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This study was aimed to investigate the preventive effects of N-acetyl-l-cysteine (NAC) against renal tubular cell injury induced by oxalate and stone formation and further explore the related mechanism. Transcriptome sequencing combined with bioinformatics analysis were performed to identify differentially expressed gene (DEG) and related pathways. HK-2 cells were pretreated with or without antioxidant NAC/with or silencing DEG before exposed to sodium oxalate. Then, the cell viability, oxidative biomarkers of superoxidase dismutase (SOD) and malondialdehyde (MDA), apoptosis and cell cycle were measured through CCK8, ELISA and flow cytometry assay, respectively. Male SD rats were separated into control group, hyperoxaluria (HOx) group, NAC intervention group, and TGF-β/SMAD pathway inhibitor group. After treatment, the structure changes and oxidative stress and CaOx crystals deposition were evaluated in renal tissues by H&E staining, immunohistochemical and Pizzolato method. The expression of TGF-β/SMAD pathway related proteins (TGF-β1, SMAD3 and SMAD7) were determined by Western blot in vivo and in vitro. CDKN2B is a DEG screened by transcriptome sequencing combined with bioinformatics analysis, and verified by qRT-PCR. Sodium oxalate induced declined HK-2 cell viability, in parallel with inhibited cellular oxidative stress and apoptosis. The changes induced by oxalate in HK-2 cells were significantly reversed by NAC treatment or the silencing of CDKN2B. The cell structure damage and CaOx crystals deposition were observed in kidney tissues of HOx group. Meanwhile, the expression levels of SOD and 8-OHdG were detected in kidney tissues of HOx group. The changes induced by oxalate in kidney tissues were significantly reversed by NAC treatment. Besides, expression of SMAD7 was significantly down-regulated, while TGF-β1 and SMAD3 were accumulated induced by oxalate in vitro and in vivo. The expression levels of TGF-β/SMAD pathway related proteins induced by oxalate were reversed by NAC. In conclusion, we found that NAC could play an anti-calculus role by mediating CDKN2B/TGF-β/SMAD axis. [ABSTRACT FROM AUTHOR]

Simple Summary: Salinity can affect the physiological, reproductive, and developmental characteristics of marine molluscs. The Chinese razor clam (Sinonovacula constricta) is a marine bivalve that inhabits estuarine and intertidal habitats where salinity is usually lower when compared to open sea waters. The species is also an economically important subject of aquaculture in China. Here, RNA sequencing was used to study the internal mechanism of salt stress response in the Chinese razor clam. Differentially expressed RNAs were identified and lncRNA/circRNA-miRNA-mRNA regulatory networks were successfully constructed. Furthermore, the expression of four candidate genes in the Chinese razor clam subjected to different salinity stress were examined by qRT-PCR. The results of this study will improve our understanding of genetic-level responses to salinity changes in the Chinese razor clam. Moreover, the acquired information may be useful for optimization of the artificial breeding of the species under aquaculture conditions. The Chinese razor clam (Sinonovacula constricta) is an important for Chinese aquaculture marine bivalve that naturally occurs across intertidal and estuarine areas subjected to significant changes in salinity level. However, the information on the molecular mechanisms related to high salinity stress in the species remain limited. In this study, nine gill samples of S. constricta treated with 20, 30, and 40 ppt salinity for 24 h were used for whole-transcriptome RNA sequencing, and a regulatory network of competing endogenous RNAs (ceRNAs) was constructed to better understand the mechanisms responsible for adaptation of the species to high salinity. A total of 83,262 lncRNAs, 52,422 mRNAs, 2890 circRNAs, and 498 miRNAs were identified, and 4175 of them displayed differential expression pattern among the three groups examined. The KEGG analyses of differentially expressed RNAs evidenced that amino acid synthesis and membrane transport were the dominant factors involved in the adaptation of the Chinese razor clam to acute salinity increase, while lipid metabolism and signaling played only a supporting role. In addition, lncRNA/circRNA-miRNA-mRNA regulatory networks (ceRNA network) showed clearly regulatory relationships among different RNAs. Moreover, the expression of four candidate genes, including tyrosine aminotransferase (TAT), hyaluronidase 4 (HYAL4), cysteine sulfinic acid decarboxylase (CSAD), and ∆1-pyrroline-5-carboxylate synthase (P5CS) at different challenge time were detected by qRT-PCR. The expression trend of TAT and HYAL4 was consistent with that of the ceRNA network, supporting the reliability of established network. The expression of TAT, CSAD, and P5CS were upregulated in response to increased salinity. This might be associated with increased amino acid synthesis rate, which seems to play an essential role in adaptation of the species to high salinity stress. In contrast, the expression level of HYAL4 gene decreased in response to elevated salinity level, which is associated with reduction Hyaluronan hydrolysis to help maintain water in the cell. Our findings provide a very rich reference for understanding the important role of ncRNAs in the salinity adaptation of shellfish. Moreover, the acquired information may be useful for optimization of the artificial breeding of the Chinese razor clam under aquaculture conditions. [ABSTRACT FROM AUTHOR]

Current cell-based bone tissue regeneration strategies cannot cover large bone defects. K-carrageenan is a highly hydrophilic and biocompatible seaweed-derived sulfated polysaccharide, that has been proposed as a promising candidate for tissue engineering applications. Whether κ-carrageenan can be used to enhance bone regeneration is still unclear. In this study, we aimed to investigate whether κ-carrageenan has osteogenic potential by testing its effect on pre-osteoblast proliferation and osteogenic differentiation in vitro. Treatment with κ-carrageenan (0.5 and 2 mg/mL) increased both MC3T3-E1 pre-osteoblast adhesion and spreading at 1 h. K-carrageenan (0.125–2 mg/mL) dose-dependently increased pre-osteoblast proliferation and metabolic activity, with a maximum effect at 2 mg/mL at day three. K-carrageenan (0.5 and 2 mg/mL) increased osteogenic differentiation, as shown by enhanced alkaline phosphatase activity (1.8-fold increase at 2 mg/mL) at day four, and matrix mineralization (6.2-fold increase at 2 mg/mL) at day 21. K-carrageenan enhanced osteogenic gene expression (Opn, Dmp1, and Mepe) at day 14 and 21. In conclusion, κ-carrageenan promoted MC3T3-E1 pre-osteoblast adhesion and spreading, metabolic activity, proliferation, and osteogenic differentiation, suggesting that κ-carrageenan is a potential osteogenic inductive factor for clinical application to enhance bone regeneration. [ABSTRACT FROM AUTHOR]

Background: Kojic acid (KA) is a widely used compound in the cosmetic, medical, and food industries, and is typically produced by Aspergillus oryzae. To meet increasing market demand, it is important to optimize KA production through seeking alternatives that are more economic than current A. oryzae-based methods. Results: In this study, we achieved the first successful heterologous production of KA in Aspergillus niger, an industrially important fungus that does not naturally produce KA, through the expression of the kojA gene from A. oryzae. Using the resulting KA-producing A. niger strain as a platform, we identified four genes (nrkA, nrkB, nrkC, and nrkD) that negatively regulate KA production. Knocking down nrkA or deleting any of the other three genes resulted in a significant increase in KA production in shaking flask cultivation. The highest KA titer (25.71 g/L) was achieved in a pH controlled batch bioreactor using the kojA overexpression strain with a deletion of nrkC, which showed a 26.7% improvement compared to the KA titer (20.29 g/L) that was achieved in shaking flask cultivation. Conclusion: Our study demonstrates the potential of using A. niger as a platform for studying KA biosynthesis and regulation, and for the cost-effective production of KA in industrial strain development. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is the most common malignant bone tumor. However, treatment strategies have not changed over the past 30 years. The relationship between OS and the immune microenvironment may provide a basis for the establishment of novel therapeutic targets. In this study, a large-scale gene expression dataset (GSE42352) was used to identify key genes in OS. A Target-OS dataset from the Cancer Genome Atlas was used as a validation set. Ecotropic viral integration site 2B (EVI2B) was significantly upregulated in OS tumor samples. Differentially expressed genes (DEGs) were identified between samples with high and low EVI2B expression in both the test and validation cohorts. The top three functions of DEGs determined by a gene set enrichment analysis (GSEA) were chemokine signaling, cytokine–cytokine receptor interaction, and Human T-cell leukemia virus 1 infection. A prognostic prediction model including EVI2B, DOCK2, and CD33 was constructed by a Cox regression analysis. This model indicated that EVI2B is an independent protective prognostic marker in OS. An analysis of immune infiltration further showed that high EVI2B expression levels were correlated with high levels of macrophage infiltration. Protein expression data derived from the Human Protein Atlas suggested EVI2B to be highly expressed in monocytes. Finally, we validated the elevated expression of EVI2B in OS cell lines and OS tissue samples; these results were consistent with those of the analyses of the GSE42352 and Target-OS datasets. Our integrative bioinformatics analysis and experimental results provide clear evidence for the prognostic value of EVI2B in OS and its close relationship with monocyte and macrophage infiltration. [ABSTRACT FROM AUTHOR]

Ube3a is a member of the E3 ubiquitin ligase HECTc family, and its role has been established in neurodevelopmental disorders. However, studies on its role in Japanese flounder are scarce. Thus, in this study, the ube3a of Japanese flounder was cloned, and its role in conferring resistance against Chinook salmon bafnivirus (CSBV) was analyzed. Japanese flounder ube3a encoded a protein containing 834 amino acids. Interestingly, its homology with the Atlantic halibut was determined to be 94%. In addition, there were differential expressions of ube3a in different tissues of Japanese flounder, with the highest expression level observed in the fin, followed by the gills and skin (P ≤ 0.05). Subcellular localization analysis revealed that Ube3a is a cytoplasmic protein. We established an in vitro CSBV infection model using Japanese flounder gill cell line (FG). After ube3a overexpression, the viral load was significantly lower than that of the control group (P ≤ 0.05). Contrastingly, after incubation of FG cells with an E3 ubiquitin ligase inhibitor, the viral load was significantly higher than in the control group (P ≤ 0.01). Then, the expression levels of nf-κb , traf3 , and tnf-α after incubation with an E3 ubiquitin ligase inhibitor were examined. The results demonstrated that ube3a may exerted a significant antiviral effect in Japanese flounder via the ubiquitination pathway. • The ube3a gene was cloned and characterized in Japanese flounder. • The mRNA expression of ube3a was changed significantly after CSBV infection. • The ube3a gene regulates CSBV through the ubiquitination pathway. • Japanese flounder ube3a is involved in the inflammatory response induced by CSBV infection. [ABSTRACT FROM AUTHOR]

• LLDT-8 reduces the percentage of HLA-DR+CD38+CD8+ T cells in SIV-infected rhesus monkeys. • LLDT-8 inhibits the proliferation related pathways and genes in SIV-infected rhesus macaques. • LLDT-8 inhibits the proliferation, activation, exhaustion, and IFN-γ production of human T cells. Chronic immune activation plays a significant role in the pathogenesis and disease progression of human immunodeficiency virus (HIV), and the existing interventions to address this issue are limited. In a phase II clinical trial, (5R)-5-hydroxytriptolide (LLDT-8) demonstrated promising potential in enhancing CD4+ T cell recovery. However, the therapeutical effects of LLDT-8 remained to be systemic explored. To assess the treatment effects of LLDT-8, we conducted flow cytometry and RNA-seq analyses on eight Chinese rhesus monkeys infected with simian immunodeficiency virus (SIV). Additionally, we performed comprehensive transcriptomic analyses, including cross-sectional and longitudinal differentially expressed gene (DEG) analysis, gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), and deconvolution analysis using peripheral blood mononuclear cell (PBMC) samples from 14-time points. These findings were further validated with RNA-seq analysis on patients who received LLDT-8 treatment, along with in vitro cellular experiments using human PBMCs. Flow cytometry analysis revealed that LLDT-8 treatment significantly reduced the percentage of HLA-DR+CD38+CD8+ T cells in SIV-infected rhesus monkeys (P < 0.001). The cross-sectional and longitudinal analysis identified 2531 and 1809 DEGs, respectively. GSEA analysis indicated that LLDT-8 treatment led to significant downregulation of proliferation-related pathways, such as E2F targets, G2M checkpoint, and mitotic spindle pathways. WGCNA analysis identified two modules and 202 hub genes associated with CD8 activation levels. Deconvolution analysis showed a significant decrease in the proportion of CD8+ T cells and activated CD4+ T cells during LLDT-8 treatment. Gene ontology results demonstrated that the common DEGs between LLDT-8-treated patients and rhesus monkeys were primarily enriched in cell activation and cell cycle progression. Furthermore, in vitro cellular experiments validated the consistent impact of LLDT-8 in inhibiting proliferation, activation (HLA-DR and CD38 expression), exhaustion (PD-1 expression), and IFN-γ production in human CD4+ and CD8+ T cells. LLDT-8 exhibited notable efficacy in alleviating immune activation in both an in vivo animal model and in vitro human cell experiments. These findings suggest that LLDT-8 may hold potential as a drug for managing systemic immune activation associated with SIV/HIV infection, warranting further prospective clinical exploration. [ABSTRACT FROM AUTHOR]

Slc2a6 is a member of the slc2 family (solute carrier 2 family) and previous reports have indicated its involvement in the inflammatory response. Slc2a6 is regulated by the NF-ĸB signaling pathway. This study investigated the differential expression of slc2a6 in the early embryonic development of Japanese flounder, revealing that the early gastrula stage had the highest level of slc2a6 expression. Moreover, slc2a6 expression was increased in vitro after stimulation by lymphocystis disease virus (LCDV), and in vivo experiments also showed significantly elevated levels in the spleen and muscle tissues following LCDV stimulation. Subcellular localization revealed that Slc2a6 was expressed in both the nucleus and cytoplasm of cells. The pcDNA3.1- slc2a6 overexpression plasmid was successfully constructed; the si- slc2a6 interfering strand was screened and samples were collected. The expression of NF-ĸB signaling pathway-related genes il-1β , il-6 , nf-ĸb , and tnf-α was evaluated in overexpressed, silenced, and LCDV-stimulated samples. The results showed that slc2a6 is involved in viral regulation in Japanese flounder by regulating innate immune responses. • The slc2a6 gene was cloned and characterized in Japanese flounder. • The mRNA expression of slc2a6 was changed significantly after LCDV infection. • Japanese flounder slc2a6 is involved in the inflammatory response induced by LCDV infection. • NF-ĸB signaling pathway regulates the role of the inflammatory factor slc2a6. [ABSTRACT FROM AUTHOR]

Morphine tolerance (MT) caused by long-term use of morphine is a major medical problem. The underlying molecular mechanisms of morphine tolerance remain unclear. Here, we establish the morphine tolerance model in mice and verify whether a novel circRNA, circRalgapa1 is involved in morphine tolerance and its specific molecular mechanism. We show that the expression of circRalgapa1 in the spinal cord is significantly down-expressed in the spinal cord of morphine-tolerant mice. CircRalgapa1 is mainly located in the neuronal cytoplasm and co-localizes with miR-873a-5p. Mechanically, circRalgapa1 acts as competing endogenous RNAs (ceRNAs) to regulate the inhibitory of miR-873a-5p on A20 (also known as tumor necrosis factor α-induced protein 3, TNFAIP3). Functionally, overexpression of circRalgapa1 by intrathecal injection of adeno-associated virus (AAV- circRalgapa1) attenuated the formation of morphine tolerance and partially reversed the development of morphine tolerance. Moreover, overexpression of miR-873a-5p blocked the effect of AAV-circRalgapa1 on alleviating morphine tolerance in mice. In conclusion, chronic morphine administration-mediated down-regulation of circRalgapa1 in the spinal cord contributes to morphine tolerance via miR-873a-5p/A20 axis in mice. Overexpression of circRalgapa1 may be a promising RNA-based therapy for morphine tolerance. Schematic diagram for the proposed mechanisms of circRalgapa1 regulates morphine tolerance.Chronic application of morphine down-regulates circRalgapa1 in the spinal cord of mice, attenuating the competitive binding with miR-873a-5p, enhancing the inhibition of miR-873a-5p to A20 mRNA, thus reducing A20 expression to induce morphine tolerance in mice. [Display omitted] • CircRalgapa1 is down-regulated during induction of morphine tolerance. • CircRalgapa1 contributes to morphine tolerance via miR-873a-5p/A20 axis. • Overexpression of circRalgapa1 attenuates the formation and development of morphine tolerance. [ABSTRACT FROM AUTHOR]

• Yak DEFB124 protein could be expressed by Escherichia coli system. • Yak DEFB124 protein could increase the abundance of intestinal probiotics Lactobacillus. • Yak DEFB124 protein has the effects of protecting the intestine and reducing intestinal damage. To investigate the characteristics and functions of yak β-defensin 124 (DEFB124), prokaryotic expression, analysis of gut microbiological and other methods were used in this study. The results showed that the sequence of yak DEFB124 gene was 306 bp in length and 207 bp in open reading frame, which encoded 68 amino acids. Yak DEFB124 protein was highly conserved and had the closest relationship with cattle. Yak DEFB124 protein was a secreted cationic β-defensin. The recombinant expression plasmid pET32a-DEFB124 was constructed, and an about 24 kDa protein was successfully expressed. Yak DEFB124 protein had inhibitory activity against Staphylococcus aureus (S. aureus), and its MIC value was 64 μg/mL. After treating with yak DEFB124 protein, the activities of alkaline phosphatase (AKP) and total superoxide dismutase (T -SOD) were higher (P < 0.01) in the jejunum tissue, but the activity of lysozyme (LZM) was lower (P < 0.01). The number of goblet cells in the duodenum, jejunum, and ileum of the mice in the DEFB124 group was increased (P < 0.01). Besides, the expressions of MUC 2 mRNA and protein were increased (P < 0.05) after the treatment with yak DEFB124 protein. Furthermore, the relative abundance of Lactobacillu s in jejunum of mice in DEFB124 group was also increased. In summary, yak DEFB124 protein could increase the number of goblet cells in mice intestine and the abundance of intestinal probiotics Lactobacillus , thereby protecting the intestinal tract and alleviating intestinal damage. [ABSTRACT FROM AUTHOR]