The role of mitochondrial dynamics and mitophagy in skeletal muscle atrophy: from molecular mechanisms to therapeutic insights.

Skeletal muscle is the largest metabolic organ of the human body. Maintaining the best quality control and functional integrity of mitochondria is essential for the health of skeletal muscle. However, mitochondrial dysfunction characterized by mitochondrial dynamic imbalance and mitophagy disruption can lead to varying degrees of muscle atrophy, but the underlying mechanism of action is still unclear. Although mitochondrial dynamics and mitophagy are two different mitochondrial quality control mechanisms, a large amount of evidence has indicated that they are interrelated and mutually regulated. The former maintains the balance of the mitochondrial network, eliminates damaged or aged mitochondria, and enables cells to survive normally. The latter degrades damaged or aged mitochondria through the lysosomal pathway, ensuring cellular functional health and metabolic homeostasis. Skeletal muscle atrophy is considered an urgent global health issue. Understanding and gaining knowledge about muscle atrophy caused by mitochondrial dysfunction, particularly focusing on mitochondrial dynamics and mitochondrial autophagy, can greatly contribute to the prevention and treatment of muscle atrophy. In this review, we critically summarize the recent research progress on mitochondrial dynamics and mitophagy in skeletal muscle atrophy, and expound on the intrinsic molecular mechanism of skeletal muscle atrophy caused by mitochondrial dynamics and mitophagy. Importantly, we emphasize the potential of targeting mitochondrial dynamics and mitophagy as therapeutic strategies for the prevention and treatment of muscle atrophy, including pharmacological treatment and exercise therapy, and summarize effective methods for the treatment of skeletal muscle atrophy.

The potential of organoids in renal cell carcinoma research.

Renal cell carcinoma, a leading cause of death in urological malignancies, arises from the nephron. Its characteristics include diversity in disease biology, varied clinical behaviors, different prognoses, and diverse responses to systemic therapies. The term 'organoids' is used to describe structures resembling tissues created through the three-dimensional cultivation of stem cells in vitro. These organoids, when derived from tumor tissues, can retain the diversity of the primary tumor, mirror its spatial tissue structure, and replicate similar organ-like functions. In contrast to conventional two-dimensional cell cultures and the transplantation of tumor tissues into other organisms, organoids derived from tumors maintain the complexity and microenvironment of the original tumor tissue. This fidelity makes them a more reliable model for the development of cancer drugs, potentially accelerating the translation of these drugs to clinical use and facilitating personalized treatment options for patients. This review aims to summarize the recent advancements in the use of organoids for studying renal cell carcinoma, focusing on their cultivation, potential applications, and inherent limitations.

Splicing factor TRA2A contributes to esophageal cancer progression via a noncanonical role in lncRNA m6 A methylation.

Transformer 2 alpha homolog (TRA2A), a member of the serine/arginine-rich splicing factor family, has been shown to control mRNA splicing in development and cancers. However, it remains unclear whether TRA2A is involved in lncRNA regulation. In the present study, we found that TRA2A was upregulated and correlated with poor prognosis in esophageal cancer. Downregulation of TRA2A suppressed the tumor growth in xenograft nude mice. Epitranscriptomic microarray showed that depletion of TRA2A affected global lncRNA methylation similarly to the key m6 A methyltransferase, METTL3, by silencing. MeRIP-qPCR, RNA pull-down, CLIP analyses, and stability assays indicated that ablation of TRA2A reduced m6 A-modification of the oncogenic lncRNA MALAT1, thus inducing structural alterations and reduced stability. Furthermore, Co-IP experiments showed TRA2A directly interacted with METTL3 and RBMX, which also affected the writer KIAA1429 expression. Knockdown of TRA2A inhibited cell proliferation in a manner restored by RBMX/KIAA1429 overexpression. Clinically, MALAT1, RBMX, and KIAA1429 were prognostic factors of worse survival in ESCA patients. Structural similarity-based virtual screening in FDA-approved drugs repurposed nebivolol, a ß1 -adrenergic receptor antagonist, as a potent compound to suppress the proliferation of esophageal cancer cells. Cellular thermal shift and RIP assay indicated that nebivolol may compete with MALAT1 to bind TRA2A. In conclusion, our study revealed the noncanonical function of TRA2A, which coordinates with multiple methylation proteins to promote oncogenic MALAT1 during ESCA carcinogenesis.

Aberrant expression of CKS2 induced by ELK1 contributes to malignant progression of pancreatic cancer.

Cyclin-dependent kinase subunit 2 (CKS2) has been reported to promote various malignancies. This study investigated the functional role of CKS2 in pancreatic cancer (PC). An analysis of abnormally expressed genes and their prognostic value for PC was performed by using the Gene Expression Profiling Interactive Analysis (GEPIA) database and performing immunohistochemical staining on 64 samples of tumor tissue. CCK-8 assays, EdU staining, colony formation assays, flow cytometry, and a xenograft tumor model were used to analyze the biological function of CKS2 in PC. Our results revealed that CKS2 was expressed at significantly higher levels in PC tissues than in adjacent normal tissues, and a high level of CKS2 expression was associated with a poor prognosis for patients with PC. Moreover, functional assays revealed that CKS2 knockdown suppressed cell proliferation, induced cell cycle S phase, G2/M phase arrest, and apoptosis in vitro, and also reduced tumor growth in vivo. In addition, CKS2 knockdown increased the levels of Bax, caspase-3, P53, P21, and GADD45α expression, but decreased Bcl-2, Cyclin B1, CDK1, Cyclin A, and Cdc25C expression. CKS2 overexpression produced the opposite effects of CKS2 knockdown. Furthermore, we found that ELK1 protein regulated transcription of the CKS2 gene. In conclusion, our findings suggest that CKS2 expression is regulated by ELK1, which could possibly serve as prognostic indicator and therapeutic target for PC.

miRNAs derived from cobra venom exosomes contribute to the cobra envenomation.

Currently, there is an increasing amount of evidence indicating that exosomes and the miRNAs they contain are crucial players in various biological processes. However, the role of exosomes and miRNAs in snake venom during the envenomation process remains largely unknown. In this study, fresh venom from Naja atra of different ages (2-month-old, 1-year-old, and 5-year-old) was collected, and exosomes were isolated through ultracentrifugation. The study found that exosomes with inactivated proteins and enzymes can still cause symptoms similar to cobra envenomation, indicating that substances other than proteins and enzymes in exosomes may also play an essential role in cobra envenomation. Furthermore, the expression profiles of isolated exosome miRNAs were analyzed. The study showed that a large number of miRNAs were co-expressed and abundant in cobra venom exosomes (CV-exosomes) of different ages, including miR-2904, which had high expression abundance and specific sequences. The specific miR-2094 derived from CV-exosomes (CV-exo-miR-2904) was overexpressed both in vitro and in vivo. As a result, CV-exo-miR-2904 induced symptoms similar to cobra envenomation in mice and caused liver damage, demonstrating that it plays a crucial role in cobra envenomation. These results reveal that CV-exosomes and the miRNAs they contain play a significant regulatory role in cobra envenomation. Our findings provide new insights for the treatment of cobra bites and the development of snake venom-based medicines.

A Novel tRNA-Derived Fragment, tRFGlnCTG, Regulates Angiogenesis by Targeting Antxr1 mRNA.

As a novel non-coding RNA with important functions corresponding to various cellular stresses, the function of tRFs in angiogenesis remains unclear. Firstly, small RNA sequencing was performed on normal and post-muscle injury mouse tibialis anterior muscle to identify and analyse differentially expressed tRF/tiRNA. tRNA GlnCTG-derived fragments (tRFGlnCTG) were found to be overexpressed in high abundance in the damaged muscle. Subsequent in vitro experiments revealed that the overexpression of tRFGlnCTG suppressed the vascular endothelial cells' viability, cell cycle G1/S transition, proliferation, migration, and tube-formation capacity. Similarly, in vivo experiments showed that the tRFGlnCTG decreased the relative mRNA levels of vascular endothelial cell markers and pro-angiogenic factors and reduced the proportion of CD31-positive cells. Finally, luciferase activity analysis confirmed that the tRFGlnCTG directly targeted the 3'UTR of Antxr1, leading to a significant reduction in the mRNA expression of the target gene. These results suggest that tRFGlnCTG is a key regulator of vascular endothelial cell function. The results provide a new idea for further exploration of the molecular mechanisms that regulate angiogenesis.

Gentamicin Induces Laminin 332 and Improves Wound Healing in Junctional Epidermolysis Bullosa Patients with Nonsense Mutations.

Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal autosomal recessively inherited blistering skin disease caused by mutations in the LAMA3, LAMB3, or LAMC2 genes. Most of these mutations are nonsense mutations that create premature termination codons that lead to impaired production of functional laminin 332, a protein needed for epidermal-dermal adherence. Gentamicin induces readthrough of nonsense mutations and restores the full-length protein in various genetic diseases. Using primary keratinocytes from three GS-JEB patients, we showed that gentamicin induced functional laminin 332 that reversed a JEB-associated, abnormal cell phenotype. In a subsequent open-label trial involving the same patients, we examined whether 0.5% gentamicin ointment applied topically to open skin wounds could promote nonsense mutation readthrough and create new laminin 332 in the patients' skin. Gentamicin-treated wounds exhibited increased expression of laminin 332 at the dermal-epidermal junction for at least 3 months and were associated with improved wound closure. There were no untoward side effects from topical gentamicin. The newly induced laminin 332 did not generate anti-laminin 332 autoantibodies in either the patients' blood or skin. Gentamicin readthrough therapy may be a treatment for GS-JEB patients with nonsense mutations.

PARP14 promotes the proliferation and gemcitabine chemoresistance of pancreatic cancer cells through activation of NF-κB pathway.

Pancreatic cancer (PC) is the most fatal gastrointestinal malignancy in the world, with a 5-year relative survival of only 8%. Poly(ADP-ribose) polymerase (PARP)14, a member of the macro-PARP subfamily proteins, has been reported to participate in various biologic and pathologic processes in multiple cancers. The roles and underlying molecular mechanisms of PARP14 in PC carcinogenesis, however, remain to be elucidated. In this study, we for the first time discovered that PARP14 was highly expressed in human primary PC specimens and significantly correlated with poor patient prognosis. Using loss-of-function studies in vitro and in vivo, we showed that the knockdown of PARP14 led to enhanced apoptosis, repressed proliferation, and gemcitabine (GEM) resistance of PC cells. Further investigations revealed that PARP14 was significantly overexpressed in GEM-resistant PC cells (SW1990/GZ). And silencing of PARP14 significantly reversed the GEM resistance of SW1990/GZ cells. To the mechanism, PARP14 could stimulate PC progression by the activation of nuclear factor-κB (NF-κB) signaling pathway. And inhibition of NF-κB signal could significantly reverse PARP14-overexpression triggered PC carcinogenesis. In conclusion, PARP14 could promote PC cell proliferation, antiapoptosis, and GEM resistance via NF-κB signaling pathway, highlighting its potential role as a therapeutic target for PC.

Diagnosis and treatment of solid-pseudopapillary tumors of the pancreas: A single institution experience with 97 cases.

BACKGROUND/OBJECTIVES: We evaluated the diagnoses and surgical management of solid pseudopapillary tumors of the pancreas (SPTP) in a single center setting. METHODS: Demographic details, clinical presentations, imaging features, surgical strategies, and pathological findings of 97 consecutive patients who underwent surgery for pathologically confirmed SPTP between 2008 and 2016 were analyzed retrospectively. RESULTS: A total of 97 patients with SPTP accounted for 2.15% of total pancreatic surgeries (N = 4508). The mean age at diagnosis was 31.6 ±â€¯13.92 years (range: 7-83 years), and males:females were 85:12. Chief complaints were abdominal pain or discomfort (n = 50). The mean transverse tumor diameter was 53.2 ±â€¯2.76 mm (range: 14-170 mm). Tumors were localized to the pancreatic head (38.1%; 37/97), neck (12.4%; 12/97), body and tail (49.5%; 48/97). There were no significant differences in age of onset, presenting symptoms, and sites between males and females. A significant difference was found in the maximum diameter of SPTP across different age groups. Partial pancreatectomy was performed for patient with tumor ≥ 5 cm, and enucleation was performed when tumor was smaller than 5 cm. Pancreatic fistula was the main complication. Immunohistochemical markers for endocrine, exocrine and epithelial factors were used for a final diagnosis. During a median follow-up of 54 months (range: 7-121 months), 84 patients were alive without evidence of recurrence, and 13 patients were lost to follow-up. CONCLUSION: SPTP surgical resection is a safe procedure with low morbidity and mortality, which is also effective even in the presence of invasiveness and metastases. Accurate initial diagnoses and follow up are essential.

Predictors of distant metastasis on exploration in patients with potentially resectable pancreatic cancer.

BACKGROUND: Patients with potentially resectable pancreatic ductal adenocarcinoma (PDAC) are frequently found to be unresectable on exploration due to small distant metastasis. This study was to investigate predictors of small distant metastasis in patients with potentially resectable PDAC. METHODS: Patients who underwent surgical exploration for potentially resectable PDAC from 2013 to 2014 were reviewed retrospectively and divided into two groups according to whether distant metastases were encountered on exploration. Then, univariate and multivariate logistic regression analyses were used to identify predictors of distant metastasis. A scoring system to predict distant metastasis of PDAC on exploration was constructed based on the regression coefficient of a multivariate logistic regression model. RESULTS: A total of 235 patients were included in this study. Mean age of the study population was 61.7 ± 10.4 years old. Upon exploration, distant metastases were found intraoperatively in 62 (26.4%) patients, while the remaining 173 were free of distant metastases. Multivariate logistic regression analysis identified that age ≤ 62 years old (p < 0.001), male sex (p = 0.011), tumor size ≥4.0 cm (p < 0.001), alanine aminotransferase level (ALT) < 125 U/L (p < 0.001), and carbohydrate antigen (CA19-9) level ≥ 385 U/mL (p < 0.001) were independent risk factors for occult distant metastasis of PDAC. A preoperative scoring system (0-8 points) for distant metastasis on exploration was constructed using these five factors. The receiver operating characteristic curves showed that the area under the curve of this score was 0.85. A score of 6 points was suggested to be the optimal cut-off value, and the sensitivity and specificity were 85% and 69%, respectively. CONCLUSIONS: Distant metastasis is still frequently encountered on exploration for patients with potentially resectable PDAC. Younger age, male sex, larger tumor size, low ALT level and high CA19-9 level are independent predictors of unexpected distant metastasis on exploration.

Elastic parameter identification of three-dimensional soft tissue based on deep neural network.

In the field of virtual surgery and deformation simulation, the identification of elastic parameters of human soft tissues is a critical technology that directly affects the accuracy of deformation simulation. Current research on soft tissue deformation simulation predominantly assumes that the elasticity of tissues is fixed and already known, leading to the difficulty in populating with the elasticity measured or identified from specific tissues of real patients. Existing elasticity modeling efforts struggle to be implemented on irregularly structured soft tissues, failing to adapt to clinical surgical practices. Therefore, this paper proposes a new method for identifying human soft tissue elastic parameters based on the finite element method and the deep neural network, UNet. This method requires only the full-field displacement data of soft tissues under external loads to predict their elastic distribution. The performance and validity of the algorithm are assessed using test data and clinical data from rhinoplasty surgeries. Experiments demonstrate that the method proposed in this paper can achieve an accuracy of over 99% in predicting elastic parameters. Clinical data validation shows that the predicted elastic distribution can reduce the error in finite element deformation simulations by more than 80% at the maximum compared to the error with traditional uniform elastic parameters, effectively enhancing the computational accuracy in virtual surgery simulations and soft tissue deformation modeling.

Advances in tumor microenvironment and underlying molecular mechanisms of bladder cancer: a systematic review.

Bladder cancer is one of the most frequent malignant tumors of the urinary system. The prevalence of bladder cancer among men and women is roughly 5:2, and both its incidence and death have been rising steadily over the past few years. At the moment, metastasis and recurrence of advanced bladder cancer-which are believed to be connected to the malfunction of multigene and multilevel cell signaling network-remain the leading causes of bladder cancer-related death. The therapeutic treatment of bladder cancer will be greatly aided by the elucidation of these mechanisms. New concepts for the treatment of bladder cancer have been made possible by the advancement of research technologies and a number of new treatment options, including immunotherapy and targeted therapy. In this paper, we will extensively review the development of the tumor microenvironment and the possible molecular mechanisms of bladder cancer.

LIM domain only 7: a novel driver of immune evasion through regulatory T cell differentiation and chemotaxis in pancreatic ductal adenocarcinoma.

With advancements in genomics and immunology, immunotherapy has emerged as a revolutionary strategy for tumor treatment. However, pancreatic ductal adenocarcinoma (PDAC), an immunologically "cold" tumor, exhibits limited responsiveness to immunotherapy. This study aimed to address the urgent need to uncover PDAC's immune microenvironment heterogeneity and identify the molecular mechanisms driving immune evasion. Using single-cell RNA sequencing datasets and spatial proteomics, we discovered LIM domain only 7 (LMO7) in PDAC cells as a previously unrecognized driver of immune evasion through Treg cell enrichment. LMO7 was positively correlated with infiltrating regulatory T cells (Tregs) and dysfunctional CD8+ T cells. A series of in vitro and in vivo experiments demonstrated LMO7's significant role in promoting Treg cell differentiation and chemotaxis while inhibiting CD8+ T cells and natural killer cell cytotoxicity. Mechanistically, LMO7, through its LIM domain, directly bound and promoted the ubiquitination and degradation of Foxp1. Foxp1 negatively regulated transforming growth factor-beta (TGF-ß) and C-C motif chemokine ligand 5 (CCL5) expression by binding to sites 2 and I/III, respectively. Elevated TGF-ß and CCL5 levels contribute to Treg cell enrichment, inducing immune evasion in PDAC. Combined treatment with TGF-ß/CCL5 antibodies, along with LMO7 inhibition, effectively reversed immune evasion in PDAC, activated the immune response, and prolonged mouse survival. Therefore, this study identified LMO7 as a novel facilitator in driving immune evasion by promoting Treg cell enrichment and inhibiting cytotoxic effector functions. Targeting the LMO7-Foxp1-TGF-ß/CCL5 axis holds promise as a therapeutic strategy for PDAC. Graphical abstract revealing LMO7 as a novel facilitator in driving immune evasion by promoting Tregs differentiation and chemotaxis, inducing CD8+ T/natural killer cells inhibition.

Tumor-resident microbiota contributes to colorectal cancer liver metastasis by lactylation and immune modulation.

The role of tumor-resident microbiota in modulating tumor immunity remains unclear. Here, we discovered an abundance of intra-tumoral bacteria, such us E.coli, residing and resulting in Colorectal cancer liver metastasis (CRLM). E.coli enhanced lactate production, which mediated M2 macrophage polarization by suppressing nuclear factor-κB -gene binding (NF-κB) signaling through retinoic acid-inducible gene 1 (RIG-I) lactylation. Lactylation of RIG-I suppressed recruitment of NF-κB to the Nlrp3 promoter in macrophages, thereby reducing its transcription. This loss of Nlrp3 affected the immunosuppressive activities of regulatory T cells (Tregs) and the antitumor activities of and CD8+ T cells. Small-molecule compound screening identified a RIG-I lactylation inhibitor that suppressed M2 polarization and sensitized CRLM to 5-fluorouracil (5-FU). Our findings suggest that tumor-resident microbiota may be a potential target for preventing and treating CRLM.

Lactate supports Treg function and immune balance via MGAT1 effects on N-glycosylation in the mitochondria.

Current research reports that lactate affects Treg metabolism, although the precise mechanism has only been partially elucidated. In this study, we presented evidence demonstrating that elevated lactate levels enhanced cell proliferation, suppressive capabilities, and oxidative phosphorylation (OXPHOS) in human Tregs. The expression levels of Monocarboxylate Transporters 1/2/4 (MCT1/2/4) regulate intracellular lactate concentration, thereby influencing the varying responses observed in naive Tregs and memory Tregs. Through mitochondrial isolation, sequencing, and analysis of human Tregs, we determined that Alpha-1,3-Mannosyl-Glycoprotein 2-Beta-N-Acetylglucosaminyltransferase (MGAT1) served as the pivotal driver initiating downstream N-glycosylation events involving progranulin (GRN) and hypoxia-upregulated 1 (HYOU1), consequently enhancing Treg OXPHOS. The mechanism by which MGAT1 was upregulated in mitochondria depended on elevated intracellular lactate that promoted the activation of XBP1s, which, in turn, supported MGAT1 transcription as well as the interaction of lactate with the translocase of the mitochondrial outer membrane 70 (TOM70) import receptor, facilitating MGAT1 translocation into mitochondria. Pre-treatment of Tregs with lactate reduced mortality in a xenogeneic graft-versus-host disease (GvHD) model. Together, these findings underscored the active regulatory role of lactate in human Treg metabolism through the upregulation of MGAT1 transcription and its facilitated translocation into the mitochondria.

The global perspective on peroxisome proliferator-activated receptor γ (PPARγ) in ectopic fat deposition: A review.

Excessive expansion of adipocytes can have unhealthy consequences as excess free fatty acids enter other tissues and cause ectopic fat deposition by resynthesizing triglycerides. This lipid accumulation in various tissues is harmful and can increase the risk of related metabolic diseases such as type II diabetes, cardiovascular disease, and insulin resistance. Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that play a key role in energy metabolism as fatty acid metabolism sensors, and peroxisome proliferator-activated receptor γ (PPARγ) is the main subtype responsible for fat cell differentiation and adipogenesis. In this paper, we introduce the main structure and function of PPARγ and its regulatory role in the process of lipogenesis in the liver, kidney, skeletal muscle, and pancreas. This information can serve as a reference for further understanding the regulatory mechanisms and measures of the PPAR family in the process of ectopic fat deposition.

tsRNA Landscape and Potential Function Network in Subcutaneous and Visceral Pig Adipose Tissue.

Noncoding RNAs (ncRNAs) called tsRNAs (tRNA-derived short RNAs) have the ability to regulate gene expression. The information on tsRNAs in fat tissue is, however, limited. By sequencing, identifying, and analyzing tsRNAs using pigs as animal models, this research reports for the first time the characteristics of tsRNAs in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). A total of 474 tsRNAs, 20 and 21 of which were particularly expressed in VAT and SAT, respectively, were found in WAT. According to the analysis of the tsRNA/miRNA/mRNA co-expression network, the tsRNAs with differential expression were primarily engaged in the endocrine and immune systems, which fall under the classification of organic systems, as well as the global and overview maps and lipid metropolis, which fall under the category of metabolism. This research also discovered a connection between the activity of the host tRNA engaged in translation and the production of tsRNAs. This research also discovered that tRF-Gly-GCC-037/tRF-Gly-GCC-042/tRF-Gly-CCC-016 and miR-218a/miR281b may be involved in the regulation of fatty acid metabolism in adipose tissue through SCD based on the tsRNA/miRNA/mRNA/fatty acid network. In conclusion, our findings enrich the understanding of ncRNAs in WAT metabolism and health regulation, as well as reveal the differences between SAT and VAT at the level of tsRNAs.

tRNA-derived small RNA, 5'tiRNA-Gly-CCC, promotes skeletal muscle regeneration through the inflammatory response.

BACKGROUND: Increasing evidence shows that tRNA-derived small RNAs (tsRNAs) are not only by-products of transfer RNAs, but they participate in numerous cellular metabolic processes. However, the role of tsRNAs in skeletal muscle regeneration remains unknown. METHODS: Small RNA sequencing revealed the relationship between tsRNAs and skeletal muscle injury. The dynamic expression level of 5'tiRNA-Gly after muscle injury was confirmed by real-time quantitative PCR (q-PCR). In addition, q-PCR, flow cytometry, the 5-ethynyl-2'-deoxyuridine (Edu), cell counting kit-8, western blotting and immunofluorescence were used to explore the biological function of 5'tiRNA-Gly. Bioinformatics analysis and dual-luciferase reporter assay were used to further explore the mechanism of action under the biological function of 5'tiRNA-Gly. RESULTS: Transcriptome analysis revealed that tsRNAs were significantly enriched during inflammatory response immediately after muscle injury. Interestingly, we found that 5'tiRNA-Gly was significantly up-regulated after muscle injury (P < 0.0001) and had a strong positive correlation with inflammation in vivo. In vitro experiments showed that 5'tiRNA-Gly promoted the mRNA expression of proinflammatory cytokines (IL-1ß, P = 0.0468; IL-6, P = 0.0369) and the macrophages of M1 markers (TNF-α, P = 0.0102; CD80, P = 0.0056; MCP-1, P = 0.0002). On the contrary, 5'tiRNA-Gly inhibited the mRNA expression of anti-inflammatory cytokines (IL-4, P = 0.0009; IL-10, P = 0.0007; IL-13, P = 0.0008) and the mRNA expression of M2 markers (TGF-ß1, P = 0.0016; ARG1, P = 0.0083). Flow cytometry showed that 5'tiRNA-Gly promoted the percentage of CD86+ macrophages (16%, P = 0.011) but inhibited that of CD206+ macrophages (10.5%, P = 0.012). Immunofluorescence showed that knockdown of 5'tiRNA-Gly increased the infiltration of M2 macrophages to the skeletal muscles (13.9%, P = 0.0023) and inhibited the expression of Pax7 (P = 0.0089) in vivo. 5'tiRNA-Gly promoted myoblast the expression of myogenic differentiation marker genes (MyoD, P = 0.0002; MyoG, P = 0.0037) and myotube formation (21.3%, P = 0.0016) but inhibited the positive rate of Edu (27.7%, P = 0.0001), cell viability (22.6%, P = 0.003) and the number of myoblasts in the G2 phase (26.3%, P = 0.0016) in vitro. Mechanistically, we found that the Tgfbr1 gene is a direct target of 5'tiRNA-Gly mediated by AGO1 and AGO3. 5'tiRNA-Gly dysregulated the expression of downstream genes related to inflammatory response, activation of satellite cells and differentiation of myoblasts through the TGF-ß signalling pathway by targeting Tgfbr1. CONCLUSIONS: These results reveal that 5'tiRNA-Gly potentially regulated skeletal muscle regeneration by inducing inflammation via the TGF-ß signalling pathway. The findings of this study uncover a new potential target for skeletal muscle regeneration treatment.

Protocol for in vitro isolation, induction, expansion, and determination of human natural regulatory T cells and induced regulatory T cells.

Regulatory T cells (Tregs) can inhibit the occurrence of autoimmune diseases and increase the activation threshold of the immune response. Here, we present schemes for the isolation and culture of natural human regulatory T cells (nTregs) and in vitro-induced Tregs (iTregs). Appropriate concentrations of TGF-ß, IL-2, retinoic acid (atRA), and rapamycin were used to promote proliferation to meet sample needs in basic research, especially in technologies such as sequencing. For complete details on the use and execution of this protocol, please refer to Lu et al. (2014a) and Gu et al. (2022).