Integration Of Proteomics Research Articles

Integrated Metabolomics and Proteomics Analysis of the Myocardium in a Mouse Model of Acute Viral Myocarditis.

Acute viral myocarditis (AVMC) is a common inflammatory disease affecting the myocardium and is often accompanied by severe metabolic disturbances. The molecular mechanisms underlying this disease are complex and not yet fully understood. Coxsackievirus B3 (CVB3)-induced AVMC mouse models were established. By integrating ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based metabolomics and data-independent acquisition (DIA)-based proteomics, we aimed to investigate the global influence of CVB3 infection on the myocardial metabolome and proteome in mice. Based on the criterion of OPLS-DA VIP > 1.0 and p value < 0.05, a total of 149 differential metabolites (DMs) were identified, including 64 upregulated and 85 downregulated metabolites. Bioinformatics analysis revealed that these DMs were mostly enriched in Global and overview maps (Metabolic pathways), Energy metabolism (Sulfur metabolism and Nitrogen metabolism), Amino acid metabolism (Taurine and hypotaurine metabolism, Lysine degradation, and Arginine and proline metabolism), and Carbohydrate metabolism (Propanoate metabolism). Differential analysis also identified 1385 differential proteins (DPs) between the two groups (|Fold Change| >1.5 and p value < 0.05), including 1092 upregulated and 293 downregulated proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DPs indicated that metabolism-related pathways were significant components of the AVMC process. Next, we mined many DPs engaged in the above metabolic pathways through an integrated analysis of KEGG pathway-based metabolomics and proteomics data. Our integrated metabolomics and proteomics analysis revealed characteristic alterations in metabolites and proteins in the myocardium of AVMC, as well as the associations between them. This not only extends the existing understanding of the molecular basis of the pathogenesis and progression of AVMC but also suggests new directions for its treatment.

Limiting cap-dependent translation increases 20S proteasomal degradation and protects the proteomic integrity in autophagy-deficient skeletal muscle

ABSTRACT Postmitotic skeletal muscle critically depends on tightly regulated protein degradation to maintain proteomic stability. Impaired macroautophagy/autophagy-lysosomal or ubiquitin-proteasomal protein degradation causes the accumulation of damaged proteins, ultimately accelerating muscle dysfunction with age. While in vitro studies have demonstrated the complementary nature of these systems, their interplay at the organism levels remains poorly understood. Here, our study reveals novel insights into this complex relationship in autophagy-deficient skeletal muscle. We demonstrated that despite a compensatory increase in proteasome level in response to autophagy impairment, 26S proteasome activity was not proportionally enhanced in autophagy-deficient skeletal muscle. This functional deficit was partly attributed to reduced ATP levels to fuel the 26S proteasome. Remarkably, we found that activation of EIF4EBP1, a crucial inhibitor of cap-dependent translation, restored and even augmented proteasomal function through dual mechanisms. First, genetically activating EIF4EBP1 enhanced both ATP-dependent 26S proteasome and ATP-independent 20S proteasome activities, thereby expanding overall protein degradation capacity. Second, EIF4EBP1 activation caused muscle fiber transformation and increased mitochondrial biogenesis, thus replenishing ATP levels for 26S proteasome activation. Notably, the improved performance of the 20S proteasome in EIF4EBP1-activated skeletal muscle was attributed to an increased abundance of the immunoproteasome, a subtype specially adapted to function under oxidative stress conditions. This dual action of EIF4EBP1 activation preserved proteomic integrity in autophagy-deficient skeletal muscle. Our findings uncover a novel role of EIF4EBP1 in improving protein quality control, presenting a promising therapeutic strategy for autophagy-related muscular disorders and potentially other conditions characterized by proteostatic imbalance.

Pharmaco-Multiomics: A New Frontier in Precision Psychiatry

The landscape of psychiatric care is poised for transformation through the integration of pharmaco-multiomics, encompassing genomics, proteomics, metabolomics, transcriptomics, epigenomics, and microbiomics. This review discusses how these approaches can revolutionize personalized treatment strategies in psychiatry by providing a nuanced understanding of the molecular bases of psychiatric disorders and individual pharmacotherapy responses. With nearly one billion affected individuals globally, the shortcomings of traditional treatments, characterized by inconsistent efficacy and frequent adverse effects, are increasingly evident. Advanced computational technologies such as artificial intelligence (AI) and machine learning (ML) play crucial roles in processing and integrating complex omics data, enhancing predictive accuracy, and creating tailored therapeutic strategies. To effectively harness the potential of pharmaco-multiomics approaches in psychiatry, it is crucial to address challenges such as high costs, technological demands, and disparate healthcare systems. Additionally, navigating stringent ethical considerations, including data security, potential discrimination, and ensuring equitable access, is essential for the full realization of this approach. This process requires ongoing validation and comprehensive integration efforts. By analyzing recent advances and elucidating how different omic dimensions contribute to therapeutic customization, this review aims to highlight the promising role of pharmaco-multiomics in enhancing patient outcomes and shifting psychiatric treatments from a one-size-fits-all approach towards a more precise and patient-centered model of care.

Novel transcripts of EMT driving the malignant transformation of oral submucous fibrosis

Oral submucous fibrosis (OSF) is a chronic, progressive, and fibrotic condition of the oral mucosa that carries an elevated risk of malignant transformation. We aimed to identify and validate novel genes associated with the regulation of epithelial-to-mesenchymal transition (EMT) in OSF. Genes regulating EMT were identified through differential gene expression analysis, using a LogFC threshold of -1 and + 1 and a padj value < 0.05, based on data from GEO datasets and the TCGA-HNSC datasets. The curated EMT genes were correlated with functional cancer states and subjected to clustering to identify candidate genes. Integration of bioinformatics and proteomics led to the discovery of the EMT genes MMP9, SPARC, and ITGA5 as novel candidates. Comprehensive pathway and immunohistochemical analyses confirmed their roles in regulating EMT in OSF, oral squamous cell carcinoma (OSCC), and OSF-associated squamous cell carcinoma (OSFSCC). The significant roles of MMP9, SPARC, and ITGA5 in fibrosis and malignancy suggest a novel mechanism in which fibrosis-associated type 2 EMT undergoes transition to type 3 EMT, driving OSF towards malignancy.

Fibrinogen Alpha Chain as a Potential Serum Biomarker for Predicting Response to Cisplatin and Gemcitabine Doublet Chemotherapy in Lung Adenocarcinoma: Integrative Transcriptome and Proteome Analyses

Cisplatin combined with gemcitabine, a doublet regimen, is the first-line treatment for patients with advanced lung adenocarcinoma (ADC); however, the treatment response remains poor. This study aimed to identify potential biomarkers for predicting response to cisplatin and gemcitabine. Tissue transcriptome and blood proteome analyses were conducted on 27 patients with lung ADC. Blood-derived proteins that reflected tissue-specific biomarkers were obtained using Venn diagrams. The candidate proteins were validated by Western blotting. Lentivirus-mediated short hairpin RNA interference was used to verify the functional roles of the candidate proteins in human A549 cells. We identified 417 differentially expressed genes, including 52 upregulated and 365 downregulated genes, and 31 differentially expressed proteins, including 26 upregulated and 5 downregulated proteins. Integrative analysis revealed the presence of alpha-1-acid glycoprotein 1 (A1AG1) and fibrinogen alpha chain (FGA or FIBA) in both the tissue and serum. FGA levels were elevated in responders compared to non-responders, and reduced serum FGA levels were correlated with resistance to this regimen. Moreover, FGA knockdown in A549 cells resulted in resistance to the doublet regimen. Our findings indicate that FGA is a tissue-specific serum protein that may function as a blood-based biomarker to predict the response of patients with lung ADC to cisplatin plus gemcitabine chemotherapy.

Pharmacological Approaches and Innovative Strategies for Individualized Patient Care.

Personalized medicine is an evolving paradigm that aims to tailor therapeutic interventions to individual patient characteristics. With a growing understanding of the genetic, epigenetic, and molecular mechanisms underlying diseases, tailored therapies are becoming more feasible and effective. This review highlights the significant advancements in personalized medicine, focusing specifically on pharmacological strategies. The article explores the integration of genomics, transcriptomics, proteomics, and metabolomics in drug development and therapy optimization. Pharmacogenomics, the customization of drug therapy based on an individual's genetic makeup, receives particular emphasis. This leads to the identification of specific biomarkers that can predict therapeutic response, drug toxicity, and susceptibility to various diseases. Together with computational tools and artificial intelligence, these advancements contribute to tailored treatment plans for patients with conditions such as cancer, cardiovascular diseases, and neurological disorders. We also highlight the challenges and ethical considerations in implementing personalized medicine, such as data privacy, cost-effectiveness, and accessibility. We outline future prospects and ongoing research in this field, highlighting the importance of collaborative efforts between researchers, clinicians, pharmacists, and regulatory authorities.

Elucidating circRNA-miRNA-mRNA competing endogenous regulatory RNA network during leaf rust pathogenesis in wheat (Triticum aestivum L.).

Advancements in bioinformatic tools and breakthroughs in high throughput RNA sequencing have unveiled the potential role of non-coding RNAs in influencing the overall expression of disease-responsive genes. Owing to the increasing need to develop resilient crop varieties against environmental constraints, our study explores the functional relationship of various non-coding RNAs in wheat during leaf rust pathogenesis. MicroRNAs (miRNAs) and circular RNAs (circRNAs) were retrieved from SAGE and RNA-Seq libraries, respectively, in the susceptible (HD2329) and resistant (HD2329 + Lr28) wheat Near-Isogenic Lines (NILs). Here we explored the previously published circRNAs for their differential expression and correlated the data with the differentially expressed miRNAs (DEMs) through various in silico methods to acquire the target miRNAs of circRNAs and the downstream target mRNAs of miRNAs. Finally, a competing endogenous RNA (ceRNAs) regulatory network was constructed and validated through RT-qPCR method. We have identified the ceRNA regulatory network of four differentially expressed circRNAs (DECs) and five DEMs to highlight their crucial roles in the robust enhancement of the temporal expression profiles of five defense responsive genes (mRNAs) in wheat NILs against leaf rust infection. The study confirms the synergistic expression of circRNAs and mRNAs with an antagonistic correlation with the expression profile of the corresponding miRNAs. The vital role of leaf rust-resistant gene Lr28 has also been highlighted for driving the efficiency of the circRNAs to upregulate target gene expression. Thus, understanding the circRNA-miRNA-target gene interaction during leaf rust pathogenesis can help to identify stress-specific regulatory biomarkers to enhance defense responses in wheat for improved resilience through multi-omics integration of transcriptomics, proteomics and metabolomics.

H3K14la drives endothelial dysfunction in sepsis-induced ARDS by promoting SLC40A1/transferrin-mediated ferroptosis.

Pulmonary endothelial cell (EC) activation is a key factor in acute respiratory distress syndrome (ARDS). In sepsis, increased glycolysis leads to lactate buildup, which induces lysine lactylation (Kla) on histones and other proteins. However, the role of protein lactylation in EC dysfunction during sepsis-induced ARDS remains unclear. Integrative lactylome and proteome analyses were performed to identify the global lactylome profile in the lung tissues of septic mice. Cut&Tag analysis was used to identify the transcriptional targets of histone H3 lysine 14 lactylation (H3K14la) in ECs. Septic mice presented elevated levels of lactate and H3K14la in lung tissues, particularly in pulmonary ECs. Suppressing glycolysis reduced both H3K14la and EC activation, suggesting a link between glycolysis and lactylation. Moreover, H3K14la was enriched at promoter regions of ferroptosis-related genes such as transferrin receptor (TFRC) and solute carrier family 40 member 1 (SLC40A1), which contributed to EC activation and lung injury under septic conditions. For the first time, we reported the role of lactate-dependent H3K14 lactylation in regulating EC ferroptosis to promote vascular dysfunction during sepsis-induced lung injury. Our findings suggest that manipulation of the glycolysis/H3K14la/ferroptosis axis may provide novel therapeutic approaches for sepsis-associated ARDS.

Exploring the molecular mechanisms of tirzepatide in alleviating metabolic dysfunction-associated fatty liver in mice through integration of metabolomics, lipidomics, and proteomics

Clinical studies have suggested that tirzepatide may also possess hepatoprotective effects; however, the molecular mechanisms underlying this association remain unclear. In our study, we performed biochemical analyses of serum and histopathological examinations of liver tissue in mice. To preliminarily explore the molecular mechanisms of tirzepatide on metabolic dysfunction-associated fatty liver disease (MAFLD), liquid chromatography-mass spectrometry (LC-MS) was employed for comprehensive metabolomic, lipidomic, and proteomic analyses in MAFLD mice fed a high-fat diet (HFD). The results demonstrated that tirzepatide significantly reduced serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), as well as hepatic triglycerides (TG) and total cholesterol (TC), indicating its efficacy in treating MAFLD. Further findings revealed that tirzepatide reduced fatty acid uptake by downregulating Cd36 and Fabp2/4, as well as enhance the mitochondrial-lysosomal function by upregulating Lamp1/2. In addition, tirzepatide promoted cholesterol efflux and reduced cholesterol reabsorption by upregulating the expression of Hnf4a, Abcg5, and Abcg8. These results suggest that tirzepatide exerts its therapeutic effects on MAFLD by reducing fatty acid uptake, promoting cholesterol excretion, and enhancing mitochondrial-lysosomal function, providing a theoretical basis for a comprehensive understanding of tirzepatide.

Signaling Transduction Network Elucidation of ACE 2 Regulating Apostichopus japonicus Autolysis by Using Integrative TMT Proteomics and Transcriptomics.

This study aims to reveal the transduction signaling network that triggers sea cucumber (Apostichopus japonicus) autolysis. The tandem mass tag (TMT) proteomics and transcriptomic techniques were used to analyze expression differences between inhibited and activated sea cucumber autolysis. Flow cytometry was used to identify apoptosis. Western blotting and RT-PCR verified the signaling pathway. The results showed that the angiotensin-converting enzyme 2 (ACE 2) activator (diminazene, DIZE) maintained the health of A. japonicus. The ACE 2 inhibitor (captopril, Capt) accelerated the autolysis. Based on the multiomics analysis, the ACE 2 activator activated the downstream NF-κB pathway to prevent the sea cucumber apoptosis. The Capt activated apoptosis initiation. Apoptosis occurred through the regulation of TNF and PI3K-Akt signaling pathways, which downregulate NF-κB. Akt was identified as an intermediate signaling protein downstream of ACE 2 that regulates autolysis in A. japonicus. Compared to A. japonicus in the ultraviolet-irradiated and Capt groups, the Akt inhibitor (perifosine, KRX) significantly reduced the expression of the PI3K-Akt and NF-κB pathways, thereby inhibiting the autolysis process. Additionally, DIZE attenuated ROS levels, thereby inhibiting the autolysis of A. japonicus. This study provides better insight into the autolysis mechanism of A. japonicus.

Impact of arsenic exposure on the hepatic metabolic molecular network in obese pregnant mice using metabolomics and proteomics

Arsenic is a ubiquitous environmental toxin that can affect normal physiological processes. Although the health impacts of arsenic have been investigated, its influence on hepatic metabolism in obese pregnant women and the underlying mechanisms remain unclear. Multi-omics analysis, including metabolomics and proteomics, can improve the understanding of arsenic-induced hepatotoxicity in obese pregnant women. This study aimed to investigate the adverse effects of gestational arsenic exposure on hepatic metabolism in high-fat-diet-induced obese pregnant mice. Following arsenic exposure during pregnancy, the liver tissue was evaluated comprehensively using metabolomics and proteomics techniques combined with pathological and biochemical analyses. Arsenic exposure not only significantly increased lipid accumulation in the livers of obese pregnant mice but also elevated inflammatory factors and oxidative stress markers. Specifically, histopathological examination revealed more steatosis, inflammatory cell infiltration, and hepatocyte ballooning in the livers of arsenic-exposed mice than in those of controls. These changes indicate that arsenic exposure exacerbates hepatic lipid accumulation and induces liver damage in the context of obesity. Metabolomic analysis provided further insight into the metabolic-level disruption caused by arsenic exposure. Significant changes were observed in lipid metabolism pathways, particularly the arachidonic acid metabolism pathway. As arachidonic acid and its metabolites play important roles in inflammation and oxidative stress, this pathway may be critical in arsenic-induced hepatotoxicity. Additionally, proteomic analysis showed differences in the expression levels of several key proteins involved in lipid synthesis, oxidative stress, and inflammatory response. Notably, oxidative-stress-related proteins, including glutathione peroxidase 4 (GPX4), were upregulated, suggesting an increased oxidative burden. In summary, there are complex interaction mechanisms among arsenic exposure, inflammatory response, and related lipid metabolism. The integration of metabolomics and proteomics aided in clarifying the molecular alterations induced by arsenic. The results show that arsenic exposure significantly affects hepatic lipid metabolism in obese pregnant mice through multiple metabolic pathways and protein regulatory mechanisms. In addition to providing new insights into the relationship between arsenic exposure and obesity as well as related metabolic diseases, this study can act as a reference for environmental health risk assessment and the formulation of public health policies. This enhanced understanding of the adverse effects of arsenic on hepatic metabolism will contribute to the development of strategies for mitigating the health risks associated with environmental toxins, particularly for vulnerable groups such as obese pregnant women.

Potential Functions and Causal Associations of GNLY in Primary Open-Angle Glaucoma: Integration of Blood-Derived Proteome, Transcriptome, and Experimental Verification.

Genome-wide association studies (GWAS) have identified multiple genetic loci associated with primary open-angle glaucoma (POAG). However, the mechanisms by which these loci contribute to POAG progression remain unclear. This study aimed to identify potential causative genes involved in the development of POAG. We utilized multi-dimensional high-throughput data, integrating proteome-wide association study(PWAS), transcriptome-wide association study (TWAS), and summary data-based Mendelian randomization (SMR) analysis. This approach enabled the identification of genes influencing POAG risk by affecting gene expression and protein concentrations in the bloodstream. The key gene was validated through enzyme-linked immunosorbent assay (ELISA) analysis. PWAS identified 86 genes associated with altered blood protein levels in POAG patients. Of these, eight genes (SFTPD, CSK, COL18A1, TCN2, GZMK, RAB2A, TEK, and GNLY) were identified as likely causative for POAG (P SMR < 0.05). TWAS revealed that GNLY was significantly associated with POAG at the gene expression level. GNLY-interacting genes were found to play roles in immune dysregulation, inflammation, and apoptosis. Clinical and cell-based validation confirmed reduced GNLY expression in POAG groups. This study reveals GNLY as a significant potential therapeutic target for managing primary open-angle glaucoma.

Deciphering Cancer Complexity: Integrative Proteogenomics and Proteomics Approaches for Biomarker Discovery.

Proteomics has revolutionized the field of cancer biology because the use of a large number of in vivo (SILAC), in vitro (iTRAQ, ICAT, TMT, stable-isotope Dimethyl, and 18O) labeling techniques or label-free methods (spectral counting or peak intensities) coupled with mass spectrometry enables us to profile and identify dysregulated proteins in diseases such as cancer. These proteome and genome studies have led to many challenges, such as the lack of consistency or correlation between copy numbers, RNA, and protein-level data. This review covers solely mass spectrometry-based approaches used for cancer biomarker discovery. It also touches on the emerging role of oncoproteogenomics or proteogenomics in cancer biomarker discovery and how this new area is attracting the integration of genomics and proteomics areas to address some of the important questions to help impinge on the biology and pathophysiology of different malignancies to make these mass spectrometry-based studies more realistic and relevant to clinical settings.

Transcriptomics, metabolomics and proteomics analyses reveal glyphosate tolerance mechanism in red swamp crayfish Procambarus clarkii.

Glyphosate (Gly), the world's most widely used herbicide in agriculture, can poison the red swamp crayfish, Procambarus clarkii, via spray drift and surface runoff into surface waters. However, there is a paucity of research on the mechanisms that affect crayfish tolerance to Gly at typical environmental concentrations. To address this research gap, we investigated the effects of Gly stress (0, 6, 12, 24, and 72h) at different concentrations (0, 1.20, 3.60, 7.20, and 10.80mg·L-1) on antioxidant enzyme activity in crayfish hepatopancreas. Furthermore, we analyzed the species' tolerance mechanism to Gly exposure at a typical environmental concentration (3.60mg·L-1) based on integrative transcriptomics, metabolomics, and proteomics. The Gly concentration and exposure time affected the crayfish's antioxidant system, and interacted with each other (P<0.01). Gly concentrations higher than 7.20mg·L-1 and exposure times longer than 48h caused oxidative stress. When the Gly concentrations were lower than 3.60mg·L-1, crayfish tolerated Gly exposure within 72h by self-regulating superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA). A multi-omics analysis revealed that crayfish upregulated the expression of amino acid metabolites (such as glutamate, proline, and lysine) and amino acid transformation-related genes (such as GlnA and P5CS) to tolerate Gly stress by enhancing the antioxidant capacity, ammonia‑nitrogen regulation, and energy supply of the organism. Metallothionein and polyadenylate-binding proteins, which are potential markers of Gly exposure, crucially influenced crayfish tolerance to Gly by synthesizing metalloenzymes and scavenging reactive oxygen species. This study revealed the Gly tolerance mechanism in crayfish and can provide a theoretical reference for commercial eco-farming in rice-crayfish integrated aquaculture systems.

Protein “purity,” proteoforms, and the albuminome: critical observations on proteome and systems complexity

IntroductionThe identification of effective, selective biomarkers and therapeutics is dependent on truly deep, comprehensive analysis of proteomes at the proteoform level.MethodsBovine serum albumin (BSA) isolated by two different protocols, cold ethanol fractionation and heat shock fractionation, was resolved and identified using Integrative Top-down Proteomics, the tight coupling of two-dimensional gel electrophoresis (2DE) with liquid chromatography and tandem mass spectrometry (LC-MS/MS).Results and discussionNumerous proteoforms were identified in both “purified” samples, across a broad range of isoelectric points and molecular weights. The data highlight several concerns regarding proteome analyses using currently popular analytical approaches and what it means to (i) purify a “protein” if the isolate consists of a wide variety of proteoforms and/or co-purifying species; and (ii) use these preparations as analytical standards or therapeutics. Failure to widely recognize and accept proteome complexity has likely delayed the identification of effective biomarkers and new, more selective drug targets. iTDP is the most logical available analytical technique to effectively provide the necessary critical depth and breadth for complex proteome analyses. Routine analyses at the level of proteoforms will provide the much-needed data for the development and validation of selective biomarkers and drugs, including biologics.

Integrative Transcriptomics and Proteomics Analysis Reveals THRSP's Role in Lipid Metabolism.

Background/Objectives: Abnormalities in lipid metabolism and endoplasmic reticulum (ER) stress are strongly associated with the development of a multitude of pathological conditions, including nonalcoholic fatty liver disease (NAFLD), diabetes mellitus, and obesity. Previous studies have indicated a potential connection between thyroid hormone responsive (THRSP) and lipid metabolism and that ER stress may participate in the synthesis of key regulators of adipogenesis. However, the specific mechanisms remain to be investigated. Methods: In this study, we explored the roles of THRSP in lipid metabolism by interfering with THRSP gene expression in mouse mesenchymal stem cells, comparing the effects on adipogenesis between control and interfered groups, and by combining transcriptomic and proteomic analysis. Results: Our results showed that the number of lipid droplets was significantly reduced after interfering with THRSP, and the expression levels of key regulators of adipogenesis, such as LPL, FABP4, PLIN1, and CIDEC, were significantly downregulated. Both transcriptomic and proteomic results showed that the differential genes (proteins) were enriched in the processes of lipolytic regulation, ER stress, cholesterol metabolism, sphingolipid metabolism, PPAR signaling pathway, and glycerophospholipid metabolism. The ER stress marker gene, ATF6, was the most significantly downregulated transcription factor. In addition, RT-qPCR validation indicated that the expression levels of PPAR signaling pathway gene SCD1; key genes of lipid droplet generation including LIPE, DGAT1, and AGPAT2; and ER stress marker gene ATF6 were significantly downregulated. Conclusions: These suggest that THRSP is involved in regulating ER stress and the PPAR signaling pathway, which is closely related to lipid synthesis and metabolism. Interfering with the expression of THRSP may be helpful in ameliorating the occurrence of diseases related to abnormalities in lipid metabolism.

Jingfang Granules alleviates the lipid peroxidation induced ferroptosis in rheumatoid arthritis rats by regulating gut microbiota and metabolism of short chain fatty acids

BackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation, bone and cartilage damage, musculoskeletal pain, swelling, and stiffness. Inflammation is one of the key factors that induce RA. Jingfang Granule (JFG) is a traditional Chinese medicine (TCM) with significant anti-inflammatory effects. Clinical studies have confirmed that JFG can be used to treat RA, but the mechanism is still vague. PurposeThis study was designed to evaluate the protective function and the mechanism of JFG on rats with RA. Study design and methodsComplete Freud's Adjuvant (CFA) was used to establish a rat RA model, and JFG or Diclofenac Sodium (Dic) was orally administered. Foot swelling and hematoxylin eosin (H&E) staining were used to test the therapeutic effect of JFG on RA treatment, while ELISA kits were used to detect serum cytokines. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), and reactive oxygen species (ROS) were used to evaluate oxidative stress levels. The integration of label-free proteomics, fecal short chain fatty acid (SCFA) targeted metabolomics, peripheral blood SCFA, medium and long chain fatty acid targeted metabolomics, and 16S rDNA sequencing of gut microbiota were used to screen the mechanism. Western blot technology was used to validate the results of multiple omics studies. Serum D-Lactic acid, lipopolysaccharide specific IgA antibody (LPS IgA), diamine oxidase (DAO), and colon Claudin 5 and ZO-1 were used to evaluate the intestinal barrier. ResultsThe results confirmed that JFG effectively protected rats from RA injury, which was confirmed by improved foot swelling and synovial pathology. At the same time, JFG reduced the levels of TNF-α, IL-1β, and IL-6 in serum by inhibiting the NLRP3 inflammasome signaling pathway and TLR4/NF-κB signaling pathway in synovial tissue. Multiple omics studies indicated that JFG increased the abundance of gut microbiota and regulated the number of gut bacteria, thereby increased the levels of Acetic acid, Propionic acid, and Butyric acid in the gut and serum of RA rats, which activated AMPK to regulate fatty acid metabolism and fatty acid biosynthesis, thereby inhibited lipid oxidative stress induced ferroptosis to improve tissue damage caused by RA. Meanwhile, JFG improved the intestinal barrier by upregulating the expresses of Claudin 5 and ZO-1, which was confirmed by low concentrations of D-Lactic acid, LPS-SIgA and DAO in serum. ConclusionsThis study confirmed that JFG improved the disturbance of fatty acid metabolism by modulating gut microbiota and the production of fecal SCFAs to activate AMPK, and then inhibited ferroptosis caused by lipid oxidative stress in synovium tissue and prevented AR injury. This study proposes for the first time to investigate the mechanism of JFG treatment for RA from the perspective of the "Gut-joint" axis, and provides a promising approach for the treatment of RA.

Integrative metabolomics and proteomics reveal the effect and mechanism of Zi Qi decoction on alleviating liver fibrosis

Liver fibrosis is a common progressive liver disease that can cause liver dysfunction and lead to serious complications. Zi Qi decoction (ZQ) is a traditional formulation that exerts pharmacological effects on the treatment of liver fibrosis. However, precise intervention mechanisms remain unclear. The aim of this study was to synergistically harness proteomics and metabolomics techniques to elucidate the specific target of ZQ and its potential mechanism of action. A carbon tetrachloride (CCl4)-induced liver fibrosis mouse model was established. Subsequently, the protective effect of ZQ on liver fibrosis mice was evaluated according to histopathological examination and biochemical indicators. Quantitative proteomics based on data independent acquisition (DIA) and non-targeted metabolomic analyses revealed the pharmacodynamic mechanism of ZQ. In addition, various cellular and molecular assays were used to detect changes in glycolysis levels in LSECs and mouse liver fibrosis models. The study results showed that ZQ significantly alleviated CCl4-induced liver injury and fibrosis in mice. DIA-based quantitative proteomics and non-targeted metabolomics analyses indicated that ZQ treatment downregulated glycolysis-related proteins such as PKM2, PFKP, and HK2, while regulating glycolysis-related metabolites and pathways. In addition, ZQ down-regulated glycolytic activity in mice with liver fibrosis and in LSECs, and inhibited CXCL1 secretion and neutrophil recruitment. ZQ inhibited LSEC glycolysis and mitigated neutrophil infiltration, thereby playing a therapeutic role in liver fibrosis.

Chronic Viral Reactivation and Associated Host Immune Response and Clinical Outcomes in Acute COVID-19 and Post-Acute Sequelae of COVID-19.

Chronic viral infections are ubiquitous in humans, with individuals harboring multiple latent viruses that can reactivate during acute illnesses. Recent studies have suggested that SARS-CoV-2 infection can lead to reactivation of latent viruses such as Epstein-Barr Virus (EBV) and cytomegalovirus (CMV), yet, the extent and impact of viral reactivation in COVID-19 and its effect on the host immune system remain incompletely understood. Here we present a comprehensive multi-omic analysis of viral reactivation of all known chronically infecting viruses in 1,154 hospitalized COVID-19 patients, from the Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study, who were followed prospectively for twelve months. We reveal significant reactivation of Herpesviridae, Enteroviridae, and Anelloviridae families during acute stage of COVID-19 (0-40 days post-hospitalization), each exhibiting distinct temporal dynamics. We also show that viral reactivation correlated with COVID-19 severity, demographic characteristics, and clinical outcomes, including mortality. Integration of cytokine profiling, cellular immunophenotyping, metabolomics, transcriptomics, and proteomics demonstrated virus-specific host responses, including elevated pro-inflammatory cytokines (e.g. IL-6, CXCL10, and TNF), increased activated CD4+ and CD8+ T-cells, and upregulation of cellular replication genes, independent of COVID-19 severity and SARS-CoV-2 viral load. Notably, persistent Anelloviridae reactivation during convalescence (≥3 months post-hospitalization) was associated with Post-Acute Sequelae of COVID-19 (PASC) symptoms, particularly physical function and fatigue. Our findings highlight a remarkable prevalence and potential impact of chronic viral reactivation on host responses and clinical outcomes during acute COVID-19 and long term PASC sequelae. Our data provide novel immune, transcriptomic, and metabolomic biomarkers of viral reactivation that may inform novel approaches to prognosticate, prevent, or treat acute COVID-19 and PASC.

Integrative Multi-PTM Proteomics Reveals Dynamic Global, Redox, Phosphorylation, and Acetylation Regulation in Cytokine-Treated Pancreatic Beta Cells

Studying regulation of protein function at a systems level necessitates an understanding of the interplay among diverse post-translational modifications (PTMs). A variety of proteomics sample processing workflows are currently used to study specific PTMs but rarely characterize multiple types of PTMs from the same sample inputs. Method incompatibilities and laborious sample preparation steps complicate large-scale physiological investigations and can lead to variations in results. The single-pot, solid-phase-enhanced sample preparation (SP3) method for sample cleanup is compatible with different lysis buffers and amenable to automation, making it attractive for high-throughput multi-PTM profiling. Herein, we describe an integrative SP3 workflow for multiplexed quantification of protein abundance, cysteine thiol oxidation, phosphorylation, and acetylation. The broad applicability of this approach is demonstrated using cell and tissue samples, and its utility for studying interacting regulatory networks is highlighted in a time-course experiment of cytokine-treated β-cells. We observed a swift response in global regulation of protein abundances consistent with rapid activation of JAK-STAT and NF-κB signaling pathways. Regulators of these pathways as well as proteins involved in their target processes displayed multi-PTM dynamics indicative of a complex cellular response stages: acute, adaptation, and chronic (prolonged stress). PARP14, a negative regulator of JAK-STAT, had multiple co-localized PTMs that may be involved in intraprotein regulatory crosstalk. Our workflow provides a high-throughput platform that can profile multi-PTMomes from the same sample set, which is valuable in unraveling the functional roles of PTMs and their co-regulation.