Antimicrobial activity and enzymatic analysis of endophytes isolated from Codonopsis pilosula.

The roots of the medicinal plant Codonopsis pilosula (Franch.) Nannf (C. pilosula) possess most medicinal supplements. In current research on C. pilosula root endophytes were isolated, identified, and evaluated for their antimicrobial activity against human pathogens such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Salmonella typhi, and Pseudomonas aeruginosa and the fungi Candida albicans and Aspergillus niger. Endophytes C.P-8 and C.P-20 exhibited very significant antimicrobial activity, the secondary metabolite of C.P-8 registered at retention time 24.075 by HPLC analysis. Significant minimum inhibitory concentration (MIC) of C.P-8 was exhibited at 250 µg/ml against S. aureus and 500 µg/ml against B. subtilis. Qualitative, quantitative analyses, and partial purification of enzymes and purity was analysed by molecular weight determined by SDS‒PAGE of enzymes produced by C.P-20, amylase-64 kDa, protease-64 kDa, chitinase-30 kDa, and cellulase-54 kDa. Optimum pH and temperature of the partially purified enzymes, was carried out. The partially purified enzymes from C.P-20 displayed maximum activity at pH 6-7 and temperatures of 40°C-45°C. Moreover, the above endophytes will be useful tools for producing active enzymes and active bioantimicrobial agents against human pathogens.

Identification of Mutation Landscape and Immune Cell Component for Liver Hepatocellular Carcinoma Highlights Potential Therapeutic Targets and Prognostic Markers.

Liver hepatocellular carcinoma (LIHC) is a primary malignancy, and there is a lack of effective treatment for advanced patients. Although numerous studies exist to reveal the carcinogenic mechanism of LIHC, few studies have integrated multi-omics data to systematically analyze pathogenesis and reveal potential therapeutic targets. Here, we integrated genomic variation data and RNA-seq profiles obtained by high-throughput sequencing to define high- and low-genomic instability samples. The mutational landscape was reported, and the advanced patients of LIHC were characterized by high-genomic instability. We found that the tumor microenvironment underwent metabolic reprograming driven by mutations accumulate to satisfy tumor proliferation and invasion. Further, the co-expression network identifies three mutant long non-coding RNAs as potential therapeutic targets, which can promote tumor progression by participating in specific carcinogenic mechanisms. Then, five potential prognostic markers (RP11-502I4.3, SPINK5, CHRM3, SLC5A12, and RP11-467L13.7) were identified by examining the association of genes and patient survival. By characterizing the immune landscape of LIHC, loss of immunogenicity was revealed as a key factor of immune checkpoint suppression. Macrophages were found to be significantly associated with patient risk scores, and high levels of macrophages accelerated patient mortality. In summary, the mutation-driven mechanism and immune landscape of LIHC revealed by this study will serve precision medicine.

MiR-377-3p inhibits atherosclerosis-associated vascular smooth muscle cell proliferation and migration via targeting neuropilin2.

Vascular smooth muscle cell (VSMC) proliferation and migration are vital to atherosclerosis (AS) development and plaque rupture. MicroRNA-377-3p (miR-377-3p) has been reported to inhibit AS in apolipoprotein E knockout (ApoE-/-) mice. Herein, the mechanism underlying the effect of miR-377-3p on alleviating AS is explored. In vivo experiments, ApoE-/- mice were fed with high-fat diet (HFD) to induce AS and treated with miR-377-3p agomir or negative control agomir (agomir-NC) on week 0, 2, 4, 6, 8, 10 after HFD feeding. MiR-377-3p was found to restore HFD-induced AS lesions and expressions of matrix metalloproteinase (MMP)-2, MMP-9, α-smooth muscle actin (α-actin) and calponin. In in vitro experiments, human VSMCs were tranfected with miR-377-3p agomir or agomir-NC, followed by treatment with oxidized low-density lipoprotein (ox-LDL). MiR-377-3p was observed to significantly inhibit ox-LDL-induced VSMC proliferation characterized by inhibited cell viability, expressions of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E and cell cycle transition from G1 to S phase accompanied with less 5-Ethynyl-2'-deoxyuridine (EdU)-positive cells. Furthermore, MiR-377-3p significantly inhibited ox-LDL-induced VSMC migration characterized by inhibited wound closure and decreased relative VSMC migration. Besides, neuropilin2 (NRP2) was verified as a target of miR-377-3p. MiR-377-3p was observed to inhibit NRP2 expressions in vivo and in vitro. Moreover, miR-377-3p significantly inhibited MMP-2 and MMP-9 expressions in human VSMCs. Additionally, miR-377-3p-induced inhibition of VSMC proliferation and migration could be attenuated by NRP2 overexpression. These results indicated that miR-377-3p inhibited VSMC proliferation and migration via targeting NRP2. The present study provides an underlying mechanism for miR-377-3p-based AS therapy.

MicroRNA-99a-5p alleviates atherosclerosis via regulating Homeobox A1.

AIMS: MicroRNAs have been demonstrated to be involved in the development of atherosclerosis. The present study aimed to evaluate the effect of miR-99a-5p and its target gene Homeobox A1 (HOXA1) in atherosclerosis. MAIN METHODS: The biological functions of miR-99a-5p on human aortic smooth muscle cells (ASMCs) were assessed by MTT, wound healing and transwell assays. The target genes of microRNAs were predicted by TargetScan and miRDB. The binding of miR-99a-5p and HOXA1 was confirmed by luciferase reporter assay. In the in vivo study, high-fat diet-induced atherosclerosis model was established in Apolipoprotein E knockout mice. Hematoxylin-eosin (H&E), oil Red O and Masson trichrome staining were performed for determination of atherosclerotic lesion. The levels of miR-99a-5p and HOXA1 mRNA were detected by real-time PCR. HOXA1 and migration-associated protein levels were detected by western blot or immunohistochemistry analysis. KEY FINDINGS: MiR-99a-5p inhibited HOXA1 expression by targeting 3'UTR of HOXA1 mRNA. Enforced HOXA1 significantly promoted the proliferation, migration, and invasion of ASMCs. Furthermore, miR-99a-5p overexpression inhibited the proliferation, migration, and invasion of ASMCs stimulated by HOXA1, whereas miR-99a-5p inhibition reversed the effects of HOXA1 knockdown on these behaviours of ASMCs. In vivo, the specific overexpression of miR-99a-5p significantly abated atherosclerotic lesions formatted, accompanied with a significant down-regulation of HOXA1 mRNA and protein expression levels. SIGNIFICANCE: We demonstrate for first time that miR-99a-5p may serve as a potential inhibitor of the atherosclerosis, and miR-99a-5p plays its role partially through targeting HOXA1.

The IL-6/STAT3 pathway regulates adhesion molecules and cytoskeleton of endothelial cells in thromboangiitis obliterans.

Thromboangiitis obliterans (TAO) (also known as Buerger's disease) is an inflammatory vascular disease that predominantly affects small- and medium-sized blood vessels of extremities. Endothelial cells play critical roles in the initiation and progression of this disease, but the underlying mechanisms remain unclear. In the present study, we demonstrate that patients with TAO had significantly higher levels of interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in their plasmas, and the involved arterial tissues expressed higher levels of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), ICAM-1 and VCAM-1. In exploring the molecular mechanisms with human aortic endothelial cells (HAECs), we found that recombinant IL-6 activated the STAT3 pathway, leading to the upregulation and overproduction of ICAM-1 and VCAM-1. RhoA (Ras homolog family member A), eNOS (endothelial nitric oxide synthase) and MMP-9 (matrix metalloproteinase-9) participated in this cellular signaling, and their interaction regulated the expression of ICAM-1 and VCAM-1. The activated STAT3 pathway by IL-6 also modulated the cytoskeleton of HAECs by regulating phosphorylation of focal adhesion kinase (FAK) and acetylation of α-tubulin through interplaying with RhoA. In summary, the present results indicate that activation of the IL-6/STAT3 pathway contributes to the pathogenesis of TAO by regulating cellular adhesion molecules and cytoskeleton of vascular endothelial cells, suggesting that targeting this pathway may provide a potential approach for the management of TAO.