Downregulation of N4-acetylcytidine modification in myeloid cells attenuates immunotherapy and exacerbates hepatocellular carcinoma progression.

BACKGROUND: N4-acetylcytidine (ac4C) is a conserved and abundant mRNA modification that controls protein expression by affecting translation efficiency and mRNA stability. Whether the ac4C modification of mRNA regulates hepatocellular carcinoma (HCC) development or affects the immunotherapy of HCC is unknown. METHODS: By constructing an orthotopic transplantation mouse HCC model and isolating tumour-infiltrated immunocytes, we evaluated the ac4C modification intensity using flow cytometry. Remodelin hydrobromide (REM), an ac4C modification inhibitor, was systematically used to understand the extensive role of ac4C modification in immunocyte phenotypes. Single-cell RNA-seq was performed to comprehensively evaluate the changes in the tumour-infiltrating immunocytes and identify targeted cell clusters. RNA-seq and RIP-seq analyses were performed to elucidate the underlying molecular mechanisms. Tyramide Signal Amplification (TSA) analysis on the HCC tissue microarray was performed to explore the clinical relatedness of our findings. RESULTS: Ac4C modification promoted M1 macrophage infiltration and reduced myeloid-derived suppressor cell MDSCs infiltration in HCC. The inhibition of ac4C modification induces PDL1 expression by stabilising mRNA in the myeloid cells, thereby attenuating the CTL-mediated tumour cell-killing ability. High infiltration of ac4C+CD11b+ cells is positively related to a better prognosis in patients with HCC. CONCLUSIONS: Ac4C modification of myeloid cells enhanced the HCC immunotherapy by suppressing PDL1 expression.

Gp78 deficiency in hepatocytes alleviates hepatic ischemia-reperfusion injury via suppressing ACSL4-mediated ferroptosis.

Ferroptosis, which is driven by iron-dependent lipid peroxidation, plays an essential role in liver ischemia-reperfusion injury (IRI) during liver transplantation (LT). Gp78, an E3 ligase, has been implicated in lipid metabolism and inflammation. However, its role in liver IRI and ferroptosis remains unknown. Here, hepatocyte-specific gp78 knockout (HKO) or overexpressed (OE) mice were generated to examine the effect of gp78 on liver IRI, and a multi-omics approach (transcriptomics, proteomics, and metabolomics) was performed to explore the potential mechanism. Gp78 expression decreased after reperfusion in LT patients and mice with IRI, and gp78 expression was positively correlated with liver damage. Gp78 absence from hepatocytes alleviated liver damage in mice with IRI, ameliorating inflammation. However, mice with hepatic gp78 overexpression showed the opposite phenotype. Mechanistically, gp78 overexpression disturbed lipid homeostasis, remodeling polyunsaturated fatty acid (PUFA) metabolism, causing oxidized lipids accumulation and ferroptosis, partly by promoting ACSL4 expression. Chemical inhibition of ferroptosis or ACSL4 abrogated the effects of gp78 on ferroptosis and liver IRI. Our findings reveal a role of gp78 in liver IRI pathogenesis and uncover a mechanism by which gp78 promotes hepatocyte ferroptosis by ACSL4, suggesting the gp78-ACSL4 axis as a feasible target for the treatment of IRI-associated liver damage.

The Effects of Mammary Gland ATIII Overexpression on the General Health of Dairy Goats and Their Anti-Inflammatory Response to LPS Stimulation.

Antithrombin III is an important anticoagulant factor with anti-inflammatory properties. However, few studies have explored its anti-inflammatory actions in ATIII overexpressed transgenic animals. In this study, the dairy goats with mammary overexpression of ATIII were used to investigate their general health, milk quality and particularly their response to inflammatory challenge. The results showed that transgenic goats have a normal phenotype regarding their physiological and biochemical parameters, including whole blood cells, serum protein levels, total cholesterol, urea nitrogen, uric acid, and total bilirubin, compared to the WT. In addition, the quality of milk also improved in transgenic animals compared to the WT, as indicated by the increased milk fat and dry matter content and the reduced somatic cell numbers. Under the stimulation of an LPS injection, the transgenic goats had elevated contents of IGA, IGM and superoxide dismutase SOD, and had reduced proinflammatory cytokine release, including IL-6, TNF-α and IFN-ß. A 16S rDNA sequencing analysis also showed that the transgenic animals had a similar compositions of gut microbiota to the WT goats under the stimulation of LPS injections. Mammary gland ATIII overexpression in dairy goats is a safe process, and it did not jeopardize the general health of the transgenic animals; moreover, the compositions of their gut microbiota also improved with the milk quality. The LPS stimulation study suggests that the increased ATIII expression may directly or indirectly suppress the inflammatory response to increase the resistance of transgenic animals to pathogen invasion. This will be explored in future studies.

Insulin-induced gene 2 protects against hepatic ischemia-reperfusion injury via metabolic remodeling.

BACKGROUND: Hepatic ischemia-reperfusion (IR) injury is the primary reason for complications following hepatectomy and liver transplantation (LT). Insulin-induced gene 2 (Insig2) is one of several proteins that anchor the reticulum in the cytoplasm and is essential for metabolism and inflammatory responses. However, its function in IR injury remains ambiguous. METHODS: Insig2 global knock-out (KO) mice and mice with adeno-associated-virus8 (AAV8)-delivered Insig2 hepatocyte-specific overexpression were subjected to a 70% hepatic IR model. Liver injury was assessed by monitoring hepatic histology, inflammatory responses, and apoptosis. Hypoxia/reoxygenation stimulation (H/R) of primary hepatocytes and hypoxia model induced by cobalt chloride (CoCl2) were used for in vitro experiments. Multi-omics analysis of transcriptomics, proteomics, and metabolomics was used to investigate the molecular mechanisms underlying Insig2. RESULTS: Hepatic Insig2 expression was significantly reduced in clinical samples undergoing LT and the mouse IR model. Our findings showed that Insig2 depletion significantly aggravated IR-induced hepatic inflammation, cell death and injury, whereas Insig2 overexpression caused the opposite phenotypes. The results of in vitro H/R experiments were consistent with those in vivo. Mechanistically, multi-omics analysis revealed that Insig2 is associated with increased antioxidant pentose phosphate pathway (PPP) activity. The inhibition of glucose-6-phosphate-dehydrogenase (G6PD), a rate-limiting enzyme of PPP, rescued the protective effect of Insig2 overexpression, exacerbating liver injury. Finally, our findings indicated that mouse IR injury could be attenuated by developing a nanoparticle delivery system that enables liver-targeted delivery of substrate of PPP (glucose 6-phosphate). CONCLUSIONS: Insig2 has a protective function in liver IR by upregulating the PPP activity and remodeling glucose metabolism. The supplementary glucose 6-phosphate (G6P) salt may serve as a viable therapeutic target for alleviating hepatic IR.

Dynamic immune cell profiling identified natural killer cell shift as the key event in early allograft dysfunction after liver transplantation.

Early allograft dysfunction (EAD) is a life-threatening and fast-developing complication after liver transplantation. The underlying mechanism needs to be better understood, and there has yet to be an efficient therapeutic target. This study retrospectively reviewed 109 patients undergoing liver transplantation, with dynamic profiling of CD3/4/8/16/19/45/56 on the peripheral immune cells (before transplant and 2-4 days after). Altogether, 35 out of the 109 patients developed EAD after liver transplantation. We observed a significant decrease in the natural killer cell proportion (NK cell shift, p = 0.008). The NK cell shift was linearly correlated with cold ischemic time (p = 0.016) and was potentially related to the recipients' outcomes. In mouse models, ischemic/reperfusion (I/R) treatments induced the recruitment of NK cells from peripheral blood into liver tissues. NK cell depletion blocked a series of immune cascades (including CD8+ CD127+ T cells) and inhibited hepatocyte injury effectively in I/R and liver transplantation models. We further found that I/R treatment increased hepatic expression of the ligands for natural killer group 2 member D (NKG2D), a primary activating cell surface receptor in NK cells. Blockade of NKG2D showed a similar protective effect against I/R injury, indicating its role in NK cell activation and the subsequent immunological injury. Our findings built a bridge for the translation from innate immune response to EAD at the bedside. Peripheral NK cell shift is associated with the incidence of EAD after liver transplantation. NKG2D-mediated NK cell activation is a potential therapeutic target.

ATRA sensitized the response of hepatocellular carcinoma to Sorafenib by downregulation of p21-activated kinase 1.

BACKGROUND: Sorafenib resistance greatly reduces the efficacy of treatments in advanced hepatocellular carcinoma (HCC) patients, but the underlying mechanisms are not thoroughly understood. All-trans retinoic acid (ATRA), an anti-leukaemia agent, has attracted considerable attention due to its role in sensitizing cells to other anticancer treatments. We aimed to investigate the combined effect of ATRA and Sorafenib on HCC and the underlying mechanisms. METHODS: CCK-8, cell sphere formation, trans-well migration, and wound-healing assays were used to analyse the biological behaviours of HCC cells in vitro. Western blotting and qRT-PCR analysis were conducted to measure the expression of p21 activated kinase 1 (PAK1) and phospho-p21 activated kinase 1 (pPAK1). Xenograft models were established to confirm the synergistic effects of ATRA and Sorafenib in vivo. TUNEL assays and immunohistochemistry were utilized to determine apoptosis, proliferation, PAK1 and pPAK1 levels in tumour tissues. RESULTS: We observed that PAK1 was overexpressed in HCC, and its expression was negatively correlated with the survival of patients. PAK1 promoted the proliferation, self-renewal and epithelial-mesenchymal transition of HCC cells. Correlation analysis indicated that the IC50 of Sorafenib was positively correlated with the level of pPAK1 in HCC cell lines. ATRA inhibited the progression of HCC and sensitized HCC response to Sorafenib by downregulation of PAK1, as shown by the calculated coefficient of drug interaction and the data obtained from xenograft models. CONCLUSIONS: Our findings indicated that instead of treatment with Sorafenib alone, the combination of ATRA and Sorafenib provides a more effective treatment for HCC patients. Video Abstract.

TLR4 Overexpression Aggravates Bacterial Lipopolysaccharide-Induced Apoptosis via Excessive Autophagy and NF-κB/MAPK Signaling in Transgenic Mammal Models.

Gram-negative bacterial infections pose a significant threat to public health. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and induces innate immune responses, autophagy, and cell death, which have major impacts on the body's physiological homeostasis. However, the role of TLR4 in bacterial LPS-induced autophagy and apoptosis in large mammals, which are closer to humans than rodents in many physiological characteristics, remains unknown. So far, few reports focus on the relationship between TLR, autophagy, and apoptosis in large mammal levels, and we urgently need more tools to further explore their crosstalk. Here, we generated a TLR4-enriched mammal model (sheep) and found that a high-dose LPS treatment blocked autophagic degradation and caused strong innate immune responses and severe apoptosis in monocytes/macrophages of transgenic offspring. Excessive accumulation of autophagosomes/autolysosomes might contribute to LPS-induced apoptosis in monocytes/macrophages of transgenic animals. Further study demonstrated that inhibiting TLR4 downstream NF-κB or p38 MAPK signaling pathways reversed the LPS-induced autophagy activity and apoptosis. These results indicate that the elevated TLR4 aggravates LPS-induced monocytes/macrophages apoptosis by leading to lysosomal dysfunction and impaired autophagic flux, which is associated with TLR4 downstream NF-κB and MAPK signaling pathways. This study provides a novel TLR4-enriched mammal model to study its potential effects on autophagy activity, inflammation, oxidative stress, and cell death. These findings also enrich the biological functions of TLR4 and provide powerful evidence for bacterial infection.

Improving the Efficiency of Precise Genome Editing with CRISPR/Cas9 to Generate Goats Overexpressing Human Butyrylcholinesterase.

The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It is the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated genome-edited primary goat fetal fibroblast cells (GFFs). Improving the double-strand break (DSB) efficiency of Cas9 in primary cells would improve the homologous repair (HR) efficiency. The low efficiency of HR remains a major hurdle in CRISPR/Cas9-mediated precise genome editing, increasing the work required to screen the genome-edited primary cell clones. In this study, we modified several essential parameters that affect the efficiency of the CRISPR/Cas9-mediated knock-in GFF cloning system, including establishing a high-efficiency transfection system for primary cells via nucleofection and optimizing homology arm (HA) length during HR. Here, we specifically inserted a recombinant human butyrylcholinesterase gene (rhBChE) into the goat fibroblast growth factor (FGF)-5 locus through the CRISPR/Cas9 system, thereby achieving simultaneous rhBChE insertion and FGF5 knock-out. First, this study introduced the Cas9, FGF5 knock-out small guide RNA, and rhBChE knock-in donors into GFFs by electroporation and obtained positive cell clones without off-target effects. Then, we demonstrated the expression of rhBChE in GFF clones and verified its function. Finally, we obtained a CRISPR/Cas9-mediated rhBChE-overexpression goat.

Fibroblast growth factor 21 (FGF21) attenuates tacrolimus-induced hepatic lipid accumulation through transcription factor EB (TFEB)-regulated lipophagy. / æˆçº¤ç»´ç»†èƒžç”Ÿé•¿å› å­21通过TFEBä»‹å¯¼çš„è„‚å™¬ç¼“è§£ä»–å ‹èŽ«å¸å¼•èµ·çš„è‚è„è„‚è´¨ç§¯èš.

Tacrolimus (TAC), also called FK506, is one of the classical immunosuppressants to prevent allograft rejection after liver transplantation. However, it has been proved to be associated with post-transplant hyperlipemia. The mechanism behind this is unknown, and it is urgent to explore preventive strategies for hyperlipemia after transplantation. Therefore, we established a hyperlipemia mouse model to investigate the mechanism, by injecting TAC intraperitoneally for eight weeks. After TAC treatment, the mice developed hyperlipemia (manifested as elevated triglyceride (TG) and low-density lipoprotein cholesterol (LDL-c), as well as decreased high-density lipoprotein cholesterol (HDL-c)). Accumulation of lipid droplets was observed in the liver. In addition to lipid accumulation, TAC induced inhibition of the autophagy-lysosome pathway (microtubule-associated protein 1 light chain 3ß (LC3B) II/I and LC3B II/actin ratios, transcription factor EB (TFEB), protein 62 (P62), and lysosomal-associated membrane protein 1 (LAMP1)) and downregulation of fibroblast growth factor 21 (FGF21) in vivo. Overexpression of FGF21 may reverse TAC-induced TG accumulation. In this mouse model, the recombinant FGF21 protein ameliorated hepatic lipid accumulation and hyperlipemia through repair of the autophagy-lysosome pathway. We conclude that TAC downregulates FGF21 and thus exacerbates lipid accumulation by impairing the autophagy-lysosome pathway. Recombinant FGF21 protein treatment could therefore reverse TAC-caused lipid accumulation and hypertriglyceridemia by enhancing autophagy.

Reproduction and viscera organ characteristics of MSTN and FGF5 dual-gene knockout sheep.

Introduction: Myostatin (MSTN) negatively regulates skeletal muscle development. However, its function in reproductive performance and visceral organs has not been thoroughly investigated. Previously, we prepared a MSTN and fibroblast growth factor 5 (FGF5) double-knockout sheep, which was a MSTN and FGF5 dual-gene biallelic homozygous (MF-/-) mutant. Methods: To understand the role of MSTN and FGF5 in reproductive performance and visceral organs, this study evaluated the ejaculation amount, semen pH, sperm motility, sperm density, acrosome integrity, rate of teratosperm, and seminal plasma biochemical indicators in adult MF-/- rams. We also compared the overall morphology, head, head-neck junction, middle segment and the transection of middle segment of spermatozoa between wildtype (WT) and MF-/- rams. Results: Our results showed that the seminal plasma biochemical indicators, sperm structure and all sperm indicators were normal, and the fertilization rate also has no significant difference between WT and MF-/- rams, indicating that the MF-/- mutation did not affect the reproductive performance of sheep. Additional analysis evaluated the histomorphology of the visceral organs, digestive system and reproductive system of MF+/- sheep, the F1 generation of MF-/-, at the age of 12 months. There was an increased spleen index, but no significant differences in the organ indexes of heart, liver, lung, kidney and stomach, and no obvious differences in the histomorphology of visceral organs, digestive system and reproductive system in MF+/- compared with WT sheep. No MF+/- sheep were observed to have any pathological features. Discussion: In summary, the MSTN and FGF5 double-knockout did not affect reproductive performance, visceral organs and digestive system in sheep except for differences previously observed in muscle and fat. The current data provide a reference for further elucidating the application of MSTN and FGF5 double-knockout sheep.

Global Long Noncoding RNA Expression Profiling of MSTN and FGF5 Double-Knockout Sheep Reveals the Key Gatekeepers of Skeletal Muscle Development.

Improving livestock and poultry growth rates and increasing meat production are urgently needed worldwide. Previously, we produced a myostatin (MSTN) and fibroblast growth factor 5 (FGF5) double-knockout (MF-/-) sheep by CRISPR Cas9 system to improve meat production, and also wool production. Both MF-/- sheep and the F1 generation (MF+/-) sheep showed an obvious "double-muscle" phenotype. In this study, we identified the expression profiles of long noncoding RNAs (lncRNAs) in wild-type and MF+/- sheep, then screened out the key candidate lncRNAs that can regulate myogenic differentiation and skeletal muscle development. These key candidate lncRNAs can serve as critical gatekeepers for muscle contraction, calcium ion transport and skeletal muscle cell differentiation, apoptosis, autophagy, and skeletal muscle inflammation, further revealing that lncRNAs play crucial roles in regulating muscle phenotype in MF+/- sheep. In conclusion, our newly identified lncRNAs may emerge as novel molecules for muscle development or muscle disease and provide a new reference for MSTN-mediated regulation of skeletal muscle development.

Shifts in intestinal microbiota and improvement of sheep immune response to resist Salmonella infection using Toll-like receptor 4 (TLR4) overexpression.

Introduction: Toll-like receptor 4 (TLR4) identifies Gram-negative bacteria or their products and plays a crucial role in host defense against invading pathogens. In the intestine, TLR4 recognizes bacterial ligands and interacts with the immune system. Although TLR4 signaling is a vital component of the innate immune system, the influence of TLR4 overexpression on innate immune response and its impact on the composition of the intestinal microbiota is unknown. Methods: Here, we obtained macrophages from sheep peripheral blood to examine phagocytosis and clearance of Salmonella Typhimurium (S. Typhimurium) in macrophages. Meanwhile, we characterized the complex microbiota inhabiting the stools of TLR4 transgenic (TG) sheep and wild-type (WT) sheep using 16S ribosomal RNA (rRNA) deep sequencing. Results: The results showed that TLR4 overexpression promoted the secretion of more early cytokines by activating downstream signaling pathways after stimulation by S. Typhimurium. Furthermore, diversity analysis demonstrated TLR4 overexpression increased microbial community diversity and regulated the composition of intestinal microbiota. More importantly, TLR4 overexpression adjusted the gut microbiota composition and maintained intestinal health by reducing the ratio of Firmicutes/Bacteroidetes and inflammation and oxidative stress-producing bacteria (Ruminococcaceae, Christensenellaceae) and upregulating the abundance of Bacteroidetes population and short-chain fatty acid (SCFA)-producing bacteria, including Prevotellaceae. These dominant bacterial genera changed by TLR4 overexpression revealed a close correlation with the metabolic pathways of TG sheep. Discussion: Taken together, our findings suggested that TLR4 overexpression can counteract S. Typhimurium invasion as well as resist intestinal inflammation in sheep by regulating intestinal microbiota composition and enhancing anti-inflammatory metabolites.

The Role of m6A Modifications in B-Cell Development and B-Cell-Related Diseases.

B cells are a class of professional antigen-presenting cells that produce antibodies to mediate humoral immune response and participate in immune regulation. m6A modification is the most common RNA modification in mRNA; it involves almost all aspects of RNA metabolism and can affect RNA splicing, translation, stability, etc. This review focuses on the B-cell maturation process as well as the role of three m6A modification-related regulators-writer, eraser, and reader-in B-cell development and B-cell-related diseases. The identification of genes and modifiers that contribute to immune deficiency may shed light on regulatory requirements for normal B-cell development and the underlying mechanism of some common diseases.

MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

CRISPR-Cas9-mediated genome editing in sheep is of great use in both agricultural and biomedical applications. While targeted gene knockout by CRISPR-Cas9 through non-homologous end joining (NHEJ) has worked efficiently, the knockin efficiency via homology-directed repair (HDR) remains lower, which severely hampers the application of precise genome editing in sheep. Here, in sheep fetal fibroblasts (SFFs), we optimized several key parameters that affect HDR, including homology arm (HA) length and the amount of double-stranded DNA (dsDNA) repair template; we also observed synchronization of SFFs in G2/M phase could increase HDR efficiency. Besides, we identified three potent small molecules, RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, that caused activation of the p53 pathway, resulting in distinct G2/M cell-cycle arrest in response to DNA damage and improved CRISPR-Cas9-mediated HDR efficiency by 1.43- to 4.28-fold in SFFs. Furthermore, we demonstrated that genetic knockout of p53 could inhibit HDR in SFFs by suppressing the expression of several key factors involved in the HDR pathway, such as BRCA1 and RAD51. Overall, this study offers an optimized strategy for the usage of dsDNA repair template, more importantly, the application of MDM2 antagonists provides a simple and efficient strategy to promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

SULT2B1-CS-DOCK2 axis regulates effector T-cell exhaustion in HCC microenvironment.

BACKGROUND AND AIMS: HCC is a malignant disease. Compared with tyrosine kinase inhibitors (the classical therapy), immune checkpoint inhibitors are more effective in the treatment of HCC, despite their limited efficacy. Among these restricted factors, exhaustion of tumor-infiltrated lymphocytes, especially CD8 + T cells, is a core event. We aimed to determine the key factors contributing to CD8 + T-cell infiltration in HCC and investigate the underlying mechanisms. APPROACH AND RESULTS: Using machine learning and multiplex immunohistochemistry analysis, we showed that dedicator of cytokinesis protein 2 (DOCK2) was a potential indicator of infiltrated CD8 + T cells in HCC. Using RNA sequencing, flow cytometry analysis, and mouse HCC models, we demonstrated that DOCK2 inactivation accounted for infiltrated CD8 + T-cell exhaustion in tumors. Using quasi-targeted metabolomics, mass spectrum, and mass cytometry by time of flight analysis, we found that cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells suppressed DOCK2 enzymatic activity of T cells. Through virtual screening, molecular docking simulation, and experiments validation, we demonstrated that tolazamide reversed DOCK2 inactivation-mediated CD8 + T-cell exhaustion and enhanced anti-programmed death-ligand 1 antibody+apatinib immunotherapeutic effects on HCC. CONCLUSIONS: This study indicates that DOCK2 controls CD8 + T-cell infiltration in HCC, and cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells promotes effector T-cell exhaustion. The findings suggest that the usage of conventional drugs affects immunotherapy efficacy in HCC patients.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned, ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer.

BACKGROUND: SCNT (somatic cell nuclear transfer) is of great significance to biological research and also to the livestock breeding. However, the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods. This indicates the potential epigenetic variations between them. DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences. In this study, ASMT (acetylserotonin-O-methyltransferase) ovarian overexpression transgenic goat was produced by using SCNT. To investigate whether there are epigenetic differences between cloned and WT (wild type) goats, WGBS (whole-genome bisulfite sequencing) was used to measure the whole-genome methylation of these animals. RESULTS: It is observed that the different mCpG sites are mainly present in the intergenic and intronic regions between cloned and WT animals, and their CG-type methylation sites are strongly correlated. DMR (differentially methylated region) lengths are located around 1000 bp, mainly distributed in the exonic, intergenic and intronic functional domains. A total of 56 and 36 DMGs (differentially methylated genes) were identified by GO and KEGG databases, respectively. Functional annotation showed that DMGs were enriched in biological-process, cellular-component, molecular-function and other signaling pathways. A total of 10 identical genes related to growth and development were identified in GO and KEGG databases. CONCLUSION: The differences in methylation genes among the tested animals have been identified. A total of 10 DMGs associated with growth and development were identified between cloned and WT animals. The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT. These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology, particularly in goats.

Toll-like receptor (TLRs) agonists and antagonists for COVID-19 treatments.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) rapidly infects humans and animals which make coronavirus disease 2019 (COVID-19) a grievous epidemic worldwide which broke out in 2020. According to data analysis of the other coronavirus family, for instance severe acute respiratory syndrome SARS coronavirus (SARS-CoV), can provide experience for the mutation of SARS-CoV-2 and the prevention and treatment of COVID-19. Toll-like receptors (TLRs) as a pattern recognition receptor (PRRs), have an indispensable function in identifying the invader even activate the innate immune system. It is possible for organism to activate different TLR pathways which leads to secretion of proinflammatory cytokines such as Interleukin 1 (IL-1), Interleukin 6 (IL-6), Tumor necrosis factor α (TNFα) and type Ⅰ interferon. As a component of non-specific immunity, TLRs pathway may participate in the SARS-CoV-2 pathogenic processes, due to previous works have proved that TLRs are involved in the invasion and infection of SARS-CoV and MERS to varying degrees. Different TLR, such as TLR2, TLR4, TLR7, TLR8 and TLR9 probably have a double-sided in COVID-19 infection. Therefore, it is of great significance for a correctly acknowledging how TLR take part in the SARS-CoV-2 pathogenic processes, which will be the development of treatment and prevention strategies.

Skeletal muscle oxidative stress and inflammation in aging: Focus on antioxidant and anti-inflammatory therapy.

With aging, the progressive loss of skeletal muscle will have negative effect on multiple physiological parameters, such as exercise, respiration, thermoregulation, and metabolic homeostasis. Accumulating evidence reveals that oxidative stress and inflammation are the main pathological characteristics of skeletal muscle during aging. Here, we focus on aging-related sarcopenia, summarize the relationship between aging and sarcopenia, and elaborate on aging-mediated oxidative stress and oxidative damage in skeletal muscle and its critical role in the occurrence and development of sarcopenia. In addition, we discuss the production of excessive reactive oxygen species in aging skeletal muscle, which reduces the ability of skeletal muscle satellite cells to participate in muscle regeneration, and analyze the potential molecular mechanism of ROS-mediated mitochondrial dysfunction in aging skeletal muscle. Furthermore, we have also paid extensive attention to the possibility and potential regulatory pathways of skeletal muscle aging and oxidative stress mediate inflammation. Finally, in response to the abnormal activity of oxidative stress and inflammation during aging, we summarize several potential antioxidant and anti-inflammatory strategies for the treatment of sarcopenia, which may provide beneficial help for improving sarcopenia during aging.

Molecular Regulation of Androgen Receptors in Major Female Reproductive System Cancers.

There are three main types of cancer in the female reproductive system, specifically ovarian cancer (OVCA), endometrial cancer (EC), and cervical cancer (CC). They are common malignant tumors in women worldwide, with high morbidity and mortality. In recent years, androgen receptors (ARs) have been found to be closely related to the occurrence, progression, prognosis, and drug resistance of these three types of tumors. This paper summarizes current views on the role of AR in female reproductive system cancer, the associations between female reproductive system cancers and AR expression and polymorphisms. AR regulates the downstream target genes transcriptional activity and the expression via interacting with coactivators/corepressors and upstream/downstream regulators and through the gene transcription mechanism of "classical A/AR signaling" or "non-classical AR signaling", involving a large number of regulatory factors and signaling pathways. ARs take part in the processes of cancer cell proliferation, migration/invasion, cancer cell stemness, and chemotherapeutic drug resistance. These findings suggest that the AR and related regulators could target the treatment of female reproductive system cancer.

The Role of RNA Modification in HIV-1 Infection.

RNA plays an important role in biology, and more than 170 RNA modifications have been identified so far. Post-transcriptional modification of RNA in cells plays a crucial role in the regulation of its stability, transport, processing, and gene expression. So far, the research on RNA modification and the exact role of its enzymes is becoming more and more comprehensive. Human immunodeficiency virus 1 (HIV-1) is an RNA virus and the causative agent of acquired immunodeficiency syndrome (AIDS), which is one of the most devastating viral pandemics in history. More and more studies have shown that HIV has RNA modifications and regulation of its gene expression during infection and replication. This review focuses on several RNA modifications and their regulatory roles as well as the roles that different RNA modifications play during HIV-1 infection, in order to find new approaches for the development of anti-HIV-1 therapeutics.