NSUN2 mediates distinct pathways to regulate enterovirus 71 replication.

Increasing evidences suggest that the methyltransferase NSUN2 catalyzes 5-methylcytosine (m5C) modifications on viral RNAs, which are essential for the replication of various viruses. Despite the function of m5C deposition is well characterized, other potential roles of NSUN2 in regulating viral replication remain largely unknown. In this study, the m5C modified residues catalyzed by NSUN2 on enterovirus 71 (EV71) RNAs were mapped. NSUN2, along with m5C modifications, played multiple roles during the EV71 life cycle. Functional m5C modified nucleotides increased the translational efficiency and stability of EV71 RNAs. Additionally, NSUN2 was found to target the viral protein VP1 for binding and promote its stability by inhibiting the ubiquitination. Furthermore, both viral replication and pathogenicity in mice were largely attenuated when functional m5C residues were mutated. Taken together, this study characterizes distinct pathways mediated by NSUN2 in regulating EV71 replication, and highlights the importance of its catalyzed m5C modifications on EV71 RNAs for the viral replication and pathogenicity.

Applications of Engineered Skin Tissue for Cosmetic Component and Toxicology Detection.

The scale of the cosmetic market is increasing every day. There are many safety risks to cosmetics, but they benefit people at the same time. The skin can become red, swollen, itchy, chronically toxic, and senescent due to the misuse of cosmetics, triggering skin injuries, with contact dermatitis being the most common. Therefore, there is an urgent need for a system that can scientifically and rationally detect the composition and perform a toxicological assessment of cosmetic products. Traditional detection methods rely on instrumentation and method selection, which are less sensitive and more complex to perform. Engineered skin tissue has emerged with the advent of tissue engineering technology as an emerging bioengineering technology. The ideal engineered skin tissue is the basis for building good in vitro structures and physiological functions in this field. This review introduces the existing cosmetic testing and toxicological evaluation methods, the current development status, and the types and characteristics of engineered skin tissue. The application of engineered skin tissue in the field of cosmetic composition detection and toxicological evaluation, as well as the different types of tissue engineering scaffold materials and three-dimensional (3D) organoid preparation approaches, is highlighted in this review to provide methods and ideas for constructing the next engineered skin tissue for cosmetic raw material component analysis and toxicological evaluation.

NS1-mediated enhancement of MVC transcription and replication promoted by KAT5/H4K12ac.

Histone modifications function in both cellular and viral gene expression. However, the roles of acetyltransferases and histone acetylation in parvoviral infection remain poorly understood. In the current study, we found the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), promoted the replication and transcription of parvovirus minute virus of canines (MVC). Notably, the expression of host acetyltransferases KAT5, GTF3C4, and KAT2A was increased in MVC infection, as well as H4 acetylation (H4K12ac). KAT5 is not only responsible for H4K12ac but also crucial for viral replication and transcription. The viral nonstructural protein NS1 interacted with KAT5 and enhanced its expression. Further study showed that Y44 in KAT5, which may be tyrosine-phosphorylated, is indispensable for NS1-mediated enhancement of KAT5 and efficient MVC replication. The data demonstrated that NS1 interacted with KAT5, which resulted in an enhanced H4K12ac level to promote viral replication and transcription, implying the epigenetic addition of H4K12ac in viral chromatin-like structure by KAT5 is vital for MVC replication.IMPORTANCEParvoviral genomes are chromatinized with host histones. Therefore, histone acetylation and related acetyltransferases are required for the virus to modify histones and open densely packed chromatin structures. This study illustrated that histone acetylation status is important for MVC replication and transcription and revealed a novel mechanism that the viral nonstructural protein NS1 hijacks the host acetyltransferase KAT5 to enhance histone acetylation of H4K12ac, which relies on a potential tyrosine phosphorylation site, Y44 in KAT5. Other parvoviruses share a similar genome organization and coding potential and may adapt a similar strategy for efficient viral replication and transcription.

Epigenetic addition of m5C to HBV transcripts promotes viral replication and evasion of innate antiviral responses.

Eukaryotic five-methylcytosine (m5C) is an important regulator of viral RNA splicing, stability, and translation. However, its role in HBV replication remains largely unknown. In this study, functional m5C sites are identified in hepatitis B virus (HBV) mRNA. The m5C modification at nt 1291 is not only indispensable for Aly/REF export factor (ALYREF) recognition to promote viral mRNA export and HBx translation but also for the inhibition of RIG-I binding to suppress interferon-ß (IFN-ß) production. Moreover, NOP2/Sun RNA methyltransferase 2 (NSUN2) catalyzes the addition of m5C to HBV mRNA and is transcriptionally downregulated by the viral protein HBx, which suppresses the binding of EGR1 to the NSUN2 promoter. Additionally, NSUN2 expression correlates with m5C modification of type I IFN mRNA in host cells, thus, positively regulating IFN expression. Hence, the delicate regulation of NSUN2 expression induces m5C modification of HBV mRNA while decreasing the levels of m5C in host IFN mRNA, making it a vital component of the HBV life cycle. These findings provide new molecular insights into the mechanism of HBV-mediated IFN inhibition and may inform the development of new IFN-α based therapies.

Predictive factors that influence the clinical efficacy of umbilical cord-derived mesenchymal stromal cells in the treatment of type 2 diabetes mellitus.

BACKGROUND: Our previous single-center, randomized, double-blinded, placebo-controlled phase 2 study evaluated the safety and effectiveness of human umbilical cord mesenchymal stromal cell (UC-MSC) transfusion for treating patients with type 2 diabetes mellitus (T2DM). Indeed, this potential treatment strategy was able to reduce insulin use by half in a considerable number of patients. However, many other patients' responses to UC-MSC transfusion were insignificant. The selection of patients who might benefit from UC-MSC treatment is crucial from a clinical standpoint. METHODS: In this post hoc analysis, 37 patients who received UC-MSC transfusions were divided into two groups based on whether their glycated hemoglobin (hemoglobin A1c, or HbA1c) level was less than 7% after receiving UC-MSC treatment. The baseline differences between the two groups were summarized, and potential factors influencing efficacy of UC-MSCs for T2DM were analyzed by univariate and multivariate logistic regression. The correlations between the relevant hormone levels and the treatment effect were further analyzed. RESULTS: At the 9-week follow-up, 59.5% of patients achieved their targeted HbA1c level. Male patients with lower baseline HbA1c and greater C-peptide area under the curve (AUCC-pep) values responded favorably to UC-MSC transfusion, according to multivariate analysis. The effectiveness of UC-MSCs transfusion was predicted by AUCC-pep (cutoff value: 14.22 ng/h/mL). Further investigation revealed that AUCC-pep was increased in male patients with greater baseline testosterone levels. CONCLUSIONS: Male patients with T2DM with greater AUCC-pep may be more likely to respond clinically to UC-MSC therapy, and further large-scale multi-ethnic clinical studies should be performed to confirm the conclusion.

Efficacy of Umbilical Cord-Derived Mesenchymal Stem Cells in the Treatment of Type 2 Diabetes Assessed by Retrospective Continuous Glucose Monitoring.

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been proved a promising clinical strategy for the treatment of diabetes, and time in range (TIR) has been demonstrated a new metric of glycemic control links to diabetes complications. To further assess the therapeutic effect of UC-MSCs on TIR, a phase II study investigating the efficacy of UC-MSCs in Chinese adults with type 2 diabetes (T2D) assessed by retrospective continuous glucose monitoring (CGM) was conducted. In this randomized and placebo-controlled trial, a total of 73 patients were randomly assigned to receive intravenous infusion of UC-MSCs (n = 37) or placebo (n = 36) 3 times at 4-week intervals and followed up for 48 weeks. The primary endpoint was the changes in TIR and glycosylated hemoglobin (HbA1c). TIR and HbA1c were both significantly improved in UC-MSCs and placebo groups after 48 weeks of therapy compared with baseline. Compared with placebo group, UC-MSCs group exhibited more pronounced changes at 9 and 48 weeks from baseline in TIR (26.54 vs. 15.84 and 21.36 vs. 6.32) and HbA1c (-1.79 vs. -0.96 and -1.36 vs. -0.51). More patients in UC-MSCs group achieved the glycemic control target of TIR ≥ 70% and HbA1c < 7% at 9 and 48 weeks than in placebo group (59.5% vs. 27.8% and 43.2% vs. 11.1%). The C-peptide area under the curve (AUCC-pep) was an independent risk factor associated with efficacy in T2D undergoing UC-MSCs intervention. These results illustrate that UC-MSCs administration via intravenous infusion is an effective approach for ameliorating TIR.

The Role of Mesenchymal Stem/Stromal Cells Secretome in Macrophage Polarization: Perspectives on Treating Inflammatory Diseases.

Mesenchymal stem/stromal cells (MSCs) have exhibited potential for treating multiple inflammation- related diseases (IRDs) due to their easy acquisition, unique immunomodulatory and tissue repair properties, and immune-privileged characteristics. It is worth mentioning that MSCs release a wide array of soluble bioactive components in the secretome that modulate host innate and adaptive immune responses and promote the resolution of inflammation. As the first line of defense, macrophages exist throughout the entire inflammation process. They continuously switch their molecular phenotypes accompanied by complementary functional regulation ranging from classically activated pro-inflammatory M1-type (M1) to alternatively activated anti-inflammatory M2-type macrophages (M2). Recent studies have shown that the active intercommunication between MSCs and macrophages is indispensable for the immunomodulatory and regenerative behavior of MSCs in pharmacological cell therapy products. In this review, we systematically summarized the emerging capacities and detailed the molecular mechanisms of the MSC-derived secretome (MSC-SE) in immunomodulating macrophage polarization and preventing excessive inflammation, providing novel insights into the clinical applications of MSC-based therapy in IRD management.

Human umbilical cord mesenchymal stromal cell small extracellular vesicle transfer of microRNA-223-3p to lung epithelial cells attenuates inflammation in acute lung injury in mice.

BACKGROUND: Acute lung injury (ALI), manifested as strong pulmonary inflammation and alveolar epithelial damage, is a life-threatening disease with high morbidity and mortality. Small extracellular vesicles (sEVs), secreted by multiple types of cells, are critical cellular communication mediators and can inhibit inflammation by transferring bioactive molecules, such as microRNAs (miRNAs). Thus, we hypothesized that sEVs derived from mesenchymal stromal cells (MSC sEVs) could transfer miRNAs to attenuate inflammation of lung epithelial cells during ALI. METHODS: C57BL/6 male mice were intratracheally administered LPS (10 mg/kg). Six hours later, the mice were randomly administered with MSC sEVs (40 µg per mouse in 150 µl of saline), which were collected by ultracentrifugation. Control group received saline administration. After 48 h, the mice were sacrificed to evaluate pulmonary microvascular permeability and inflammatory responses. In vitro, A549 cells and primary human small airway epithelial cells (SAECs) were stimulated with LPS with or without MSC sEVs treatment. RESULTS: In vitro, MSC sEVs could also inhibit the inflammation induced by LPS in A549 cells and SAECs (reducing TNF-α, IL-1ß, IL-6 and MCP-1). Moreover, MSC sEV treatment improved the survival rate, alleviated pulmonary microvascular permeability, and inhibited proinflammatory responses (reducing TNF-α, IL-1ß, IL-6 and JE-1) in ALI mice. Notably, miR-223-3p was found to be served as a critical mediator in MSC sEV-induced regulatory effects through inhibition of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) in lung epithelial cells. CONCLUSIONS: Overall, these findings suggest that MSC sEVs may offer a novel promising strategy for ALI.

Bone mesenchymal stromal cell-derived small extracellular vesicles inhibit inflammation and ameliorate sepsis via delivery of microRNA-21a-5p.

BACKGROUND AIMS: Sepsis is a potentially life-threatening disease that results from a severe systemic inflammatory response due to infection. Mesenchymal stromal cell-derived small extracellular vesicles (MSC sEVs) are able to transfer bioactive molecules and have been demonstrated to play an important role in the pathophysiological process of sepsis. Herein the authors aimed to investigate the potential role and downstream molecular mechanism of MSC sEVs in sepsis. METHODS: MSC sEVs were acquired by ultracentrifugation and then injected into a cecal ligation and puncture mouse model. The efficacy of MSC sEVs in both in vitro and in vivo models of sepsis was evaluated. RESULTS: MSC sEV therapy improved survival, reduced sepsis-induced inflammation, attenuated pulmonary capillary permeability and improved liver and kidney function in septic mice. In addition, the authors found that microRNA-21a-5p (miR-21a-5p) was highly enriched in MSC sEVs, could be transferred to recipient cells, inhibited inflammation and increased survival in septic mice. Furthermore, the authors demonstrated that MSC sEV miR-21a-5p suppressed inflammation by targeting toll-like receptor 4 and programmed cell death 4. The therapeutic efficacy of MSC sEVs was partially abrogated by transfection with miR-21a-5p inhibitors. CONCLUSIONS: Collectively, the authors' data suggest that miR-21a-5p-bearing MSC sEVs may be a prospective and effective sepsis therapeutic strategy.

N4-acetylcytidine regulates the replication and pathogenicity of enterovirus 71.

Chemical modifications are important for RNA function and metabolism. N4-acetylcytidine (ac4C) is critical for the translation and stability of mRNA. Although ac4C is found in RNA viruses, the detailed mechanisms through which ac4C affects viral replication are unclear. Here, we reported that the 5' untranslated region of the enterovirus 71 (EV71) genome was ac4C modified by the host acetyltransferase NAT10. Inhibition of NAT10 and mutation of the ac4C sites within the internal ribosomal entry site (IRES) suppressed EV71 replication. ac4C enhanced viral RNA translation via selective recruitment of PCBP2 to the IRES and boosted RNA stability. Additionally, ac4C increased the binding of RNA-dependent RNA polymerase (3D) to viral RNA. Notably, ac4C-deficient mutant EV71 showed reduced pathogenicity in vivo. Our findings highlighted the essential role of ac4C in EV71 infection and provided insights into potential antiviral treatments.

Exosomes Derived From Umbilical Cord Mesenchymal Stem Cells Treat Cutaneous Nerve Damage and Promote Wound Healing.

Wound repair is a key step in the treatment of skin injury caused by burn, surgery, and trauma. Various stem cells have been proven to promote wound healing and skin regeneration as candidate seed cells. Therefore, exosomes derived from stem cells are emerging as a promising method for wound repair. However, the mechanism by which exosomes promote wound repair is still unclear. In this study, we reported that exosomes derived from umbilical cord mesenchymal stem cells (UC-MSCs) promote wound healing and skin regeneration by treating cutaneous nerve damage. The results revealed that UC-MSCs exosomes (UC-MSC-Exo) promote the growth and migration of dermal fibroblast cells. In in vitro culture, dermal fibroblasts could promote to nerve cells and secrete nerve growth factors when stimulated by exosomes. During the repair process UC-MSC-Exo accelerated the recruitment of fibroblasts at the site of trauma and significantly enhanced cutaneous nerve regeneration in vivo. Interestingly, it was found that UC-MSC-Exo could promote wound healing and skin regeneration by recruiting fibroblasts, stimulating them to secrete nerve growth factors (NGFs) and promoting skin nerve regeneration. Therefore, we concluded that UC-MSC-Exo promote cutaneous nerve repair, which may play an important role in wound repair and skin regeneration.

Efficacy and safety of umbilical cord-derived mesenchymal stem cells in Chinese adults with type 2 diabetes: a single-center, double-blinded, randomized, placebo-controlled phase II trial.

BACKGROUND: To determine the efficacy and safety of umbilical cord-derived mesenchymal stem cells (UC-MSCs) in Chinese adults with type 2 diabetes mellitus (T2DM). METHODS: In this single-center, double-blinded, randomized, placebo-controlled phase II trial, 91 patients were randomly assigned to receive intravenous infusion of UC-MSCs (n = 45) or placebo (n = 46) three times with 4-week intervals and followed up for 48 weeks from October 2015 to December 2018. The primary endpoint was the percentage of patients with glycated hemoglobin (HbA1c) levels of < 7.0% and daily insulin reduction of ≥ 50% at 48 weeks. Additional endpoints were changes of metabolic control, islet ß-cell function, insulin resistance, and safety. RESULTS: At 48 weeks, 20% of the patients in the UC-MSCs group and 4.55% in the placebo group reached the primary endpoint (p < 0.05, 95% confidence interval (CI) 2.25-28.66%). The percentage of insulin reduction of the UC-MSCs group was significantly higher than that of the placebo group (27.78% versus 15.62%, p < 0.05). The levels of HbA1c decreased 1.31% (9.02 ± 1.27% to 7.52 ± 1.07%, p < 0.01) in the UC-MSCs group, and only 0.63% in the placebo group (8.89 ± 1.11% to 8.19 ± 1.02%, p˃0.05; p = 0.0081 between both groups). The glucose infusion rate (GIR) increased significantly in the UC-MSCs group (from 3.12 to 4.76 mg/min/kg, p < 0.01), whereas no significant change was observed in the placebo group (from 3.26 to 3.60 mg/min/kg, p ˃ 0.05; p < 0.01 between both groups). There was no improvement in islet ß-cell function in both groups. No major UC-MSCs transplantation-related adverse events occurred. CONCLUSIONS: UC-MSCs transplantation could be a potential therapeutic approach for Chinese adults with T2DM. Trial registration This study was registered on ClinicalTrials.gov (identifier: NCT02302599).

Hypoxic culture of umbilical cord mesenchymal stem cell-derived sEVs prompts peripheral nerve injury repair.

Introduction: Repair and regeneration of the peripheral nerve are important for the treatment of peripheral nerve injury (PNI) caused by mechanical tears, external compression injuries and traction injuries. Pharmacological treatment can promote the proliferation of fibroblasts and Schwann cells (SCs), which longitudinally fill the endoneurial canal and form Bungner's band, helping the repair of peripheral nerves. Therefore, the development of new drugs for the treatment of PNI has become a top priority in recent years. Methods: Here, we report that small extracellular vesicles (sEVs) produced from umbilical cord mesenchymal stem cells (MSC-sEVs) cultured under hypoxia promote repair and regeneration of the peripheral nerve in PNI and may be a new therapeutic drug candidate. Results: The results showed that the amount of secreted sEVs was significantly increased in UC-MSCs compared with control cells after 48 h of culture at 3% oxygen partial pressure in a serum-free culture system. The identified MSC-sEVs could be taken up by SCs in vitro, promoting the growth and migration of SCs. In a spared nerve injury (SNI) mouse model, MSC-sEVs accelerated the recruitment of SCs at the site of PNI and promoted peripheral nerve repair and regeneration. Notably, repair and regeneration in the SNI mouse model were enhanced by treatment with hypoxic cultured UC-MSC-derived sEVs. Discussion: Therefore, we conclude that hypoxic cultured UC-MSC-derived sEVs may be a promising candidate drug for repair and regeneration in PNI.

Liraglutide inhibits the progression of prediabetes in rats by reducing Raf-1 kinase inhibitor protein.

BACKGROUND: The cleavage product of Raf-1 kinase inhibitor protein (RKIP), hippocampal cholinergic neurostimulating peptide (HCNP) is involved in the promotion of insulin secretion. Studies have shown that liraglutide can inhibit the progression of prediabetes. This study aims to investigate whether the above effects of liraglutide are related to RKIP and HCNP. METHODS: Insulin-1 (INS-1) cells were divided into control group (CON), HCNP group, and HCNP + darifenacin group (H-DAR). The three groups were cultured with Roswell Park Memorial Institute (RPMI) 1640, synthetic HCNP (50 pg/mL) and RPMI 1640, and HCNP + RPMI 1640 + darifenacin respectively. Subsequently, twelve 12- to 14-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were randomly divided into 2 groups: the placebo group (PBO) and the liraglutide treatment group (LIRA). Six Long Evans Tokushima Otsuka (LETO) rats were used as the control group (CON). The LIRA group was given liraglutide 200 µg/kg intraperitoneally twice a day. After 12 weeks, body weight, fasting blood glucose, 2 hours postprandial blood glucose, and insulin resistance index were recorded. Western blot was used to detect expression level of C-RKIP, N-RKIP, and extracellular signal-regulated kinase of phosphorylation (p-ERK). Real-time quantitative polymerase chain reaction (qRT-PCR) to detect pancreatic tissue choline acetyltransferase (ChAT) and M3 cholinergic receptor (M3R) gene expression levels. RESULTS: At glucose concentrations of 5.6 and 16.7 mmol/L, the insulin content in the HCNP group was higher than that in the CON and H-DAR groups (all P<0.01). The body weight and fasting serum insulin (FINS) of rats in the PBO group were higher than those in the LIRA group and the CON group (P<0.01). The relative content of C-RKIP protein in the PBO group was higher than that in the LIRA and CON groups (P<0.01). The relative content of N-RKIP protein and p-ERK protein was lower than that in the LIRA and CON group (P<0.05 and P<0.01, respectively). ChAT and M3R gene expression levels in PBO group were lower than those in LIRA and CON group (P<0.01). CONCLUSIONS: Liraglutide promotes the production of HCNP, can increase ChAT activity, activate M3R, and further promote the secretion of insulin.

Anti-obesity effect and mechanism of mesenchymal stem cells influence on obese mice.

Mesenchymal stem cells (MSCs) can be obtained from almost all tissues and present promising therapeutic effects for metabolic diseases. Human adipose-derived MSCs (hASCs) have recently been widely studied due to their easy access and low immunity. Thus, we intended to figure out the effects and potential mechanism of hASCs on obesity in high-fat-diet (HFD)-induced obese mice. Following 16 weeks of being fed HFD, hASCs were intravenously injected. Two weeks later, body weight, body composition, and energy expenditure were evaluated. Additionally, the phenotypes of macrophages infiltrating adipose tissue were analyzed. The results revealed that hASCs administration significantly reduced adipose tissue weight, adipocyte size, and fat mass and exerted beneficial effects in serum lipid profile. This anti-obesity effect was mediated by the increased O2 consumption, CO2 production, and energy expenditure, which was further evidenced by the upregulation of uncoupling protein-1 (UCP-1) and metabolism-associated genes. Furthermore, hASCs infusion increased the amount of alternatively activated (M2) macrophages in adipose tissue, and the expression of pro-inflammatory cytokines-related genes was reduced. Taken together, these results indicated that hASCs suppressed obesity by increasing UCP-1 expression and enhancing energy expenditure, and this effect might be due to the increased M2 macrophages.

Human mesenchymal stromal cells small extracellular vesicles attenuate sepsis-induced acute lung injury in a mouse model: the role of oxidative stress and the mitogen-activated protein kinase/nuclear factor kappa B pathway.

BACKGROUND AIMS: Acute lung injury (ALI) secondary to sepsis is a complex disease associated with high morbidity and mortality. Mesenchymal stem cells (MSCs) and their conditioned medium have been demonstrated to reduce alveolar inflammation, improve lung endothelial barrier permeability and modulate oxidative stress in vivo and in vitro. Recently, MSCs have been found to release small extracellular vesicles (sEVs) that can deliver functionally active biomolecules into recipient cells. The authors' study was designed to determine whether sEVs released by MSCs would be effective in sepsis-induced ALI mice and to identify the potential mechanisms. METHODS: A total of 6 h after cercal ligation and puncture, the mice received saline, sEV-depleted conditioned medium (sEVD-CM) or MSC sEVs via the tail vein. RESULTS: The administration of MSC sEVs improved pulmonary microvascular permeability and inhibited both histopathological changes and the infiltration of polymorphonuclear neutrophils into lung tissues. In addition, the activities of antioxidant enzymes were significantly increased in the group treated with sEVs compared with the saline and sEVD-CM groups, whereas lipid peroxidation was significantly decreased. Furthermore, sEVs were found to possibly inhibit phosphorylation of the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) pathway and degradation of IκB but increase the activities of nuclear factor erythroid 2-related factor 2 and heme oxygenase 1. CONCLUSIONS: These findings suggest that one of the effective therapeutic mechanisms of sEVs against sepsis-induced ALI may be associated with upregulation of anti-oxidative enzymes and inhibition of MAPK/NF-κB activation.

Methyltransferase-like 3 Modulates Severe Acute Respiratory Syndrome Coronavirus-2 RNA N6-Methyladenosine Modification and Replication.

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an ongoing global public crisis. Although viral RNA modification has been reported based on the transcriptome architecture, the types and functions of RNA modification are still unknown. In this study, we evaluated the roles of RNA N6-methyladenosine (m6A) modification in SARS-CoV-2. Our methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and Nanopore direct RNA sequencing (DRS) analysis showed that SARS-CoV-2 RNA contained m6A modification. Moreover, SARS-CoV-2 infection not only increased the expression of methyltransferase-like 3 (METTL3) but also altered its distribution. Modification of METTL3 expression by short hairpin RNA or plasmid transfection for knockdown or overexpression, respectively, affected viral replication. Furthermore, the viral key protein RdRp interacted with METTL3, and METTL3 was distributed in both the nucleus and cytoplasm in the presence of RdRp. RdRp appeared to modulate the sumoylation and ubiquitination of METTL3 via an unknown mechanism. Taken together, our findings demonstrated that the host m6A modification complex interacted with viral proteins to modulate SARS-CoV-2 replication. IMPORTANCE Internal chemical modifications of viral RNA play key roles in the regulation of viral replication and gene expression. Although potential internal modifications have been reported in SARS-CoV-2 RNA, the function of the SARS-CoV-2 N6-methyladenosine (m6A) modification in the viral life cycle is unclear. In the current study, we demonstrated that SARS-CoV-2 RNA underwent m6A modification by host m6A machinery. SARS-CoV-2 infection altered the expression pattern of methyltransferases and demethylases, while the expression level of methyltransferase-like 3 (METTL3) and fat mass and obesity-associated protein (FTO) was linked to the viral replication. Further study showed that METTL3 interacted with viral RNA polymerase RNA-dependent RNA polymerase (RdRp), which influenced not only the distribution but also the posttranslational modification of METTL3. Our study provided evidence that host m6A components interacted with viral proteins to modulate viral replication.

Combined Treatment with Bone Marrow-Derived Mesenchymal Stem Cells and Exendin-4 Promotes Islet Regeneration in Streptozotocin-Induced Diabetic Rats.

This study was designed to assess whether the combination of the glucagon-like peptide-1 (GLP-1) analog exendin-4 (Ex4) and bone marrow-derived mesenchymal stem cell (BM-MSC) could enhance ß-cell action in streptozotocin (STZ)-induced diabetic rats. Forty male Sprague-Dawley rats were randomly assigned to five groups: the normal control group (Normal), diabetes mellitus (DM) group, MSC-treated group (MSC), Ex4-treated group (Ex4), and MSC plus Ex4-treated group (MSC+Ex4). Body weight, blood glucose level, intraperitoneal glucose tolerance test, and in vitro glucose-stimulated insulin secretion were used to assess the treatment efficacy. The expression level of insulin, glucagon, pancreatic duodenal homeobox-1 (PDX-1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), glucagon-like peptide-1 receptor (GLP-1R), and forkhead transcription factor 1 (FoxO1) was estimated by immunofluorescence analysis. Proliferation was assessed by Ki67 staining, and apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in ß-cells. Glucose-induced insulin secretion in the MSC+Ex4 group was significantly increased compared to that in the MSC group in vitro and in vivo. Compared to that of the other groups, the number of insulin-immunopositive cells was increased in both the MSC and MSC+Ex4 groups. However, ß-cell proliferation and apoptosis in the MSC group and MSC+Ex4 group were not significantly different. Importantly, the expression level of PDX-1, MafA, FoxO1, and GLP-1R in ß-cells in the MSC+Ex4 group was significantly higher than those in the MSC group. The numbers of insulin+ glucagon+ double positive cells and glucagon+ GLP-1+ double positive cells were significantly increased after MSC treatment and MSC+Ex4 combined treatment, suggesting the enhanced function of newly formed islet ß-cells. Our findings showed that the combination of MSC and Ex4 enhanced the function of newly formed ß-cells in STZ-induced diabetic rats by acting on multiple insulin transcription factors. Thus, combined MSC and Ex4 therapy provides a feasible approach for future diabetes treatments.

Low-Dose Decitabine Assists Human Umbilical Cord-Derived Mesenchymal Stem Cells in Protecting ß Cells via the Modulation of the Macrophage Phenotype in Type 2 Diabetic Mice.

BACKGROUND: Progressive ß-cell dysfunction, a major characteristic of type 2 diabetes (T2D), is closely related to the infiltration of inflammatory macrophages within islets. Mesenchymal stem cells (MSCs) have been identified to alleviate ß-cell dysfunction by modulating macrophage phenotype in T2D, but the restoration of ß-cells by a single MSC infusion is relatively transient. Decitabine (DAC) has been reported to polarize macrophages towards the anti-inflammatory phenotype at low doses. We therefore investigated whether low-dose decitabine could enhance the antidiabetic effect of MSCs and further promote the restoration of ß-cell function. METHODS: We induced a T2D mice model by high-fat diets and streptozotocin (STZ) injection. Mice were divided into five groups: the normal group, the T2D group, the DAC group, the MSC group, and the MSC plus DAC group (MD group). We examined the blood glucose and serum insulin levels of mice 1, 2, and 4 weeks after MSC and/or DAC treatment. Dynamic changes in islets and the phenotype of intraislet macrophages were detected via immunofluorescence. In vitro, we explored the effect of MSCs and DAC on macrophage polarization. RESULTS: The blood glucose and serum insulin levels revealed that DAC prolonged the antidiabetic effect of MSCs to 4 weeks in T2D mice. Immunofluorescence staining demonstrated more sustainable morphological and structural amelioration in islets of the MD group than in the MSC group. Interestingly, further analysis showed more alternatively activated macrophages (M2, anti-inflammatory) and fewer classically activated macrophages (M1, proinflammatory) in islets of the MD group 4 weeks after treatment. An in vitro study demonstrated that DAC together with MSCs further polarized macrophages from the M1 to M2 phenotype via the PI3K/AKT pathway. CONCLUSION: These data unveiled that DAC prolonged the antidiabetic effect of MSCs and promoted sustainable ß-cell restoration, possibly by modulating the macrophage phenotype. Our results offer a preferable therapeutic strategy for T2D.

Berberine: A Traditional Natural Product With Novel Biological Activities.

CONTEXT: Having been used for thousands of years to treat gastrointestinal diseases, the natural isoquinoline alkaloid, berberine, has exhibited a wide spectrum of biochemical and pharmacological effects in studies of recent years. OBJECTIVE: The review intended to examine the many novel bioactivities of berberine, including antidiabetic, anticancer, neuroprotective, anti-inflammatory, and anti-atherosclerotic actions. DESIGN: The research team searched the MEDLINE database using PubMed, using different keyword combinations, including berberine AND diabetes, berberine AND cancer, berberine AND (neuron OR brain), berberine AND inflammation, and "berberine AND atherosclerosis to find studies evaluating the various effects exerted berberine. CONCLUSION: Berberine is a promising multipotent agent to combat diabetes, cancer, Alzheimer's disease, and other diseases.