Ubiquitin-mediated Proteolysis Research Articles

Molecular basis for C-degron recognition by CRL2APPBP2 ubiquitin ligase

E3 ubiquitin ligases determine the specificity of eukaryotic protein degradation by selective binding to destabilizing protein motifs, termed degrons, in substrates for ubiquitin-mediated proteolysis. The exposed C-terminal residues of proteins can act as C-degrons that are recognized by distinct substrate receptors (SRs) as part of dedicated cullin-RING E3 ubiquitin ligase (CRL) complexes. APPBP2, an SR of Cullin 2-RING ligase (CRL2), has been shown to recognize R-x-x-G/C-degron; however, the molecular mechanism of recognition remains elusive. By solving several cryogenic electron microscopy structures of active CRL2APPBP2 bound with different R-x-x-G/C-degrons, we unveiled the molecular mechanisms underlying the assembly of the CRL2APPBP2 dimer and tetramer, as well as C-degron recognition. The structural study, complemented by binding experiments and cell-based assays, demonstrates that APPBP2 specifically recognizes the R-x-x-G/C-degron via a bipartite mechanism; arginine and glycine, which play critical roles in C-degron recognition, accommodate distinct pockets that are spaced by two residues. In addition, the binding pocket is deep enough to enable the interaction of APPBP2 with the motif placed at or up to three residues upstream of the C-end. Overall, our study not only provides structural insight into CRL2APPBP2-mediated protein turnover but also serves as the basis for future structure-based chemical probe design.

Integrative profiling of metabolome and transcriptome of skeletal muscle after acute exercise intervention in mice.

This study aims to explore the molecular regulatory mechanisms of acute exercise in the skeletal muscle of mice. Male C57BL/6 mice were randomly assigned to the control group, and the exercise group, which were sacrificed immediately after an acute bout of exercise. The study was conducted to investigate the metabolic and transcriptional profiling in the quadriceps muscles of mice. The results demonstrated the identification of 34 differentially expressed metabolites (DEMs), with 28 upregulated and 6 downregulated, between the two groups. Metabolic pathway analysis revealed that these DEMs were primarily enriched in several, including the citrate cycle, propanoate metabolism, and lysine degradation pathways. In addition, the results showed a total of 245 differentially expressed genes (DEGs), with 155 genes upregulated and 90 genes downregulated. KEGG analysis indicated that these DEGs were mainly enriched in various pathways such as ubiquitin mediated proteolysis and FoxO signaling pathway. Furthermore, the analysis revealed significant enrichment of DEMs and DEGs in signaling pathways such as protein digestion and absorption, ferroptosis signaling pathway. In summary, the identified multiple metabolic pathways and signaling pathways were involved in the exercise-induced physiological regulation of skeletal muscle, such as the TCA cycle, oxidative phosphorylation, protein digestion and absorption, the FoxO signaling pathway, ubiquitin mediated proteolysis, ferroptosis signaling pathway, and the upregulation of KLF-15, FoxO1, MAFbx, and MuRF1 expression could play a critical role in enhancing skeletal muscle proteolysis.

Circulatory extracellular vesicle derived miR-195-5p promotes cellular apoptosis and suppresses cell proliferation in the buffalo endometrial primary cell culture

In pregnant animals, communication between the mother and conceptus occurs via extracellular vesicles (EVs) that carry several biomolecules such as nucleic acids (miRNAs, mRNAs), proteins, and lipids. At the time of implantation, the endometrium undergoes several morphological and physiological changes, such as angiogenesis, apoptosis, and cell proliferation regulation at the implantation site, to attain a receptive state. This study was conducted to detect pregnancy-specific miRNAs derived from extracellular vesicles in the systemic circulation of Bubalus bubalis (water buffalo) and to assess their functional significance in the modulation of endometrial primary cells. The extracellular vesicles were isolated from the blood plasma using a precipitation-based method and further characterized by various methods such as Differential light scattering, Nanoparticle tracking assay, Western blot, and transmission electron microscopy. The relative expression of the selected extracellular vesicles associated miRNAs (EV-miRNA) at different intervals (days 15, 19, 25, and 30) post artificial insemination (AI) was analyzed using RT-qPCR, and expression of miR-195-5p was found to be significantly higher (P < 0.01) in pregnant animals on day 19 post AI (implantation window) as compared to day 15 post AI. The elevated expression might indicate the involvement of this miRNA in the maternal-conceptus cross-talk occurring during the implantation period. The KEGG pathway enrichment and Gene Ontology analyses of the miR-195-5p target genes revealed that these were mostly involved in the PI3-Akt, MAPK, cell cycle, ubiquitin-mediated proteolysis, and mTOR signaling pathways, which are related to the regulation of cell proliferation. Transfecting the in vitro cultured cells with miR-195-5p mimic significantly suppressed (P < 0.05) the expression of its target genes such as YWHAQ, CDC27, AKT-3, FGF-7, MAPK8, SGK1, VEGFA, CACAND1, CUL2, MKNK1, and CACAN2D1. Furthermore, the downregulation of the miR-195-5p target genes was positively correlated with a significant increase in the apoptotic rate and a decrease in the proliferation. In conclusion, the current findings provide vital information on the presence of EV miR-195-5p in maternal circulation during the implantation window indicating its important role in the modulation of buffalo endometrium epithelial cells via promoting cell death. Altogether, the milieu of miR-195-5p may serve as a novel and potential molecular factor facilitating the implantation of the early embryo during the establishment of pregnancy in buffaloes. Thus, miR-195-5p may be identified as a unique circulatory EV biomarker related to establishing pregnancy in buffaloes as early as day 19 post-AI.

Large-scale RNAseq analysis provide a new insight into the critical genes and regulatory networks of tiller development mediated by gibberellin in sugarcane

Tillering plays a key role in sugarcane growth, development, and cane yield. Gibberellin (GA) and its biosynthesis inhibitor, paclobutrazol, have been used to manipulate the tiller production in sugarcane planting. However, the key genes and regulatory networks of sugarcane tiller development remain unclear. In this study, we applied GA3 and paclobutrazol to soak the sugarcane seeds for generating samples with varying tiller numbers. A total of 54 samples from multiple tissues of three tillering stages were employed for transcriptome sequencing using two full-length transcript isoform sequences datasets as reference. We observed significant downregulation of two genes within the GA metabolism pathway in response to the GA synthesis inhibitor. Additionally, we noted a substantial enrichment of genes associated with photosynthesis following treatment with GA3 or paclobutrazol. Using all the differentially expressed genes (DEGs) between each sample group, 41 gene modules were generated in the coexpression network. The associations between modules and traits were performed. The Gene Ontology (GO) enrichment, KEGG pathway enrichment, and hub genes analysis were implemented in the modules correlated with traits, especially, the modules correlated with GA3 content and tiller number were emphasized. Notably, the pathway of ubiquitin-mediated proteolysis were enriched in both the GA3 content positive correlated module and the tiller number negative correlated module, while an E3 ubiquitin ligase gene, RZFP34, was the top hub gene in these two modules, indicating the vital role of ubiquitin-mediated proteasomal degradation system in tillering regulation in sugarcane. Our study provides a comprehensive insight into regulatory networks involved in GA-mediated regulation of tiller development in sugarcane, which would lay the foundation of hormone regulation to promote sugarcane production.

Rose long noncoding RNA lncWD83 promotes flowering by modulating ubiquitination of the floral repressor RcMYC2L.

Long noncoding RNAs (lncRNAs) play important roles in various signaling pathways in vascular plants. However, the crosstalk between lncRNAs and E3 ubiquitin ligases has been barely reported. In this study, we demonstrate that the lncRNA lncWD83 from rose (Rosa chinensis) 'Old blush' activates flowering by modulating the ubiquitination of the floral repressor MYC2 LIKE (RcMYC2L). Flowering was substantially delayed in rose by virus-induced gene silencing of lncWD83. In an in vitro pull-down assay, lncWD83 associated with PLANT U-BOX PROTEIN 11 (PUB11), a U-box-containing E3 ubiquitin ligase. Seedlings with knocked down RcPUB11 transcripts phenocopied the later-flowering phenotype of lncWD83-silenced seedlings. RcMYC2L physically interacted with RcPUB11 and was ubiquitinated in an RcPUB11-dependent manner in vitro. Accordingly, silencing RcMYC2L fully reversed the later-flowering phenotype resulting from RcPUB11 knockdown. Furthermore, RcMYC2L bound to G-box-related motifs in the FLOWERING LOCUS T (RcFT) promoter and repressed its transcription. However, RcPUB11 alleviated this repression of RcFT expression via proteasomal degradation of RcMYC2L, and lncWD83 enhanced this degradation by associating with RcPUB11. Therefore, lncWD83 promotes flowering by modulating the ubiquitination of the floral repressor RcMYC2L in rose plants. These findings reveal a distinct regulatory mechanism for an lncRNA in facilitating ubiquitin-mediated proteolysis to regulate rose flowering.

Identification, characterization and expression profiles of E2 and E3 gene superfamilies during the development of tetrasporophytes in Gracilariopsis lemaneiformis (Rhodophyta)

E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases play important roles in the growth and development of plants and animals. To date, the systematic analysis of E2 and E3 genes in Rhodophyta is limited. In this study, 14 E2 genes and 51 E3 genes were identified in Gracilariopsis lemaneiformis, an economically important red alga. E2 genes were classified into four classes according to the structure of the conserved domain, UBC. E3 genes were classified into 12 subfamilies according to individual conserved domains. A phylogenetic tree of seven algae species showed that functional differentiation of RING-type E3s was the highest, and the similarity between orthologous genes was high except in Chlamydomonas reinhardtii and Chara braunii. RNA-seq data analysis showed significant differential expression levels of E2 and E3 genes under the life stages of tetraspore formation and release, especially GlUBCN and GlAPC3. According to GO and KEGG analysis of two transcriptomes, GlUBCN and GlAPC3 were involved in ubiquitin-mediated proteolysis, and other subunits of the anaphase promoting complex or cyclosome (APC/C) and its activators GlCDC20 and GlCDH1 were also enriched into this process. The CDH1 and CDC20 in 981 were down-regulated during tetraspores formation and release, with the down-regulation of CDH1 being particularly significant; CDH1 and CDC20 in WLP-1, ZC, and WT were up-regulated during tetraspores formation and release, with CDC20 being more significantly up-regulated. Therefore, GlCDH1, rather than GlCDC20, in ‘981’ might play the leading role in the activation of the APC/C, and GlCDC20 might play the leading role rather than GlCDH1 in strains WLP-1, ZC and wild type. The low fertility of cultivar 981 might be highly correlated with the inactivity of activators CDH1 and CDC20. This study provided a basic and comprehensive understanding of characteristic of E2 and E3 genes in Gp. lemaneiformis and set a foundation for further understanding of E2 ubiquitin conjugating enzymes and E3 ubiquitin ligase in regulating tetrasporophytes development of Gp. lemaneiformis.

Extracellular vesicle-coupled miRNA profiles of chicken seminal plasma and their potential interaction with recipient cells

The presence of EVs in seminal plasma (SPEVs) suggests their involvement on fertility via transmitting information between the original cells and recipient cells. SPEVs-coupled miRNAs have been shown to affect sperm motility, maturation, and capacitation in mammals, but rarely in poultry species. The present study aims to reveal the profile of SPEVs miRNAs and their potential effect on sperm storage and function in poultry. The SPEVs was successfully isolated from 4 different chicken breeds by ultracentrifugation and verified. Deep sequencing of SPEVs small RNA library of each breed identified 1077 miRNAs in total and 563 shared ones. The top 10 abundant miRNAs (such as miR-10-5p, miR-100-5p, and miR-10a-5p etc.) accounted for around 60% of total SPEVs miRNA reads and are highly conserved across species, predisposing their functional significance. Target genes prediction and functional enrichment analysis indicated that the most abundantly expressed miRNAs may regulate pathways like ubiquitin-mediated proteolysis, endocytosis, mitophagy, glycosphingolipid biosynthesis, fatty acid metabolism, and fatty acid elongation. The high abundant SPEVs-coupled miRNAs were found to target 107 and 64 functionally important mRNAs in the potential recipient cells, sperm and sperm storage tubules (SST) cells, respectively. The pathways that enriched by target mRNAs revealed that the SPEVs-coupled miRNA may rule the fertility by affecting the sperm maturation and regulating the female's immune response and lipid metabolism. In summary, this study presents the distinctive repertoire of SPEVs-coupled miRNAs, and extends our understanding about their potential roles in sperm maturation, capacitation, storage, and fertility, and may help to develop new therapeutic strategies for male infertility and sperm storage.

The mechanisms involved in byssogenesis in Pteria penguin under different temperatures

Byssus is important for marine bivalves to adhere robustly to diverse substrates and resist environmental impacts. The winged pearl oyster, Pteria penguin, can reattach or not reattach to the same environment, which leaves the development and survival of the oyster population at risk. In this study, diverse methods were employed to evaluate the byssus quality and explore the mechanism of byssus secretion at different temperatures. The results demonstrated that oysters maintained their byssus properties at different temperatures through polyphenol oxidase (PPO) and reactive oxygen species (ROS) variation. They were both higher at 27 °C than at 21 °C. Furthermore, PPO activities of WB27 (31.78 U/g ± 1.50 U/g) were significantly higher than NB27, WB21, and NB21. Sectional observation revealed three types of vesicles, from which a novel vesicle might participate in byssogenesis as a putative metal storage particle. Moreover, cytoskeletal proteins may cooperate with cilia to transport byssal proteins, which then facilitate byssus formation under the regulation of upstream signals. Transcriptome analysis demonstrated that protein quality control, ubiquitin-mediated proteolysis, and cytoskeletal reorganization-related genes contributed to adaptation to temperature changes and byssus fabrication, and protection-related genes play a critical role in byssogenesis, byssus toughness, and durability. These results were utilized to create a byssogenesis mechanism model, to reveal the foot gland and vesicle types of P. penguin and provide new insights into adaptation to temperature changes and byssus fabrication in sessile bivalves.

A combined transcriptomic and physiological approach to understanding the adaptive mechanisms to cope with oxidative stress in Fusarium graminearum.

In plant-pathogen interactions, oxidative bursts are crucial for plants to defend themselves against pathogen infections. Rapid production and accumulation of reactive oxygen species kill pathogens directly and cause local cell death, preventing pathogens from spreading to adjacent cells. Meanwhile, the pathogens have developed several mechanisms to tolerate oxidative stress and successfully colonize plant tissues. In this study, we investigated the mechanisms responsible for resistance to oxidative stress by analyzing the transcriptomes of six oxidative stress-sensitive strains of the plant pathogenic fungus Fusarium graminearum. Weighted gene co-expression network analysis identified several pathways related to oxidative stress responses, including the DNA repair system, autophagy, and ubiquitin-mediated proteolysis. We also identified hub genes with high intramodular connectivity in key modules and generated deletion or conditional suppression mutants. Phenotypic characterization of those mutants showed that the deletion of FgHGG4, FgHGG10, and FgHGG13 caused sensitivity to oxidative stress, and further investigation on those genes revealed that transcriptional elongation and DNA damage responses play roles in oxidative stress response and pathogenicity. The suppression of FgHGL7 also led to hypersensitivity to oxidative stress, and we demonstrated that FgHGL7 plays a crucial role in heme biosynthesis and is essential for peroxidase activity. This study increases the understanding of the adaptive mechanisms to cope with oxidative stress in plant pathogenic fungi. IMPORTANCE Fungal pathogens have evolved various mechanisms to overcome host-derived stresses for successful infection. Oxidative stress is a representative defense system induced by the host plant, and fungi have complex response systems to cope with it. Fusarium graminearum is one of the devastating plant pathogenic fungi, and understanding its pathosystem is crucial for disease control. In this study, we investigated adaptive mechanisms for coping with oxidative stress at the transcriptome level using oxidative stress-sensitive strains. In addition, by introducing genetic modification technique such as CRISPR-Cas9 and the conditional gene expression system, we identified pathways/genes required for resistance to oxidative stress and also for virulence. Overall, this study advances the understanding of the oxidative stress response and related mechanisms in plant pathogenic fungi.

Integrated transcriptomic and proteomic analyses of plerocercoid and adult Spirometra mansoni reveal potential important pathways in the development of the medical tapeworm

BackgroundSpirometra mansoni can parasitize animals and humans through food and water, causing parasitic zoonosis. Knowledge of the developmental process of S. mansoni is crucial for effective treatment; thus, it is important to characterize differential and specific proteins and pathways associated with parasite development.MethodsIn this study, we performed a comparative proteomic analysis of the plerocercoid and adult stages using a tandem mass tag-based quantitative proteomic approach. Additionally, integrated transcriptomic and proteomic analyses were conducted to obtain the full protein expression profiles of different life cycle stages of the tapeworm.ResultsApproximately 1166 differentially expressed proteins (DEPs) were identified in adults versus plerocercoids, of which 641 DEPs were upregulated and 525 were downregulated. Gene Ontology (GO), Clusters of Orthologous groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that most DEPs related to genetic information processing and metabolism of energy in adults seem to be more activated. In the plerocercoid stage, compared to metabolism, genetic information processing appears more dynamic. Protein-protein interaction (PPI) revealed six key proteins (phosphomannomutase, glutathione transferase, malate dehydrogenase, cytoplasmic, 40S ribosomal protein S15, ribosomal protein L15 and 60S acidic ribosomal protein P2) that may play active roles in the growth and development of S. mansoni. Finally, the combination of transcriptomic and proteomic data suggested that three pathways (ubiquitin-mediated proteolysis, phagosome and spliceosome) and five proteins closely related to these pathways might have a significant influence in S. mansoni.ConclusionsThese findings contribute to increasing the knowledge on the protein expression profiles of S. mansoni and provide new insights into functional studies on the molecular mechanisms of the neglected medical tapeworm.Graphical

Immunological characterization and diagnostic models of RNA N6-methyladenosine regulators in Alzheimer's disease

Alzheimer's disease (AD) is the most prevalent form of dementia, and it displays both clinical and molecular variability. RNA N6-methyladenosine (m6A) regulators are involved in a wide range of essential cellular processes. In this study, we aimed to identify molecular signatures associated with m6A in Alzheimer's disease and use those signatures to develop a predictive model. We examined the expression patterns of m6A regulators and immune features in Alzheimer’s disease using the GSE33000 dataset. We examined the immune cell infiltration and molecular groups based on m6A-related genes in 310 Alzheimer's disease samples. The WGCNA algorithm was utilized to determine differently expressed genes within each cluster. After evaluating the strengths and weaknesses of the random forest model, the support vector machine model, the generalized linear model, and eXtreme Gradient Boosting, the best machine model was selected. Methods such as nomograms, calibration curves, judgment curve analysis, and the use of independent data sets were used to verify the accuracy of the predictions made. Alzheimer's disease and non-disease Alzheimer's groups were compared to identify dysregulated m6A-related genes and activated immune responses. In Alzheimer's disease, two molecular clusters linked to m6A were identified. Immune infiltration analysis indicated substantial variation in protection between groups. Cluster 1 included processes like the Toll-like receptor signaling cascade, positive regulation of chromatin binding, and numerous malignancies; cluster 2 included processes like the cell cycle, mRNA transport, and ubiquitin-mediated proteolysis. With a lower residual and root mean square error and a larger area under the curve (AUC = 0.951), the Random forest machine model showed the greatest discriminative performance. The resulting random forest model was based on five genes, and it performed well (AUC = 0.894) on external validation datasets. Accuracy in predicting Alzheimer's disease subgroups was also shown by analyses of nomograms, calibration curves, and decision curves. In this research, we methodically outlined the tangled web of connections between m6A and AD and created a promising prediction model for gauging the correlation between m6A subtype risk and AD pathology.

A novel and independent survival prognostic model for OSCC: the functions and prognostic values of RNA-binding proteins.

Oral squamous cell carcinoma (OSCC), exhibiting high morbidity and malignancy, is the most common type of oral cancer. The abnormal expression of RNA-binding proteins (RBPs) plays important roles in the occurrence and progression of cancer. The objective of the present study was to establish a prognostic assessment model of RBPs and to evaluate the prognosis of OSCC patients. Gene expression data in The Cancer Genome Atlas (TCGA) were analyzed by univariate Cox regression analysis model that established a novel nine RBPs, which were used to build a prognostic risk model. A multivariate Cox proportional regression model and the survival analysis were used to evaluate the prognostic risk model. Moreover, the receive operator curve (ROC) analysis was tested further the efficiency of prognostic risk model based on data from TCGA database and Gene Expression Omnibus (GEO). Nine RBPs' signatures (ACO1, G3BP1, NMD3, RNGTT, ZNF385A, SARS, CARS2, YARS and SMAD6) with prognostic value were identified in OSCC patients. Subsequently, the patients were further categorized into high-risk group and low-risk in the overall survival (OS) and disease-free survival (DFS), and external validation dataset. ROC analysis was significant for both the TCGA and GEO. Moreover, GSEA revealed that patients in the high-risk group significantly enriched in many critical pathways correlated with tumorigenesis than the low, including cell cycle, adheres junctions, oocyte meiosis, spliceosome, ERBB signaling pathway and ubiquitin-mediated proteolysis. Collectively, we developed and validated a novel robust nine RBPs for OSCC prognosis prediction. The nine RBPs could serve as an independent and reliable prognostic biomarker and guiding clinical therapy for OSCC patients.

Transcriptomic Analysis of the Response of the Toxic Dinoflagellate Prorocentrum lima to Phosphorous Limitation.

Some dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Phosphorus (P) is a limiting macronutrient for dinoflagellate growth in the ocean. Previous studies have been focused on the physiological response of dinoflagellates to ambient P changes. However, the whole-genome's molecular mechanisms are poorly understood. In this study, RNA-Seq was utilized to compare the global gene expression patterns of a marine diarrheic shellfish poisoning (DSP) toxin-producing dinoflagellate, Prorocentrum lima, grown in inorganic P-replete and P-deficient conditions. A total of 148 unigenes were significantly up-regulated, and 30 unigenes were down-regulated under 1/4 P-limited conditions, while 2708 unigenes were significantly up-regulated, and 284 unigenes were down-regulated under 1/16 P-limited conditions. KEGG enrichment analysis of the differentially expressed genes shows that genes related to ribosomal proteins, glycolysis, fatty acid biosynthesis, phagosome formation, and ubiquitin-mediated proteolysis are found to be up-regulated, while most of the genes related to photosynthesis are down-regulated. Further analysis shows that genes encoding P transporters, organic P utilization, and endocytosis are significantly up-regulated in the P-limited cells, indicating a strong ability of P. lima to utilize dissolved inorganic P as well as intracellular organic P. These transcriptomic data are further corroborated by biochemical and physiological analyses, which reveals that under P deficiency, cellular contents of starch, lipid, and toxin increase, while photosynthetic efficiency declines. Our results indicate that has P. lima evolved diverse strategies to acclimatize to low P environments. The accumulation of carbon sources and DSP toxins could provide protection for P. lima to cope with adverse environmental conditions.

Proliferation-Related Features of the Human Mesenchymal Stem Cells Derived from Palatine Tonsils, Adipose Tissues, and Bone Marrow.

Mesenchymal stem cells (MSCs) are widely used in regenerative medicine and cell-based transplantations. However, an in-depth comparison of the different MSC origins is lacking. This study aimed to compare the expression of adipose-derived (AMSCs), bone marrow-derived (BMSCs), and tonsil-derived (TMSCs) and evaluate whether TMSCs are good alternatives for AMSCs or BMSCs. We analyzed the expression levels of 47,000 transcripts in AMSCs (n = 4), BMSCs (n = 4), and TMSCs (n = 4) using GeneChip. Microarray data were analyzed using the LIMMA package to compare the TMSCs, AMSCs, and BMSCs. Hub genes were analyzed using STRING and Cytoscape. To ascertain the functional roles of AURKA and AURKB, small interfering RNA (siRNA) molecules specifically targeting AURKA and AURKB mRNA were synthesized and employed to induce knockdown of AURKA and AURKB in TMSC and AMSC. We analyzed the expression level of OCT4, SOX-2, and NANOG genes in TMSC and AMSCs by cell culture and real-time PCR. We identified commonly increased 256 and decreased 160 genes in TMSCs from the differentially expressed genes (DEGs) between the TMSCs, AMSCs, and BMSCs. In the DEG-based protein-protein interaction and gene set enrichment analysis, hub genes (AURKA, AURKB, CDC20, and BUB1) highly expressed in TMSCs were enriched for development- and progression-related oocyte meiosis, the cell cycle, and ubiquitin-mediated proteolysis. In vitro analysis demonstrated that cells with downregulated expression of AURKA and AURKB exhibited a significant reduction in proliferation compared to control cells. However, silencing of the genes did not affect the differentiation capacity in TMSCs and AMSCs. Our study compared MSCs of different origins to better understand the similarities and differences among these cell types.

FAM64A aggravates proliferation, invasion, lipid droplet formation, and chemoresistance in gastric cancer: A biomarker for aggressiveness and a gene therapy target.

FAM64A is a mitogen-induced regulator of the metaphase and anaphase transition. Here, we found that FAM64A messenger RNA (mRNA) and protein expression levels were higher in gastric cancer tissue than in normal mucosa (p < .05). FAM64A methylation was negatively correlated with FAM64A mRNA expression (p < .05). The differentially expressed genes of FAM64A were mainly involved in digestion, potassium transporting or exchanging ATPase, contractile fibers, endopeptidase, and pancreatic secretion (p < .05). The FAM64A-related genes were principally categorized into ubiquitin-mediated proteolysis, cell cycle, chromosome segregation and mitosis, microtubule binding and organization, metabolism of amino acids, cytokine receptors, lipid droplet, central nervous system, and collagen trimer (p < .05). FAM64A protein expression was lower in normal gastric mucosa than intestinal metaplasia, adenoma, and primary cancer (p < .05), negatively correlated with older age, T stage, lymphatic and venous invasion, tumor, node, metastasis stage, and dedifferentiation (p < .05), and associated with a favorable overall survival of gastric cancer patients. FAM64A overexpression promoted proliferation, antiapoptosis, migration, invasion, and epithelial-mesenchymal transition via the EGFR/Akt/mTOR/NF-κB, while the opposite effect was observed for FAM64A knockdown. FAM64A also induced chemoresistance directly or indirectly through lipid droplet formation via ING5. These results suggested that upregulation of FAM64A expression might induce aggressive phenotypes, leading to gastric carcinogenesis and its subsequent progression. Thus, FAM64A could be regarded as a prognosis biomarker and a target for gene therapy.

Potential Involvement of DNA Methylation in Hybrid Sterility in Hermaphroditic Argopecten Scallops.

DNA methylation is an important epigenetic modification factor in regulating fertility. Corresponding process remains poorly investigated in hermaphroditic scallops. The interspecific F1 hybrids between the hermaphroditic bay scallops (Argopecten irradians) and Peruvian scallops (Argopecten purpuratus) exhibited significant heterosis in yield, but sterility in hybrids obstructs the utilization of the genetic resources. However, the determination mechanism of hybrid sterility in the hermaphroditic Argopecten scallops is still unclear. In this study, the effect of DNA methylation in the hybrid sterility of hermaphroditic Argopecten scallops was explored. The results showed that the mean methylation level was higher in sterile hybrids than fertile hybrids, especially on chromosome 11 of the paternal parent. A total of 61,062 differentially methylated regions (DMRs) were identified, containing 3619 differentially methylated genes (DMGs) and 1165 differentially methylated promoters that are located in the DMRs of CG sequence context. The hyper-methylated genes were enriched into five KEGG pathways, including ubiquitin-mediated proteolysis, ECM-receptor interaction, non-homologous end-joining, notch signaling, and the mismatch repair pathways. The DMGs might induce hybrid sterility by inhibition of oogenesis and egg maturation, induction of apoptosis, increased ROS, and insufficient ATP supply. Our results would enrich the determination mechanism of hybrid sterility and provide new insights into the utilization of the genetic resources of the interspecific hybrids.

Immune infiltration and drug specificity analysis of different subtypes based on functional status in angioimmunoblastic T-cell lymphoma

Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphoma (PTCL) strongly correlated with worse clinical outcomes. However, the role of characteristic pathway-related genes in patients with AITL (e.g., subtype typing and pathogenesis) remains unknown. In this study, we intended to understand the potential role and prognostic value of characteristic pathways in AITL and identified a model for subtype identification based on pathway-related functional status. Transcriptomic (RNA-seq) data were obtained from the Gene Expression Omnibus database for three sets of tumor tissues from AITL patients. AITL was divided into three clusters based on the pathway profile of patients and the best clustering k = 3, and differentially expressed genes (DEGs) in the three clusters were analyzed. The top 45 important variables associated with characteristic pathways, such as Huntington's disease, VEGF signaling pathway, nucleotide excision repair, ubiquitin-mediated proteolysis, purine metabolism, olfactory transduction, etc., were used to construct a subtype identification model. The model was experimentally validated and proved to possess good predictive efficacy. In addition, pathway-related subtype typing was significantly associated with different immune cell infiltration in AITL. Further analysis revealed that the drug IC50 values predicted also differed markedly among the different subtypes, thus further identifying some subtype-specific drugs. Our study indicates a potential role of characteristic pathways in AITL staging for the first time, provides novel insights for future research targeting AITL, and points to potential therapeutic options for patients with different subtypes of AITL.

Inhibition of CDC20 potentiates anti-tumor immunity through facilitating GSDME-mediated pyroptosis in prostate cancer

BackgroundIncreasing evidence suggests that immunotherapy, especially immune checkpoint inhibitors (ICIs), has the potential to facilitate long-term survival in various cancer besides prostate cancer. Emerging evidence indicated that pyroptosis, an immunogenic form of cell death, could trigger an anti-tumor immune microenvironment and enhance the effectiveness of immunotherapy. Nevertheless, the mechanism underlying the regulation of pyroptosis signaling in prostate cancer remains unclear.MethodsThe differential expression of human E3 ligases in prostate cancer was integratedly analyzed from five independent public datasets. Moreover, the immunohistochemistry analysis of a tissue microarray derived from prostate cancer patients confirmed the results from the bioinformatic analysis. Furthermore, prostate cancer cell lines were evaluated via the next-generation RNA sequencing to assess transcriptomic profile upon CDC20 depletion. Next, qRT-PCR, Western blotting, cycloheximide assay, immunoprecipitation, and ubiquitination assay were employed to explore the correlation and interaction between CDC20 and GSDME. Both immune-deficient and immune-competent murine models were utilized to examine the anti-tumor efficacy of CDC20 inhibition with or without the anti-PD1 antibodies, respectively. To analyze the immune microenvironment of the xenografts, the tumor tissues were examined by immunohistochemistry and flow cytometry.ResultsThe analysis of multiple prostate cancer cohorts suggested that CDC20 was the most significantly over-expressed E3 ligase. In addition, CDC20 exerted a negative regulatory effect on the pyroptosis pathway by targeting GSDME for ubiquitination-mediated proteolysis in a degron-dependent manner. Knockdown of CDC20 leads to increased GSDME abundance and a transition from apoptosis to pyroptosis in response to death signals. Furthermore, in our syngeneic murine models, we found that depletion of CDC20 significantly enhances the anti-tumor immunity by promoting the infiltration of CD8+ T lymphocytes dependent on the existence of GSDME, as well as reducing myeloid immune cells. More importantly, Apcin, a small molecular inhibitor that targets CDC20, exhibited synergistic effects with anti-PD1-based immunotherapy in murine models of prostate cancer.ConclusionsOverall, these findings provide new insights into the upstream regulation of GSDME-mediated pyroptosis by CDC20, which specifically interacts with GSDME and facilitates its ubiquitination in a degron-dependent manner. Importantly, our data highlight novel molecular pathways for targeting cellular pyroptosis and enhancing the effectiveness of anti-PD1-based immunotherapy.

Oral Spirochete Treponema denticola Intraoral Infection Reveals Unique miR-133a, miR-486, miR-126-3p, miR-126-5p miRNA Expression Kinetics during Periodontitis.

miRNAs are major regulators of eukaryotic gene expression and host immunity, and play an important role in the inflammation-mediated pathways in periodontal disease (PD) pathogenesis. Expanding our previous observation with the global miRNA profiling using partial human mouth microbes, and lack of in vivo studies involving oral spirochete Treponema denticola-induced miRNAs, this study was designed to delineate the global miRNA expression kinetics during progression of periodontitis in mice infected with T. denticola by using NanoString nCounter® miRNA panels. All of the T. denticola-infected male and female mice at 8 and 16 weeks demonstrated bacterial colonization (100%) on the gingival surface, and an increase in alveolar bone resorption (p < 0.0001). A total of 70 miRNAs with at least 1.0-fold differential expression/regulation (DE) (26 upregulated and 44 downregulated) were identified. nCounter miRNA expression profiling identified 13 upregulated miRNAs (e.g., miR-133a, miR-378) and 25 downregulated miRNAs (e.g., miR-375, miR-34b-5p) in T. denticola-infected mouse mandibles during 8 weeks of infection, whereas 13 upregulated miRNAs (e.g., miR-486, miR-126-5p) and 19 downregulated miRNAs (miR-2135, miR-142-3p) were observed during 16 weeks of infection. One miRNA (miR-126-5p) showed significant difference between 8 and 16 weeks of infection. Interestingly, miR-126-5p has been presented as a potential biomarker in patients with periodontitis and coronary artery disease. Among the upregulated miRNAs, miR-486, miR-126-3p, miR-126-5p, miR-378a-3p, miR-22-3p, miR-151a-3p, miR-423-5p, and miR-221 were reported in human gingival plaques and saliva samples from periodontitis and with diabetes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed various functional pathways of DE miRNAs, such as bacterial invasion of epithelial cells, Ras signaling, Fc gamma R-mediated phagocytosis, osteoclast differentiation, adherens signaling, and ubiquitin mediated proteolysis. This is the first study of DE miRNAs in mouse mandibles at different time-points of T. denticola infection; the combination of three specific miRNAs, miR-486, miR-126-3p, and miR-126-5p, may serve as an invasive biomarker of T. denticola in PD. These miRNAs may have a significant role in PD pathogenesis, and this research establishes a link between miRNA, periodontitis, and systemic diseases.

MicroRNA transcriptome analysis of chicken embryo fibroblast cells infected with Newcastle disease virus variants

Variations in the pathogenicity of Newcastle disease virus (NDV), the agent causing Newcastle disease, are associated with variants of different virulence. A few studies have characterized the expression of microRNAs (miRNAs) in NDV-infected avian cells. Here, the expression of miRNAs in chicken embryo fibroblasts (CEFs) infected with Herts/33 and LaSota NDV strains (highly virulent and nonvirulent, respectively) was determined using RNA sequencing. miRNAs involved in NDV infection included 562 previously documented and 184 novel miRNAs. miRNA target genes involved transcription factors, cell apoptosis, ubiquitin-mediated proteolysis, and protein processing in the endoplasmic reticulum. Potential target genes associated with autophagy were verified by qRT-PCR. No studies have documented the miRNA profiles of CEFs infected with NDVs variants. This study adds to our knowledge of the cellular miRNAs involved in NDV infection and the complex molecular mechanisms mediating virus-host interactions. The results of this study will aid the development of strategies against the chicken virus.