune diseases, but also has a major effect in immune surveillance of tumors Recently, we identified a novel pathway of immune regulation in which sulfatide-activation of type II NKT cells results in anergy induction in type I NKT cells Type 2 diabetes patients have low serum levels of sulfatide Scid mice. The ability of these isoforms to activate NKT cells and modulate their cytokine secretion profiles were also investigated. Our results demonstrate that treatment of NOD mice with C24:0 sulfatide preferentially protects against the transfer of T1D, and suggest that this isoform may have therapeutic value in clinical trials in human subjects at risk for T1D and/or in patients newly diagnosed with T1D.Although treatment of NOD mice with brain sulfatide protects them from T1D Maricic et al., in preparation], a mouse model of multiple sclerosis + T cell proliferation than C16:0 and C24:0, we injected higher doses (50 mg/dose or 100 mg/dose) of C16:0 and C24:0 into NOD mice according to the same multi-dose protocol and still found that these higher doses provided only very low (10–20%) protection of the mice from the spontaneous development of T1D. In contrast, when NOD female mice  were administered three weekly injections (i.p.) of 20 mg of sulfatide, significant protection of these NOD mice from T1D was achieved . Compared to C16:0, C24:0 and aGalCer both delayed the onset of T1D appreciably in recipient NOD.Scid mice, with aGalCer yielding the longest delay  or varying doses (5–50 mg/ml) of C16:0 or C24:0 in the presence of different ratios of mitomycin C treated CD4– T cells  were measured after 72 h. The CD4– T cells were used as APCs, and the optimum concentations of aGalCer and the sulfatides used were determined in preliminary experiments. Similar responses were obtained for a 100 ng/ml dose of aGalCer and a 50 mg/ml dose of C16:0  ratio of 100∶1 in vitro for varying times (12–72 h) with either control vehicle or an optimum dose (50 mg/ml) of C16:0 or C24:0. Interestingly, C16:0 stimulated significantly higher secretion of IFN-g than C24:0, particularly at the peak of the C16:0 response at 24 h , NOD mice were challenged with different diabetogenic peptides derived from the islet antigens glutamic acid dehydrogenase (GAD), heat shock protein 60 (hsp60), or insulin. Ten days after antigenic challenge, PLN T cell proliferative and cytokine responses to the respective antigen were analyzed. The frequency of both IFN-g and IL-4-secreting T cells in Elispot assays of responses to the insulin 9–23, GAD206-220, GAD524-543 and hsp277 peptides were significantly decreased in sulfatide-treated mice , C and DC16:0 and C24:0 Sulfatide do not Stimulate iNKT Cell Early Activation and Expansion+ T cells differ in their level of protection from T1D conferred upon transfer to NOD.Scid recipients  or aGalCer (4 mg) that yielded optimum protection from the transfer of T1D. At 2 h post-injection of NOD mice  with vehicle, sulfatide (C16:0 or C24:0) or aGalCer, splenocytes were analyzed by FACS for the frequency of iNKT cells  that express the CD69 early activation surface marker. Compared to vehicle-stimulated iNKT cells, CD69 surface expression was increased about 4-fold on aGalCer stimulated iNKT cells while no significant increase in CD69 expression was detected on iNKT cells exposed to C16:0 or C24:0 (Our observation that C16:0 and C24:0 activated donor NOD CD4cipients  raised tor C24:0 . Similar+ T cells and iNKT cells in the PLN of the sulfatide treated mice (data not shown). Thus, unlike aGalCer, C16:0 and C24:0 sulfatide do not stimulate the expansion or early activation of iNKT cells, consistent with the ability of sulfatide to function as a ligand for type II NKT but not type I iNKT cells.Next, we monitored the kinetics of iNKT cell expansion in the spleen and PLN, two sites of activation of iNKT cells that mediate protection from T1D, using the same protocol as described for the iNKT cell expansion studies discussed above. While the frequency of aGalCer stimulated iNKT cells in the spleen was about 0.6% at 2 h post-injection (control vehicle induced a frequency of 0.2%), this frequency decreased significantly by 4 h and 12 h after injection , consist+ T cells are localized predominantly in the liver, and only a minor subpopulation (∼0.1%) is present in the spleen. In NOD mice, sulfatide/CD1d-tetramer+ cells are barely detectable in the spleen at any age (6–20 weeks) examined and irrespective of the onset of T1D. Since sulfatide is highly enriched in the membranes of islet beta cells, we reasoned that it may be presented as an antigen to type II NKT cells localized in the PLN during the development of destructive insulitis in diabetic NOD mice. In this scenario, the frequency of sulfatide/CD1d-tetramer+ cells should be enriched in the draining PLN. To determine whether sulfatide-reactive type II NKT cells accumulate in the draining PLN during the onset of T1D, PLN-derived mononuclear cells isolated from NOD mice (aged 6–20 weeks) were stained with either sulfatide/CD1d- or αGalCer/CD1d-tetramers for the identification of type II and type I NKT cells, respectively. We found that sulfatide-reactive T cells are present at a significantly higher frequency (0.36%) of infiltrating T cells in the PLN than in the liver or spleen (<0.1%)  . InteresRecent studies have shown that the restoration of an iNKT cell deficiency in NOD mice following their activation with aGalCer protects against T1D + T cells from NOD mice to transfer protection from T1D. Prior analyses of stimulation of CD4+ T cell in vitro proliferative responses showed that a robust response to sulfatide C16:0 and C24:0 required about a 500-fold higher concentration (50 mg/ml) of C16:0 or C24:0 than that of aGalCer (100 ng/ml)  . Therefo0 ng/ml) , further+ T cells stimulated with a high dose of C16:0 or CD24:0 sulfatide demonstrated a more rapid and preferential secretion of IFN-γ secretion by C16:0 stimulated CD4+ T cells. The latter C16:0 induced response peaked at 24 h post stimulation. In contrast, the IFN-g response of C24:0 stimulated cells did not peak until 48 h post-stimulation, and this level of secretion was about 3-fold lower than that obtained for the C16:0 induced response at 24 h. Importantly, C24:0 sulfatide stimulated cells secreted large amounts of IL-10 and reached a maximal response at 48 h post-stimulation at which time the level of IL-10 secretion by C16:0 stimulated T cells was not significantly different from the control vehicle response. These results implicate a role for IL-10 in C24:0 sulfatide-mediated protection of NOD mice against T1D. IL-10 is an anti-inflammatory cytokine that elicits immunosuppression, and when expressed in the pancreas, IL-10 mediates elevated regulatory T (Treg) cell activity and protection from T1D Cytokines produced by glycolipid stimulated iNKT cells can regulate the phenotype and function of interacting APCs + T cells undergo reduced proliferation  potently stimulate type II NKT cells. In contrast, sulfatide isoforms with either a shorter fatty acyl chain length (≤ C18:0) or saturation of the long fatty acyl chain (C24:0) are weak stimulators of type II NKT cells, and fatty acid hydroxylation abolishes the response Maricic et al., in preparation), concanavalin A-induced hepatitis In conclusion, our findings demonstrate that while bovine brain-derived sulfatide consists of several molecular species that vary in fatty acid chain length, unsaturation and hydroxylation, treatment of NOD mice with the long fatty acid chain sulfatide isoform results in the activation of type II NKT cells that may mediate significant protection from T1D. Consistently, long fatty acid sulfatide isoforms, including C24:1 and C24:0, are immunodominant in mice and can bind with high avidity to murine CD1d molecules NOD/Del, NOD.Scid and NOD.CD1d−/− mice were bred and maintained in a specific pathogen-free barrier facility under the direction of the Animal Care and Veterinary Services at the Robarts Research Institute, Western University  and according to the Canadian Council for Animal Care guidelines. NOD mice were also purchased from The Jackson Laboratory/Taconic Farms and kept under specific pathogen-free conditions in our colony at the Torrey Pines Institute for Molecular Studies . These experiments were performed in compliance with federal and institutional guidelines and have been approved by the Institutional Animal Care and Use Committee of the Torrey Pines Institute for Molecular Studies.The Animal Care and Veterinary Services Committee at Western University specifically approved this study (protocol number 2008-025). The incidence of T1D in female NOD mice in our colony is ≥80% by 30 weeks.Mice were monitored for hyperglycemia beginning at 15 weeks of age in the spontaneous model and at 8 weeks of age in the adoptive transfer model by measurement of BGL twice weekly, as described. Mice were considered diabetic when two consecutive BGL readings of >11.3 mmol/l were obtained.Synthetic KRN7000  was kindly provided by Kirin Pharmaceutical Research Laboratories , solubilized in water and injected i.p. into mice (4 mg/dose). Native sulfatide was isolated from pig brain as described 6) and PLN lymphocytes (0.5×106) were prepared in PBS containing 2% FBS (PBS/FBS) Scid mice (n = 10/treatment group).The aGalCer and sulfatide (C24:0 or C16:0 isoform) glycolipids (100 mg) were suspended in PBS 5 cells for proliferation analysis, 5×106 cells for cytokine analysis) were cultured  in triplicate with glycolipid (100 ng/ml) or vehicle, as described 3H]-thymidine  for the last 18 h of culture, harvested and incorporated radioactivity was quantified using a 1450 Microbeta scintillation counter (Perkin Elmer). All cultures were maintained in complete RPMI (RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Hyclone), 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 0.05 mM 2-ME (Invitrogen Life Technologies). A standard sandwich ELISA was performed to analyze mouse cytokines in culture supernatants using paired antibody kits for IL-4, IL-10, IL-2, IFN-g (BD Biosciences). Streptavidin-horse radish peroxidase (HRP) conjugate and development solution for BD OptiEIA Reagent Set A (BD Bioscience) were used for signal detection at a dual wavelength of 450/570 nm using a Benchmark Microplate Reader (Bio-Rad).Spleen- and PLN-derived lymphocytes (5×106 cells/ml) were cultured for 48 h in 24-well plates either with medium alone or with different islet antigen-derived peptides (20 mg/ml). Nitrocellulose plates (Millipore) were coated overnight at 4°C with anti-IFN-g or anti-IL-4 Abs. After blocking the coated plates, Ag-stimulated cells were added at graded concentrations for 24 h at 37°C. The wells were then incubated with biotin-conjugated anti-IFN-g or anti-IL-4 mAbs followed by incubation with avidin peroxidase (Vector Laboratories). Spots were developed by the addition of 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich), and were counted using a computerized image analysis system and the image analyzer program, NIH Image 1.61.The frequency of IFN-g and IL-4-producing cells were enumerated by cellular ELISPOT essentially as described earlier + T cell isolation kit (Miltenyi Biotec) was used to isolate CD4+ splenocytes by negative selection with a purity of 93%, as assayed by flow cytometry. The CD4– T cells (15×106/ml in PBS/FBS) obtained were treated  with mitomycin C (100 ml/ml) and used as a source of APCs in proliferative responses.A mouse CD4Single-cell suspensions of splenocytes and PLN lymhocytes were treated  with an anti-FcgR mAb and then stained with an FITC-anti-TCR mAb (H57–597) and APC-labeled empty or PBS-57-loaded CD1 tetramers (provided by NIH Tetramer Core Facility), PE CD69 (H1.2F3), and PE-rat IgG2b isotype control (eB149/10H5). Sulfatide/CD1d-tetramers were generated as described earlier (2). Flow cytometry was performed using FACSCalibur and CellQuest software (BD Biosciences) or FACSCanto II and FACSDiva software. Analyses were conducted using FlowJo software . Intracellular cytokine  staining was performed using a BD Cytofix/Cytoperm buffer set (BD Biosciences) Results are expressed as mean ± standard error of the mean (SE). Statistical analyses were performed using the Student’s t test or log rank test (for T1D incidence) . Differences were considered statistically significant at P values ≤0.05.