Search

Background: Insulin synthesis and cell proliferation are under tight regulation in pancreatic β-cells to maintain glucose homeostasis. Dysfunction in either aspect leads to development of diabetes. PERK (EIF2AK3) loss of function mutations in humans and mice exhibit permanent neonatal diabetes that is characterized by insufficient β-cell mass and reduced proinsulin trafficking and insulin secretion. Unexpectedly, we found that Perk heterozygous mice displayed lower blood glucose levels. Methodology: Longitudinal studies were conducted to assess serum glucose and insulin, intracellular insulin synthesis and storage, insulin secretion, and β-cell proliferation in Perk heterozygous mice. In addition, modulation of Perk dosage specifically in β-cells showed that the glucose homeostasis phenotype of Perk heterozygous mice is determined by reduced expression of PERK in the β-cells. Principal Findings: We found that Perk heterozygous mice first exhibited enhanced insulin synthesis and secretion during neonatal and juvenile development followed by enhanced β-cell proliferation and a substantial increase in β-cell mass at the adult stage. These differences are not likely to entail the well-known function of PERK to regulate the ER stress response in cultured cells as several markers for ER stress were not differentially expressed in Perk heterozygous mice. Conclusions: In addition to the essential functions of PERK in β-cells as revealed by severely diabetic phenotype in humans and mice completely deficient for PERK, reducing Perk gene expression by half showed that intermediate levels of PERK have a profound impact on β-cell functions and glucose homeostasis. These results suggest that an optimal level of PERK expression is necessary to balance several parameters of β-cell function and growth in order to achieve normoglycemia. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral pathogen that causes acute respiratory illnesses in young pigs. Since 1987, PRRSV has contributed substantial economic losses to the swine industry. Elevation of proinflammatory cytokines in PRRSV-infected pigs is thought to contribute to PRRSV pathogenesis. In this study, PRRSV VR-2385, a Type 2 strain with moderate virulence, was found to induce phosphorylation of signal transducer and activator of transcription 1 (STAT1) at serine 727 (pSTAT1-S727) in MARC-145 cells. No phosphorylated STAT1 at tyrosine 701 was detected, which indicates that the pSTAT1-S727 elevation was interferon-independent. The PRRSV-induced pSTAT1-S727, however, was dose-dependent and its levels increased with infection time. IngelVac PRRS MLV strain had a minimal effect on pSTAT1-S727. Compared to MLV-infected cells, VR-2385 infection caused significantly higher level of expression of proinflammatory cytokines, including interleukin 1 beta (IL-1beta) and IL-8. The VR-2385-induced pSTAT1-S727 and cytokine expression were reduced after SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), or methylthioadenosine (MTA), a methyl transferase inhibitor, was added to the cells. The SB203580 and MTA-mediated inhibition suggested that the virus-induced pSTAT1-S727 was dependent on p38 MAPK pathway. In primary porcine alveolar macrophages (PAMs), VR-2385 also induced pSTAT1-S727 and expression of proinflammatory cytokines and chemokines, including IL-1beta, IL-8, chemokine ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 10 (CXCL10). Similarly, SB203580 treatment of PAM cells blocked the elevation of pSTAT1-S727 and cytokine expression. Overexpression of individual viral proteins showed that non-structural protein 12 (nsp12) was able to induce elevation of pSTAT1-S727 and the expression of IL-1β and IL-8. These results indicated that PRRSV VR-2385 induces pSTAT1-S727 and the expression of proinflammatory cytokines, which contributes to the insight of PRRSV pathogenesis. [ABSTRACT FROM AUTHOR]

Recent studies indicate that soluble β-amyloid (sAβ) oligomers, rather than their fibrillar aggregates, contribute to the pathogenesis of Alzheimer's disease (AD), though the mechanisms of their neurotoxicity are still elusive. Here, we demonstrate that sAβ derived from 7PA2 cells exert a much stronger effect on the regulation of a set of functionally validated microRNAs (miRNAs) in primary cultured neurons than the synthetic insoluble Aβ fibrils (fAβ). Synthetic sAβ peptides at a higher concentration present comparable effect on these miRNAs in our neuronal model. Further, the sAβ-induced miR-134, miR-145 and miR-210 expressions are fully reversed by two selective N-methyl-d-aspartate (NMDA) receptor inhibitors, but are neither reversed by insulin nor by forskolin, suggesting an NMDA receptor-dependent, rather than PI3K/AKT or PKA/CREB signaling dependent regulatory mechanism. In addition, the repression of miR-107 expression by the sAβ containing 7PA2 CM is likely involved multiple mechanisms and multiple players including NMDA receptor, N-terminally truncated Aβ and reactive oxygen species (ROS). [ABSTRACT FROM AUTHOR]

The MYB proteins comprise one of the largest families of transcription factors (TFs) in plants. Although several MYB genes have been characterized to play roles in secondary metabolism, the MYB family has not yet been identified in apple. In this study, 229 apple MYB genes were identified through a genome-wide analysis and divided into 45 subgroups. A computational analysis was conducted using the apple genomic database to yield a complete overview of the MYB family, including the intron-exon organizations, the sequence features of the MYB DNA-binding domains, the carboxy-terminal motifs, and the chromosomal locations. Subsequently, the expression of 18 MYB genes, including 12 were chosen from stress-related subgroups, while another 6 ones from other subgroups, in response to various abiotic stresses was examined. It was found that several of these MYB genes, particularly MdoMYB121, were induced by multiple stresses. The MdoMYB121 was then further functionally characterized. Its predicted protein was found to be localized in the nucleus. A transgenic analysis indicated that the overexpression of the MdoMYB121 gene remarkably enhanced the tolerance to high salinity, drought, and cold stresses in transgenic tomato and apple plants. Our results indicate that the MYB genes are highly conserved in plant species and that MdoMYB121 can be used as a target gene in genetic engineering approaches to improve the tolerance of plants to multiple abiotic stresses. [ABSTRACT FROM AUTHOR]

Melanoma differentiation-associated gene-5 (MDA5) recognizes distinct subsets of viruses including Encephalomyocarditis virus (EMCV) of picornavirus family, but the molecular mechanisms underlying the specificity of the viral recognition of MDA5 in immune cells remain obscure. DHX29 is a RNA helicase required for the translation of 5’ structured mRNA of host and many picornaviruses (such as EMCV). We identify that DXH29 as a key RNA co-sensor, plays a significant role for specific recognition and triggering anti-EMCV immunity. We have observed that DHX29 regulates MDA5-, but not RIG-I-, mediated type I interferon signaling by preferentially interacting with structured RNAs and specifically with MDA5 for enhancing MDA5-dsRNA binding affinity. Overall, our results identify a critical role for DHX29 in innate immune response and provide molecular insights into the mechanisms by which DHX29 recognizes 5’ structured EMCV RNA and interacts with MDA5 for potent type I interferon signaling and antiviral immunity. [ABSTRACT FROM AUTHOR]

The pine aphid Cinara pinitabulaeformis Zhang et Zhang is the main pine pest in China, it causes pine needles to produce dense dew (honeydew) which can lead to sooty mold (black filamentous saprophytic ascomycetes). Although common chemical and physical strategies are used to prevent the disease caused by C. pinitabulaeformis Zhang et Zhang, new strategies based on biological and/or genetic approaches are promising to control and eradicate the disease. However, there is no information about genomics, proteomics or transcriptomics to allow the design of new control strategies for this pine aphid. We used next generation sequencing technology to sequence the transcriptome of C. pinitabulaeformis Zhang et Zhang and built a transcriptome database. We identified 80,259 unigenes assigned for Gene Ontology (GO) terms and information for a total of 11,609 classified unigenes was obtained in the Clusters of Orthologous Groups (COGs). A total of 10,806 annotated unigenes were analyzed to identify the represented biological pathways, among them 8,845 unigenes matched with 228 KEGG pathways. In addition, our data describe propagative viruses, nutrition-related genes, detoxification related molecules, olfactory related receptors, stressed-related protein, putative insecticide resistance genes and possible insecticide targets. Moreover, this study provides valuable information about putative insecticide resistance related genes and for the design of new genetic/biological based strategies to manage and control C. pinitabulaeformis Zhang et Zhang populations. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small, short noncoding RNAs that modulate the expression of numerous genes by targeting their mRNA. Numerous abnormal miRNA expression patterns are observed in various human malignancies, and certain miRNAs can act as oncogenes or tumor suppressors. Astrocytoma, the most common neuroepithelial cancer, represents the majority of malignant brain tumors in humans. In our previous studies, we found that the downregulation of miR-181b-5p in astrocytomas is associated with a poor prognosis. The aim of the present study was to investigate the functional role of miR-181b-5p and its possible target genes. miR-181b-5p was significantly downregulated in astrocytoma specimens, and the reduced expression of miR-181b-5p was inversely correlated with the clinical stage. The ectopic expression of miR-181b-5p inhibited proliferation, migration and invasion and induced apoptosis in astrocytoma cancer cells in vitro. The NOVA1 (neuro-oncological ventral antigen 1) gene was further identified as a novel direct target of miR-181b-5p. Specifically, miR-181b-5p bound directly to the 3'-untranslated region (UTR) of NOVA1 and suppressed its expression. In clinical specimens, NOVA1 was overexpressed, and its protein levels were inversely correlated with miR-181b-5p expression. Furthermore, the changing level of NOVA1 was significantly associated with a poor survival outcome. Similar to restoring miR-181b-5p expression, downregulating NOVA1 inhibited cell growth, migration and invasion. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. Our results indicate that miR-181b-5p is a tumor suppressor in astrocytoma that inhibits tumor progression by targeting NOVA1. These findings suggest that miR-181b-5p may serve as a novel therapeutic target for astrocytoma. [ABSTRACT FROM AUTHOR]

Histone demethylases have emerged as important players in developmental processes. Jumonji domain containing-3 (Jmjd3) has been identified as a key histone demethylase that plays a critical role in the regulation of gene expression; however, the in vivo function of Jmjd3 in embryonic development remains largely unknown. To this end, we generated Jmjd3 global and conditional knockout mice. Global deletion of Jmjd3 induces perinatal lethality associated with defective lung development. Tissue and stage-specific deletion revealed that Jmjd3 is dispensable in the later stage of embryonic lung development. Jmjd3 ablation downregulates the expression of genes critical for lung development and function, including AQP-5 and SP-B. Jmjd3-mediated alterations in gene expression are associated with locus-specific changes in the methylation status of H3K27 and H3K4. Furthermore, Jmjd3 is recruited to the SP-B promoter through interactions with the transcription factor Nkx2.1 and the epigenetic protein Brg1. Taken together, these findings demonstrate that Jmjd3 plays a stage-dependent and locus-specific role in the mouse lung development. Our study provides molecular insights into the mechanisms by which Jmjd3 regulates target gene expression in the embryonic stages of lung development. [ABSTRACT FROM AUTHOR]

Resistance to TKI treatment is a major obstacle in effective treatment of NSCLC. Besides EGFR mutation status, the mechanisms involved are largely unknown. Some evidence supports a role for microRNA 21 in modulating drug sensitivity of chemotherapy but its role in NSCLC TKI resistance still remains unexplored. This study aimed to investigate whether NSCLC miR-21 mediated resistance to TKIs also results from Pten targeting. Here, we show miR-21 promotes cancer by negatively regulating Pten expression in human NSCLC tissues: high miR-21 expression levels were associated with shorter DFS in 47 NSCLC patients; high miR-21/low Pten expression levels indicated a poor TKI clinical response and shorter overall survival in another 46 NSCLC patients undergoing TKI treatment. In vitro assays showed that miR-21 was up-regulated concomitantly to down-regulation of Pten in pc-9/GR cells in comparison with pc-9 cells. Moreover, over-expression of miR-21 significantly decreased gefitinib sensitivity by down-regulating Pten expression and activating Akt and ERK pathways in pc-9 cells, while miR-21 knockdown dramatically restored gefitinib sensitivity of pc-9/GR cells by up-regulation of Pten expression and inactivation of AKT and ERK pathways, in vivo and in vitro. We propose alteration of miR-21/Pten expression as a novel mechanism for TKI resistance in NSCLC cancer. Our findings provide a new basis for using miR 21/Pten-based therapeutic strategies to reverse gefitinib resistance in NSCLC. [ABSTRACT FROM AUTHOR]

Background & Aims: The cellular immunity has a profound impact on the status of hepatitis C virus (HCV) infection. However, the response of cellular immunity on the virological response in patients with antiviral treatment remains largely unclear. We aimed to clarify the response of peripheral T cells and monocytes in chronic hepatitis C patients with antiviral treatment. Methods: Patients with chronic hepatitis C were treated either with interferon alpha-2b plus ribavirin (n = 37) or with pegylated interferon alpha-2a plus ribavirin (n = 33) for up to 24 weeks. Frequencies of peripheral regulatory T-cells (Tregs), programmed death-1 (PD-1) expressing CD4+ T-cells or CD8+ T-cells and toll-like receptor (TLR) 3 expressing CD14+ monocytes were evaluated by flow cytometry in patients at baseline, 12 and 24 weeks following treatment and in 20 healthy controls. Results: Frequencies of Tregs, PD-1 and TLR3 expressing cells were higher in patients than those in control subjects (P<0.05). Patients with complete early virological response (cEVR) showed lower Tregs, PD-1 expressing CD4+ or CD8+ T-cells than those without cEVR at 12 weeks (P<0.05). Patients with low TLR3 expressing CD14+ monocytes at baseline had a high rate of cEVR (P<0.05). Conclusions: Low peripheral TLR3 expressing CD14+ monocytes at baseline could serve as a predictor for cEVR of antiviral therapy in chronic HCV-infected patients. The cEVR rates were significantly increased in the patients with reduced circulating Tregs, PD-1 expressing CD4+ or CD8+ T-cells. Trial Registration: Chinese Clinical Trial Registry ChiCTR10001090. [ABSTRACT FROM AUTHOR]

Astrocytomas are common malignant intracranial tumors that comprise the majority of adult primary central nervous system tumors. MicroRNAs (miRNAs) are small, non-coding RNAs (20–24 nucleotides) that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs. In our previous studies, we found that the downregulation of miR-106a-5p in astrocytomas is associated with poor prognosis. However, its specific gene target(s) and underlying functional mechanism(s) in astrocytomas remain unclear. In this study, we used mRNA microarray experiments to measure global mRNA expression in the presence of increased or decreased miR-106a-5p levels. We then performed bioinformatics analysis based on multiple target prediction algorithms to obtain candidate target genes that were further validated by computational predictions, western blot analysis, quantitative real-time PCR, and the luciferase reporter assay. Fas-activated serine/threonine kinase (FASTK) was identified as a direct target of miR-106a-5p. In human astrocytomas, miR-106a-5p is downregulated and negatively associated with clinical staging, whereas FASTK is upregulated and positively associated with advanced clinical stages, at both the protein and mRNA levels. Furthermore, Kaplan-Meier analysis revealed that the reduced expression of miR-106a-5p or the increased expression of FASTK is significantly associated with poor survival outcome. These results further supported the finding that FASTK is a direct target gene of miR-106a-5p. Next, we explored the function of miR-106a-5p and FASTK during astrocytoma progression. Through gain-of-function and loss-of-function studies, we demonstrated that miR-106a-5p can significantly inhibit cell proliferation and migration and can promote cell apoptosis in vitro. The knockdown of FASTK induced similar effects on astrocytoma cells as those induced by the overexpression of miR-106a-5p. These observations suggest that miR-106a-5p functions as a tumor suppressor during the development of astrocytomas by targeting FASTK. [ABSTRACT FROM AUTHOR]

Hypoxic/ischemic injury to kidney is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs are differentially involved in hypoxic/ischemic events and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering the microRNA (miRNA) expression in kidney under hypoxic condition. We selected 31 miRNAs based on microarray data for quantitative PCR analysis. Among them, 14 miRNAs were significantly altered after prolonged hypoxia, DOR activation or a combination of both. We found that 1) DOR activation alters miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters miRNA expression depending on the duration of hypoxia; and 3) DOR activation can modify hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by over 70%, while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100%, which was reversed following DOR activation. These first data suggest that hypoxia comprehensively modifies the miRNA profile within the kidney, which can be mimicked or modified by DOR activation. Ascertaining the targeted pathways that regulate the diverse cellular and molecular functions of miRNA may provide new insights into potential therapies for hypoxic/ischemic injury of the kidney. [ABSTRACT FROM AUTHOR]

Background: A large number of human tumor-associated antigens that are recognized by CD8+ T cells in a human leukocyte antigen class I (HLA-I)-restricted fashion have been identified. Special AT-rich sequence binding protein 1 (SATB1) is highly expressed in many types of human cancers as part of their neoplastic phenotype, and up-regulation of SATB1 expression is essential for tumor survival and metastasis, thus this protein may serve as a rational target for cancer vaccines. Methodology/Principal Findings: Twelve SATB1-derived peptides were predicted by an immuno-informatics approach based on the HLA-A*02 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from HLA-A*02+ healthy donors and/or HLA-A*02+ cancer patients. The recognition of HLA-A*02+ SATB1-expressing cancer cells was also tested. Among the twelve SATB1-derived peptides, SATB1565–574 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and cancer patients. Importantly, SATB1565–574-specific T cells recognized and killed HLA-A*02+ SATB1+ cancer cells in an HLA-I-restricted manner. Conclusions/Significance: We have identified a novel HLA-A*02-restricted SATB1-derived peptide epitope recognized by CD8+ T cells, which, in turn, recognizes and kills HLA-A*02+ SATB1+ tumor cells. The SATB1-derived epitope identified may be used as a diagnostic marker as well as an immune target for development of cancer vaccines. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The disease is characterized by various cytogenetic and molecular abnormalities with distinct prognoses and gene expression profiles. Emerging evidence has suggested that circulating microRNAs (miRNAs) could serve as noninvasive biomarkers for cancer detection; however, little is known about circulating miRNA profiles in AML patients. In this study, a genome-wide serum miRNA expression analysis was performed using Solexa sequencing for initial screen, followed by validation with real-time PCR assays. The analysis was conducted on training and verification sets of serum samples from 140 newly diagnosed AML patients and 135 normal adult donors. After a two-phase selection and validation process, 6 miRNAs, miR-10a-5p, miR-93-5p, miR-129-5p, miR-155-5p, miR-181b-5p and miR-320d, were found to have significantly different expression levels in AML compared with control serum samples. Furthermore, unsupervised clustering analysis revealed the remarkable ability of the 6-miRNA profile to differentiate between AML patients and normal controls. The areas under the ROC curve for the selected miRNAs ranged from 0.8129 to 0.9531. More importantly, miR-181b-5p levels in serum were significantly associated with overall survival. These data demonstrated that the expression patterns of circulating miRNAs were systematically altered in AML and miR-181b-5p may serve as a predictor for overall survival in AML patients. [ABSTRACT FROM AUTHOR]

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3’ untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5’, central or 3’ region of mature miR-223 suppressed miR-223 targeting the 3’UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3’UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3’UTRs have similar binding sites for miR-223 with IGF1R 3’UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting. [ABSTRACT FROM AUTHOR]