Ubiquitin-mediated Proteolysis Research Articles

Analyzing immune cell infiltrates in skeletal muscle of infantile-onset Pompe disease using bioinformatics and machine learning

Pompe disease, a severe lysosomal storage disorder, is marked by heart problems, muscle weakness, and respiratory difficulties. This study aimed to identify novel markers for infantile-onset Pompe disease by analyzing key genes and immune cells infiltrating affected skeletal muscles, building on existing research linking its progression to the immune cell infiltration. The datasets GSE38680 and GSE159062 were downloaded and differential expression genes (DEGs) were identified. Potential markers were screened using support vector machine recursive feature elimination (SVM-RFE) analysis and least absolute shrinkage and selection operator (LASSO) regression models. To analyze DEGs and immune processes, 22 immune cell types in affected tissues were examined using CIBERSORT. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment in the calcium signaling pathway, phosphatidylinositol signaling system, ubiquitin-mediated proteolysis and JAK-STAT signaling pathway. Machine learning identified GPNMB, CALML6 and TRIM7 as key genes. Immune cell infiltration analysis showed that the expression levels of the above three genes were closely related to immune cells in our target population under study, including T cells regulatory (Tregs), monocytes, macrophages M0, resting and activated dendritic cells, naive and memory B cells. Overall, our study results may provide new clues for exploring markers related to Pompe disease by highlighting key genes and their relationship with immune infiltration, offering insights into the disease’s development. However, previous studies have shown limited evidence of significant immune cell infiltration in muscle biopsies from Pompe patients, and this lack of direct evidence necessitates further investigation and experimental validation in laboratory settings to confirm these associations.

Construction of a Wilms tumor risk model based on machine learning and identification of cuproptosis-related clusters

BackgroundCuproptosis, a recently identified type of programmed cell death triggered by copper, has mechanisms in Wilms tumor (WT) that are not yet fully understood. This research focuses on examining the link between WT and Cuproptosis-related genes (CRGs), with the goal of developing a predictive model for WT.MethodsFour gene expression datasets related to WT were sourced from the GEO database. Subsequently, expression profiles of CRGs were extracted for differential analysis and immune infiltration studies. Utilizing 105 WT samples, clusters related to Cuproptosis were identified. This involved analyzing associated immune cell infiltration and conducting functional enrichment analysis. Disease-characteristic genes were pinpointed using weighted gene co-expression network analysis. Finally, the WT risk prediction model was constructed by four machine learning methods: random forest, support vector machine (SVM), generalized linear and extreme gradient strength model. The best-performing machine learning model was chosen, and a nomogram was created. The effectiveness of this predictive model was validated using methods such as the calibration curve, decision curve analysis, and by appiying it to the TARGET-GTEx dataset.ResultsThirteen differentially expressed Cuproptosis-related genes were identified. The infiltration level of CD8 + T cells in WT children was lower than that in Normal tissue (NT) children, and the level of M0 infiltration of macrophages and T follicular helper cells was higher than that in NT children. In addition, two clusters of cuproptosis-related WT were identified. Enrichment analysis results indicated that genes in cluster 2 were primarily involved in cell division, nuclear division regulation, DNA biosynthesis process, ubiquitin-mediated proteolysis. The SVM model was judged to be the optimal model using 5 genes. Its accuracy was confirmed through a calibration curve and decision curve analysis, demonstrating satisfactory performance on the TARGET-GTEx validation dataset. Additional analysis revealed that these five genes exhibited high expression in both the TARGET-GTEx validation dataset and sequencing data.ConclusionThis research established a link between WT and Cuproptosis. It developed a predictive model for assessing the risk of WT and pinpointed five key genes associated with the disease.

Deciphering Mechanisms of Adipocyte Differentiation in Abdominal Fat of Broilers.

The excessive deposition of abdominal fat tissue (AFT) in broilers has emerged as a major concern in the poultry industry. Despite some progress in recent years, the molecular mechanisms underlying AFT development remain ambiguous. The current study combined RNA-seq with transposase-accessible chromatin sequencing (ATAC-seq) to map the dynamic profiling of chromatin accessibility and transcriptional reprogramming in AFT adipocyte differentiation in broilers at day 3 (D3) and D14. Our results found that the levels of CDK1 and PCNA were down-regulated at D14, D28, and D42 compared to D3, while the levels of C/EBPα and FABP4 were up-regulated at D14 and D42 compared to D3. Meanwhile, PPARγ was significantly up-regulated at D28 and D42. RNA-seq of AFT identified 1705 up-regulated and 1112 down-regulated differential expression genes (DEGs) between D3 and D14. Pathways based on up-regulated DEGs mainly enriched some pathways related to adipocyte differentiation, while down-regulated DEGs pointed to DNA replication, cell cycle, and gap junction. Gene set enrichment analysis (GSEA) revealed that DNA replication and the cell cycle were down-regulated at D14, while the insulin signaling pathway was up-regulated. In the OA-induced immortalized chicken preadipocyte (ICP2) model, protein dynamic changes were consistent with AFT from D3 to D14. Same pathways were enriched in ICP2. In addition, based on overlapped DEGs from AFT and ICP2, enriched pathways related to adipocyte differentiation or proliferation mentioned above were all involved. A total of 1600 gain and 16727 loss differential peaks (DPs) were identified in ICP2 by ATAC-seq. Predicted genes from DPs at the promoter regions were enriched in glycerophospholipid metabolism, TGF-β signaling, FoxO signaling, and ubiquitin-mediated proteolysis. DNA motifs predicted 159 transcription factors (TFs) based on gain and loss peaks from the promoter regions, where 1 and 10 TFs were overlapped with up or down TFs from DEGs. Overall, this study presents a framework for the comprehension of the epigenetic regulatory mechanisms of adipocyte differentiation and identifies candidate genes and potential TFs involved in AFT adipocyte differentiation in broilers.

Differences in Immune Cell-Derived Circular RNA Between Fulminant Type 1 Diabetes and Type 1 Diabetes.

Circular RNAs (circRNAs) are associated with fulminant type 1 diabetes (FT1D) and type 1 diabetes (T1D). However, the differences in circRNAs between FT1D and T1D remain unclear. Our objective is to identify peripheral blood mononuclear cells (PBMCs)-derived circRNAs in FT1D and T1D and explore their potential functions. PBMCs were extracted from six patients with FT1D and age-, sex-, and duration-matched T1D patients. The Arraystar Human circRNA Array was utilised to obtain circRNA expression profiles. Seven aberrantly expressed circRNAs were selected for determining expression levels in another independent cohort (FT1D subjects n=35, T1D subjects n=70, and controls n=100) using real-time quantitative PCR (RT-qPCR). Biological functions, circRNA-miRNA-mRNA networks, and the coding potential of circRNAs were predicted through bioinformatics analysis. In total, 145 differentially expressed circRNAs were identified in FT1D and T1D, with 63 upregulated and 82 downregulated circRNAs. Hsa_circRNA_038288, hsa_circRNA_104405, and hsa_circRNA_405498 were successfully validated among the 7 aberrantly expressed circRNAs selected to determine expression levels. Bioinformatics analysis revealed that majority of the parent genes of circRNAs are enriched in signalling pathways such as RNA transport, ubiquitin-mediated proteolysis, and focal adhesion. Hsa_circRNA_038288 appears to play a role in 51 circRNA-miRNA-mRNA signalling pathways associated with immune regulation and diabetes. Additionally, hsa_circRNA_038288 potentially exhibits coding potential and is involved in the progression of both FT1D and T1D. Significant differences in immune cell-derived circRNAs were found between FT1D and T1D patients, offering novel insights into the molecular mechanisms that distinguish FT1D from T1D.

Vitamin C Alleviates Heat-Stress-Induced Damages in Pig Thoracic Vertebral Chondrocytes via the Ubiquitin-Mediated Proteolysis Pathway

Heat stress can impair organismal growth by inducing ubiquitination, proteasome-mediated degradation, and subsequent cellular damage. Vitamin C (VC) has been shown to potentially mitigate the detrimental effects of abiotic stresses on cells. Nevertheless, the impact of heat stress on growth plate chondrocytes remains unclear, and the underlying protective mechanisms of VC in these cells warrant further investigation. In this study, we focused on pig thoracic vertebral chondrocytes (PTVCs) that are crucial for promoting the body’s longitudinal elongation and treated them with 41 °C heat stress for 24 h, under varying concentrations of VC. Our findings reveal that, while oxidative stress induced by heat triggers apoptosis and inhibits the ubiquitin-mediated proteolysis pathway, the addition of VC alleviates heat-stress-induced oxidative stress and apoptosis, mitigates cell cycle arrest, and promotes cellular viability. Furthermore, we demonstrate that VC enhances the ubiquitin-proteasome proteolysis pathway by promoting the expression of ubiquitin protein ligase E3A, which thereby stabilizes the ubiquitin-mediated degradation machinery, alleviates the apoptosis, and enhances cell proliferation. Our results suggest the involvement of the ubiquitin-mediated proteolysis pathway in the effects of VC on PTVCs under heat stress, and offer a potential strategy to make use of VC to ensure the skeletal growth of animals under high temperature pressures in summer or in tropical regions.

Plasma acellular transcriptome contains Parkinson's disease signatures that can inform clinical diagnosis.

We aimed to identify plasma cell-free transcripts (cfRNA) associated with Parkinson's disease (PD) that also have a high predictive value to differentiate PD from healthy controls. Leveraging two independent populations from two different movement disorder centers we identified 2,188 differentially expressed cfRNAs after meta-analysis. The identified transcripts were enriched in PD relevant pathways, such as PD (p=9.26×10 -4 ), ubiquitin-mediated proteolysis (p=7.41×10 -5 ) and endocytosis (p=4.21×10 -6 ). Utilizing in-house and publicly available brain, whole blood, and acellular plasma transcriptomic and proteomic PD datasets, we found significant overlap across dysregulated biological species in the different tissues and the different biological layers. We developed three predictive models containing increasing number of transcripts that can distinguish PD from healthy control with an area under the ROC Curve (AUC) ≥0.85. Finally, we showed that several of the predictive transcripts significantly correlate with symptom severity measured by UPDRS-III. Overall, we have demonstrated that cfRNA contains pathological signatures and has the potential to be utilized as biomarker to aid in PD diagnostics and monitoring.

Comprehensive Proteomic Profiling of Human Myocardium Reveals Signaling Pathways Dysregulated in Hypertrophic Cardiomyopathy

BackgroundHypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Signaling pathways that link genetic sequence variants to clinically overt HCM and progression to severe forms of HCM remain unknown. ObjectivesThe purpose of this study was to identify signaling pathways that are differentially regulated in HCM, using proteomic profiling of human myocardium, confirmed with transcriptomic profiling. MethodsIn this multicenter case-control study, myocardial samples were obtained from cases with HCM and control subjects with nonfailing hearts. Proteomic profiling of 7,289 proteins from myocardial samples was performed using the SomaScan assay (SomaLogic). Pathway analysis of differentially expressed proteins was performed, using a false discovery rate <0.05. Pathway analysis of proteins whose concentrations correlated with clinical indicators of severe HCM (eg, reduced left ventricular ejection fraction, atrial fibrillation, and ventricular tachyarrhythmias) was also executed. Confirmatory analysis of differentially expressed genes was performed using myocardial transcriptomic profiling. ResultsThe study included 99 HCM cases and 15 control subjects. Pathway analysis of differentially expressed proteins revealed dysregulation of the Ras–mitogen-activated protein kinase, ubiquitin-mediated proteolysis, angiogenesis-related (eg, hypoxia-inducible factor-1, vascular endothelial growth factor), and Hippo pathways. Pathways known to be dysregulated in HCM, including metabolic, inflammatory, and extracellular matrix pathways, were also dysregulated. Pathway analysis of proteins associated with clinical indicators of severe HCM and of differentially expressed genes supported these findings. ConclusionsThe present study represents the most comprehensive (>7,000 proteins) and largest-scale (n = 99 HCM cases) proteomic profiling of human HCM myocardium to date. Proteomic profiling and confirmatory transcriptomic profiling elucidate dysregulation of both newly recognized (eg, Ras–mitogen-activated protein kinase) and known pathways associated with pathogenesis and progression to severe forms of HCM.

BZW2 is a potential regulator of non-small cell lung cancer progression

BackgroundPersonalized targeted therapy has become an important strategy for cancer treatment owing to its remarkable therapeutic efficacy and safety. However, drug resistance remains the primary cause of treatment failure. Basic leucine zipper and W2 domain 2 (BZW2), which is aberrantly expressed in cancer, has been implicated in tumor progression and may serve as a new therapeutic target. Therefore, the role of BZW2 in non-small cell lung cancer (NSCLC) requires further investigation. MethodsThe expression and genetic alterations of BZW2 in pan-cancers were explored using The Cancer Genome Atlas (TCGA) PanCancer databases. The mRNA and protein levels of BZW2 in patients with NSCLC were verified in our cohort. Functional experiments including CCK8, colony formation, and transwell assays were performed to evaluate the impact of BZW2 on the proliferative, migratory, and invasive capacities of SK-MES-1 cells. Gene Set Enrichment Analysis was used to identify underlying biological processes and pathways. Single-cell RNA (scRNA) sequencing data were employed to investigate the tumor microenvironment of NSCLC and the co-expression of BZW2 and stemness-related genes. ResultsDysregulated BZW2 expression was observed in various malignant tumors. BZW2 expression was found to be significantly elevated in NSCLC. BZW2 depletion inhibited the growth, mobility, and invasive abilities of lung squamous cell carcinoma SK-MES-1 cells. BZW2 may be related to signaling pathways such as nucleotide excision repair, ubiquitin-mediated proteolysis, and the P53 signaling pathway. Biological processes, including translational initiation, tRNA processing, and RNA methylation, were observed to be enriched in the high-BZW2 group. Furthermore, there was a positive correlation between BZW2 and the m6A- and m5C-related genes. scRNA analysis revealed a co-expression relationship between BZW2 and stemness-related genes such as CD44, SOX9, and CD133. ConclusionsElevated BZW2 expression is associated with the proliferation, migration, and invasion of NSCLC, and BZW2 may be a potential therapeutic target for NSCLC.

MetaDegron: multimodal feature-integrated protein language model for predicting E3 ligase targeted degrons.

Protein degradation through the ubiquitin proteasome system at the spatial and temporal regulation is essential for many cellular processes. E3 ligases and degradation signals (degrons), the sequences they recognize in the target proteins, are key parts of the ubiquitin-mediated proteolysis, and their interactions determine the degradation specificity and maintain cellular homeostasis. To date, only a limited number of targeted degron instances have been identified, and their properties are not yet fully characterized. To tackle on this challenge, here we develop a novel deep-learning framework, namely MetaDegron, for predicting E3 ligase targeted degron by integrating the protein language model and comprehensive featurization strategies. Through extensive evaluations using benchmark datasets and comparison with existing method, such as Degpred, we demonstrate the superior performance of MetaDegron. Among functional features, MetaDegron allows batch prediction of targeted degrons of 21 E3 ligases, and provides functional annotations and visualization of multiple degron-related structural and physicochemical features. MetaDegron is freely available at http://modinfor.com/MetaDegron/. We anticipate that MetaDegron will serve as a useful tool for the clinical and translational community to elucidate the mechanisms of regulation of protein homeostasis, cancer research, and drug development.

Integrating network pharmacology and bioinformatics to explore the mechanism of Xiaojian Zhongtang in treating major depressive disorder: An observational study.

Major depressive disorder (MDD) is a common mental illness. The traditional Chinese medicine compound Xiaojian Zhongtang (XJZT) has a good therapeutic effect on MDD, but the specific mechanism is not clear. The aim of this study is to explore the molecular mechanism of XJZT in the treatment of MDD through network pharmacology and bioinformatics. The traditional Chinese medicine system pharmacology database was used to screen the chemical components and targets of XJZT, while the online Mendelian inheritance in man, DisGeNET, Genecards, and therapeutic target database databases were used to collect MDD targets and identify the intersection targets of XJZT and MDD. A "drugs-components-targets" network was constructed using the Cytoscape platform, and the STRING was used for protein-protein interaction analysis of intersecting targets. Gene Ontology and Kyoto encyclopedia of genes and genomes analysis of intersecting targets was performed using the DAVID database. Obtain serum and brain transcriptome datasets of MDD from the gene expression omnibus database, and perform differentially expressed genes, weighted gene co-expression network analysis, gene set enrichment analysis, and receiver operating characteristic analysis. A total of 127 chemical components and 767 targets were obtained from XJZT, among which quercetin, kaempferol, and maltose are the core chemical components, and 1728 MDD targets were screened out, with 77 intersecting targets between XJZT and MDD. These targets mainly involve AGE-RAGE signaling pathway in diabetic complexes, epidermal growth factor receptor tyrosine kinase inhibitor resistance, and HIF-1 signaling pathway, and these core targets have strong binding activity with core components. In addition, 1166 differentially expressed genes were identified in the MDD serum transcriptome dataset, and weighted gene co-expression network analysis identified the most relevant gene modules (1269 genes), among which RAC-alpha serine/threonine-protein kinase (AKT1), D(4) dopamine receptor (DRD4), and kynurenine 3-monooxygenase (KMO) were target genes for the treatment of MDD with XJZT, these 3 genes are mainly related to the ubiquitin-mediated proteolysis, arachidonic acid (AA) metabolism, and Huntington disease pathways, and the expression of AKT1, DRD4, and KMO was also found in the MDD brain transcriptome dataset, which is significantly correlated with the occurrence of MDD. We have identified 3 key targets for XJZT treatment of MDD, including AKT1, KMO, and DRD4, and they can be regulated by the key components of XJZT, including quercetin, maltose, and kaempferol. This provides valuable insights for the early clinical diagnosis and development of therapeutic drugs for MDD.

Low-Protein Diet Inhibits the Synovial Tissue Macrophage Pro-Inflammatory Polarization Via NRF2/SIRT3/SOD2/ROS Pathway in K/BxN Rheumatoid Arthritis Mice.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by pain, swelling, stiffness, and impaired function. Attenuating inflammation is a crucial objective in RA management. Diet and nutrition are believed to influence RA symptomatology, with a low-protein diet being one potential nutritional strategy, although its underlying mechanisms remain to be fully elucidated. In this research, serum derived from arthritic transgenic K/BxN mice was administered to naive mice to establish a K/BxN rheumatoid arthritis model. Physiological assessments and histological staining were performed to evaluate joint pathology. (Enzyme-linked immunosorbent assay) ELISA was used to measure inflammatory cytokines. Flow cytometry and immunofluorescence were applied to characterize macrophage phenotypes. Transcriptomic analysis elucidated molecular pathways under the effect of a low-protein diet and verified by immunoblotting. Mitochondrial reactive oxygen species (ROS) was detected by Mito-SOX. Protein expression was silenced through the application of siRNA transfection. Our results indicate that a low-protein diet significantly alleviates disease symptoms and decreases pro-inflammatory cytokine levels in synovial fluid. Furthermore, this dietary intervention inhibits M1 macrophage polarization while promoting a shift towards the M2 phenotype. Transcriptomic analysis revealed that the beneficial effects of the low-protein diet in alleviating rheumatoid arthritis are closely linked to the NRF2 pathway. In vitro, low protein treatment can promote the activity of NRF2 via inhibiting the ubiquitin mediated proteolysis and activate the NRF2/SIRT3/SOD2 pathway to inhibit the production of ROS, which will further inhibit the M1 macrophage polarization. NRF2 knockdown can abolish the effects of low-protein treatment, indicating that the inhibition of M1 polarization and the anti-inflammatory response induced by low-protein treatment are dependent on NRF2. In summary, our findings propose that low-protein diet can inhibit synovial macrophage M1 polarization via activating NRF2/SIRT3/SOD2 pathway to reduce mitochondrial ROS production. This mechanism effectively decreases synovial inflammation and alleviates RA symptoms.

Methyl jasmonate induced tolerance effect of Pinus koraiensis to Bursaphelenchus xylophilus.

Methyl jasmonate (MeJA) can affect the balance of hormones and regulate the disease resistance of plants. Exploring the application and mechanism of MeJA in inducing the tolerance of Pinus koraiensis to pine wood nematode (PWN) infection is of great significance for developing new strategies for pine wilt disease control. Different concentrations (0.1, 1, 5 and 10 mm) of MeJA treatment groups showed differences in relative tolerance index and relative anti-nematode index of P. koraiensis seedlings to PWN infection. The treatment of 5 mm MeJA solution induced the best tolerance effect, followed by the 1 mm MeJA solution. Transcriptome analysis indicated that many plant defense-related genes upregulated after treatment with 1, 5 and 10 mm MeJA solutions. Among them, genes such as jasmonate ZIM domain-containing protein, phenylalanine ammonia-lyase and peroxidase also continuously upregulated after PWN infection. Metabolome analysis indicated that jasmonic acid (JA) was significantly increased at 7 days postinoculation with PWN, and after treatment with both 1 and 5 mm MeJA solutions. Integrated analysis of transcriptome and metabolome indicated that differences in JA accumulation might lead to ubiquitin-mediated proteolysis, and expression changes in trans-caffeic acid and trans-cinnamic acid-related genes, leading to the abundance differences of these two metabolisms and the formation of multiple lignin and glucosides. MeJA treatment could activate the expression of defense-related genes that correlated with JA, regulate the abundance of defense-related secondary metabolites, and improve the tolerance of P. koraiensis seedlings to PWN infection. © 2024 Society of Chemical Industry.

Multi-Omics Exploration of the Mechanism of Curcumol to Reduce Invasion and Metastasis of Nasopharyngeal Carcinoma by Inhibiting NCL/EBNA1-Mediated UBE2C Upregulation.

Nasopharyngeal carcinoma (NPC) is closely linked to Epstein-Barr virus (EBV) infection. Curcumae Rhizoma, a traditional Chinese herb, has shown antitumor effects, primarily through its component curcumol (Cur), which has been shown to reduce NPC cell invasion and migration by targeting nucleolin (NCL) and Epstein-Barr Virus Nuclear Antigen 1 (EBNA1). We constructed an EBV-positive NPC cell model using C666-1 cells and performed transcriptomics studies after treatment with curcumol, which revealed a significant enrichment of ubiquitin-mediated proteolysis, the PI3K-AKT and mTOR signaling pathways, cell cycle and apoptosis involved in tumor invasion and migration. To investigate the importance of NCL and EBNA1 in curcumol-resistant EBV-positive NPC, we performed a multi-omics study using short hairpin NCL (shNCL) and shEBNA1 EBV-positive NPC cells, and the proteomics results showed enrichment in complement and coagulation cascades and ubiquitin-mediated proteolysis signaling pathways. Here, we focused on ubiquitin-conjugating enzyme E2C (UBE2C), which plays an important role in the ubiquitin-mediated proteolysis signaling pathway. In addition, metabolomics revealed that UBE2C is highly associated with 4-Aminobutanoic acid (GABA). In vitro studies further validated the function of the key targets, suggesting that UBE2C plays an important role in NCL and EBNA1-mediated curcumol resistance to nasopharyngeal carcinoma invasion and metastasis.

Delineation of the substrate recognition domain of MARCHF6 E3 ubiquitin ligase in the Ac/N-degron pathway and its regulatory role in ferroptosis

Nα-terminal acetylation in eukaryotic proteins creates specific degradation signals (Ac/N-degrons) targeted for ubiquitin-mediated proteolysis via the Ac/N-degron pathway. Despite the identification of key components of the Ac/N-degron pathway over the past 15 years, the precise recognition domain (Ac/N domain) remains unclear. Here, we defined the Ac/N domain of the endoplasmic reticulum MARCHF6 E3 ubiquitin ligase through a systematic analysis of its cytosol-facing regions using alanine-stretch mutagenesis, chemical crosslinking-based co-immunoprecipitation–immunoblotting, and split-ubiquitin assays in human and yeast cells. The Ac/N domain of MARCHF6 exhibits preferential binding specificity to Nα-terminally acetylated proteins and peptides over their unacetylated counterparts, mediating the degradation of Ac/N-degron-bearing proteins, such as the G-protein regulator RGS2 and the lipid droplet protein PLIN2. Furthermore, abolishing the recognition of Ac/N-degrons by MARCHF6 stabilized RGS2 and PLIN2, thereby increasing the resistance to ferroptosis, an iron-dependent lipid peroxidation-mediated cell death. These findings provide mechanistic and functional insights into how MARCHF6 serves as a rheostatic modulator of ferroptosis by recognizing Ac/N-degron substrates via its Ac/N domain and non-Ac/N-degron substrates via distinct recognition sites.

Antibiotic-induced gut microbiota disruption promotes vascular calcification by reducing short-chain fatty acid acetate

BackgroundVascular calcification is a common vascular lesion associated with high morbidity and mortality from cardiovascular events. Antibiotics can disrupt the gut microbiota (GM) and have been shown to exacerbate or attenuate several human diseases. However, whether antibiotic-induced GM disruption affects vascular calcification remains unclear.MethodsAntibiotic cocktail (ABX) treatment was utilized to test the potential effects of antibiotics on vascular calcification. The effects of antibiotics on GM and serum short-chain fatty acids (SCFAs) in vascular calcification mice were analyzed using 16 S rRNA gene sequencing and targeted metabolomics, respectively. Further, the effects of acetate, propionate and butyrate on vascular calcification were evaluated. Finally, the potential mechanism by which acetate inhibits osteogenic transformation of VSMCs was explored by proteomics.ResultsABX and vancomycin exacerbated vascular calcification. 16 S rRNA gene sequencing and targeted metabolomics analyses showed that ABX and vancomycin treatments resulted in decreased abundance of Bacteroidetes in the fecal microbiota of the mice and decreased serum levels of SCFAs. In addition, supplementation with acetate was found to reduce calcium salt deposition in the aorta of mice and inhibit osteogenic transformation in VSMCs. Finally, using proteomics, we found that the inhibition of osteogenic transformation of VSMCs by acetate may be related to glutathione metabolism and ubiquitin-mediated proteolysis. After adding the glutathione inhibitor Buthionine sulfoximine (BSO) and the ubiquitination inhibitor MG132, we found that the inhibitory effect of acetate on VSMC osteogenic differentiation was weakened by the intervention of BSO, but MG132 had no effect.ConclusionABX exacerbates vascular calcification, possibly by depleting the abundance of Bacteroidetes and SCFAs in the intestine. Supplementation with acetate has the potential to alleviate vascular calcification, which may be an important target for future treatment of vascular calcification.

Sleep disturbances associated with DEAF1 pathogenic variants.

Neurodevelopmental disorders and sleep disturbances share genetic risk factors. DEAF1 genetic variants are associated with rare syndromes in which sleep disturbances are commonly reported, yet the specific sleep disorders in these patients, and the molecular mechanisms underlying this association, are unknown. We aimed to pinpoint specific biological processes that may be disrupted by pathogenic variants in this gene, comparing a list of DEAF1 regulatory target genes with a list of insomnia-associated genes, and using the intersect gene list as the input for pathway enrichment analysis. Thirty-nine DEAF1 regulatory targets were also identified as insomnia-associated genes, and the intersecting gene list was found to be strongly associated with immune processes, ubiquitin-mediated proteolysis pathways and regulation of the cell cycle. This preliminary study highlights pathways that may be disrupted by DEAF1 pathogenic mutations and might be putative factors underlying the manifestation of insomnia in patients harboring such variants.

Potential Diagnostic Biomarkers of tRNA-Derived Small RNAs in PBMCs for Nonproliferative Diabetic Retinopathy in Patients With Type 2 Diabetes Mellitus.

This study aimed to reveal the altered expressions of transfer RNA (tRNA)-derived small RNAs (tsRNAs) in peripheral blood mononuclear cells and identify potential diagnostic biomarkers for nonproliferative diabetic retinopathy (NPDR) from patients with type 2 diabetes mellitus. Fifty-three patients diagnosed with type 2 diabetes mellitus were enrolled, including 25 patients with NPDR and 28 patients without diabetic retinopathy (DR) as the control group. A small RNA microarray was performed to screen the differentially expressed tsRNAs. Reverse transcriptase quantitative polymerase chain reaction was used to validate the significantly altered tsRNAs in a screening cohort and a verification cohort. The target genes, their enriched functions, and signaling pathways were predicted by bioinformatics analyses. In total, 668 upregulated and 485 downregulated tsRNAs were found in the NPDR group by microarray. Eight tsRNAs were validated preliminarily to be altered significantly by reverse transcriptase quantitative polymerase chain reaction, and their target genes were enriched in cellular macromolecule metabolic process and ubiquitin-mediated proteolysis. The verification experiments confirmed the increased levels of 5'tiRNA-35-PheGAA-8, tRF3-28-PheGAA-1, and tRF3b-PheGAA-6, and the decreased levels of mt-tRF3-19-ArgTCG, mt-tRF3-20-ArgTCG, and mt-tRF3-21-ArgTCG in patients with NPDR, which may serve as potential biomarkers with clinical significance. The study recognized the tsRNA expression changes in peripheral blood mononuclear cells from patients with NPDR and discovered potential diagnostic biomarkers that hold clinical significance. The significantly altered tsRNAs identified in the study may serve as potential diagnostic biomarkers for patients with NPDR as well as possible molecular targets of the occurrence and development of DR.

Unraveling the molecular pathogenesis of Type 2 Diabetes and its impact on female infertility: A bioinformatics and systems biology approach

Type 2 diabetes mellitus (T2D) has been linked with female infertility (FI). Nevertheless, our understanding of the molecular hallmarks and underlying mechanisms remains elusive. This research article aimed to find the hub genes, pathways, transcription factors, and miRNA involved. For this study, softwares like cytoscape, string, Enrichr, FFL loop, etc., were utilized. This research article employed differentially expressed genes (DEGs) to identify multiple biological targets to understand the association between T2D and female infertility (FI). Between T2D and FI, we found 3869 differentially expressed genes. We have also analyzed different pathways like thyroid hormone signaling pathways, AGE-RAGE signaling pathways in diabetic complications and ubiquitin-mediated proteolysis through pathway analysis. Moreover, hub genes MED17, PRKCG, THRA, FOXO1, NCOA2, PLCG2, COL1A1, CXCL8, PRPF19, ANAPC5, UBE2I, XIAP and KEAP1 have been identified. Additionally, these hub genes were subjected to identify the miRNA-mRNA regulation network specific to T2D-associated female infertility. In the FFL study (Feed Forward Loop), transcription factor (SP1, NFKB1, RELA and FOX01), miRNA (has-mir-7-5p, has-let-7a-5p, hsa-mir-16-5p, hsa-mir-155-5p, has-mir-122-5p, has-let-7b-5p, has-mir-124-3p, has-mir-34a-5p, has-mir-130a-3p, has-let-7i-5p, and hsa-mir-27a-3p) and six genes (XIAP, THRA, NCOA2, MED17, FOXO1, and COL1A1) among the thirteen key genes were recognized as regulator and inhibitor. Our analysis reveals that these genes can serve as a significant biomarker for female infertility linked with Type 2 Diabetes, through the prioritization of candidate genes. This study gives us insight into the molecular and cellular mechanism of T2D-associated FI. This finding helps in developing novel therapeutic approaches and will improve efficacy and reduce side effects of the treatment. This research requires further experimental investigation of the principal targets.

Exploring the Heat-Responsive miRNAs and their Target Gene Regulation in Ruditapes philippinarum Under Acute Heat Stress.

This study aimed to investigate the inherent molecular regulatory mechanisms of Ruditapes philippinarum in response to extremely high-temperature environments and to enhance the sustainable development of the R. philippinarum aquaculture industry. In this study, we established a differential expression profile of miRNA under acute heat stress and identified a total of 46 known miRNAs and 80 novel miRNAs, three of which were detected to be significantly differentially expressed. We analyzed the functions of target genes regulated by differentially expressed miRNAs (DEMs) of R. philippinarum. The findings of the KEGG enrichment analysis revealed that 29 enriched pathways in the group were subjected to acute heat stress. Notably, fatty acid metabolism, FoxO signaling pathway, TGF-β signaling pathway, and ubiquitin-mediated proteolysis were found to play significant roles in response to acute heat stress. We established a regulatory map of DEMs and their target genes in response to heat stress and constructed the miRNA-mRNA regulation network. This study provides valuable insights into the response of R. philippinarum to high temperature, helping to understand its underlying molecular regulatory mechanisms under high-temperature stress.

Mck1-mediated proteolysis of CENP-A prevents mislocalization of CENP-A for chromosomal stability in Saccharomyces cerevisiae.

Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to noncentromeric regions contributes to chromosomal instability in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers are associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and chromosomal instability under normal physiological conditions. Mck1-mediated phosphorylation of Skp, Cullin, F-box-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4, and reduced Cse4-Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus sites (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, chromosomal instability, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the chromosomal instability phenotype of GAL-cse4-3A strain, suggesting that the chromosomal instability phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and chromosomal instability. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.