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Severe fever with thrombocytopenia syndrome virus (SFTSV) infection typically causes acute fever, thrombocytopenia and leucopenia, presenting with a high case fatality rate. The pathogenesis of SFTSV infection, however, is not well described. It was hypothesized that endothelial dysfunction might play part in the disease process. In current study, we retrospectively analyzed the clinical manifestations among a large group of confirmed SFTS cases and found evidence of plasma leakage and vascular endothelial injury. Then we established a SFTSV infection cell model and determined the infectivity and stimulation of SFTSV on vascular endothelial cells in vitro. The hyperpermeability of endothelial cells directly induced by SFTSV was confirmed by electrical resistance and dextran diffusion assay. The virus induced alterations of cell junctions and cytoskeleton was also revealed. It's suggested that vascular endothelial cell injury and barrier function damage were induced after SFTSV infection, which is a vital but neglected pathogenesis of SFTS. [ABSTRACT FROM AUTHOR]

Abstract: Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand–receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation. [Copyright &y& Elsevier]

Abstract: Basic fibroblast growth factor (bFGF) is a known neuroprotectant against a number of brain injury conditions such as cerebral ischemia. However, bFGF also regulates a plethora of brain developmental processes and functions as a strong mitogen. Therefore, unregulated long-term expression of bFGF in brain may potentially be tumorigenic, limiting its utility in brain therapy. Here, we report the successful construction of an adenoviral vector (Ad-5HRE-bFGF) expressing bFGF under the regulation of five hypoxia-responsive elements (5HRE) and a minimal cytomegalovirus promoter (CMVmp). Following hypoxia treatment in a hypoxic chamber with less than 1% of oxygen, Ad-5HRE-bFGF induced a significant and time-dependent expression of bFGF protein and the fluorescent tag, humanized GFP (hrGFP) protein, in infected PC12 cells. In contrast, normoxia treatment evoked extremely low level of bFGF and hrGFP expression, demonstrating that the 5HRE-CMVmp cassette was effective in regulating the expression of bFGF gene in response to hypoxia. More importantly, bFGF expressed by the Ad-5HRE-bFGF viral vector under the regulation of hypoxia was significantly neuroprotective against PC12 cell death evoked by serum deprivation. Taken together, these studies demonstrated the feasibility to express bFGF in a hypoxia-regulated fashion to provide neuroprotection. The Ad-5HRE-bFGF can be further developed as an effective tool to provide neuroprotection against hypoxia-induced brain diseases, such as cerebral ischemia. [Copyright &y& Elsevier]

Abstract: A convenient silver staining method for protein in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8ng/band in approximately 1h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1h with a detection limit of 0.2ng/band. [Copyright &y& Elsevier]

Peroxiredoxin 6 (Prx6) is an important member of the peroxiredoxin family that plays critical roles in protecting host against the toxicity of oxidative stress and participates in cell signaling. Herein, we report Prx6 gene from red swamp crayfish, Procambarus clarkii. The cDNA fragment of PcPrx6 was 660 bp, encoding a 219 amino acid residues protein. The quantitative real time PCR analysis showed ubiquitous expression of PcPrx6 mRNA in the tested tissues. The challenge with peptidoglycan and Poly I:C remarkably suppressed the mRNA level of PcPrx6 in hepatopancreas at 3, 12, 48 h compared with the PBS control. However, the expression level significantly increased after 36 h of their treatment. The knockdown of PcPrx6 by small interference RNA significantly enhanced the transcript levels of Toll pathway-responsive genes at 24 h. Recombinant PcPrx6 protein was purified using affinity chromatography and analyzed for its biological role. The results revealed that the recombinant PcPrx6 protein manifested the ability to protect supercoiled DNA damage from oxidative stress elicited by mixed function oxidative assay. Altogether, PcPrx6 may have multiple functional roles in the physiology of P. clarkii , since it negatively regulates the Toll signaling transduction and protects supercoiled DNA damage from oxidative stress. • Peroxiredoxin 6 from Procambarus clarkii was identified and characterized for the first time. • The PcPrx6 expression in the hepatopancreas was up-regulated post PGN and Poly I:C challenge. • We investigated the antioxidant role of recombinant PcPrx6 protein in vitro by mixed-function oxidase assay. • RNA interference of PcPrx6 affected the gene expression of members of the Toll pathway. [ABSTRACT FROM AUTHOR]

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive and simple technique for the negative detection of lipopolysaccharides (LPSs) following polyacrylamide gel electrophoresis (PAGE) using eosin B (EB) was developed. After electrophoresis, gels were fixed, stained, and developed within 30min to achieve transparent and colorless LPS bands under opaque gel matrix background. As low as 20 to 40ng of total LPSs could be detected, which is 4-fold more sensitive than those of the widely used silver stain developed by Fomsgaard and coworkers and imidazole–zinc (IZ) negative stain. For its sensitivity and brevity, this stain may be a practical method for LPS determination in the routine laboratory. [Copyright &y& Elsevier]

Abstract: We describe a visible dye-based staining method for DNA in polyacrylamide gels using ethyl violet (EV). The novel method is a background-free, sensitive, economical, and simple procedure involving only staining and washing steps that can be completed within 30 min. As little as 0.8–1.6ng of φX174 DNA/HaeIII can be detected by EV, which is about eightfold more sensitive than Nile blue (NB) stain and twofold less sensitive than ethidium bromide (EB) stain. [Copyright &y& Elsevier]