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Gene cloning, expression, and enzyme kinetics analysis of Eimeria tenella 2- methylcitrate synthase.Veterinary Parasitology Jun 2024In prokaryotes and lower eukaryotes, 2-methylcitrate cycle (2-MCC) is the main pathway for propionate decomposition and transformation, but little is known about the...
In prokaryotes and lower eukaryotes, 2-methylcitrate cycle (2-MCC) is the main pathway for propionate decomposition and transformation, but little is known about the 2-MCC pathway of Eimeria tenella. The analysis of genomic data found that the coding gene of 2- methylcitrate synthase (EC 2.3.3.5, PrpC) exists in E. tenella, which is a key enzyme of 2-MCC pathway. Through the search analysis of the database (ToxoDB), it was found that ETH_ 00026655 contains the complete putative sequence of EtprpC. In this study, we amplified the ORF sequence of EtprpC based on putative sequence. Then, prokaryotic expression, enzyme activity and kinetic analysis was performed. The results showed that the EtprpC ORF sequence was 1272 bp, encoding a 46.3 kDa protein comprising 424 amino acids. Enzyme activity assays demonstrate linearity between the initial reaction rate (OD/min) and EtPrpC concentration (ranging from 1.5 to 9 µg/reaction), with optimal enzyme activity observed at 41°C and pH 8.0. The results of enzymatic kinetic analysis showed that the Km of EtPrpC for propionyl-CoA, oxaloacetic acid, and acetyl-CoA was 5.239 ± 0.17 mM, 1.102 ± 0.08 μM, and 5.999 ± 1.24 μM, respectively. The Vmax was 191.11 ± 19.1 nmol/min/mg, 225.48 ± 14.4 nmol/min/mg, and 370.02 ± 25.8 nmol/min/mg when EtPrpC concentration at 4, 6, and 8 μg, respectively. Although the ability of EtPrpC to catalyze acetyl-CoA is only 0.11% of its ability to catalyze propionyl-CoA, it indicates that the 2-MCC pathway in E. tenella is similar to that in bacteria and may have a bypass function in the TCA cycle. This study can provide the theoretical foundation for the new drug targets and the development of new anticoccidial drugs.
Topics: Eimeria tenella; Kinetics; Cloning, Molecular; Citrate (si)-Synthase; Amino Acid Sequence; Citrates
PubMed: 38704976
DOI: 10.1016/j.vetpar.2024.110193 -
Archives of Microbiology May 2024A Gram-stain-positive aerobic, rod-shaped, spore-producing bacterium forming colonies with convex elevation and a smooth, intact margin was isolated from a freshwater...
Ureibacillus aquaedulcis sp. nov., isolated from freshwater well and reclassification of Lysinibacillus yapensis and Lysinibacillus antri as Ureibacillus yapensis comb. nov. and Ureibacillus antri comb. Nov.
A Gram-stain-positive aerobic, rod-shaped, spore-producing bacterium forming colonies with convex elevation and a smooth, intact margin was isolated from a freshwater sample collected from a well situated in an agricultural field. The 16S rRNA gene sequence of the isolated strain BA0131 showed the highest sequence similarity to Lysinibacillus yapensis ylb-03 (99.25%) followed by Ureibacillus chungkukjangi 2RL3-2 (98.91%) and U. sinduriensis BLB-1 (98.65%). The strain BA0131 was oxidase and catalase positive and urease negative. It also tested positive for esculin hydrolysis and reduction of potassium nitrate, unlike its phylogenetically closest relatives. The predominant fatty acids in strain BA0131 included were anteiso-C, iso-C, iso-C, iso-C and the major polar lipids comprised were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The respiratory quinones identified in strain BA0131 were MK8 (H2) (major) and MK8 (minor). The strain BA0131 shared the lowest dDDH values with L. yapensis ylb-03 (21%) followed by U. chungkukjangi 2RL3-2 (24.2%) and U. sinduriensis BLB-1 (26.4%) suggesting a closer genetic relationship U. sinduriensis BLB-1. The ANI percentage supported the close relatedness with U. sinduriensis BLB-1 (83.61%) followed by U. chungkukjangi 2RL3-2 (82.03%) and U. yapensis ylb-03 (79.57%). The core genome-based phylogeny constructed using over 13,704 amino acid positions and 92 core genes revealed the distinct phylogenetic position of strain BA0131 among the genus Ureibacillus. The distinct physiological, biochemical characteristics and genotypic relatedness data indicate the strain BA0131 represents a novel species of the genus Ureibacillus for which the name Ureibacillus aquaedulcis sp. nov. (Type strain, BA0131 = MCC 5284 = JCM 36475) is proposed. Additionally, based on extensive genomic and phylogenetic analyses, we propose reclassification of two species, L. yapensis and L. antri, as U. yapensis comb. nov. (Type strain, ylb-03 = JCM 32871 = MCCC 1A12698) and U. antri (Type strain, SYSU K30002 = CGMCC 1.13504 = KCTC 33955).
Topics: Phylogeny; RNA, Ribosomal, 16S; Fatty Acids; DNA, Bacterial; Fresh Water; Bacterial Typing Techniques; Base Composition; Bacillaceae; Sequence Analysis, DNA; Phospholipids
PubMed: 38698177
DOI: 10.1007/s00203-024-03970-0 -
Frontiers in Neurology 2024Ischemic stroke (IS) is a cerebrovascular disease that can be disabling and fatal, and there are limitations in the clinical treatment and prognosis of IS. It has been...
Analysis of the expression level and predictive value of CLEC16A|miR-654-5p|RARA regulatory axis in the peripheral blood of patients with ischemic stroke based on biosignature analysis.
INTRODUCTION
Ischemic stroke (IS) is a cerebrovascular disease that can be disabling and fatal, and there are limitations in the clinical treatment and prognosis of IS. It has been reported that changes in the expression profile of circRNAs have been found during injury in ischemic stroke, and circRNAs play an important role in the IS cascade response. However, the specific mechanisms involved in the pathogenesis of IS are not yet fully understood, and thus in-depth studies are needed.
METHODS
In this study, one circRNA dataset (GSE161913), one miRNA dataset (GSE60319) and one mRNA dataset (GSE180470) were retrieved from the Gene Expression Omnibus (GEO) database and included, and the datasets were differentially expressed analyzed by GEO2R and easyGEO to get the DEcircRNA, DEmiRNA and DEmRNA, and DEmRNA was enriched using ImageGP, binding sites were predicted in the ENCORI database, respectively, and the competitive endogenous RNA (ceRNA) regulatory network was visualized by the cytoscape software, and then selected by MCC scoring in the cytoHubba plugin Hub genes. In addition, this study conducted a case-control study in which blood samples were collected from stroke patients and healthy medical examiners to validate the core network of ceRNAs constructed by biosignature analysis by real-time fluorescence quantitative qRT-PCR experiments.
RESULTS
A total of 233 DEcircRNAs, 132 DEmiRNAs and 72 DEmRNAs were screened by bioinformatics analysis. circRNA-mediated ceRNA regulatory network was constructed, including 148 circRNAs, 43 miRNAs and 44 mRNAs. Finally, CLEC16A|miR-654-5p|RARA competitive endogenous regulatory axis was selected for validation by qRT-PCR, and the validation results were consistent with the bioinformatics analysis.
DISCUSSION
In conclusion, the present study establishes a new axis of regulation associated with IS, providing new insights into the pathogenesis of IS.
PubMed: 38682035
DOI: 10.3389/fneur.2024.1353275 -
Frontiers in Immunology 2024Over the past decades, immune dysregulation has been consistently demonstrated being common charactoristics of endometriosis (EM) and Inflammatory Bowel Disease (IBD) in...
INTRODUCTION
Over the past decades, immune dysregulation has been consistently demonstrated being common charactoristics of endometriosis (EM) and Inflammatory Bowel Disease (IBD) in numerous studies. However, the underlying pathological mechanisms remain unknown. In this study, bioinformatics techniques were used to screen large-scale gene expression data for plausible correlations at the molecular level in order to identify common pathogenic pathways between EM and IBD.
METHODS
Based on the EM transcriptomic datasets GSE7305 and GSE23339, as well as the IBD transcriptomic datasets GSE87466 and GSE126124, differential gene analysis was performed using the limma package in the R environment. Co-expressed differentially expressed genes were identified, and a protein-protein interaction (PPI) network for the differentially expressed genes was constructed using the 11.5 version of the STRING database. The MCODE tool in Cytoscape facilitated filtering out protein interaction subnetworks. Key genes in the PPI network were identified through two topological analysis algorithms (MCC and Degree) from the CytoHubba plugin. Upset was used for visualization of these key genes. The diagnostic value of gene expression levels for these key genes was assessed using the Receiver Operating Characteristic (ROC) curve and Area Under the Curve (AUC) The CIBERSORT algorithm determined the infiltration status of 22 immune cell subtypes, exploring differences between EM and IBD patients in both control and disease groups. Finally, different gene expression trends shared by EM and IBD were input into CMap to identify small molecule compounds with potential therapeutic effects.
RESULTS
113 differentially expressed genes (DEGs) that were co-expressed in EM and IBD have been identified, comprising 28 down-regulated genes and 86 up-regulated genes. The co-expression differential gene of EM and IBD in the functional enrichment analyses focused on immune response activation, circulating immunoglobulin-mediated humoral immune response and humoral immune response. Five hub genes (SERPING1、VCAM1、CLU、C3、CD55) were identified through the Protein-protein Interaction network and MCODE.High Area Under the Curve (AUC) values of Receiver Operating Characteristic (ROC) curves for 5hub genes indicate the predictive ability for disease occurrence.These hub genes could be used as potential biomarkers for the development of EM and IBD. Furthermore, the CMap database identified a total of 9 small molecule compounds (TTNPB、CAY-10577、PD-0325901 etc.) targeting therapeutic genes for EM and IBD.
DISCUSSION
Our research revealed common pathogenic mechanisms between EM and IBD, particularly emphasizing immune regulation and cell signalling, indicating the significance of immune factors in the occurence and progression of both diseases. By elucidating shared mechanisms, our study provides novel avenues for the prevention and treatment of EM and IBD.
Topics: Humans; Endometriosis; Female; Inflammatory Bowel Diseases; Protein Interaction Maps; Transcriptome; Computational Biology; Gene Expression Profiling; Databases, Genetic; Gene Regulatory Networks; Biomarkers; Gene Expression Regulation
PubMed: 38660311
DOI: 10.3389/fimmu.2024.1339647 -
Heliyon Apr 2024Kaposi's sarcoma (KS) is the second most common tumor in human immunodeficiency virus (HIV) infected patients worldwide. While many miRNAs have been confirmed to be...
Kaposi's sarcoma (KS) is the second most common tumor in human immunodeficiency virus (HIV) infected patients worldwide. While many miRNAs have been confirmed to be involved in KS biological processes, no relevant studies have combined miRNA and mRNA expression profiles using KS patient tissue biopsies. In this study, we performed transcriptome sequencing on tumor and normal tissues from four KS patients and identified differentially expressed mRNA and miRNA, further performed target gene prediction and enrichment analysis. 19,551 target-mRNAs were identified by predicting 106 miRNAs, with 553 overlapping with 571 significantly differentially expressed mRNAs. Enrichment analysis showed significant involvement of the Ubiquitin-mediated proteolysis pathway. Additionally, the miRNA-mRNA interaction network was established, and the topological score of Cytohubba's algorithm was calculated for comparison with three other datasets. The Mutual Clustering Coefficient (MCC) scoring ranking placed ZBTB34, NFIB, and RORA as the top three mRNAs, while hsa-miR-16-5p, hsa-miR-27a-3p, hsa-miR-340-5p, hsa-miR-182-5p, and hsa-miR-186-5p ranked as the top five miRNAs. Hsa-miR-101-3p is the only miRNA that appears both in the top 10 MCC scores and at the intersection of the other two datasets. Finally, qRT-PCR was used to validate the findings at the cellular level. In summary, the miRNA analysis results indicated that hsa-miR-101-3p could be used as a potential diagnostic or therapeutic marker in future studies. Moreover, the mRNA analysis results suggested that the histone binding pathways involved in mRNAs and ubiquitin-related biological processes were closely associated with KS and could serve as promising biomarkers for the diagnosis and treatment of this disease.
PubMed: 38660282
DOI: 10.1016/j.heliyon.2024.e29502 -
Scientific Reports Apr 2024Long extrachromosomal circular DNA (leccDNA) regulates several biological processes such as genomic instability, gene amplification, and oncogenesis. The identification...
Long extrachromosomal circular DNA (leccDNA) regulates several biological processes such as genomic instability, gene amplification, and oncogenesis. The identification of leccDNA holds significant importance to investigate its potential associations with cancer, autoimmune, cardiovascular, and neurological diseases. In addition, understanding these associations can provide valuable insights about disease mechanisms and potential therapeutic approaches. Conventionally, wet lab-based methods are utilized to identify leccDNA, which are hindered by the need for prior knowledge, and resource-intensive processes, potentially limiting their broader applicability. To empower the process of leccDNA identification across multiple species, the paper in hand presents the very first computational predictor. The proposed iLEC-DNA predictor makes use of SVM classifier along with sequence-derived nucleotide distribution patterns and physicochemical properties-based features. In addition, the study introduces a set of 12 benchmark leccDNA datasets related to three species, namely Homo sapiens (HM), Arabidopsis Thaliana (AT), and Saccharomyces cerevisiae (SC/YS). It performs large-scale experimentation across 12 benchmark datasets under different experimental settings using the proposed predictor, more than 140 baseline predictors, and 858 encoder ensembles. The proposed predictor outperforms baseline predictors and encoder ensembles across diverse leccDNA datasets by producing average performance values of 81.09%, 62.2% and 81.08% in terms of ACC, MCC and AUC-ROC across all the datasets. The source code of the proposed and baseline predictors is available at https://github.com/FAhtisham/Extrachrosmosomal-DNA-Prediction . To facilitate the scientific community, a web application for leccDNA identification is available at https://sds_genetic_analysis.opendfki.de/iLEC_DNA/.
Topics: DNA, Circular; Humans; Saccharomyces cerevisiae; Arabidopsis; Computational Biology; Nucleotides; Support Vector Machine
PubMed: 38658614
DOI: 10.1038/s41598-024-57457-5 -
BMC Cancer Apr 2024Therapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and...
Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cancer (mCRPC) by integrating the VTX-1 CTC...
BACKGROUND
Therapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported.
METHODS
To characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay.
RESULTS
AR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA.
CONCLUSION
VTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.
Topics: Humans; Male; Androgen Receptor Antagonists; Biomarkers, Tumor; Cell-Free Nucleic Acids; Exosomes; Neoplastic Cells, Circulating; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms, Castration-Resistant; Protein Isoforms; Receptors, Androgen; RNA, Messenger
PubMed: 38627648
DOI: 10.1186/s12885-024-12139-3 -
AMB Express Apr 2024Tuberculosis (TB) poses significant challenges due to its high transmissibility within populations and intrinsic resistance to treatment, rendering it a formidable...
Tuberculosis (TB) poses significant challenges due to its high transmissibility within populations and intrinsic resistance to treatment, rendering it a formidable respiratory disease with a substantial susceptibility burden. This study was designed to identify new potential therapeutic targets for TB and establish a diagnostic model. mRNA expression data for TB were from GEO database, followed by conducting differential expression analysis. The top 50 genes with differential expression were subjected to GO and KEGG enrichment analyses. To establish a PPI network, the STRING database was utilized, and hub genes were identified utilizing five algorithms (EPC, MCC, MNC, Radiality, and Stress) within the cytoHubba plugin of Cytoscape software. Furthermore, a hub gene co-expression network was constructed using the GeneMANIA database. Consistency clustering was performed on hub genes, and ssGSEA was utilized to analyze the extent of immune infiltration in different subgroups. LASSO analysis was employed to construct a diagnostic model, and ROC curves were used for validation. Through the analysis of GEO data, a total of 159 genes were identified as differentially expressed. Further, GO and KEGG enrichment analyses revealed that these genes were mainly enriched in viral defense, symbiotic defense, and innate immune response-related pathways. Hub genes, including DDX58, IFIT2, IFIH1, RSAD2, IFI44L, OAS2, OAS1, OASL, IFIT1, IFIT3, MX1, STAT1, and ISG15, were identified using cytoHubba analysis of the PPI network. The GeneMANIA analysis unmasked that the co-expression rate of hub genes was 81.55%, and the physical interaction rate was 12.27%. Consistency clustering divided TB patients into two subgroups, and ssGSEA revealed different degrees of immune infiltration in different subgroups. LASSO analysis identified IFIT1, IFIT2, IFIT3, IFIH1, RSAD2, OAS1, OAS2, and STAT1 as eight immune-related key genes, and a diagnostic model was constructed. The ROC curve demonstrated that the model exhibited excellent diagnostic performance. DDX58, IFIT2, IFIH1, RSAD2, IFI44L, OAS2, OAS1, OASL, IFIT1, IFIT3, MX1, STAT1, and ISG15 were hub genes in TB, and the diagnostic model based on eight immune-related key genes exhibited good diagnostic performance.
PubMed: 38615114
DOI: 10.1186/s13568-024-01691-7 -
Poultry Science Jun 2024As a Chinese local chicken breed, Hongshan chickens have 2 kinds of tail feather phenotypes, normal and taillessness. Our previous studies showed that taillessness was a...
As a Chinese local chicken breed, Hongshan chickens have 2 kinds of tail feather phenotypes, normal and taillessness. Our previous studies showed that taillessness was a sex-linked dominant trait. Abnormal development of the tail vertebrae could be explained this phenomenon in some chicken breeds. However, the number of caudal vertebrae in rumpless Hongshan chickens was normal, so rumplessness in Hongshan chicken was not related to the development of the caudal vertebrae. Afterwards, we found that rumplessness in Hongshan was due to abnormal development of tail feather rather than abnormal development of caudal vertebrae. In order to understand the genetic foundation of the rumplessness of Hongshan chickens, we compared and reanalyzed 2 sets of data in normal and rumpless Hongshan chickens from our previous studies. By joint analysis of genome-wide selection signature analysis and genome-wide association approach, we found that 1 overlapping gene (EDIL3) and 16 peak genes (ENSGALG00000051843, ENSGALG00000053498, ENSGALG00000054800, KIF27, PTPRD, ENSGALG00000047579, ENSGALG00000041052, ARHGEF28, CAMK4, SERINC5, ENSGALG00000050776, ERCC8, MCC, ADAMTS19, ENSGALG00000053322, CHRNA8) located on the Z chromosome was associated with the rumpless trait. The results of this study furtherly revealed the molecular mechanism of the rumpless trait in Hongshan chickens, and identified the candidate genes associated with this trait. Our results will help to improve the shape of chicken tail feathers and to rise individual economic value in some specific market in China.
Topics: Animals; Chickens; Male; Female; Feathers; Tail; Genome-Wide Association Study; Phenotype; China
PubMed: 38603937
DOI: 10.1016/j.psj.2024.103685 -
BMC Genomics Apr 2024Posterior capsular opacification (PCO) is the main reason affecting the long-term postoperative result of cataract patient, and it is well accepted that fibrotic PCO is...
BACKGROUND
Posterior capsular opacification (PCO) is the main reason affecting the long-term postoperative result of cataract patient, and it is well accepted that fibrotic PCO is driven by transforming growth factor beta (TGFβ) signaling. Ferroptosis, closely related to various ocular diseases, but has not been explored in PCO.
METHODS
RNA sequencing (RNA-seq) was performed on both TGF-β2 treated and untreated primary lens epithelial cells (pLECs). Differentially expressed genes (DEGs) associated with ferroptosis were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to investigate their biological function. Additionally, protein-to-protein interactions among selected ferroptosis-related genes by PPI network and the top 10 genes with the highest score (MCC algorithm) were selected as the hub genes. The top 20 genes with significant fold change values were validated using quantitative real-time polymerase chain reaction (qRT-PCR).
RESULTS
Our analysis revealed 1253 DEGs between TGF-β2 treated and untreated pLECs, uncovering 38 ferroptosis-related genes between two groups. Among these 38 ferroptosis-related genes,the most prominent GO enrichment analysis process involved in the response to oxidative stress (BPs), apical part of cell (CCs),antioxidant activity (MFs). KEGG were mainly concentrated in fluid shear stress and atherosclerosis, IL-17 and TNF signaling pathways, and validation of top 20 genes with significant fold change value were consistent with RNA-seq.
CONCLUSIONS
Our RNA-Seq data identified 38 ferroptosis-related genes in TGF-β2 treated and untreated pLECs, which is the first observation of ferroptosis related genes in primary human lens epithelial cells under TGF-β2 stimulation.
Topics: Humans; Transforming Growth Factor beta2; Transcriptome; Epithelial-Mesenchymal Transition; Ferroptosis; Blotting, Western; Capsule Opacification; Epithelial Cells
PubMed: 38594623
DOI: 10.1186/s12864-024-10244-y