Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1.

BACKGROUND: Circular RNAs (circRNAs) are highly enriched in the central nervous system and have been implicated in neurodegenerative diseases. However, whether and how circRNAs contribute to the pathological processes induced by traumatic brain injury (TBI) has not been fully elucidated. METHODS: We conducted a high-throughput RNA sequencing screen for well-conserved, differentially expressed circRNAs in the cortex of rats subjected to experimental TBI. Circular RNA METTL9 (circMETTL9) was ultimately identified as upregulated post-TBI and further characterized by RT-PCR and agarose gel electrophoresis, Sanger sequencing, and RNase R treatment. To examine potential involvement of circMETTL9 in neurodegeneration and loss of function following TBI, circMETTL9 expression in cortex was knocked-down by microinjection of a shcircMETTL9 adeno-associated virus. Neurological functions were evaluated in control, TBI, and TBI-KD rats using a modified neurological severity score, cognitive function using the Morris water maze test, and nerve cell apoptosis rate by TUNEL staining. Pull-down assays and mass spectrometry were conducted to identify circMETTL9-binding proteins. Co-localization of circMETTL9 and SND1 in astrocytes was examined by fluorescence in situ hybridization and immunofluorescence double staining. Changes in the expression levels of chemokines and SND1 were estimated by quantitative PCR and western blotting. RESULTS: CircMETTL9 was significantly upregulated and peaked at 7 d in the cerebral cortex of TBI model rats, and it was abundantly expressed in astrocytes. We found that circMETTL9 knockdown significantly attenuated neurological dysfunction, cognitive impairment, and nerve cell apoptosis induced by TBI. CircMETTL9 directly bound to and increased the expression of SND1 in astrocytes, leading to the upregulation of CCL2, CXCL1, CCL3, CXCL3, and CXCL10, and ultimately to enhanced neuroinflammation. CONCLUSION: Altogether, we are the first to propose that circMETTL9 is a master regulator of neuroinflammation following TBI, and thus a major contributor to neurodegeneration and neurological dysfunction.

Feature selection and classification of noisy proteomics mass spectrometry data based on one-bit perturbed compressed sensing.

MOTIVATION: The classification of high-throughput protein data based on mass spectrometry (MS) is of great practical significance in medical diagnosis. Generally, MS data are characterized by high dimension, which inevitably leads to prohibitive cost of computation. To solve this problem, one-bit compressed sensing (CS), which is an extreme case of quantized CS, has been employed on MS data to select important features with low dimension. Though enjoying remarkably reduction of computation complexity, the current one-bit CS method does not consider the unavoidable noise contained in MS dataset, and does not exploit the inherent structure of the underlying MS data. RESULTS: We propose two feature selection (FS) methods based on one-bit CS to deal with the noise and the underlying block-sparsity features, respectively. In the first method, the FS problem is modeled as a perturbed one-bit CS problem, where the perturbation represents the noise in MS data. By iterating between perturbation refinement and FS, this method selects the significant features from noisy data. The second method formulates the problem as a perturbed one-bit block CS problem and selects the features block by block. Such block extraction is due to the fact that the significant features in the first method usually cluster in groups. Experiments show that, the two proposed methods have better classification performance for real MS data when compared with the existing method, and the second one outperforms the first one. AVAILABILITY AND IMPLEMENTATION: The source code of our methods is available at: https://github.com/tianyan8023/OBCS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

[Effects of Annexin A2 gene transcription down-regulation on human hepatoma cell biological behaviour].

OBJECTIVE: To investigate the effects of Annexin A2 (ANXA2) deficiency on the malignant biological behaviour of hepatoma cells. METHODS: The human hepatocellular carcinoma (HCC) cells lines MHCC97-H, HepG2, SMMC-7721, SMMC-7402 and L02 were evaluated. The expression and distribution of ANXA2 were analysed by western blotting, real-time PCR, immunofluorescence and immunohistochemistry.Cell cycle was assessed by flow cytometry and propidium iodide staining. Effects of ANXA2 silencing on invasion and migration potential were assessed by transwell assay and wound healing assay, respectively. Proliferative potential was assessed by CCK-8 kit in vitro and xenograft tumour-growth assay in vivo. The t-test, chi square test, rank sum test, q-test and F-test were used for statistical analyses. RESULTS: The expression level of ANXA2 was markedly higher in the MHCC97-H cells with high metastasis potential than in the HepG2, SMMC-7721, SMMC-7402 and L02 cells. The efficiency of shRNA-mediated ANXA2 deficiency was more than 80%. Immunofluorescence analysis of the MHCC97-H cells indicated that ANXA2 expression was mainly localized to the cellular membrane and cytoplasm, with some nuclear localization. Down-regulation of ANXA2 led to S-phase arrest of HCC cells (q =8.001, P =0.002) and an inhibition of proliferation (q =17.140, P less than 0.01), migration (q =12.808, P less than 0.01) and invasion potential (q =9.069, P =0.002). Xenograft tumour-growth assay indicated that shRNA targeting of ANXA2 led to lower tumour weight (q =11.968, P < 0.001) and down-regulated ANXA2 expression (Z =2.530, P =0.011). CONCLUSION: Down-regulation of Annexin A2 gene transcription effectively changes the biological behaviours of hepatoma cells, and may represent a potential target of HCC molecular therapies.

[Inhibitory effects of intervention of the TNFa/NF-kappaB signaling pathway activation on hepatoma cell proliferation].

OBJECTIVE: To investigate the inhibitory effects of intervention of the tumor necrosis factor-alpha (TNFa)/nuclear factor-kappa B (NF-kappaB) signaling pathway activation on hepatoma cell proliferation and to explore its mechanism. METHODS: A rodent hepatoma model was established by feeding N-2-fluorenylacetamide (2-N-FAA) to male Sprague-Dawley rats. Human subjects with various liver diseases were enrolled in the study, and serum and peripheral blood nuclear cells were collected for analysis. HepG2 cells were cultured in vitro and treated with anti-TNFa (monoclonal antibody, mAb) to down-regulate its expression or transfected with siRNA targeting the p65 subunit of NF-kappaB to inhibit its activation. The liver cell line L02 was used as a control. Changes in protein and gene expression levels of NF-kappaB and TNFa were analyzed by Western blotting or enzyme-linked immunosorbent assay and real-time PCR, respectively. Changes in the cell cycle or apoptosis were evaluated by flow cytometry or Annexin-V/PI double-labeling assay, respectively. RESULTS: TNFa and NF-kappaB expression showed increasing trends during the malignant transformation of rat hepatocytes, and the differential expression patterns showed association with histopathological alterations in the hepatocytes. Following treatment with the TNFa mAb, the HepG2 cells showed a higher percentage of apoptotic cells than the untreated control cells (21.45% +/- 4.07% vs. 5.63% +/- 0.93%, q =10.07, P less than 0.01).There was a significant difference in the rate of cells in the G0/G1 phase in the p65-siRNA transfected cells (66.23% +/- 1.29% vs. untreated control cells: 59.00% +/- 1.02%, q =10.98, P less than 0.01). The decreased expression of TNFa and NF-kappaB in cell culture supernatants was positively correlated with the dose of treatment (r =0.89, P less than 0.01), with the most robust decreases being achieved with the highest concentrations ( P less than 0.01). NF-kappaB expression was significantly higher in the HepG2 cells than in the L02 cells, and transfection of p65-siRNA reduced the mRNA (93%) and protein (62%) levels and increased the cell apoptosis index (to 85%). CONCLUSION: Proliferation of hepatoma cells may be significantly inhibited by intervening in the activation of the TNFa/NF-kappaB signaling pathway, which promotes cell apoptosis and blocks cell cycling.

Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure.

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Optimizing instruction scheduling and register allocation for register-file-connected clustered VLIW architectures.

Clustering has become a common trend in very long instruction words (VLIW) architecture to solve the problem of area, energy consumption, and design complexity. Register-file-connected clustered (RFCC) VLIW architecture uses the mechanism of global register file to accomplish the inter-cluster data communications, thus eliminating the performance and energy consumption penalty caused by explicit inter-cluster data move operations in traditional bus-connected clustered (BCC) VLIW architecture. However, the limit number of access ports to the global register file has become an issue which must be well addressed; otherwise the performance and energy consumption would be harmed. In this paper, we presented compiler optimization techniques for an RFCC VLIW architecture called Lily, which is designed for encryption systems. These techniques aim at optimizing performance and energy consumption for Lily architecture, through appropriate manipulation of the code generation process to maintain a better management of the accesses to the global register file. All the techniques have been implemented and evaluated. The result shows that our techniques can significantly reduce the penalty of performance and energy consumption due to access port limitation of global register file.

Hyperbaric oxygen therapy promotes consciousness, cognitive function, and prognosis recovery in patients following traumatic brain injury through various pathways.

Objective: The aim of this study was to investigate the clinical curative effect of hyperbaric oxygen (HBO) treatment and its mechanism in improving dysfunction following traumatic brain injury (TBI). Methods: Patients were enrolled into control and HBO groups. Glasgow coma scale (GCS) and coma recovery scale-revised (CRS-R) scores were used to measure consciousness; the Rancho Los Amigos scale-revised (RLAS-R) score was used to assess cognitive impairment; the Stockholm computed tomography (CT) score, quantitative electroencephalography (QEEG), and biomarkers, including neuron-specific enolase (NSE), S100 calcium-binding protein beta (S100ß), glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF), were used to assess TBI severity. The patients were followed up 6 months after discharge and assessed with the Glasgow outcome scale-extended (GOSE), functional independence measure (FIM), and the disability rating scale (DRS). Results: The CRS-R scores were higher in the HBO group than the control group at 10 days after treatment. The RLAS-R scores were higher in the HBO group than the control group at 10 and 20 days after treatment. The Stockholm CT scores were significantly lower in the HBO group than the control group at 10 days after treatment. HBO depressed the (δ + θ)/(α + ß) ratio (DTABR) of EEG, with lower δ band relative power and higher α band relative power than those in the control group. At 20 days after treatment, the expression of NSE, S100ß, and GFAP in the HBO group was lower than that in controls, whereas the expression of BDNF, NGF, and VEGF in the HBO group was higher than that in controls. Six months after discharge, the HBO group had lower DRS scores and higher FIM and GOSE scores than the control group significantly. Conclusions: HBO may be an effective treatment for patients with TBI to improve consciousness, cognitive function and prognosis through decreasing TBI-induced hematoma volumes, promoting the recovery of EEG rhythm, and modulating the expression of serum NSE, S100ß, GFAP, BDNF, NGF, and VEGF.

Influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza.

BACKGROUND: Severe influenza is characterized by cytokine storm and multiorgan failure with edema. The aim of this study was to define the impact of the cytokine storm on the pathogenesis of vascular hyperpermeability in severe influenza. METHODS: Weanling mice were infected with influenza A WSN/33(H1N1) virus. The levels of proinflammatory cytokines, tumor necrosis factor (TNF) alpha, interleukin (IL) 6, IL-1beta, and trypsin were analyzed in the lung, brain, heart, and cultured human umbilical vein endothelial cells. The effects of transcriptional inhibitors on cytokine and trypsin expressions and viral replication were determined. RESULTS: Influenza A virus infection resulted in significant increases in TNF-alpha, IL-6, IL-1beta, viral hemagglutinin-processing protease trypsin levels, and viral replication with vascular hyperpermeability in lung and brain in the first 6 days of infection. Trypsin upregulation was suppressed by transcriptional inhibition of cytokines in vivo and by anti-cytokine antibodies in endothelial cells. Calcium mobilization and loss of tight junction constituent, zonula occludens-1, associated with cytokine- and trypsin-induced endothelial hyperpermeability were inhibited by a protease-activated receptor-2 antagonist and a trypsin inhibitor. CONCLUSIONS: The influenza virus-cytokine-protease cycle is one of the key mechanisms of vascular hyperpermeability in severe influenza.

Pathogenic Functions of Tumor Necrosis Factor Receptor-Associated Factor 6 Signaling Following Traumatic Brain Injury.

Neuroinflammation contributes to delayed (secondary) neurodegeneration following traumatic brain injury (TBI). Tumor necrosis factor receptor-associated factor 6 (TRAF6) signaling may promote post-TBI neuroinflammation, thereby exacerbating secondary injury. This study investigated the pathogenic functions of TRAF6 signaling following TBI in vivo and in vitro. A rat TBI model was established by air pressure contusion while lipopolysaccharide (LPS) exposure was used to induce inflammatory-like responses in cultured astrocytes. Model rats were examined for cell-specific expression of TRAF6, NF-κB, phosphorylated (p)-NF-κB, MAPKs (ERK, JNK, and p38), p-MAPKs, chemokines (CCL2 and CXCL1), and chemokine receptors (CCR2 and CXCR2) by immunofluorescence, RT-qPCR, western blotting, and ELISA, for apoptosis by TUNEL staining, and spatial cognition by Morris water maze testing. These measurements were compared between TBI model rats receiving intracerebral injections of TRAF6-targeted RNAi vector (AAV9-TRAF6-RNAi), empty vector, MAPK/NF-κB inhibitors, or vehicle. Primary astrocytes were stimulated with LPS following TRAF6 siRNA or control transfection, and NF-κB, MAPKs, chemokine, and chemokine receptor expression levels evaluated by western blotting and ELISA. TRAF6 was expressed mainly in astrocytes and neurons of injured cortex, peaking 3 days post-TBI. Knockdown by AAV9-TRAF6-RNAi improved spatial learning and memory, decreased TUNEL-positive cell number in injured cortex, and downregulated expression levels of p-NF-κB, p-ERK, p-JNK, p-p38, CCL2, CCR2, CXCL1, and CXCR2 post-TBI. Inhibitors of NF-κB, ERK, JNK, and p38 significantly suppressed CCL2, CCR2, CXCL1, and CXCR2 expression following TBI. Furthermore, TRAF6-siRNA inhibited LPS-induced NF-κB, ERK, JNK, p38, CCL2, and CXCL1 upregulation in cultured astrocytes. Targeting TRAF6-MAPKs/NF-κB-chemokine signaling pathways may provide a novel therapeutic approach for reducing post-TBI neuroinflammation and concomitant secondary injury.

[Mechanisms of multi-organ failure in severe influenza].

Severe influenza is characterized by cytokine storm and multi-organ failure with edema. We found that the "influenza virus-cytokine-trypsin/MMP-9 cycle" in the endothelial cells is one of the key mechanisms of vascular hyperpermeability, the major pathogen of multi-organ failure. Upregulated TNF-alpha, IL-6 and IL-beta induce ectopic pancreatic trypsin and pro-MMP-9 in the endothelial cells and in various organs. Trypsin mediates the viral hemagglutinin processing, which is crucial for viral entry and multicycles of replication. In addition, trypsin is the most potent pro-MMP-9 convertase to form active MMP-9 and both proteases synergistically destruct matrix around blood vessels. In addition upregulated trypsin triggers through its receptor, PAR-2, the modification of cellular functions, such as increase in calcium mobilization and mitochondrial membrane permeability, suppression of ATP generation and loss of tight junction constituent, zonula occludens-1. High risk patients who have impaired mitochondrial fuel utilization will easy get into energy crisis, resulting in vascular hyperpermeability in severe influenza.

Chemosensitization of HepG2 cells by suppression of NF-κB/p65 gene transcription with specific-siRNA.

AIM: To investigate small interfering RNA (siRNA)-mediated inhibition of nuclear factor-kappa B (NF-κB) activation and multidrug-resistant (MDR) phenotype formation in human HepG2 cells. METHODS: Total RNA was extracted from human HepG2 or LO2 cells. NF-κB/p65 mRNA was amplified by nested reverse transcription polymerase chain reaction and confirmed by sequencing. NF-κB/p65 was analyzed by immunohistochemistry. Specific-siRNA was transfected to HepG2 cells to knock down NF-κB/p65 expression. The effects on cell proliferation, survival, and apoptosis were assessed, and the level of NF-κB/p65 or P-glycoprotein (P-gp) was quantitatively analyzed by enzyme-linked immunosorbent assay. RESULTS: HepG2 cells express NF-κB/p65 and express relatively less phosphorylated p65 (P-p65) and little P-gp. After treatment of HepG2 cells with different doses of doxorubicin, the expression of NF-κB/p65, P-p65, and especially P-gp were dose-dependently upregulated. After HepG2 cells were transfected with NF-κB/p65 siRNA (100 nmol/L), the expression of NF-κB/p65, P-p65, and P-gp were downregulated significantly and dose-dependently. The viability of HepG2 cells was decreased to 23% in the combination NF-κB/p65 siRNA (100 nmol/L) and doxorubicin (0.5 µmol/L) group and 47% in the doxorubicin (0.5 µmol/L) group (t = 7.043, P < 0.001). CONCLUSION: Knockdown of NF-κB/p65 with siRNA is an effective strategy for inhibiting HepG2 cell growth by downregulating P-gp expression associated chemosensitization and apoptosis induction.

Up-regulation of ectopic trypsins in the myocardium by influenza A virus infection triggers acute myocarditis.

AIMS: Influenza A virus (IAV) infection markedly up-regulates ectopic trypsins in various organs, viral envelope glycoprotein processing proteases, which are pre-requisites for virus entry and multiplication. We investigated the pathological roles of trypsin up-regulation in the progression of IAV-induced myocarditis, cytokine induction, and viral replication in the hearts, and also investigated the protective effects of trypsin inhibitor on cardiac dysfunction in vivo and selective knockdown of trypsin on IAV-induced cellular damage in cardiomyoblasts. METHODS AND RESULTS: The relationship of the expression among IAV RNA, trypsins, matrix metalloproteinase (MMP)-9, MMP-2, pro-inflammatory cytokines interleukin (IL)-6, IL-1ß, and tumour necrosis factor-α was analysed in mice hearts and cardiomyoblasts after IAV infection. The severity of myocarditis was most noticeable during Day 6-9 post-infection, along with peak expression of viral RNA, trypsins, particularly trypsin2, MMPs, and cytokines. Cardiac ATP levels were the lowest at Day 9. Up-regulated trypsins, viral protein, and tissue-injured loci in the myocardium were closely localized. Trypsin inhibitor aprotinin treatment in vivo and selective trypsin1- and trypsin2-knockdown, particularly the latter, in H9c2 cardiomyoblasts significantly suppressed viral replication, up-regulation of MMPs, and production of active MMP-9 and cytokines, resulting in marked protection against cellular damage, ATP depletion, and apoptosis. IAV infection-induced cardiac dysfunction monitored by echocardiography was improved significantly by aprotinin treatment. CONCLUSIONS: IAV-induced trypsins, particularly trypsin2, in the myocardium trigger acute viral myocarditis through stimulation of IAV replication, proMMP-9 activation, and cytokine induction. These results suggest that up-regulation of trypsins is one of the key host pathological findings in IAV-induced myocarditis.

Mechanisms of matrix metalloproteinase-9 upregulation and tissue destruction in various organs in influenza A virus infection.

Severe influenza is characterized clinicopathologically by multiple organ failure, although the relationship amongst virus and host factors that influence this morbid outcome and the underlying mechanisms of action remain unclear. The present study identified marked upregulation of matrix metalloproteinase (MMP)-9 and pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in various organs after intranasal infection of influenza A WSN virus. MMP-9 and TNF-alpha were upregulated in the lung, the site of initial infection, as well as in the brain and heart. The infection-induced MMP-9 upregulation was inhibited by anti-TNF-alpha antibodies and by anti-oxidative reagents pyrrolidine dithiocarbamate and N-acetyl-L-cysteine, which inhibit activation of nuclear factor kappa B (NF-kappaB), as well as by nordihydroguaiaretic acid, which inhibits activation of activator protein 1 (AP-1). In addition, MMP-9 upregulation via TNF-alpha was also suppressed by inhibitors of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 and p38, and partly by a c-Jun N-terminal kinase inhibitor. These results indicated that the influenza-induced MMP-9 upregulation in various organs is mediated through MAPK-NF-kappaB- and/or AP-1-dependent mechanisms. Strategies that neutralize TNF-alpha as well as inhibitors of MAPK-NF-kappa B- and/or AP-1-dependent pathways may be useful for suppressing the MMP-9 effect and thus preventing multiple organ failure in severe influenza.

Host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses.

Influenza A virus (IAV) is one of the most common infectious pathogens in humans and causes considerable morbidity and mortality. The recent spread of highly-pathogenic avian IAV H5N1 viruses has reinforced the importance of pandemic preparedness. In the pathogenesis of IAV infection, cellular proteases play critical roles in the process of viral entry into cells that subsequently leads to tissue damage in the infected organs. Since there are no processing protease for the viral membrane fusion glycoprotein hemagglutinin precursor (HA(0)) in IAV, entry of the virus into cells is determined primarily by the host cellular HA(0) processing proteases that proteolytically activate membrane fusion activity. HA(0) of seasonal human IAV has the consensus cleavage site motif Q(E)-T/X-R and is selectively processed by at least seven different trypsin-type processing proteases identified to-date in animal model experiments using mouse-adapted IAV or gene expression system in MDCK cells. As is the case for the highly pathogenic avian influenza (HPAI) A virus, endoproteolytic processing of the HA(0) occurs through ubiquitous cellular processing proteases, which selectively recognize the multi-basic consensus cleavage site motifs, such as R-X-K/R-R, and K-X-K/R-R. The cleavage enzymes for the R-X-K/R-R motif, but not K-X-K/R-R motif, have been reported to be furin and pro-protein convertase (PC)5/6 in the trans-Golgi network. Here we report new members of type II transmembrane serine proteases of the cell membrane, mosaic serine protease large form (MSPL) and its splice variant TMPRSS13, which recognize and cleave both R-X-K/R-R and K-X-K/R-R motifs without calcium. Furthermore, IAV infection significantly up-regulates a latent ectopic pancreatic trypsin, one of the potent HA processing proteases, and pro-matrix metalloprotease-9, in various organs. These proteases may synergistically damage the blood-brain barrier in the brain and basement membrane of blood vessels in various organs, resulting in severe edema and multiple organ failure. In this review, we discuss these proteases as new drug target molecules for IAV treatment acting by inhibition of IAV multiplication and prevention of multiple organ failure, other than anti-viral agents, viral neuraminidase inhibitors.