Accordingly, Pin1 has been linked to numerous phosphorylation-controlled signaling pathways and cellular processes such as cell cycle progression, proliferation, and differentiation. In addition, Pin1 plays a pivotal role in DNA damage-triggered cell fate decisions. Whereas moderate DNA damage is balanced by DNA repair, cells confronted with massive genotoxic stress are eliminated by the induction of programed cell death or cellular senescence. In this review, we summarize and discuss the current knowledge on how Pin1 specifies cell fate through regulating key players of the apoptotic and the repair branch of the DNA-damage response.The peptidyl–prolyl  The PPIases) .Three distinct families of PPIases that can facilitate prolyl isomerization have been identified so far. Among those are the family of Cyclophilins (Cyp), FK506-binding proteins (FKBPs), and the parvulins –7. Both cis and trans conformation is dependent on the phosphorylation state of the Ser/Thr–Pro motif, which is the target sequence of Pin1  , 45. PhoTP53 gene are frequent in many cancer types and have a huge impact on the clinical outcome of those cancers , which exerts transcription-dependent and independent functions –60. AparInterestingly, it has been demonstrated that DNA damage-induced p53-dependent apoptosis requires functional p63 and p73 . Along tp53 activity needs to be tightly regulated in unstressed cells to prevent an unscheduled activation of the apoptotic response. To this end, p53 is sustained at low levels under physiological conditions, which is achieved by constant proteasomal degradation of p53 mediated by the E3 ubiquitin ligase MDM2. On the other hand, also association with iASPP inhibits p53 by preventing its binding to promoters of pro-apoptotic target genes . ChIP exHomeodomain-interacting protein kinase-2 is a central activator of the apoptotic cell death in development and in response to cellular stress and also acts as a tumor suppressor targeted by cellular and viral oncogenes , 65–70. in trans at Thr880/Ser882 through an intermolecular mechanism activates its kinase activity and apoptotic function upon genotoxic stress. Thr880/Ser882 autophosphorylation takes place at an early phase of HIPK2 activation and decreases when HIPK2 is fully stabilized. Furthermore, pThr880/pSer882 is followed by Pro residues and thus represent bona fide Pin1 binding sites. In fact, Pin1 binds HIPK2 through this phospho-motif and alters the conformation of the autophosphorylated HIPK2 isoform and mediates stabilization by inhibiting its proteasomal degradation. Loss of Pin1 by genetic deletion or RNA interference results in a lack of DNA damage-induced HIPK2 stabilization and apoptosis induction  is able to induce p53-dependent apoptosis via a Pin1-mediated mechanism . Activatc from the mitochondria  and non-homologous end-joining (NHEJ). HR represents a highly accurate, error-free repair mechanism, whereas NHEJ is more error-prone. In mammalian cells, DSBs produced for instance by IR are mostly repaired by NHEJ, however, when DNA replication is impeded this leads to stalled replication fork collapses and resulting DSBs are resolved by the HR pathway . The NHEG2 phase .2 phase (2 phase. Of note, since Pin1 overexpression is frequently observed in cancer, this mechanism is presumably involved in the increased genomic instability observed in human cancer cells. Taken together, Pin1 obviously plays a critical role in coordinating DNA repair pathway choice by suppressing HR and promoting the NHEJ pathway.A recent study demonstrated that Pin1 plays an important role in the DNA repair pathway , 107. Us2 phase –110. IntThe identification of Pin1 and the clarification of its underlying enzymology have greatly put forward our knowledge on mechanisms of signal transduction. The intense research activities on Pin1 during the last years generated fascinating novel insight in the function and regulation of this remarkable enzyme and its role in cell signaling and disease. Pin1 provides a highly sophisticated, elegant means to translate pSer/pThr–Pro phosphorylation marks into conformational changes and thus in altered protein function. Since Ser/Thr–Pro phosphorylation is the most abundant post-translational modification in mammalian cells, it appears not very surprising that Pin1 emerged as a central specifier in signal transduction. Despite these facts, however, there is still much to be learned about the biochemistry and biology of Pin1.For example, numerous Pin1 substrates including p53, HIPK2, and CtIP harbor several pSer/pThr–Pro sites, which critically contribute to Pin1 binding. This raises a number of questions: how is the exact stoichiometry of the Pin1–substrate complexes? Does Pin1 isomerize all pSer/pThr–Pro bonds in its substrates or only a subset? In addition, it is currently unclear whether Pin1 also cross-talks to phosphatases. The timed removal of the pSer/pThr–Pro marks by a given phosphatase would contribute to lock a substrate in a particular conformational state by preventing a backward isomerization reaction.Even though there is evidence that Pin1 is subject to regulation by post-translational modifications including phosphorylation and SUMOylation, it currently remains unclear whether Pin1 function is also regulated in response to DNA damage. Such regulation might facilitate functional dissection of the DNA damage-associated functions and the cell growth regulatory and mitotic activities.Beyond its impact on CtIP function, Pin1 has been found to interact with numerous additional factors implicated in DNA repair , 107 sugFinally, Pin1 appears to play important roles both in oncogenic and tumor suppressive signaling pathways. To exploit Pin1 function in diseases such as cancer it will be a major effort in the future to dissect the molecular determinants in order to design small molecules for specific interference with its oncogenic and mitotic functions, but to conserve its pro-apoptotic activities, which are necessary for the efficacy of genotoxic stress-inducing cancer therapies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salmonella can lead to changes in the host signalling pathways in gallbladder cancer.We earlier proposed a genetic model for gallbladder carcinogenesis and its dissemination cascade. However, the association of gallbladder cancer and ‘inflammatory stimulus’ to drive the initial cascade in the model remained unclear. A recent study suggested infection with Salmonella serotypes  using a computational subtraction pipeline based on the HPVDetector, we recently described. Based on our evaluation of 26 whole exome gallbladder primary tumours and matched normal samples: association of typhoidal Salmonella species were found in 11 of 26 gallbladder cancer samples, and non-typhoidal Salmonella species in 12 of 26 gallbladder cancer, with 6 samples were found co-infected with both.We examined the whole exomes of 26 primary gall bladder tumour and paired normal samples for presence of 143 HPV (Human papilloma virus) types along with 6 common Salmonella species along with typhoidal strains in gallbladder cancer. Salmonella infection in the chronic carrier state fits the role of the ‘inflammatory stimulus’ in the genetic model for gallbladder carcinogenesis that may play a role in gallbladder cancer analogous to Helicobacter pylori in gastric cancer.We present the first evidence to support the association of non-typhoidal The online version of this article (doi:10.1186/s13027-016-0057-x) contains supplementary material, which is available to authorized users. Salmonella infection of the gallbladder can lead to changes in the host signalling pathways triggering cellular transformations resembling the evolution of gallbladder cancer [Salmonella typhi and S. paratyphi with the risk of gallbladder cancer [S. typhimurium, S choleraesuis) that elicits a stronger host immune response compared to the typhoidal species and are linked to a systemic illness have as yet not reported to be associated with gallbladder disease or cancer [Salmonella infection is also considerably severe [Gallbladder cancer with its attendant poor prognosis , despiter cancer . This isr cancer –7. Howevr cancer . Interesy severe .in situ to invasive cancer? Here in this study we examined the exomes of primary gall bladder tumour and paired normal samples for presence of 6 common Salmonella serotypes with available genome information  using a computational subtraction pipeline based on the HPVDetector tool [We have previously proposed a model for gallbladder carcinogenesis based on the current understanding of the tumour biology and associated inflammation . Howevertor tool Twenty six fresh frozen primary tumour and matched normal tissues were obtained from the tissue repository of Tata Memorial hospital (TMH). The Institutional Review Board (IRB) and the Ethics Committee (EC) of Tata Memorial Centre (TMC) - Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)  approved the project (#104). Since this was a retrospective analysis, the IRB and the EC waived the need for an informed consent. Patients were randomly selected based on the availability of fresh frozen tissues. The patient characteristics including age, gender, gall stone status and histopathology were recorded.Salmonella detection has been previously described [Salmonella sequences using read specific primers. The PCR conditions - 94 °C for 1 min, 59 °C for 30s, 72 °C for 45 s – for 30 cycles. The primer sequences are detailed in Additional file The PCR method used for escribed . Nested Salmonella traces, the HPVDetector pipeline was used, as described previously [Salmonella species genomes in addition of the HPVDetector nce data set of 143 HPV types, as downloaded from the National Centre for Biotechnology Information (NCBI), using BWA  aligner (v0.6.2). All reference sequences were annotated and concatenated to compose a multi-fasta sequences using bio-perl modules. The alignment files were parsed using UNIX shell program to detect the types of Salmonella represented by at least one read that aligned to a particular Salmonella type with high confidence.Exome capture and library preparation was performed using Agilent Sure select in-solution (low-input capture-500 ng) target enrichment technology. Genomic DNA was sheared and size selected (150–200 bp) and ligated to adaptors and run on Illumina Nextseq 500 platform to generate 150 bp paired end reads at a coverage of 100X and above. To detect eviously . BrieflySalmonella DNA using pan primers, as described earlier [Salmonella (data not shown). As a next step, whole exome data for these 26 samples  were analysed to detect Salmonella traces using HPVDetector pipeline, modified to include additional genome sequence of 6 common Salmonella isolates. The computational approach, in brief, subtracts all reads that align to human genome and aligns remaining reads to HPV and Salmonella reference database from NCBI. While HPV16 was detected in 1 gallbladder sample, Salmonella isolates were found across multiple samples: S. typhi Ty2 (3 samples), S. typhi CT18 (6 samples), S. typhimurium LT2 (10 samples), S. choleraesuis SCB67 (5 samples), S. paratyphi TCC (3 samples), and S. paratyphi SPB7 (7 samples). In total, Salmonella reads were found in 19 of 26 gallbladder tissues . Interestingly, 10 of 19 samples were co-infected with multiple isolates independent of gallstone status or gender of the patients, as shown in Fig. We performed PCR based analysis of 26 gallbladder tumour and paired normal samples to detect the presence of  earlier . None ofX2 multiple alignment as shown in Additional file Salmonella isolates from NCBI  database. 114 reads of multiple Salmonella isolates were found in 19 of 26 samples analyzed. 47 of 114 reads of Salmonella ORF (open reading frame) were identified as encoding for bacterium genes known to be involved in metabolism and those related to the toxin-antitoxin system. Rest of the reads aligned to the Salmonella ribosomal genes, understandably due to their relatively higher abundance , as described earlier [Salmonella reads using the HPVDetector pipeline. Distinct Salmonella reads were detected at as low as 10X whole exome coverage that increased linearly  to simulate random sequence, but retaining composition of nucleotides and genome complexity, using an in-house perl script, as described earlier . We foun earlier . The resSalmonella read sequences identified by HPVDetector in 4 of 16 Salmonella positive samples using Sanger sequencing (Additional file We have attempted to validate the presence of Salmonella and HPV DNA sequences in gallbladder tumours and paired normals, The high incidence of Salmonella sequence found in 16 of 26 samples analysed in the study suggests a possible role of Salmonella infection in gallbladder cancer analogous to Helicobacter pylori in gastric cancer and Fusobacterium in colon cancer [Salmonella species in 11 of 26 gallbladder cancer samples, consistent with as known earlier. In addition, we present the first evidence to support the association of even non-typhoidal Salmonella species in 12 of 26 gallbladder cancer, with 6 of 26 samples co-infected with typhoidal as well as non typhoidal Salmonella isolates.We examined n cancer , 14. We Salmonella infection to stimulate a host response, it is likely that these bacteria are able to provide the continued ‘inflammatory stimulus’ necessary for carcinogenesis. Recent reports suggest that Salmonella infections promote malignant transformation in genetically predisposed mice, murine gall bladder organoids and fibroblasts with TP53 mutations [Salmonella positive samples harboured TP53 mutations while we did not observe TP53 mutations in Salmonella negative samples. Salmonella isolates in the chronic carrier state thus fits the role of the ‘inflammatory stimulus’ in the genetic model for gallbladder carcinogenesis and its dissemination cascade, which may trigger transformation through chronic inflammation, but not for maintenance of tumourigenesis [Systemic inflammation is known to be associated with a poor prognosis in gallbladder cancer . Owing tigenesis .Salmonella species often underestimating the contribution of the non-typhoidal isolates that show an inherent higher resistance to the standard antibiotics [Salmonella species in our study highlights that in typhoid-, as well as gallbladder cancer-endemic countries such as India and other similar countries, efforts must be directed not only at treating typhoid fever, but also diagnosing and appropriately managing non-typhoidal Salmonella species. This simple approach could reduce the chronic carrier state of these species in humans, which by our hypothesis may be contributing to the inflammatory stimulus driving gallbladder carcinogenesis. Thus, this simple strategy may help reduce in the incidence of gallbladder cancer. While this study validates and extends the association of Salmonella with gall bladder carcinoma, further study is required to establish the causality of infection to the disease.The focus of treatment in typhoid-endemic countries such as India has historically been solely on eliminating typhoidal ibiotics  resultin


Electron backscattered diffraction (EBSD) analysis reveals that olivine grains co-existing with exsolved phases inside chromite grains and occurring on chromite grain boundaries have a single pronounced crystallographic preferred orientation (CPO). This suggests that olivine preserves the CPO of a high-pressure polymorph (wadsleyite) before the high-pressure polymorph of chromite began to invert and exsolve. Chromite also shows a significant CPO. Thus, the fine-grained high-pressure phases were deformed by dislocation creep in the MTZ. Grain growth in inverted chromite produced an equilibrated microstructure during exhumation to the surface, masking at first sight its MTZ deformation history. These unique observations provide a window into the deep Earth, and constraints for interpreting geophysical signals and their geodynamic implications in a geologically robust context.Investigations of the Mantle Transition Zone  by deformation experiments and geophysical methods suggest that the MTZ has distinct rheological properties, but their exact cause is still unclear due to the lack of natural samples. Here we present the first direct evidence for crystal-plastic deformation by dislocation creep in the MTZ using a chromitite from the Luobusa peridotite (E. Tibet). Chromite grains show exsolution of diopside and SiO Seismological studies of Earth’s mantle reveal three distinct changes in seismic velocity, at depths of 410, 660 and 2700 km; the interval between 410 and 660 km is termed the Mantle Transition Zone (MTZ). The first two changes are explained by phase transitions in olivine, the dominant mineral of the upper mantle; olivine to wadsleyite at 410 km, and ringwoodite to Mg-silicate perovskite (bridgmanite) plus ferropericlase at 660 km. Tomographic images of cold slabs in the lower mantle, and the displacements of the 410-km and 660-km discontinuities beneath subduction zones imply whole-mantle convection1in situ)45Luobusa is one of several large peridotite massifs along the Yarlung-Zangbo suture zone of southern Tibet, which marks the boundary between the Indian and Asian blocksRecently, a basic model for the subduction, MTZ UHP metamorphism and exhumation of the Tibetan peridotites has been presented2O4) structure (>15 GPa at TZ temperatures of 1400–1500 °C)810et al. 122Cr2O5 + Cr2O3 eskolaite assemblage (12–15 GPa) before the CF structure is formed. However, these experiments may not be relevant to real chromite, which contains significant levels of aluminum. Multianvil experiments using natural Al-rich Luobusa chromitite + diopside or SiO2 as a starting materials 141522O5 with significant Ca or Si substitution on the Al site. Although further experiments are required, the intermediate phase is orthorhombic in all cases (as is the CF-type). Here we refer to the group of suggested high-pressure chromite phases as HPP (High Pressure Polymorph)-chromite.The chromites commonly show exsolution of diopside, enstatite and coesite4 14 GPa, 00 °C1415The apparent deep recycling of some podiform chromitites (and their host peridotites) implies previously unrecognized types of mantle dynamics, and requires reinterpretation of the history of the lithospheric mantle in some ophiolites. This contribution presents an electron backscattered diffraction (EBSD) study of UHP massive chromitites from the Luobusa peridotite , shedding light on the deformation behavior of high-pressure phases at MTZ conditions. We provide the first direct evidence of crystal-plastic deformation, evidenced by mineral CPOs and substructures interpreted to have formed by dislocation creep in the UHP phases wadsleyite (now olivine) and HPP-chromite, now normal spinel-structured chromite.98; The massive chromitite studied here is similar to previously-reported samplesvs 526 μm) with slightly higher median aspect ratios (1.73 vs 1.70) and lower abundance of olivine (1.6 area% vs 2.2 area%) than zone 2  is shown in dio//<111>chr, [010] dio//<110> chr, and [001] dio//<112> chr , further supports our interpretation that the olivine CPO was produced by deformation under UHP conditions.The strong CPO of the olivine indicates deformation by dislocation creep, as crystal alignment during compaction and/or flow of partially molten systems produces a weaker CPO4850wds//(100)ol and [010]wds//[001]ol, (2) (021)wds//(100)ol, (011)wds//(010)ol and [100]wds//[001]ol, respectively. Although these topotaxial relationships are the result of the phase transformation from olivine to wadsleyite, crystallographic considerations predict that they are expected also to be applicable to that from wadsleyite to olivine, because the close-packed oxygen layers of olivine and wadsleyite cause these topotaxial relations. Integration of our data indicates that the observed (010)[001] fabric preserves the (011)[100] fabric of wadsleyite despite the transformation. Such a fabric is most consistent with simple shear deformation of “dry” wadsleyite under MTZ conditionsOlivines hosted in chromite  have a (010)[001] fabric, which we interpret as reflecting deformation by dislocation creep within the MTZ. This implies that these olivine grains were deformed and aligned while in one of the higher-P polymorph states. Wadsleyite (or β-olivine) is volumetrically the main component of the upper transition zone, between 410 and 520 km depths, and is not stable at shallower depths. Deformation experiments on wadsleyite show several active slip systems535455565655The significant CPO of included and interstitial olivine grains in chromitite implies the activation of the (011)[100] slip system in wadsleyite during crystal-plastic deformation by dislocation creep under MTZ P-T conditions. The transformation from an orthorhombic HPP-chromite to cubic chromite during exhumation drove the exsolution of diopside and coesite, and the wadsleyite grains transformed to olivine during this process, while preserving the wadsleyite CPO. Significant grain-boundary migration under static conditions during passive exhumation produced equilibrated microstructures but preserved the core-rim structure with exsolved diopside only in the cores , as wellChemical compositions of host chromite and olivine inclusions in the thin section were analyzed with Cameca SX-100 electron microprobe at Geochemical Analysis Unit (GAU) at the Australian Research Council Center (ARC) of Excellence for Core to Crust Fluid Systems (CCFS) in Macquarie University, Australia). Quantitative analyses were performed with an accelerating voltage of 15 kV, 12 nA beam current. Crystallographic orientation measurements were obtained using the SEM-EBSD facilities at GAU at the ARC Center of Excellence for CCFS  and Australian Centre for Microscopy and Microanalysis . The EBSD patterns were generated by the interaction of a vertical electron beam with a polished thin section, tilted at 70° to the horizontal in a scanning electron microscope (Zeiss EVO MA15 and Zeiss Ultra Plus). The operating conditions were a voltage of 20 kV, a current of 8.2 nA and working distance of 12–13 mm. The diffraction pattern was projected onto a phosphor screen and recorded using a digital CCD camera. The resulting image was then processed and indexed in terms of crystal orientation using the AZtec software distributed by Oxford Instruments. Maps were acquired with sampling step size of 20 μm, 1–5 μm or 200 nm. Raw indexation rates were >85%. Data treatment allowed the rare non-indexed pixels to be filled, if up to six identical neighbors existed with this orientation. We present the resulting data in the form of color-coded maps and pole figures. For the maps that show crystal orientation changes relative to the specific direction of the sample reference frame, full red, green, and blue colors are assigned to the grains whose <100>, <110> or <111> axes are parallel to the projection of the inverse pole figure. Intermediate orientations are colored as a mixture of the primary axes. For our analysis, we define (i) a grain as an area that is completely surrounded by boundaries with a misorientation of 10, and (ii) a grain size of equivalent circle diameter calculated by grain area. One point per grain data are plotted on pole figures using programs developed by D. Mainprice of Université Montpelier II, France.How to cite this article: Satsukawa, T. et al. Messengers from the deep: Fossil wadsleyite-chromite microstructures from the Mantle Transition Zone. Sci. Rep.5, 16484; doi: 10.1038/srep16484 (2015).


In summary, our framework methodology is a useful tool to disclose specific bioprocesses and molecular targets that can be attributed to a certain disease. Our results indicated that targeted inhibition of autophagy pathways may perturb the recurrence of uveitis.Human uveitis is a type of T cell-mediated autoimmune disease that often shows relapse–remitting courses affecting multiple biological processes. As a cytoplasmic process, autophagy has been seen as an adaptive response to cell death and survival, yet the link between autophagy and T cell-mediated autoimmunity is not certain. In this study, based on the differentially expressed genes (GSE19652) between the recurrent versus monophasic T cell lines, whose adoptive transfer to susceptible animals may result in respective recurrent or monophasic uveitis, we proposed grouping annotations on a subcellular layered interactome framework to analyze the specific bioprocesses that are linked to the recurrence of T cell autoimmunity. That is, the subcellular layered interactome was established by the Cytoscape and Cerebral plugin based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The analysis showed that significant bioprocesses with autophagy were orchestrated in the cytoplasmic layered interactome and that mTOR may have a regulatory role in it. Furthermore, by setting up recurrent and monophasic uveitis in Lewis rats, we confirmed by transmission electron microscopy that, in comparison to the monophasic disease, recurrent uveitis  It is estimated that approximately 10% of the blindness cases in the United States have been caused by uveitisCerebral (Cell Region-Based Rendering and Layout)and ClueGOinteractome by distributing the node proteins into different layers of their respective subcellular localizationsComplex cellular activity is coordinately regulated by protein interaction networks. In recent years, technological advances in the area of omics research have been successful in demonstrating biological networks that closely relate to phenotypic changesin vivo by transmission electron microscopy (TEM).Autophagy is an essential homeostatic process in which cells break down their own components through lysosomal machineryAfter a total of 592 differentially expressed genes were obtained when the recurrent T cell line was compared with the monophasic T cell line, the CLICK algorithm was initially used to separate such genes into two clusters: 346 up-regulated genes, 244 down-regulated genes, and two ignored genes that could not be clustered. The 590 genes were then incorporated into the FunCoup database to construct a context-specific interactome in which single node and small components (less than five connected nodes) were removed and only the largest component was left. The adjusted interactome included 339 nodes; of these, 191 were up-regulated and 148 were down-regulated . These wTo assess the layered interactomes in the context of the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, the DAVID functional annotation tool was applied to identify significantly over-represented biological processes. In addition, the ClueGO plugin was applied to group these functional annotations and recognize the relationships among these processes (Tables S1–S3 in p = 0.0055) and “G1/S transition of mitotic cell cycle”  in the plasma membrane indicated events that were related to increased T cell receptor activation and T cell proliferation (p<0.0085), may characterize epigenetic alterations that may lead to large spatial and temporal changes in gene regulations in autoimmunityParallel to the five inter-related annotation groups in the cytoplasm, the groups in the plasma membrane and nucleus indicated significantly active processes that were involved in T cell activation and autoimmune responses. For example, the up-regulated “T cell activation”  was annotated directly, the other four groups of annotations , and down-regulated “mitochondrial membrane organization”  imply that the recurrent T cell line may undergo organelle dissociation, which was reported to initiate isolation of membranesp = 0.0002) indicated that the recurrent T cell line may undergo mitochondrial dissociation to accommodate bioenergetic requirements upon TCR engagementp = 0.0247), a highly energetically favorable process, was also steadily down-regulated. In addition, “membrane protein ectodomain proteolysis”  was also a highly autophagy-associated event. Collectively, in the cytoplasm layer, all these annotation-defined processes indicated that the recurrent T cell line may experience an energy shortage state, and autophagy orchestrated multiple organelle activity vigorously.Autophagy is a catabolic process that can be divided into four processes: isolation membrane initiation, elongation and closure, cargo selection, and autophagolysosome maturation . Strikins groups –7; 4 shoCtsd, Gbf1, and Map1lc3a , sphingomyelin phosphodiesterase 1 acid lysosomal (Smpd1), mammalian target of rapamycin (mTOR), progesterone receptor membrane component 2 (Pgrmc2), and kinesin light chain 1 (Klc1)–showed a direct functional interaction with all of the three seeds and were organized in a network, with the seed gene-encoded proteins located in the center -specific Th1 responses were severely compromised in Beclin 1-deficient mice (Beclin 1 was required for the initiation of autophagasome formation), whereas Th17 responses were only modestly affectedPrevious work by Gerhild Wildner and co-workers, using the same data we analyzed in this assay, found that relapsing–remitting T cells appeared to have increased activation, survival, and enhanced Th1 cytokine (IFN-γ) production compared with monophasic T cells, which appeared to be more Th17 proneWhile dysregulated or malfunctioned TregsThe framework we proposed here, grouped annotations on the subcellular layered interactome, can decipher the most relevant biological process, which may point to a novel way to enhance the understanding of regulatory mechanisms behind a gene expression experiment. This report is the first to show that the recurrent EAU T cell line has an autophagy phenotype. Our results also indicate that targeting at mTOR pathways may perturb the pathology of recurrent autoimmune responses.In order to decipher subtle differences, especially the difference in the initiation element in the specific effector phenotype of recurrent (R14) and monophasic uveitis (PDSAg) T cell lines, as Gerhild Wildner and co-workers intended to do in their previous microarray dataset analysis Briefly, so as to establish PDSAg- and R14 -specific T cell lines, Gerhild Wildner first set up active immunization by injection of peptide PDSAg derived from the sequence of bovine S-Ag , and peptide R14 from human IRBP , to induce monophasic and recurrent uveitis in Lewis rats. In brief, Lewis rats were immunized subcutaneously into both hind legs with a total volume of 200 µl emulsion containing 15 µg peptide R14 or PDSAg, both in complete Freund's adjuvant and fortified with Mycobacterium tuberculosis strain H37RA to a final concentration of 2.5 mg/ml. Uveitis was graded clinically and the clinical course were shown in 3H-thymidine incorporation. These procedures ensure that the established cell lines are the auto-antigen specific T cells. PDSAg-specific T cell lines were testified to induce monophasic uveitis disease process, whereas R14-specific T cell lines were proved to induce recurrent uveitis process through adoptively transfer, yet the R14-mediated relapses are generally less severe and unpredictable with respect to clinical onset and intensity Then, lymph nodes were collected 10 days post active immunization and PDSAg- and R14 -specific T cell lines were generated by alternating cycles of antigen stimulation in the presence of Antigen Presenting Cells . After restimulation, the APC were removed from the cell cultures using a Ficoll density gradient centrifugation and the remaining T cells were stored at −80°C until RNA preparation. In order to obtain antigen specific T cell lines, 2–4 re-stimulations of the cultured lymphocytes were performed. The antigen specificity of T cell lines was then tested by proliferation assays measured by t test filter (p<0.05). The differential genes were further clustered by using the CLICK algorithmThe differentially expressed genes between relapsing–remitting and monophasic T cell lines were identified using Student's Rattus norvegicus. Therefore, we used the FunCoup database to set up the initial interactome of the supposed proteins encoded by the differentially expressed genes. FunCoup is a statistical framework of data integration for identifying functional coupling between proteins. It transfers information from model organisms via orthologs found using the In Paranoid programThere are few protein interaction databases that are available for P test was applied to identify significantly over-expressed terms (p<0.05). To avoid being overwhelmed by a long list of terms, ClueGO, another Cytoscape plugin, was used to combine the highly associated terms to reduce redundancy and to reflect the relationships among these selected terms based on the similarity of their related genes. In brief, a binary gene-term matrix with the selected terms and their associated genes was created; then based on this matrix, a term–term similarity matrix was calculated using chance-corrected kappa statistics to determine the association strength between the terms. Grouped annotations were created by iterative merging of initially defined groups based on the predefined kappa score threshold (>0.3)To determine the underlying events in the different layers of the interactomes, differential genes in the different layers were annotated in the context of the GO and KEGG databases, using the DAVID functional annotation tool; the Bonferroni Female Lewis rats  of specified pathogen-free grade were purchased from Peking Vital River Laboratory Animal Ltd. . All rats were fed and maintained in specified pathogen-free conditions and processed according to the guidelines of Care and Use of Laboratory Animals published by the China National Institute of Health. The rats were injected with a combination of intramuscular ketamine hydrochloride 50 mg/kg and xylazine hydrochloride 5 mg/kg to induce anesthesia and analgesia. All experimental procedures adhered to the Association for Research in Vision and Ophthalmology Statement for the use of animals in ophthalmic and vision research. The study was approved by the Animal Ethics Committee of Harbin Medical University, China (Permit Number: HMUIRB20140016).Peptides from human IRBP-R16  and R14  were used to induce monophasic and recurrent uveitis. Three batches of immunization, each including 14 Lewis rats (randomly selected rats for monophasic [n = 7] and recurrent [n = 7] uveitis induction) were used. The immune process followed that of our previously published paper Animals were monitored daily. Clinical signs were scored by slit-lamp biomicroscopy according to the following criteriaThe entire eye balls of euthanized animals at the peak period at 14 days post immunization (dpi) with both of the disease models (three rats per group) were freshly dissected and fixed in 1% freshly made paraformaldehyde with 2% glutaraldehyde for 24 h before the corneas were cut and lens were carefully removed. Samples were fixed for 2 h in 1% osmium tetroxide and dehydrated in graded ethanol and embedded in araldite. Semi-thin sections of the eyes were cut to locate the optic nerve parts of the block, as the inflammation in these layers was the most serious. Then, three ultra-thin sections per rat were cut and stained with uranylacetate and lead citrate and then observed with TEM for 20 fields/section .Figure S1Time course of clinical disease from rats immunized with R-16-CFA (A) and rats after immunization with R-14-CFA (B). The curves show the inflammation of single eyes; grey symbols, right eyes and black symbols, left eyes. Each diagram represents one individual animal.(TIF)Click here for additional data file.Figure S2Increased autophagy activity in recurrent uveitis T cells compared with monophasic uveitis T cells. TEM showed more autophagy vacuoles (AV) in infiltrating T cells in recurrent uveitis compared with monophasic uveitis. The T cell autophagy observed by TEM was quantified by either percentage of AV-positive T cells (A) or the total number of AV (B). Data represent three rats per group, three sections per rat; 20 fields/section (p<0.05).(TIF)Click here for additional data file.Figure S3The activated microglias in plexiform layers of the retina in monophasic uveitis. TEM showed that no infiltrated or activated microglias could be observed in the plexiform layers of recurrent uveitis (A), while activated microglias could be observed in the plexiform layers of monophasic uveitis (B) (indicated by arrows). The number of infiltrating microglias observed by TEM was quantified (C). Data represent three rats per group, three sections per rat; 20 fields/section .(TIF)Click here for additional data file.File S1Supporting tables. Table S1, Annotation grouped in plasma membrane layers. The Cytoscape and Cerebral plugin were used to establish the subcellular layered interactome based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. Such grouped annotations in the plasma membrane layers were listed to recognize the relationships among these processes. Table S2, Annotation grouped in cytoplasm layers. The Cytoscape and Cerebral plugin were used to establish the subcellular layered interactome based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. Such grouped annotations in the cytoplasm layers were listed to recognize the relationships among these processes. Table S3, Annotation grouped in nucleus layers. The Cytoscape and Cerebral plugin were used to establish the subcellular layered interactome based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. Such grouped annotations in the nucleus layers were listed to recognize the relationships among these processes.(XLS)Click here for additional data file.


Statins are a class of oral drugs that are widely used for treatment of hypercholesterolemia. Recent clinical data suggest that chronic use of these drugs increases the frequency of new onset diabetes. Studies to define the risks of statin-induced diabetes and its underlying mechanisms are clearly necessary. We explored the possible mechanism of statin induced insulin resistance using a well-established cell based model and simvastatin as a prototype statin. Our data show that simvastatin induces insulin resistance in a cholesterol biosynthesis inhibition independent fashion but does so by a fatty acid mediated effect on insulin signaling pathway. These data may help design strategies for prevention of statin induced insulin resistance and diabetes in patients with hypercholesterolemia. Statins are a class of drugs that are used for lowering of plasma cholesterol. There are several marketed statins and they work by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, an intracellular enzyme which plays a central role in production of cholesterol1234678Statins are used as front line therapy for lowering plasma cholesterol and prevention of CVD. Several clinical studies have shown that statins are very effective in reducing death and disability due to CVD10111213141516181920232+-dependent insulin secretion or by interfering with isoprenylation of guanosine triphosphate(GTP)–binding proteinsGiven that statins are chronically used by millions of patients worldwide, it is important to understand the mechanism of increased incidence of diabetesData reported in another study attempted to examine the mechanism in patients. This study connects the effect of simvastatin treatment with levels of Uncoupling protein (UCP3)in vitro studies mentioned above used either adipocytes or pancreatic β-cells as model systems to understand the mechanism of statin induced diabetes. Since a major hallmark of type 2 diabetes is insulin resistance, we decided to use muscle cell based system to understand the mechanism. Muscle accounts for major portion of whole body glucose uptake and any impairment in glucose uptake by this tissue may result in onset of diabetes. Our results show that a novel fatty acid dependent, cholesterol synthesis independent mechanism of statins may lead to insulin resistance and increased incidence of diabetes.The in vitro model, we explored its effects on the uptake of glucose using a L6 myotubes model  . To makeWe then explored if simvastatin may have caused general defect(s) in signaling pathways related to glucose uptake by depleting the cellular membranes of cholesterol. To this end, we examined if the drug had any impact on PPAR gamma mediated glucose uptake, a critical pathway in glucose regulation in cells and tissues.Cells were treated either with simvastatin or pioglitazone alone or with a combination of the two drugs. Pioglitazone is a known PPAR gamma activator. As shown in Similarly when the cells were stimulated with insulin, simvastatin alone decreased (~50% decrease compared to DMSO treated insulin stimulated cells) glucose uptake. However in cells that were treated with both simvastatin and pioglitazone, the inhibitory effect of the simvastatin was relieved by pioglitazone and the increase in glucose uptake was similar to that obtained by treatment by pioglitazone alone . These dWe also explored if simvastatin treatment had any impact on the expression of glucose transporter-4 (Glut-4) protein. Glut-4 is a transport protein which in response to insulin treatment transports glucose by translocation. Our results showed (data not shown) that simvastatin treatment had no effect on Glut-4 protein expression suggesting that reduced expression of Glut-4 protein is not the mechanism of simvastatin’s inhibitory effect.608 in IRS-1 and glucose uptake by Glut-4 transporter translocation. Conversely, inhibition of its phosphorylation of Tyr608 in IRS-1 impedes glucose uptake. To explore simvastatin’s effects on IRS-1 phosphorylation, cells were treated with and without simvastatin and were incubated with insulin for 20 min followed by measurement of IRS-1 phosphorylation at Tyr608 by western blotting. As shown in 608 (~50%) and pAKT (~70%) compared to DMSO treated control cells  mediated step. It is well established that events such as accumulation of cellular free fatty acids (FFA) increase intracellular level of diacyl glycerol (DAG), which allosterically activates PKCof IRS-1 .Furthermore, we incubated the cells in presence or absence of a pan PKC inhibitor  and acetyl coA carboxylase 1 (ACC1)We next questioned if addition of exogenous fatty acids could mimic the inhibition of insulin-mediated glucose by simvastatin. We cultured cells in presence of simvastatin alone or palmitic acid alone (150 μM) for 48 h and measured glucose uptake. Indeed, incubation of cells with palmitic acid resulted in significant inhibition  inhibitorWe then measured the FFAs concentration in the muscle cells treated with simvastatin or C-75 alone or in combination. Upon inhibition of FAS, there was ~50% reduction in FFAs levels in the control cells . On the Collectively these studies support the suggestion that simvastatin induced buildup of free fatty acids in the cells could be responsible for its inhibitory effect.Since the mechanism of statin induced diabetes is not completely understood, we examined potential causes using a muscle cell based model system. Our data shown here demonstrate the following observations: a) Simvastatin inhibits insulin stimulated glucose uptake, b) This effect of simvastatin is not rescued by addition of mevalonate suggesting it is unrelated to cholesterol biosynthesis, c) Simvastatin does not inhibit pioglitazone mediated glucose uptake pathways but specifically inhibits insulin signaling by IRS-1 phosphorylation, d) The effect may be mediated by buildup of FFA in the cell by simvastatin inhibition of HMG CoA Reductase.Based on our data we hypothesize that simvastatin may cause insulin resistance through a novel fatty acid based mechanism independent of its cholesterol lowering effects. We hypothesized that by blocking HMG CoA Reductase, simvastatin may lead to accumulation of acetyl CoA, a precursor of fatty acid synthesis. Fatty acids are synthesized from acetyl-CoA and malonyl-CoA precursors. So a potential consequence of treatment of cells with this drug is that it could lead to intracellular buildup of fatty acids.Our proposed mechanism of statin induced insulin resistance is different from what has been reported by others21Another publication has summarized the various putative mechanisms by which statins may cause insulin resistanceOur proposed mechanism may be only one of many mechanisms by which statins therapy increases the risk of diabetes. Nevertheless it is important to consider mechanism based on muscle cells since defects in glucose uptake by muscle alone can cause insulin resistance and diabetes. The mechanism proposed here will now need to be further corroborated in human studies using muscle biopsies’ of statin treated patients. A robust understanding of the mechanism will help to design strategies to prevent statin induced insulin resistance.2. When L6 myoblasts reached confluence, the medium was switched to the differentiation medium containing DMEM and 2% horse serum, which was changed every alternative day. After 4 additional days, the differentiated L6 cells had fused into myotubes. L6 myotubes were used for treatment as indicated in figures. All the chemicals were bought from Sigma  or otherwise stated.L6 cells  were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 100 μg/mL penicillin and streptomycin, at 37 °C in an atmosphere of 5% CO2--2-deoxyglucose (2-NBDG)  has been used to assess glucose uptake in L6 myoblasts307) , phospho-IRS-1(Ser616(h)/Ser612(m)) , phospho-IRS-1(Tyr612(h)/Tyr608(m)) , total IRS-1, pAkt (Akt Ser 473) , total AKT  and total Actin  using specific antibodies and appropriate HRP-conjugated secondary antibody. The chemiluminescence signals were captured on photographic films using an enhanced chemiluminescence detection system .After completion of respective treatments, myotubes were scraped and cellular protein lysate was prepared using radio-immunoprecipitation assay (RIPA) lysis buffer. Protein matched lysate were resolved on to 10% SDS–polyacrylamide gel, transferred to PVDF membrane , blocked with 5% nonfat milk in TRIS buffered saline , 150 mM NaCl) for 1 hour and probed phospho-IRS-1. Briefly, cells were lysed in 1% Triton X-100 in chloroform (w/v). The samples were centrifuged at 13000 × g for 10 min to remove insoluble debris. Organic phase was collected and allowed to air dry on 50 t test was used. For comparisons of 2 or more groups Two-Way ANOVA was used followed by Bonferroni’s post-hoc analysis, p < 0.05 was considered as significant.Values were expressed as mean±SD. For comparison between 2 groups, the unpaired Student’s How to cite this article: Kain, V. et al. Simvastatin may induce insulin resistance through a novel fatty acid mediated cholesterol independent mechanism. Sci. Rep.5, 13823; doi: 10.1038/srep13823 (2015).


Ceramide is the central core of the sphingolipid pathway, and its production has been observed following various treatments that can induce several different cellular effects including growth arrest, DNA damage, apoptosis, differentiation, and senescence. Ceramides are generally produced through the sphingomyelin hydrolysis and catalyzed by the enzyme sphingomyelinase (SMase) in mammals. Presently, there are many known SMases and they are categorized into three groups acid SMases (aSMases), alkaline SMases , and neutral SMases (nSMases). The yeast homolog of mammalians neutral SMases is inositol phosphosphingolipid phospholipase C. Yeasts generally have inositol phosphosphingolipids instead of sphingomyelin, which may act as a homolog of mammalian sphingomyelin. In this review, we shall explain the structure and function of inositol phosphosphingolipid phospholipase C1, its localization inside the cells, mechanisms, and its roles in various cell responses during replication stresses and diseases. This review will also give a new basis for our understanding for the mechanisms and nature of the inositol phosphosphingolipid phospholipase C1/nSMase.Sphingolipids play a very crucial role in many diseases and are well-known as signaling mediators in many pathways. Sphingolipids are produced during the  Saccharomyces cerevisiae has been used by various researchers as a model for studying the sphingolipid biosynthesis because of its ease in sphingolipid metabolism as compared to mammalian systems . Se. Se16].  enzymes , 18. In of Lcb2p , 20. Thef csg2Δ) . In mousf csg2Δ) . These sf csg2Δ) . S. cerevisiae to generate phytosphingosine. Ceramide synthases, Lag1 and Lac1, later acylate phytosphingosine while in mammalian cells dihydrosphingosine is straightforwardly acylated into dihydroceramide by the action of enzymes Lac1p and Lag1p. Longevity assurance gene cognate 1 (LAC1) and longevity assurance gene 1 (LAG1) encode the protein Lac1p and Lag1p, respectively. The yeast enzymes Lac1p and Lag1p have high homology to mammalian six Lass's (longevity assurance)/CerS1-6 [CERS1-6/LASS1-6) that mainly localized into the ER. In mammals, dihydroceramide desaturase enzyme converts dihydroceramide into ceramide. In yeast, the following complex sphingolipids are synthesized from phytoceramide such as mannosylinositol phosphorylceramide (MIPC), inositol phosphorylceramide (IPC), and mannosyldiinositol phosphorylceramide (M(IP)2C). In case of mammals many complex sphingolipids, such as sphingomyelin, galactosylceramide, and glucosylceramide, are synthesized. Sphingosine is generated by the removal of an acetyl group from the ceramide through the action of many ceramidases. Ceramide phosphate is produced by the phosphorylation of ceramide via the sphingolipid pathway enzyme ceramide kinase which transfers phosphate group to ceramide [Differences in the sphingolipid metabolism pathways between yeast and mammals begin to arise after the above two similar steps. Dihydrosphingosine is generally hydroxylated by the enzyme suppressor of rsv161/syringomycin response protein 2 (Sur2p/Syr2p), in/CerS1-6 . CerS1-6ceramide , 23. YER019w decodes the IPS-PLC and possesses homology to bacterial neutral sphingomyelinase [ S. cerevisiae dramatically increased N-SMase as well activities of all IPSs including (M(IP)2C), IPC, and MIPC and has a size of ~55 kDa [ Cryptococcus neoformans IPC-PLC activity completely lost in case D114A and K119A mutations suggesting that these two amino acids are very crucial for the catalytic activity of Cryptococcus neoformans inositol phosphosphingolipid phospholipase C1 [Sawai et al. reported that geneyelinase . They nayelinase . They al ~55 kDa . Green f ~55 kDa . The sec ~55 kDa . Mutatio ~55 kDa . In Crypipase C1 . SWE1 and cyclin-dependent kinase 1 (Cdk1) were found to control not only the cell cycle of yeast cells but also the morphological defects of inositol phosphosphingolipid phospholipase C depleted cells [Many groups have already shown the relationship between ceramides and DNA damage response after the ionizing radiation . The croed cells . Lastly,ed cells , 32, 33. Cryptococcus neoformans (Cn). The Cryptococcus neoformans is a serious menace to immunocompromised, especially for the person suffering from the disease HIV and immunocompetent individuals also [ S. cerevisiae [ Cryptococcus [ Leishmania [ S. cerevisiae makes it more sensitive to hydroxyurea and methyl methanesulfonate and causes arrest of cell cycle and leads to morphological defects in cell shapes. In Cryptococcus neoformans inositol phosphosphingolipid phospholipase C plays a crucial role in hydroxyurea and methyl methanesulfonate tolerance. Hydroxyurea also affect the cell division in inositol phosphosphingolipid phospholipase C knockdown cells as compared to wild type cells, this leads to morphological changes (formation of cell chains and lawns kind of morphology) in inositol phosphosphingolipid phospholipase C depleted cells. Hydroxyurea also affects cell wall synthesis in these pathogens and actin polymerization during Cryptococcus neoformans cell division which could be a potential drug for treatment of these cells. We also observed that hydroxyurea treatment inhibits the growth of inositol phosphosphingolipid phospholipase C delta cells in the mouse. This clearly indicates that hydroxyurea treatment with inositol phosphosphingolipid phospholipase C deletion has a synergistic effect and this increasing host survival occurs due to decreasing organ fungal load [ Cryptococcus neoformans wild type cells nearly about tenfold from the initial inoculums as compared to untreated mice. Amazingly on the other hand the number of colony forming units of Cryptococcus neoformans inositol phosphosphingolipid phospholipase C depleted cells decreased more than thousandfold in hydroxyurea drug treated mice as compared to untreated mice [Inositol phosphosphingolipid phospholipase C one side plays a crucial role during the checkpoint while other ways have also shown that sphingolipid metabolism is closely linked to the virulence of pathogenesis ofals also . As we drevisiae  Cryptocotococcus  and Leisishmania , suggestgal load . In the ted mice .Leishmania parasites cause a spectrum of diseases very well-known as leishmaniasis and infect more than twelve million people around the world. Leishmania also possesses inositol phosphosphingolipid phospholipase C homolog that is known as inositol phosphosphingolipid phospholipase C-Like (ISCL). Zhang et al. described that Leishmania parasites like cryptococcus inositol phosphosphingolipid phospholipase C-Like possesses neutral SMase activity and plays an essential role in sphingolipid degradation and Leishmania major virulence. They also observed that in BALB/c mice, knockdown of inositol phosphosphingolipid phospholipase C-like completely abolished acute disease pathology. Inositol phosphosphingolipid phospholipase C-like is also necessary for the production of IPC but is not indispensable for the production of ethanolamine (EtN) [ne (EtN) . Cryptococcus and Leishmania. It will also be interesting to know how these pathways play the crucial role during DNA replication and checkpoint response. Another interesting area will be to explore the role of its mammalian homolog in cell division and apoptosis in various cancer models where it plays a crucial role. Knockdown of inositol phosphosphingolipid phospholipase C in combination with other drugs could be a novel tool for the treatment of pathogen induced diseases.It is well-known that after hydroxyurea drug treatment the level of inositol phosphosphingolipid phospholipase C increases into the cell but the exact mechanism behind this process is not uncovered yet . It will


Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China.Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes  most widely used in the production of  Oryza sativa) has strong seed-shattering and seed dormancy and is highly competitive with cultivated rice (O. sativa). At present, there is no effective and selective chemical control available to cull the weedy rice population, given that both cultivated and weedy rice are the same species and share similar morphological and physiological characteristics1Oryza rufipogon) and cultivated rice457indica-japonica subspecies101112Weedy rice  cytoplasmic male sterile line is the most widely used to breed japonica hybrid riceA wild abortive (WA) cytoplasmic male sterile line was first developed and became the most widely used to breed indica or japonica subspecies according to Cheng’s Index, which measures six rice characteristicsindica-japonica hybridization. Of the 12 weedy rice samples from Liaoning province, eight accessions were japonica and four were japonica-cline types. Conversely, of the 24 weedy rice accessions from Jiangsu and Guangdong, eight accessions were indica and 16 were indica-cline type  can be considered intermediate forms between indica and japonica rices.Prior to assaying 322 weedy rice accessions collected across China for CMS genes, 36 accessions independently obtained from three major rice-producing provinces experiencing serious weedy rice infestations  were assigned to ine type . Thus, tindica-type WA and japonica-type BT maintainer lines LTPB and S28B, respectively, as pollen donors, . No hybrids were obtained from the combinations of three accessions from Guangdong (GD) with the S28B line  due to seed abortion before grain filling. All the obtained hybrid progeny were fertile according to affinity detection tests except for GD9 × S28B, which had very low fertility (indica subspecies. Weedy rice from Liaoning (LN) hybridized better with S28B than with LTPB, reinforcing the notion that weedy rice from this region is closely related to japonica. Conversely, weedy rice accessions from Jiangsu (JS) and Guangdong were apparently more closely related to indica because hybridization was more effective with LTPB than with S28B. Most weedy rice accessions, however, were widely compatible because they were cross-compatible with both typical japonica and indica rice, and the difference between the affinities was not considerable. The classification of weedy accessions based on hybridization affinity was consistent with the classification obtained with Cheng’s indices  accessions and either the ertility . Therefo indices .The seed setting rates of the hybrid progeny of weedy rice accessions and the sterile line S28A as the female parent (S28A × 36WR) were all less than 5%, which suggested that these weedy rice accessions lack the Rf gene for CMS-BT. It is unlikely that they also have the CMS-BT because they set seed normally. As artificial  hybridizations were cross-incompatible, and given that maintainer line S28B shares the same nuclear constitution as S28A, the infertility of these hybrid progeny might involve other factors in addition to the absence of the Rf genes for CMS-BT in the weedy rice parents.2-KI solution was not sufficiently clear to ensure accuracy, because the pollen of BT type male sterile lines typically has spherical abortion characteristics or a stained abortion phenotype. The size and staining depth of pollen grains were diversiform (Assay of pollen viability in hybrid progeny (S28A × 36WR) by Iersiform .The reliability of the p3p4 marker, specific for CMS-BT, was verified and it was determined that it only amplified with the DNA fragment from material containing the CMS-BT gene . None ofNine hybrid combinations between the male-sterile line LTPA as female parent and weedy rices JS3 and JS10 (Jiangsu) and GD2, GD4, GD7, GD9, GD10, GD11, and GD12 (Guangdong) as male parents were fertile as determined by pollen viability and seed setting of their hybrid progenies  of interest, segregated sterile plants whose female parents were GD9, GD10, GD11, GD12  was considered validated if it reliably yielded a band only for the CMS-WA gene that was contained by the plant  segregated sterile plants  or were identified as japonica-cline (four accessions) but none were derived from hybrid rice. Previous studies have determined that weedy rice accessions from this northern province are genetically narrowly diverse (within populations) and are closely related to cultivated japonica rice but distant to indica rice111313indica , also in agreement with previous studies12japonica cultivarsindica rice has been grown in the past and is still planted adjacent to japonica fields by some farmers. These conditions may help explain the intermediate nature of a portion of the tested weedy rices, which may have evolved through gene flow and hybridization. In Guangdong, where weedy rice is a serious problem exacerbated by increasing adoption of direct seedingindica type. Weedy rice most likely originated from local cultivated rice and not from the japonica-type wild rice found in the provinceIndividuals from a selected collection of 36 weedy rice accessions - 12 from each of three Chinese provinces - were assigned to O. rufipogon) has been exploited in the production of the majority of three-line indica-type hybrid rice cultivars in ChinaO. rufipogon is still present2428Sixteen weedy rices , representing 4.5% of the total 358 accessions analyzed, contained CMS-WA. The accuracy of this finding is supported by both genetic and molecular marker tests. The CMS-WA that was originally selected from a sterile mutant wild rice plant (2 generation) would theoretically fall between 0.25 (3:1) and 0.062 (15:1). If the two Rf genes become homozygous or if there is a sufficient load of pollen containing Rf genes from cultivated or weedy rice (frequent in regions of hybrid rice cultivation), plants carrying the CMS-WA will become fertile and consequently the unfitness conferred by CMS-WA would be relieved. This would rule out the notion that the outbreak of weedy rice in China can be attributed to the ample cultivation of hybrid rice. Although CMS-BT has been exploited in japonica hybrid rice production, it was not detected in any of the 36 weedy rice accessions from Liaoning, Jiangsu and Guangdong. The area dedicated to cultivating BT-type hybrid rice is limited, thus decreasing the probability of detecting weedy rice accessions containing the CMS-BT.The majority of the 358 accessions came from the three provinces  where weedy rice infestations are most severe. However, given the considerable number of accessions tested and their disparate geographical origins, it appears that weedy rices directly related to hybrid rice are a relatively small proportion of those currently infesting rice fields in China. There is a chance that the CMS trait is unfit for weedy rice and segregates out of weedy populations, further reducing the frequency of weedy rice individuals carrying the CMS gene. However, the CMS-WA phenotype can be restored by either of two non-allelic, dominant Rf genesThe evolution of some weedy rice accessions from cultivated hybrid rice has probably been a complex process. The CMS-WA in weedy rice could have come from hybrid rice through direct or indirect evolution.Direct evolution entails the possibility that some individuals of segregant populations of hybrid rice becoming weedy rice through gene recombination. The heterosis in hybrid rice can be considered only transitory in the scheme of weedy rice evolution, but gene recombination in progeny should have a long-standing effect on adaptation to novel environments by the creation of new gene combinations upon which selection can act. A case study has shown that hybrid progeny of different rice varieties could evolve into weedy rice like individuals, and their occurrence was more frequent when the parent accessions were more genetically distant1 hybrid are lost upon segregation in the F2 generation−1 of rice seeds can reach the soil after harvesting due to natural shattering and losses during harvestingEstablishment of hybrid rice progeny in the field depends mostly on volunteers. Farmers do not save the seeds of hybrid rice for future planting, as the benefits of the heterosis in the Findica cultivars contain the CMS gene14363738391, F2, and F3 progeny of hybrids between weedy rice and cultivated rice were at least as fit as the weedy rice parents, indicating that the hybrids have the potential to evolve into new types of weedy rice164243Indirect evolution of weedy rice would be mediated by gene flow between hybrid rice or its progeny and other rice varieties, wild relatives or already existing weedy types. The outcome of this process would be determined by the outcrossing frequency, the direction of the gene flow and the genetic background of the accompanying rice forms in the paddy field itself or in adjacent fields within the range of gene flow. A cultivar could serve as a bridge for the movement of the CMS gene from hybrid rice to weedy rice; recently it has been determined that some Gene flow between cultivated rice and weedy rice can occur in both directions, with an outcrossing rate usually below 1%1035451635464818There is also a risk of hybrid rice seed contamination with weedy rice seed, because the majority of hybrid rice seed is produced in open farm fields and not at experimental farms. A significant proportion of plants in a seed production field are a sterile line that must be cross-pollinated. Thus, without proper isolation or in the presence of weedy rice arising from dormant seed, there is opportunity for gene flow to occur with the consequence of off-types (weedy rice) being planted along when the obtained hybrid rice seed is sown2135The proportion of weedy rice accessions found to have a direct relationship to hybrid rice by this study is much lower than expected, considering the high potential of hybrid rice to evolve into weedy rice. This propensity is likely because hybrid rice has heterozygous segregating progeny (volunteers) with a potentially higher outcrossing rate. Additionally, comparative studies of weedy and cultivated rice in China have shown that either weedy rice has a close relationship with companion cultivated rice or that the crop was involved in weedy rice evolution through gene introgression10111213Furthermore, rice production in China has long depended on abundant landraces selected and kept by farmers since crop domestication began. In recent decades, landraces have been gradually replaced by improved varieties. During the Seventh Five-Year Plan period (1986-1990) of China, 34663 rice landraces were collected and stored, of which 8963 - 26% of the collection - had a red pericarp. Several red pericarp landraces were tall, matured early and were tolerant of poor soils and droughtWeedy rice continuously evolves with the cultivated rice it infests. Since hybrid rice cultivation is very recent, weedy rice evolving from it should be at an early stage of evolution. It is expected that with increased planting of hybrid rice, opportunities for weedy rice to directly or indirectly evolve from hybrid rice and containing the CMS gene will also increase. Long-term dynamics of weedy rice evolution from hybrid rice would also be modulated by outcrossing rates and by the contribution of Rf genes that are not always homozygous. Special attention should be paid if in the future, transgenic hybrid rice, particularly carrying herbicide resistance traits, is released, as its progeny might also have high outcrossing rates, and the new traits will alter the dynamics of weedy rice evolution under the selective pressure of the herbicide.Thirty-six weedy rice accessions initially collected (12 each) in Liaoning, Jiangsu and Guangdong provinces were selected from the weedy rice germplasm bank of the Weeds Research Laboratory at Nanjing Agricultural University (NJAU) and used in field experiments for CMS detection . An addijaponica and indica affinity of the 36 weedy rice accessions was determined through morphological characterization and analysis of artificial crosses between the weedy forms and appropriate maintainer lines for pollen viability and seed setting. Artificial hybrids with sterile lines were also used to determine the presence of CMS and Rf genes in the weedy types.Our experimental approach to determine whether hybrid rice is implicated as a direct source of some of the weedy rice biotypes currently infesting rice fields in China is illustrated in indica and japonica rice, respectively, to kill the stamens without injuring the pistils. One-third to a half of each glume was carefully cut to expose the pistils. A paper pollination bag with both ends open was placed over each prepared panicle and closed at the bottom with a clip. While maintaining the opening at the upper end, pollen of the designated donor was sprinkled on the pistils, and the bag was closed.The warm water emasculation method was used for artificial hybridization2-KI staining, either immediately or after being temporarily preserved in formaldehyde acetic acid solution. The stained samples were mounted on glass slides and observed by optical microscopy . Three florets from each individual plant were selected. Each floret was observed in five vision fields containing approximately 100 pollen grains each. The ratio of stained to unstained pollen grains was determined. A healthy panicle was selected from each plant and self-crossed by enclosure in a porous non-woven fabric bag before flowering for determining seed set at maturity.The fertility of experimental materials was assessed by a pollen viability test and by determining the rate of seed set. Mature florets, with filaments about half the length of the glumes, were selected to detect pollen viability by Icms (cmsF: 5′-ACTTTTTGTTTTTGTGTAGG-3′ and cmsR: 5′-TGCCATATGTCGCTTAGACTTTAC-3′) were used. Polymerase chain reaction (PCR) was performed in an automated PCR apparatus (BioRAD PTC300) using a 25 μL amplification reaction system as follows: 1.0 μL DNA template at 10 ng μL−1, 12.5 μL of Premix Taq containing 0.625 U of DNA polymerase, 0.4 mM of each dNTPs and 2 × Taq buffer , 1.0 μL each of the forward and reverse primers at 10 μM and 9.5 μL of distilled water. The PCR procedure included an initial denaturation for 5 min at 94 °C; 30 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min; extension for 10 min at 72 °C and maintenance at 4 °C.Fresh leaves were taken from each individual plant for total DNA extraction using plant genomic DNA rapid extraction kit . CMS-BT marker p3p4 (p3: 5′- ATGGCAAATCTGGTCCGATG-3′ and p4: 5′-ACTTACTTAGGAAAGACTAC-3′) and CMS-WA marker indica and japonica subspecies is important to better understand their genetic relationships with the accompanying cultivated rice. Subspecies identification was accomplished using the Cheng’s indexClassification of weedy rice accessions into indica-japonica cross-incompatibility or lack of the Rf genes in weedy rice. Thus, it was important to classify the accessions into the two subspecies and to test the affinity with typical indica (LTPB) and japonica (S28B) rices before detecting the Rf genes by artificial hybridization. The 36 weedy rice accessions were selected as female parents, and indica type WA maintainer line LTPB and japonica BT type maintainer line S28B were selected as pollen donors (72 hybrid combinations). Ten plants of each combination were used to determine pollen viability and seed setting rate. The differences in fertility of F1 hybrids  were used to determine the incompatibility between weedy rice accessions and sterile lines. These were used in the detection of the Rf genes to exclude false negatives and also to categorize the subspecies of weedy rice as indica- or japonica-like. Seed produced by selfing the F1 hybrids was used to detect CMS.Sterile hybrids in crosses between sterile lines and weedy rice can occur because of 1 hybrids were determined. Weedy rice individuals without the Rf genes (those whose offspring pollen aborted and did not set seed) were eliminated because it was highly unlikely for them to carry the CMS-BT or CMS-WA.Artificial hybridization is a reliable way to detect the presence of Rf and CMS genes in weedy rice accessions. Plants of the 36 weedy rice accessions were selected as male parents; WA type sterile line LTPA and BT sterile line S28A were used as female parents (resulting in 72 hybrid combinations). Pollen viability and seed setting rate of these F2 hybrid generation of a BT type male sterile line and a restorer line will only produce fertile offspring. Gene orf79 is responsible for CMS-BTorf79 from the BT sterile line and its hybrid progeny, but cannot amplify a fragment in a maintainer line because it lacks CMS-BTThe CMS-BT is gametophytic, and the F2 progeny of which weedy rice was the female parent (containing the Rf genes for CMS-WA) and LTPB was male parent was determined. At least two hundred plants from each hybridized combination were randomly selected for tests of pollen viability and seed setting rate. Those plants identified as sterile were considered to be derived from the progeny of weedy rice accessions used as female parents containing the CMS-WA.The fertility of Fcms can amplify a 386 bp fragment from the WA sterile line and the derived hybrid, but not from the maintainer linecms marker was determined with WA sterile lines, WA maintainer lines and the weedy rice accessions confirmed to contain the CMS-WA. Artificial hybridization was also used to further guarantee the accuracy of detection with the molecular marker. Weedy rice accessions bearing the cms molecular marker for CMS-WA were used as female parents in crosses with LTPB as pollen parents. Fertility determination in the segregating F2 generation (at least two hundred plants from each hybridized combination) was used to assay whether the CMS-WA existed in the weedy rice parents.The molecular marker WA352 the gene responsible for CMS-WA was clonedWA352 to detect the possible presence of CMS-WA in all 358 weedy rice samples.In the course of analyzing the data and preparing the manuscript, How to cite this article: Zhang, J. et al. Cytoplasmic-genetic male sterility gene provides direct evidence for some hybrid rice recently evolving into weedy rice. Sci. Rep.5, 10591; doi: 10.1038/srep10591 (2015).


Background. Inflammatory gene expression in peripheral blood mononuclear cells (PBMCs) is altered in chronic Hepatitis C Virus (HCV) infection. Duration of changes after pegylated interferon- (peg-IFN-) based HCV treatment is unclear. Methods. PBMC mRNA expression of 184 inflammatory response genes was analyzed  from peg-IFN treatment nonresponders , sustained virologic responders , and spontaneous clearers . Logistic regression was used for comparison. Results. Median time from last treatment was 2 and 2.7 years in SVR and NR, respectively (p = NS). Mean mRNA counts were significantly different for 42 and 29 genes comparing SVR to SC patients and NR to SC, respectively, and no genes comparing SVR to NR. Differential expression of 24 genes was significantly different in both SVR and NR groups compared to SC. Among these 24 acute and chronic inflammatory cascade genes, significant upregulation was noted for proinflammatory transcription regulators Fos, CEBPB, and MyD88 in SVR and NR compared to SC. HDAC4 was significantly downregulated in SVR and NR compared to the SC group. Conclusions. PBMC inflammatory gene expression patterns in SVR resemble NR more than SC patients. A generalized inflammatory response persists in PBMCs long after successful peg-IFN treatment for HCV infection. Hepatitis C Virus (HCV) infection affects over 185 million individuals worldwide and approximately 5.2 million in the US. HCV is a major cause of severe disease in the US, with mortality rates of 4.7 per 100,000 reported in 2010 [Prior to December 2013, the standard of care for HCV consisted of 24–48 weeks of pegylated interferon (peg-IFN) and ribavirin (RBV) with a HCV protease inhibitor added for genotype 1 infection . After DHowever, an estimated 3% of patients will relapse after achieving SVR with reemergence of the same virus after peg-IFN/RBV treatment . It is nIt has been demonstrated that genes involved in the inflammatory response are dysregulated in the setting of chronic HCV infection and liver disease with resulting differences in circulating cytokine profiles , 23. HowWe sought to determine if there was a persistent inflammatory response in patients who attained SVR after interferon treatment. To this end, we used a multiplex array to investigate PBMC gene expression in patients previously treated for HCV to determine if differences in mRNA transcription persisted despite successful treatment.The study was approved by Stanford University institutional review board and was conducted under guidelines established by the Declaration of Helsinki. Written informed consent was obtained from all patients.α antibodies, and antineoplastic agents), and could not attend study visits, or from whom sufficient human nucleic acid could not be isolated for analysis, were excluded.We performed a cross-sectional study of patients with a history of HCV infection from the Veterans Affairs Palo Alto Health Care System (VAPAHCS). Potential study participants were identified from a clinical case registry of previously HCV antibody tested and/or treated patients at the VAPAHCS and were recruited into the study from June 2010 to September 2014. Two arms of patients were recruited. The first arm included patients with HCV infection who had completed interferon-containing HCV treatment between 6 months and 5 years prior to study enrollment. These patients were categorized as either SVR or rebound/relapse/nonresponse (NR). SVR patients were defined as having an undetectable HCV viral load  at least 24 weeks after completing therapy. NR patients were defined as any patient who received HCV therapy and either never had an undetectable HCV viral load or had a recurrence of viremia either during or subsequent to treatment. Also included were VAPAHCS patients who were exposed to HCV and spontaneously cleared the virus (SC). SC patients were defined as antibody positive by commercial assay  but who had 2 undetectable HCV RNA viral load results at least 1 month apart without any treatment. Patients who had HIV coinfection, were on any systemic immunomodulatory medications  score as described by Vallet-Pichard et al. was calculated using each patient's age, aspartate aminotransferase (AST) and Alanine transaminase (ALT) serum levels, and platelet count. Any patient with a FIB-4 score greater than 3.25 was considered to have significant fibrosis comparable to a FibroTest score of F3-F4 . An age 6–5 × 106 cells using AllPrep DNA/RNA Mini Kits  as per manufacturer instructions and placed into 50 μL of RNase-free water. Concentration of total RNA was measured by spectrophotometry and then concentrated or diluted to a target concentration of between 20 and 60 ng/μL.All patients included in the analysis had at least one blood draw at the time of enrollment. A smaller subset of patients in the SVR and NR groups underwent a second blood draw 3–6 weeks later as an internal control to ensure that study measurements were stable over time. PBMCs were isolated from blood collected in Cell Preparation Tubes (CPT)  per manufacturer instructions and stored at −80°C. Total RNA was extracted from 2.0 × 10http://dx.doi.org/10.1155/2015/958231). The nCounter system allows for direct detection and counting of nucleic acid via reporter probes appended with multiple fluorophore barcodes and biotinylated capture-probes that attach to microscopic beads, which are then affixed to lanes in a translucent cartridge and read in an optical scanner [Gene expression was directly measured via counts of corresponding messenger RNA (mRNA) in each sample using an nCounter  GX human inflammation kit, which is a multiplex assay for 184 genes involved in the human inflammatory response . The arithmetic mean plus 2 standard deviations (SDs) of the internal negative controls in each sample was subtracted from the gene expression count to ensure that any nonspecific mRNA detection was excluded. Values below zero were included as zero for the analysis. The geometric mean of the 6 internal positive controls was used to normalize the data so that comparisons could be made across samples and to minimize distortion from batch effects. Gene expressions were then normalized using the geometric mean of 5 housekeeping genes  chosen due to their consistent expression in PBMC. Batch effects were assessed with a confirmatory mixed batch of samples selected from each prior run with adjustments made as appropriate. The in-group means of gene expression count were compared via two-tailed pairwise analysis with nonparametric distribution assumed. Given the number of simultaneous tests and an expected increase in type 1 error, a Bonferroni corrected threshold p = 0.047). Due to VAPAHCS patient demographics, whites and males were overrepresented in this study when compared to the overall US HCV population, although there were no significant differences between groups. As expected, FIB-4 scores were significantly higher in the NR group compared to SC or SVR (p = 0.001). The FIB-4 score was similar between SVR and SC patients. Age adjusted Charlson Comorbidity scores were significantly different between the three groups (p < 0.001); however, the difference appeared to be driven primarily by the amount of nonsevere liver disease (1 point) in the SVR group and cirrhotic liver disease in the NR group (3 points). Rates of prescription statin and NSAID use were similar between groups . Between the SVR and NR group, there was a significantly higher percentage of genotype 1 patients in the SVR group (p = 0.027), which was unexpected and likely due to local treatment patterns in the last 5 years at VAPAHCS after the introduction of HCV protease inhibitors in 2011. Time from last interferon treatment  and type of treatment (p = 0.116) were not significantly different between groups.In the final analysis, 55 patients  met both inclusion and exclusion criteria and were included in the study. After an initial database search and contact by mail, 79 patients were initially screened by phone for inclusion in the study. Of those, 3 patients were excluded due to refusal to participate, 5 patients were excluded due to receipt of interferon prior to the study period (before 12/2008), 8 were unable to participate in their initial visit, 1 patient was consented but was unable to complete blood draw, 1 patient was consented but RNA was not recovered from his samples, 3 patients were excluded due to the presence of immunomodulatory medications, and 3 patients were excluded due to the presence of HIV. Demographic and clinically relevant data including HCV genotype, FIB-4 and age adjusted Charlson Comorbidity scores, and statin or NSAID use are reported in α and β. A full list of assessed genes and expression is available in supplemental Table 1.As many inflammatory mediators are not primarily transcribed in PBMC, not all genes assessed had significant transcription as detected by our primary assay. Of 184 inflammatory mediator genes assessed, 127 had significant expression levels (mean copy numbers >10 in any group) for reliable detection after normalization with negative controls, positive controls, and housekeeping genes and were included in further analysis. This null expression group included important inflammatory mediators primarily expressed in neutrophils, endothelial cells, tissue based macrophages, or other tissues, including IL-1A, IL-4, IL-6, and interferons When analyzed as individual samples, it was observed that overall pattern of gene expression between SVR and NR patients was highly similar, while SC patients differed from the other two groups. A heat map of gene expression with clustering using a Spearman correlation is shown in R2 of 0.98, while the correlation between SVR and SC gene expression was reduced with an R2 of 0.88.A scatter plot using logistic regression is shown in Using a significance threshold of <0.001 (corrected for multiple testing), mean mRNA counts were significantly different for 42 genes when comparing SVR to SC patients, 29 genes comparing NR to SC, and no genes comparing SVR to NR. Differential expression of 24 genes was significantly different in both SVR and NR groups when compared to SC and is shown in Tables  Fos  and Jun  were both elevated as was CEBPB (CCAAT/enhancer-binding protein beta) . AP-1 is a known transcription factor for Toll-like receptor 4 (TLR4), which had a mild but nonsignificant elevation of transcription in the SVR and NR groups compared to SC . Significant elevations in transcription of TLR4 downstream effector Myeloid differentiation primary response gene 88 (MyD88) were also seen  [ IL-10 was not highly expressed in any group . However, there was a significant elevation in both SVR and NR versus SC, perhaps consistent with feedback regulation from stimulation of the MyD88 pathway [HDAC4) were downregulated in SVR and NR compared to the SC group  [Specific expression patterns were suggestive of increased transcription factor activation for promoters of cellular proliferation in PBMCs and resulting innate immune activity in NR and SVR patients versus SC patients. Although all 24 genes that were differentially expressed in the same direction in NR and SVR patients versus SC patients are potentially important inflammatory cascade regulators and effectors, a few of these are worth special mention as they may be of particular importance in interferon mediated immune response. It is of note that AP-1 componentsor both) , 31. The pathway . It is oor both) . Fos, CEBPB, and MyD88 are more transcriptionally active in both SVR and chronically viremic patients, while adaptive immune mediators such as HDAC4 are downregulated [α, other secondary pathways soon become the main mediators of effector response after sustained therapy and maybe responsible for the benefit of the pegylated form of the molecule [We found that the pattern of PBMC gene expression in SVR patients, who received peg-IFN treatment a median of 2 years prior, more closely resembles NR patients with active HCV infection than SC patients who have been exposed to HCV but are uninfected and who have no history of chronic viral infection or prolonged peg-IFN exposure. Given that there is ongoing viremia in NR patients and SVR patients have achieved clinical cure, it may be expected that NR patients would have a different pattern of expression in PBMCs from SVR patients; however, we found no difference. This indicates that there is a durable inflammatory transcriptional response in PBMCs that persists long after treatment. The specific pattern of gene expression demonstrates that genes related to innate immune activation such asegulated –33. The molecule .A potential explanation for this pattern of activation may be liver disease itself and prior injury from chronic HCV infection. Lack of relative improvement in fibrosis after treatment may be an important predictor of outcomes in SVR patients . Gene exAnother possible explanation is a durable effect from long-term exogenous peg-IFN exposure. There is no significant body of prior literature suggesting this. However, HCV infected patients who achieve SVR receive much longer exposure to exogenous peg-IFN therapy than any other patient group, and this pattern of activation may mimic chronic viral infection, particularly when compared to NR group that also had an extended duration  of exogenous peg-IFN. The addition of a treatment naive chronically HCV infected comparison group may be useful in future studies to explore this possibility. A similar study in patients who receive interferon-free treatment regimens and obtain SVR may also resolve this hypothesis.The explanation for this data with the most salient clinical consequences is either intra- or extrahepatic reservoirs of residual HCV infection. As demonstrated by Hara et al., late relapse in many cases is likely due to reemergence of prior infection rather than reinfection with a different strain . As note IFN-α and TNF-α in “resolved patients” versus chronically infected patients. However, the “resolved group” included both SVR and SC patients grouped together, and the chronic infection group included both treatment experienced and treatment naive patients [ IFN-α transcripts and no significant difference between groups in TNF-α. Radkowski et al. analyzed 25 cytokine transcripts in 49 patients with SVR divided into a group with residual infection as defined by very low levels of HCV in serum or PBMC detected with a home-brew ultrasensitive RT-PCR assay or by presence of NS3 protein via immunofluorescence in PBMC versus those without. There was a high rate (46.9%) of residual viremia found in this study, and in those patients there were significant elevations in IL-6, IL-8, IL-12,TNF-α, and MIP-1β (CCL4) when compared to SVR patients without evidence of residual virus [ IL-12, TNF-α, or CCL4 and did not detect significant transcription of IL-6 in any group. We did find, however, that IL-8 appeared upregulated in both SVR and NR patients compared to SC with fold changes of 7.49 and 5.73 , although these changes did not reach the threshold for significance in the final analysis.Two previous studies have used RT-PCR to assay PBMC gene expression in SVR patients and postulated residual infection as the etiology. Pham et al.  assessedpatients . Our stuOur study differs from prior work in several ways, using an entirely different methodology to assay gene expression of many more genes simultaneously as well as a fundamentally different way of defining comparison groups, which may explain differences in results. Ours is the only study to assay expression of upstream genes whose products regulate overall transcription patterns rather than only transcripts for effector cytokines, which are not always reliably expressed in PBMCs as opposed to other tissue compartments.Our study had several important limitations. The demographics of our patient population are not representative of the US HCV infected population at large in terms of gender or race; therefore, generalizability may be limited. The small numbers in each comparison group may not have allowed sufficient power to show a difference and increased the likelihood of type II error. As with any multiplex detection system reliant on binding to probes, uniform annealing conditions across 184 probe sets with different sequences may lead to falsely higher counts in probe/target sets with higher affinity and falsely lower or null counts in probe/target sets with lower affinity. Another important limitation is that the time since infection for the SC patients is unknown. This data is not obtainable due to the fact that acute infection with HCV is most often asymptomatic and a positive HCV antibody with negative serum HCV RNA PCR was identified in our SC sample on routine screening for at risk populations after the acute phase of infection. There is also a large distribution (6 months to 5 years) in the time since treatment for both the NR and SVR patients; however, the median time since treatment is not significantly different between groups; therefore, we believe these groups are comparable. In addition, our study is limited by assaying the total blood PBMC population as opposed to specific mononuclear cell subpopulations or liver tissue where important changes may be taking place. However, we believe changes in PBMC mRNA expression are important for our understanding of the systemic immune response after HCV treatment.Further investigation is warranted to explain why interferon treated patients who obtain SVR and a functional cure have a similar pattern of macrophage and lymphocyte gene expression compared to those with chronic HCV infection who did not respond to treatment and also had prior exogenous interferon exposure. This is an important question in the DAA era of HCV therapy, as retreatment is now possible for patients with late relapse, and further monitoring of peg-IFN treated patients with SVR may be useful to identify late relapse. This may also be clinically important for monitoring peg-IFN treated patients for long-term consequences of prior infection such as progression to cirrhosis and HCC as well as immunogenic consequences of HCV infection such as cryoglobulinemia and porphyria cutanea tarda. Further studies investigating the same phenomenon in patients treated with interferon-free regimens are warranted.Patients who achieved clinical cure of HCV infection with peg-IFN based regimens appear to have PBMC gene expression patterns that closely resemble chronically HCV infected patients who have failed treatment and are markedly different from patients who spontaneously clear their HCV infection. It is therefore reasonable to conclude that an inflammatory response persists in PBMCs long after successful peg-IFN treatment for HCV infection. Further study is warranted to better understand why this is occurring.The supplementary material consists of a spreadsheet containing the normalized gene expression counts for each gene investigated for each individual sample. Included are the counts for the housekeeping genes along with positive and negative controls used in normalization. The rightmost columns represent the p-values for pairwise comparisons between groups of each gene and are color coded by level of significance.