MRMPath and MRMutation, Facilitating Discovery of Mass Transitions for Proteotypic Peptides in Biological Pathways Using a Bioinformatics Approach

Introduction Advances in various proteomics technologies, especially high-throughput and reproducibility, have enabled the systematic exploration of the circulating thrombosis proteome. This includes dissecting biological systems and pathways imperative to thrombosis, such as platelet activation, coagulation cascade, complement system, and endothelial cells. These insights strengthen our understanding of the cause and effect of thrombosis and improve precision medicine by identifying better biomarkers and biomarker panels, which may aid clinicians in decision-making in venous thromboembolism (VTE) and other thrombotic patients. This progress has the potential to reduce thrombosis-related morbidity and mortality, ultimately improving patient quality of life. Areas covered This review highlights recent advances and applications of mass spectrometry and affinity-based proteomics in thrombosis over the past three years (2022–2024), focusing on the thrombotic proteome signature related to VTE. Expert opinion Plasma proteomics, predominantly driven by mass spectrometry and affinity-based proteomics, has shown promise in identifying novel disease biomarkers and pathways. With the recent advances in the field, proteomics holds the potential to revolutionize precision medicine. As thrombosis is an intravascular disease, analysis of the blood proteome can capture environmental, genetic, and epigenetic contributors to risk variation in thrombosis, revealing novel protein biomarkers for diagnosis and risk prediction and new biological pathways.

Objective To investigate the differential expressions of long non-coding RNAs (lncRNAs) and mRNAs in early phases of subarachnoid hemorrhage (SAH) and explore the possible new mechanisms which may regulate early brain injury (EBI). Methods A total of 6 C57BL/6J male wild-type mice were divided into sham-operated group and SAH group (n=3). SAH models were induced by endovascular perforation. Twenty-four hours after operation, all animals were sacrificed, brain samples were removed rapidly, and total RNAs were extracted. After quality control, total RNAs were amplified and transcribed into fluorescent cRNA, and then performed RNA-seq by an Illumina HiSeqTM2500. Reads were aligned to the mouse transcriptome with Tophat2 software. The whole sample expression levels were presented by RPKM (expected number of Reads Per Kilobase of transcript sequence per Millions base pairs Sequenced). The differential expressions of mRNAs were analyzed by biological pathways, and all the differentially expressed mRNAs were selected for Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Quantitative real-time (qRT)-PCR was used to confirm some differentially expressed mRNAs. Results The expression profiles of lncRNAs and mRNAs of sham-operated and SAH mice brain tissues were successfully analyzed by RNA-Seq. In these 6 samples, 617 lncRNAs (103 being upregulated and 514 being downregulated) and 444 mRNAs (387 being upregulated and 54 being downregulated) were differentially expressed in three pair brain tissues. GO and KEGG analysis indicated that differentially expressed mRNAs were involved in a variety of biological processes, including inflammation. The qRT-PCR analysis of 4 randomly selected lncRNAs (fantom3_C730003K16 and fantom3_I830129C17 up-regulation expressions; fantom3_A430024L20 and fantom3_C330006P03 down-regulation expressions) confirmed the accuracy of RNA-seq. Conclusion LncRNAs significantly differentially express in the SAH brain tissues and the sham-operated brain tissues; LncRNA is a promising potential therapeutic target for the prognosis, diagnosis and treatment of SAH. Key words: Subarachnoid hemorrhage; Early brain injury; Long non-coding RNA; Inflammation; Next-generation sequencing

Sphingolipid ceramide N-deacylase (SCDase, EC 3.5.1.69) is a hydrolytic enzyme isolated from Pseudomonas sp. TK 4. In addition to its primary deacylation function, this enzyme is able to reacylate lyso-sphingolipids under specific conditions. We immobilised this enzyme on magnetic macroporous cellulose and used it to semisynthesise C17:0 glucosylceramide and C17:0 sulphatide, which are required internal standards for quantification of the corresponding glycosphingolipids (GSL) by tandem mass spectrometry. A high rate of conversion was achieved for both lipids (80% for C17:0 sulphatide and 90% for C17:0 glucosylceramide). In contrast to synthesis with a soluble form of the enzyme, use of immobilised SCDase significantly reduced the contamination of the sphingolipid products with other isoforms, so further purification was not necessary. Our method can be effectively used for the simple preparation of specifically labelled sphingolipids of high isoform purity for application in mass spectrometry.

Major depressive disorder (MDD) is frequently comorbid with mild cognitive impairment (MCI), yet its molecular basis remains unclear. This study aimed to identify shared differentially expressed genes (DEGs) and biological pathways that may underlie the comorbidity between MDD and MCI. Using integrative bioinformatics approaches applied to transcriptomic datasets, we sought to uncover molecular biomarkers that could inform early diagnosis and provide novel targets for mechanism-based therapeutic strategies. Transcriptomic datasets from MDD (GSE58430) and MCI (GSE140831) patients were analyzed to identify DEGs. Functional enrichment analyses were performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Protein-protein interaction (PPI) networks were constructed to identify core genes. A total of 301 DEGs were shared between MDD and MCI. Gene Ontology and KEGG enrichment analyses revealed key biological processes involved in neuroinflammation, oxidative stress, synaptic dysfunction, and apoptotic signaling. The PPI network analysis identified nine hub genes with high connectivity: HSP90AB1, CDC42, NFKB1, CD8A, CALM3, PARP1, CD44, H2BC21, and MYH9. These findings reveal shared molecular biomarkers and pathways linking MDD and MCI, providing insights into their comorbidity. The identified core genes, particularly PARP1 and CDC42, may serve as novel targets for early diagnosis and mechanism-based therapeutic strategies in psychiatry and neurodegenerative disorders.

To investigate whether RIXI-overexpressing transgenic plants influence the expression of other genes, we analyzed transcriptomic changes between R7 (RIXI-overexpression transgenic line 7) plants and WT (wild-type) plants using deep RNA sequencing combined with digital gene expression profile analysis. The differentially expressed genes between the WT and R7 libraries were identified by DEG-Seq (differentially expressed genes from RNA-seq) software. The profiling analysis revealed that the overexpression of RIXI in rice resulted in numerous changes in gene expressions, including upregulation of 391 genes and downregulation of 905 genes. These differentially expressed genes were categorized into 30 groups with broad functions using gene ontology (GO) assignments. Among the 30 groups, five groups (single-organism metabolic process, biological regulation, anion binding, small molecule binding and nucleotide binding) had more differentially expressed genes. Biological pathways affected by RIXI overexpression were mapped using the detected genes to reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG). The differentially expressed genes were assigned to 98 KEGG pathways, and four enriched pathways were identified: metabolic, biosynthesis of secondary metabolites, plant-pathogen interaction and plant hormone signal transduction. The measurement of agronomic traits of R7 showed that the overexpression of RIXI did not influence the growth and development of rice. Thus, we conclude that the xylanase inhibitor gene RIXI may play a role in activation of a complex signal transduction network in response to various biotic and abiotic stresses, but does not have a negative influence on growth and development of rice plants.

Green tea polyphenols boost gut-microbiota-dependent mitochondrial TCA and urea cycles in Sprague–Dawley rats

Identification of ST3GAL6-AS1 Interactive Protein in Multiple Myeloma

BackgroundEndometrial cancer (EC) is one of the most common gynaecological malignancies, and its incidence has been rising over the past decade. Tetrandrine, a bisbenzylisoquinoline alkaloid, has been isolated from a vine used in traditional Chinese medicine, Stephania tetrandra. However, the key mechanism of tetrandrine in EC is still unclear.PurposeThis research was designed to predict the molecular mechanisms of tetrandrine against EC based on network pharmacology and to further verify these predictions by in vitro experiments.MethodsThe potential therapeutic targets of tetrandrine against EC were predicted by using public databases. Afterwards, the protein–protein interaction (PPI) network of the common targets was constructed, and the key gene targets were obtained. Biological function and pathway enrichment analyses were performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Furthermore, molecular docking and in vitro experiments were carried out to verify the predictions. The cell counting kit‑8 (CCK‑8) assay, Hoechst 33258 staining, flow cytometry analysis, qRT-PCR, Western blot analysis and an immunofluorescence assay were performed.ResultsOur findings identified 111 potential therapeutic targets of tetrandrine against EC. We obtained 7 key gene targets from the PPI network analysis. Furthermore, GO enrichment analysis indicated that these targets were mainly associated with metabolic processes, responses to stimulus, and biological regulation. The KEGG pathway analysis showed that the common targets were mainly distributed in the PI3K/Akt signalling pathway. A potential interaction of tetrandrine with Akt1 was revealed by molecular docking. In addition, in vitro experiments showed that tetrandrine significantly inhibited cell proliferation and induced apoptosis in Ishikawa and HEC-1-B cells in dose- and time-dependent manners. The results also revealed that tetrandrine can downregulate the expression of Bcl-2 and upregulate the expression of Bax at the mRNA level. The mRNA levels of Akt were not significantly different in the various tetrandrine (0, 10 and 20µM) groups. However, Western blot analysis demonstrated that the protein expression ratios of p-Akt/Akt decreased at the protein level. The results were further confirmed by immunofluorescence assays.ConclusionBased on bioinformatic analysis and experimental verification, our findings demonstrated that tetrandrine exerted tumour-suppressive effects on EC by regulating the PI3K/Akt signalling pathway.

Esophageal Squamous Cell Carcinoma (ESCC) remains a significant global health challenge, underscoring the urgent need for the development of innovative therapeutic approaches. Ranunculus ternatus Thunb., a traditional herbal medicine, has shown potential anticancer properties, but its pharmacological mechanisms against ESCC remain poorly understood. This study aimed to elucidate the molecular mechanisms of the ethyl acetate extract of Ranunculus ternatus Thunb. (RTE) against ESCC by integrating network pharmacology analysis with experimental validation. Potential targets of RTE and ESCC were identified through network pharmacology using public databases. A Protein-Protein Interaction (PPI) network was constructed to identify key targets, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to explore biological processes and signaling pathways. The predicted mechanisms were experimentally validated using in vitro assays, including cell proliferation analysis and western blot assay in ESCC cell lines. Network pharmacology analysis identified 274 potential targets, with 14 key genes implicated in the therapeutic effects of RTE. GO analysis revealed significant involvement in the inflammatory response and apoptotic signaling pathways. KEGG pathway analysis highlighted the MAPK, Relaxin, and PI3K/Akt signaling pathways as critical mechanisms. In vitro experiments demonstrated that RTE significantly inhibited the proliferation of EC-109 and TE-13 cells by modulating the MAPK/ERK and PI3K/Akt pathways. This study provides a comprehensive understanding of the molecular mechanisms underlying the anticancer effects of RTE against ESCC. These findings underscore the potential of RTE as a promising natural compound for ESCC treatment.

Identification of Hub genes associated with infection of three lung cell lines by SARS-CoV-2 with integrated bioinformatics analysis.

IntroductionMost of the studies on alcohol dependence (AD) emphasize its impact on the nervous system and organ functions of the human body, but its molecular mechanism is largely unknown so far. This study determines serum protein changes in alcohol-dependent patients for the identification of biomarkers and the revelation of molecular mechanisms behind alcohol dependence.MethodsSerum samples from seven newly diagnosed alcohol-dependent patients and four healthy controls are subjected to researcher-conducted proteomic analyses using data-independent acquisition (DIA) mass spectrometry. Functional enrichment analysis of gene activity and biological pathways is performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to reveal key processes related to alcohol dependence. The expression of Small Nuclear Ribonucleoprotein Polypeptide B (SNRPB) is evaluated to assess its potential as a biomarker, and survival analysis is performed to investigate its clinical relevance.ResultsThe mass spectrometry detected a total of 1,249 and 1,020 proteins in the serum from alcohol-dependent patients and healthy controls, respectively. Biomarker analysis identified 195 proteins as potential markers with a differentially expressed pattern. Among them, 99 proteins are upregulated and 96 proteins downregulated in alcohol-dependent patients compared with controls. The GO and KEGG pathway analysis revealed that alcohol-dependent patients exhibit enhanced activity in pathways related to adenosine triphosphate (ATP)-dependent chromatin remodeling and immune responses but reduced activity in metabolic pathways. Alcohol-dependent patients exhibited significantly higher levels of the SNRPB protein, suggesting its potential role in immune system regulation and cell growth control. Survival analysis results indicated that higher levels of SNRPB are linked to worse outcomes in liver cancer patients, specifically those with liver hepatocellular carcinoma (LIHC), suggesting it could be a useful biomarker for alcohol-related diseases.ConclusionThis study provides valuable insights into the systemic protein changes in alcohol dependence, identifying several potential biomarkers for early diagnosis and therapeutic targeting. The upregulation of SNRPB suggests its role as a potential biomarker in clinical applications.

Objective Banxia Xiexin decoction (BXD) is widely used in the treatment of gastrointestinal and other digestive diseases. This study is based on network pharmacology to explore the molecular mechanism of BXD in the treatment of colon cancer. Methods The bioactive components and potential targets of BXD were obtained from public database. The protein-protein interaction (PPI) network of the potential targets of BXD for colon cancer was constructed based on the STRING database, cytoscape software, gene ontology (GO), and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of the PPI network. Finally, we established a xenograft nude mouse model to verify the effect of BXD in colon cancer treatment. Results We have acquired a total of 55 bioactive components and 136 cross-targets of BXD. The results of enrichment analysis suggested that the oxidate stress and diet were the key factors of colon cancer occurrence, and AGE-RAGE signaling pathway plays an essential role in the treatment of colon cancer with BXD. Animal experiments revealed that BXD could suppress tumor growth and induce tumor cell apoptosis in the xenograft nude mouse model with HCT116 cells. Conclusion This study uncovered that BXD inhibits the malignant progression of colon cancer that may be related to multiple compounds (berberine, quercetin, baicalein, etc.), multiple targets (Bcl2, Bax, IL6, TNFα, CASP3, etc.), and multiple pathways (human cytomegalovirus infection, AGE-RAGE signaling pathway in diabetic complications, etc.).

Genetic studies of single gene variants have been criticized as providing a simplistic characterization of the genetic basis of illness risk that ignores the effects of other variants within the same biological pathways. Of candidate biological pathways for schizophrenia (SZ), the cell adhesion molecule (CAM) pathway has repeatedly been linked to both psychosis and neurocognitive dysfunction. Here we tested, using risk allele scores derived from the Schizophrenia Psychiatric Genome-Wide Association Study Consortium (PGC-SCZ), whether alleles within the CAM pathway were correlated with poorer neuropsychological function in patients. In total, 424 patients with psychosis were assessed in areas of cognitive ability typically found to be impaired in SZ: intelligence quotient, memory, working memory and attentional control. CAM pathway genes were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Alleles within these genes identified as significantly associated with SZ risk in the PGC-SCZ were then used to calculate a CAM pathway-based polygenic risk allele score for each patient and these scores were tested for association with cognitive ability. Increased CAM pathway polygenic risk scores were significantly associated with poorer performance on measures of memory and attention, explaining 1-3% of variation on these measures. Notably, the most strongly associated single nucleotide polymorphism (SNP) in the CAM pathway (rs9272105 within HLA-DQA1) explained a similar amount of variance in attentional control, but not memory, as the polygenic risk analysis. These data support a role for the CAM pathway in cognitive function, both at the level of individual SNPs and the wider pathway. In so doing these data highlight the value of pathway-based polygenic risk score studies as well as single gene studies for understanding SZ-associated deficits in cognition.

Aging is acknowledged as the most significant risk factor for cardiovascular disease (CVD). This study sought to identify and validate potential aging-related genes associated with CVD by using bioinformatics. The confluence of the limma test, weighted correlation network analysis (WGCNA), and 2129 aging and senescence-associated genes led to the identification of aging-related differential expression genes (ARDEGs). By using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), potential biological roles and pathways of ARDEGs were identified. To find the significantly different functions between CVD and non-cardiovascular disease (nCVD) and to reckon the processes score, enrichment analysis of all genes was carried out using gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA). By using GO and KEGG, potential biological roles and pathways of ARDEGs were identified. To evaluate the immune cell composition of the immune microenvironment, we performed an immune infiltration analysis on the dataset from the training group. We were able to acquire four ARDEGs (PTGS2, MMP9, HBEGF, and FN1). Aging, cellular senescence, and nitric oxide signal transduction were selected for biological function analysis. The diagnostic value of the four ARDEGs in distinguishing CVD from nCVD samples was deemed to be favorable. This research identified four ARDEGs that are associated with CVD. This study provides insight into prospective novel biomarkers for aging-related CVD diagnosis and progression monitoring.

Objective To investigate the mechanism of development and rupture of intracranial aneurysm (IA) on the protein level and to search for target protein. Methods Specimens were collected from 3 cases of IA undergoing microsurgical clipping and STA (superficial temporal artery) in 3 cases as controls undergoing craniotomy through pterional approach from October 2015 to May 2016 at Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University. All those samples were analyzed by bioinformatics and proteomics. The GO (gene ontology) analysis was performed for study of biological process, cell component and molecular function. PPI (protein-protein interaction) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used to analyze pathway and differentially expressed proteins. Results Compared between the IA and STA groups, there were 106 differentially expressed proteins identified. Among them, 20 were significantly dysregulated including 6 down-regulated proteins and 14 up-regulated ones. The GO analysis showed that differentially expressed proteins in IA group were mainly related to cellular biosynthetic process, and membrane-bounded organelles represented the most significant cellular structural difference between the IA and STA groups. Further analysis through PPI and KEGG pathway showed that the most significantly up-regulated protein was THBS4, which played a key role in extracellular matrix interaction; and the most significantly down-regulated protein was PTGS1, which was important for platelet activation. Conclusions Label-free quantitative mass spectrometry and bioinformatics could be effective ways to analyze the proteins in IA tissues. THBS could be a key protein in IA progression and a potential target for intervention. Key words: Intracranial aneurysm; Label-free; Proteomics; Computational biology