Proteomic Alterations Research Articles

Metalloproteomic analysis of liver proteins isolated from broilers fed with different sources and levels of copper and manganese

Supplementing minerals beyond dietary requirements can increase the risk of toxicity and mineral excretion, making the selection of more bioavailable sources crucial. Thus, this work aimed to use metalloproteomics tools to investigate possible alterations in the hepatic proteome of broilers fed with diets containing two sources (sulfate and hydroxychloride) and two levels of copper (15 and 150 ppm) and manganese (80 and 120 ppm), totaling four treatments: low Cu/Mn SO4, high Cu/Mn SO4, low Cu/Mn (OH)Cl and high Cu/Mn (OH)Cl. The difference in abundance of protein spots and copper and manganese concentrations in liver and protein pellets were analyzed by analysis of variance with significance level of 5%. The Cu and Mn concentrations determined in liver and protein pellets suggested greater bioavailability of hydroxychloride sources. We identified 19 Cu-associated proteins spots, 10 Mn-associated protein spots, and 5 Cu and/or Mn-associated protein spots simultaneously. The analysis also indicated the induction of heat shock proteins and detoxification proteins in broilers fed with high levels of copper and manganese, suggesting the involvement of these proteins in metal tolerance and stress.

Oral supplementation of gut microbial metabolite indole-3-acetate alleviates diet-induced steatosis and inflammation in mice

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. There is growing evidence that dysbiosis of the intestinal microbiota and disruption of microbiota-host interactions contribute to the pathology of NAFLD. We previously demonstrated that gut microbiota-derived tryptophan metabolite indole-3-acetate (I3A) was decreased in both cecum and liver of high-fat diet-fed mice and attenuated the expression of inflammatory cytokines in macrophages and Tnfa and fatty acid-induced inflammatory responses in an aryl-hydrocarbon receptor (AhR)-dependent manner in hepatocytes. In this study, we investigated the effect of orally administered I3A in a mouse model of diet-induced NAFLD. Western diet (WD)-fed mice given sugar water (SW) with I3A showed dramatically decreased serum ALT, hepatic triglycerides (TG), liver steatosis, hepatocyte ballooning, lobular inflammation, and hepatic production of inflammatory cytokines, compared to WD-fed mice given only SW. Metagenomic analysis show that I3A administration did not significantly modify the intestinal microbiome, suggesting that I3A’s beneficial effects likely reflect the metabolite’s direct actions on the liver. Administration of I3A partially reversed WD-induced alterations of liver metabolome and proteome, notably, decreasing expression of several enzymes in hepatic lipogenesis and β-oxidation. Mechanistically, we also show that AMP-activated protein kinase (AMPK) mediates the anti-inflammatory effects of I3A in macrophages. The potency of I3A in alleviating liver steatosis and inflammation clearly demonstrates its potential as a therapeutic modality for preventing the progression of steatosis to non-alcoholic steatohepatitis (NASH).

Oral supplementation of gut microbial metabolite indole-3-acetate alleviates diet-induced steatosis and inflammation in mice.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. There is growing evidence that dysbiosis of the intestinal microbiota and disruption of microbiota-host interactions contribute to the pathology of NAFLD. We previously demonstrated that gut microbiota-derived tryptophan metabolite indole-3-acetate (I3A) was decreased in both cecum and liver of high-fat diet-fed mice and attenuated the expression of inflammatory cytokines in macrophages and Tnfa and fatty acid-induced inflammatory responses in an aryl-hydrocarbon receptor (AhR)-dependent manner in hepatocytes. In this study, we investigated the effect of orally administered I3A in a mouse model of diet-induced NAFLD. Western diet (WD)-fed mice given sugar water (SW) with I3A showed dramatically decreased serum ALT, hepatic triglycerides (TG), liver steatosis, hepatocyte ballooning, lobular inflammation, and hepatic production of inflammatory cytokines, compared to WD-fed mice given only SW. Metagenomic analysis show that I3A administration did not significantly modify the intestinal microbiome, suggesting that I3A's beneficial effects likely reflect the metabolite's direct actions on the liver. Administration of I3A partially reversed WD-induced alterations of liver metabolome and proteome, notably, decreasing expression of several enzymes in hepatic lipogenesis and β-oxidation. Mechanistically, we also show that AMP-activated protein kinase (AMPK) mediates the anti-inflammatory effects of I3A in macrophages. The potency of I3A in alleviating liver steatosis and inflammation clearly demonstrates its potential as a therapeutic modality for preventing the progression of steatosis to non-alcoholic steatohepatitis (NASH).

Proteomic alterations associated with the formation of monocyte extracellular trap induced by Candidaalbicans hyphae.

Background: The interaction between the host and Candida albicans is dynamic and intricate. We performed proteomic analysis to explore monocyte-C.albicans hyphae interaction. Materials & methods: Primary human monocytes were stimulated by heat-killed C.albicans hyphae and their proteins were profiled by tandem liquid chromatography with mass spectrometry (LC-MS/MS). Results: Based on the protein database of different species for analysis, we found that stimulation of monocytes by hyphae was accompanied by upregulation of histones and activation of extracellular traps (ETs) formation pathway. Meanwhile, monocyte ETs (MoETs) were evoked by synthesis or alteration of C. albicans cell wall proteins expression during the morphological switch to hyphal. Conclusion: MoETs formation is linked to cell wall proteins of C. albicans hyphae.

Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae.

Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.

Neoadjuvant chemo- or chemo-radiation-therapy of pancreatic ductal adenocarcinoma differentially shift ECM composition, complement activation, energy metabolism and ribosomal proteins of the residual tumor mass.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, often diagnosed at stages that dis-qualify for surgical resection. Neoadjuvant therapies offer potential tumor regression and improved resectability. Although features of the tumor biology (e.g., molecular markers) may guide adjuvant therapy, biological alterations after neoadjuvant therapy remain largely unexplored. We performed mass spectrometry to characterize the proteomes of 67 PDAC resection specimens of patients who received either neoadjuvant chemo (NCT) or chemo-radiation (NCRT) therapy. We employed data-independent acquisition (DIA), yielding a proteome coverage in excess of 3500 proteins. Moreover, we successfully integrated two publicly available proteome datasets of treatment-naïve PDAC to unravel proteome alterations in response to neoadjuvant therapy, highlighting the feasibility of this approach. We found highly distinguishable proteome profiles. Treatment-naïve PDAC was characterized by enrichment of immunoglobulins, complement and extracellular matrix (ECM) proteins. Post-NCT and post-NCRT PDAC presented high abundance of ribosomal and metabolic proteins as compared to treatment-naïve PDAC. Further analyses on patient survival and protein expression identified treatment-specific prognostic candidates. We present the first proteomic characterization of the residual PDAC mass after NCT and NCRT, and potential protein candidate markers associated with overall survival. We conclude that residual PDAC exhibits fundamentally different proteome profiles as compared to treatment-naïve PDAC, influenced by the type of neoadjuvant treatment. These findings may impact adjuvant or targeted therapy options.

Intestine proteomic and metabolomic alterations in dogs infected with Toxocara canis

Toxocariasis is an important zoonotic parasitic disease. Toxocaris canis adults live and reproduce in the intestinal tract of dogs and other canine hosts, and the infectious eggs are continuously excreted in feces, which causes environmental contamination and has an important public health significance. In this study, TMT proteomic and untargeted metabolomic methods were used to explore the physiological and pathological effects on the intestinal tract of dogs which infected with T. canis, and a series of bioinformatics analyses were conducted to identify differentially expressed proteins (DEPs) and differentially expressed metabolites (DEMs). The proteomics results showed that 198 DEPs were mainly enriched in the immune system and signal transduction pathway, and involved in the regulation of the occurrence and development of cancer and infectious diseases. T. canis could disrupt intestinal permeability by increasing the expression of proteins such as zinc finger protein DZIP1L and myosin heavy chain 10. Additionally, T. canis infection could also inhibit the host immune response by decreasing the expression of MHC-II, NF-κB, DLA and other immune-related molecules. While, the metabolomics results revealed that the expression of oxoglutaric acid, glutamate, d-aspartate, arginine, taurochenodeoxycholic acid and taurocholic acid which participated in tricarboxylic acid (TCA) cycle, glycolysis/gluconeogenesis, bile secretion, biosynthesis of amino acids pathway were significantly decreased. The correlation results of proteomics and metabolomics showed that DEPs and DEMs were mainly co-enriched in bile secretion pathway to regulate intestinal peristalsis. Analyzing DEPs and DEMs will not only provide insights into the mechanisms of host parasite interaction, but also aid in identifying potential targets for therapy and diagnosis, thus setting the groundwork for effectively preventing and managing toxocariasis.

Longitudinal plasma proteomics reveals biomarkers of alveolar-capillary barrier disruption in critically ill COVID-19 patients

The pathobiology of respiratory failure in COVID-19 consists of a complex interplay between viral cytopathic effects and a dysregulated host immune response. In critically ill patients, imatinib treatment demonstrated potential for reducing invasive ventilation duration and mortality. Here, we perform longitudinal profiling of 6385 plasma proteins in 318 hospitalised patients to investigate the biological processes involved in critical COVID-19, and assess the effects of imatinib treatment. Nine proteins measured at hospital admission accurately predict critical illness development. Next to dysregulation of inflammation, critical illness is characterised by pathways involving cellular adhesion, extracellular matrix turnover and tissue remodelling. Imatinib treatment attenuates protein perturbations associated with inflammation and extracellular matrix turnover. These proteomic alterations are contextualised using external pulmonary RNA-sequencing data of deceased COVID-19 patients and imatinib-treated Syrian hamsters. Together, we show that alveolar capillary barrier disruption in critical COVID-19 is reflected in the plasma proteome, and is attenuated with imatinib treatment. This study comprises a secondary analysis of both clinical data and plasma samples derived from a clinical trial that was registered with the EU Clinical Trials Register (EudraCT 2020–001236–10, https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001236-10/NL) and Netherlands Trial Register (NL8491, https://www.trialregister.nl/trial/8491).

The CUL5 E3 ligase complex negatively regulates central signaling pathways in CD8+ T cells

CD8+ T cells play an important role in anti-tumor immunity. Better understanding of their regulation could advance cancer immunotherapies. Here we identify, via stepwise CRISPR-based screening, that CUL5 is a negative regulator of the core signaling pathways of CD8+ T cells. Knocking out CUL5 in mouse CD8+ T cells significantly improves their tumor growth inhibiting ability, with significant proteomic alterations that broadly enhance TCR and cytokine signaling and their effector functions. Chemical inhibition of neddylation required by CUL5 activation, also enhances CD8 effector activities with CUL5 validated as a major target. Mechanistically, CUL5, which is upregulated by TCR stimulation, interacts with the SOCS-box-containing protein PCMTD2 and inhibits TCR and IL2 signaling. Additionally, CTLA4 is markedly upregulated by CUL5 knockout, and its inactivation further enhances the anti-tumor effect of CUL5 KO. These results together reveal a negative regulatory mechanism for CD8+ T cells and have strong translational implications in cancer immunotherapy.

Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer's Disease Across Distinct Brain Regions.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex etiology influenced by confounding factors such as genetic polymorphisms, age, sex, and race. Traditionally, AD research has not prioritized these influences, resulting in dramatically skewed cohorts such as three times the number of Apolipoprotein E (APOE) ε4-allele carriers in AD relative to healthy cohorts. Thus, the resulting molecular changes in AD have previously been complicated by the influence of apolipoprotein E disparities. To explore how apolipoprotein E polymorphism influences AD progression, 62 post-mortem patients consisting of 33 AD and 29 controls (Ctrl) were studied to balance the number of ε4-allele carriers and facilitate a molecular comparison of the apolipoprotein E genotype. Lipid and protein perturbations were assessed across AD diagnosed brains compared to Ctrl brains, ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s and APOE4- for non-ε4 carriers), and differences in ε3ε3 and ε3ε4 Ctrl brains across two brain regions (frontal cortex (FCX) and cerebellum (CBM)). The region-specific influences of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction and cell proteostasis at the core of AD pathophysiology in the post-mortem brains, indicating these two processes may be influenced by genotypic differences and brain morphology.

Multi-omics reveals changed energy metabolism of liver and muscle by caffeine after mice swimming.

In recent years, numerous studies have investigated the effects of caffeine on exercise, and provide convincing evidence for its ergogenic effects on exercise performance. However, the precise mechanisms underlying these ergogenic effects remain unclear. In this study, an exercise swimming model was conducted to investigate the effects of orally administered with caffeine before swimming on the alterations of proteome and energy metabolome of liver and muscle after swimming. We found proteins in liver, such as S100a8, S100a9, Gabpa, Igfbp1 and Sdc4, were significantly up-regulated, while Rbp4 and Tf decreased after swimming were further down-regulated in caffeine group. The glycolysis and pentose phosphate pathways in liver and muscle were both significantly down-regulated in caffeine group. The pyruvate carboxylase and amino acid levels in liver, including cysteine, serine and tyrosine, were markedly up-regulated in caffeine group, exhibiting a strong correlation with the increased pyruvic acid and oxaloacetate levels in muscle. Moreover, caffeine significantly decreased the lactate levels in both liver and muscle after swimming, potentially benefiting exercise performance.

Potential neuroprotective effects of anti‐cytokine antibodies against lipopolysaccharide‐induced microglial inflammation

Aims/Purpose: Microglia‐mediated neuroinflammation is involved in many neurodegenerative diseases, including glaucoma and diabetic retinopathy. This study examined the effects of anti‐cytokine antibodies on LPS‐induced inflammatory response of microglial cells, and explored the underlying molecular mechanisms by proteomic analysis.Methods: A lipopolysaccharide (LPS)‐induced neuroinflammation model was established in murine BV‐2 microglial cells in vitro. The expression of microglial cell markers (CD68 and CD206) were detected by immunocytochemistry. BV‐2 was stimulated with LPS (1 μg/mL) with or without anti‐tumour necrosis factor‐alpha (anti‐TNF‐α, 2 μg /ml) or anti‐interleukin‐1 beta (anti‐IL‐1β, 4 μg /ml) for 24 h. LPS‐induced inflammation level was analysed using the Griess assay to determine nitric oxide (NO) concentration. Proteome alterations were characterized employing the mass spectrometry‐based proteomics and bioinformatics analyses.Results: Immunocytochemistry confirmed the expression of microglial markers in BV‐2 cells. LPS stimulation induced excessive activation of BV‐2, with significant increase in NO generation (p < 0.0001). Anti‐IL‐1β significantly attenuated 34% of the LPS‐induced NO generation (p < 0.0001). Interestingly, anti‐TNF‐α showed a stronger anti‐inflammatory effect by reducing 57% of LPS‐induced NO generation (p < 0.0001). Bioinformatics analyses revealed that differentially expressed proteins involved in inflammation (p = 3.8 × 10−5) and apoptotic (p = 3.2 × 10−11) signalling pathways were significantly activated by LPS. Particularly, LPS‐mediated upregulation of STAT1 (p = 8.6 × 10−6) was restored by anti‐IL‐1β and anti‐TNF‐α (p = 3.1 × 10−4 and p = 2.7 × 10−3, respectively). Anti‐IL‐1β induced proteome alterations involved in anti‐inflammatory signalling pathways and promote glial cell polarization towards neuroprotective M2 phenotype (p = 5.3 × 10−6), while anti‐TNF‐α treatment significantly regulated proteins involved in immune response of macrophages (p = 2.7 × 10−8).Conclusions: In summary, anti‐TNF‐α and anti‐IL‐1β antibodies alleviated LPS‐induced neuroinflammation and altered proteins involved in neuroprotective pathways. Hence, anti‐cytokine antibodies have therapeutic potential as neuroprotective agents.

Alterations in the plasma proteome persist ten months after recovery from mild to moderate SARS-CoV-2 infection.

Limited data are available describing the effects of SARS-CoV-2 breakthrough infections on the plasma proteome. PCR-positive SARS-CoV-2 patients, enrolled in a natural history study, underwent analysis of the plasma proteome. A prospective cohort of 66 unvaccinated and 24 vaccinated persons with different degrees of infection severity were evaluated acutely (within 40 days of symptom onset), and at three and ten months. Comparisons based on vaccination status alone and unsupervised hierarchical clustering were performed. A second cohort of vaccinated Omicron patients were evaluated acutely and at ten months. Acutely, unvaccinated patients manifested overexpression of proteins involved in immune and inflammatory responses, while vaccinated patients exhibited adaptive immune responses without significant inflammation. At three and ten months, only unvaccinated patients had diminished but sustained inflammatory (C3b, CCL15, IL17RE) and immune responses (DEFA5,TREM1). Both groups had underexpression of pathways essential for cellular function, signaling, and angiogenesis (AKT1, MAPK14, HSPB1) across phases. Unsupervised clustering, based on protein expression, identified four groups of patients with variable vaccination rates demonstrating that additional clinical factors influence the plasma proteome. The proteome of vaccinated Omicron patients did not differ from vaccinated pre-Omicron patients. Vaccination attenuates the inflammatory response to SARS-CoV-2 infection across phases. However, at ten months after symptom onset, changes in the plasma proteome persist in both vaccinated and unvaccinated individuals, which may be relevant to post-acute sequelae of SARS-CoV-2 and other viral infections associated with post-acute infection syndromes.

Investigation of pathogenesis of the pterygium by transcriptomic and proteomic analysis

Aims/Purpose: Extrinsic factors, including ultraviolet rays, are well‐known risk factors in the pathogenesis of pterygia. However, the biomolecular intrinsic factors that could serve as therapeutic targets have not been fully elucidated. Therefore, this study aimed to investigate comprehensive changes in the proteome and transcriptome of conjunctival pterygium tissues from human patients using multi‐omics analysis.Methods: A prospective cross‐sectional study enrolled 30 patients diagnosed with pterygium who underwent surgery from May 2020 to December 2020. During surgery, normal conjunctiva and conjunctival tissues with pterygium were collected. Nano liquid chromatography with tandem mass labelling spectrometry and next‐generation sequencing were performed on the obtained specimens. Proteins with significantly expressed mRNAs between pterygium and control were identified, and their biological processes were analysed using bioinformatic tools.Results: Through the integration of transcriptomic and proteomic analyses, a total of 1288 proteins were identified, which were also confirmed by mRNA expression. Hierarchical clustering heatmap analysis revealed distinct clusters corresponding to each group, and principle component analysis demonstrated a prominent separation between groups. Among the identified proteins, 185 were determined as differentially expressed proteins (DEPs), with 154 upregulated and 31 downregulated in pterygium compared to the control group. Protein–protein interaction network analysis revealed six distinct interconnected networks of DEPs, and gene ontology enrichment analysis highlighted biological processes associated with extracellular matrix organization, insulin‐like growth factor‐related pathways, and the immune system.Conclusions: This study demonstrates proteomic alterations in conjunctival tissues of patients with pterygium, which are corroborated by RNA expression analysis. The identified significant proteins in pterygium are associated with specific biological pathways and could be potential therapeutic targets. The integrated proteo‐transcriptomic analysis provides essential insights into the pathogenesis of pterygium, contributing to the identification of novel treatment strategies.

Anti‐cytokine antibodies attenuated neuroglial degeneration under hypoxic stress

Aims/Purpose: Hypoxia is a vital cause of neurodegenerative diseases such as glaucoma and retinal ischemia. We investigated the potential neuroprotective effects of anti‐cytokine antibodies against hypoxic damage.Methods: An in vitro hypoxia model was established using cobalt chloride (CoCl2) stimulation in microglial cells (BV‐2). Immunofluorescence staining was performed to detect the expression markers of different microglial phenotypes in BV‐2. Different concentrations of CoCl2 (0–150 μM) were tested to generate dose–response curves. Following exposure to CoCl2 at an approximate IC50 of 0.1 mM, BV‐2 were treated with anti‐tumour necrosis factor‐alpha (anti‐TNF‐α) or anti‐ interleukin‐1 beta (anti‐IL‐1β) antibodies for 24 h. Cell viability was determined by MTS assay. Proteome changes in BV‐2 were analysed with the mass spectrometry‐based proteomics approach.Results: Immunocytochemistry revealed the expression of M1 marker consisting CD68 and M2 marker CD206 in the BV‐2 cells. Hypoxia induced by mimetic CoCl2 led to a concentration‐dependent viability impairment in BV‐2 and cells treated with 0.1 mM CoCl2 for 24 h showed decreased viability of ~46%‐ 50%. Interestingly, the application of anti‐TNF‐α (2ug/ml) and anti‐IL‐1β (4ug/ml) antibodies significantly improved the BV‐2 cell viability to 80% (p < 0.0001) and 76% (p < 0.0001), respectively. Proteomics and bioinformatics analyses of the differentially expressed proteins revealed that CoCl2 induced proteome alterations involved in mitochondrial dysfunction signalling pathway (p = 1.10 × 10−3), which were eliminated by the treatment with both anti‐TNF‐α and anti‐IL‐1β. Besides, CoCl2‐induced activation of endoplasmic reticulum (ER) stress response (p = 3.33 × 10−9) was alleviated by anti‐IL‐1β and anti‐TNF‐α treatment (p = 1.7 × 10−7 and p = 8.82 × 10−7, respectively). Particularly, anti‐TNF‐α antibody significantly downregulated proteins involved in apoptosis (p = 1.58 × 10−10). Anti‐IL‐1β induced Nrf2‐mediated antioxidant response and exerts protection against oxidative stress (p = 1.00 × 10−6).Conclusions: Antibodies targeting cytokines provide neuroprotective effects on microglial cells from hypoxic stress. These findings demonstrated the potential use of anti‐cytokines in the treatment of neurodegenerative diseases.

Gene Ontology and pathway enrichment analysis of the upregulated biological Proteins in Single-Line Transgenic Pod Borer Resistant Cowpea

Aim: This research seeks to clarify the proteomic alterations linked to PBR cowpea in comparison to its non-transgenic isoline. Gene Ontology and pathway enrichment analyses were conducted to pinpoint critical biological processes, molecular functions, and cellular components that exhibit upregulation in the transgenic cowpea. Methods: Seeds from both cowpea lines were finely ground, and proteins were extracted utilizing Tris-EDTA and LDS buffers. The proteins were then separated and identified through mass spectrometry (MS), with peptide sequences being matched against protein sequence databases. Results: The enrichment analysis indicated upregulation in pathways associated with pollen tube adhesion, cell-cell adhesion, and biological processes pertinent to both intraspecies and interspecies interactions with no significant difference. Importantly, pathways linked to defense mechanisms against fungi and other organisms were notably enriched, suggesting an enhanced ability to detect and respond to biotic stresses. The results of this study does not underscore any substantial proteomic modifications in the transgenic cowpea. Conclusion: These findings offer valuable targets for genetic engineering and breeding initiatives aimed at creating cowpea varieties with enhanced pest resistance, thus improving crop yield and sustainability. Future investigations should concentrate on validating these proteins and pathways through functional assays and examining their roles in resistance under field conditions.

Quantitative proteomic analysis reveals regulatory networks of extracellular matrix receptor interaction pathways in endothelial cells after myocardial infarction

Cardiac fibrosis, a significant pathological alteration following myocardial infarction (MI), remains enigmatic with respect to the role of cardiac endothelial cells (ECs). To elucidate the proteomic shifts in cardiac ECs accompanying MI-induced cardiac fibrosis, a standard MI mice model was established through ligation of the left anterior descending branch. Following 14 days of effective modeling, we isolated primary ECs from the hearts of both sham and MI models utilizing the CD31 microbeads sorting technique. Quantitative proteomics and bioinformatics methodologies, including tandem mass spectrometry, were employed to discern proteomic alterations in the primary endothelial cells of the experimental groups. Comprehensive analyses, including Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, functional enrichment analysis, and functional enrichment cluster analysis, revealed an up-regulation of proteins associated with extracellular matrix-receptor interaction pathway in cardiac fibrosis post-MI. Subsequent Western blot analysis confirmed the up-regulation of specific proteins involved in this pathway, namely collagen type VI alpha 2 (Col6α2), vitronectin (Vtn), and integrin beta (Itgβ). We conclude that the expression levels of Col6α2, Vtn, and Itgβ in primary ECs during the early stage of cardiac fibrosis, 14 days post-MI, were significantly elevated compared to the sham group (P < 0.05). This observation suggests that ECM-receptor interaction could potentially influence the progression of cardiac fibrosis following MI.

Induction of proteome changes involved in the cloning of mcr-1 and mcr-2 genes in Escherichia coli DH5-α strain to evaluate colistin resistance

Plasmid genes, termed mobile colistin resistance-1 (mcr-1) and mobile colistin resistance-2 (mcr-2), are associated with resistance to colistin in Escherichia coli (E. coli). These mcr genes result in a range of protein modifications contributing to colistin resistance. This study aims to discern the proteomic characteristics of E. coli-carrying mcr-1 and mcr-2 genes. Furthermore, it evaluates the expression levels of various proteins under different conditions (with and without colistin). Plasmid extraction was performed using an alkaline lysis-based plasmid extraction kit, whereas polymerase chain reaction was used to detect the presence of mcr-1 and mcr-2 plasmids. The E. coli DH5α strain served as the competent cell for accepting and transforming mcr-1 and mcr-2 plasmids. We assessed proteomic alterations in the E. coli DH5α strain both with and without colistin in the growth medium. Proteomic data were analysed using mass spectrometry. The findings revealed significant protein changes in the E. coli DH5α strain following cloning of mcr-1 and mcr-2 plasmids. Of the 20 proteins in the DH5α strain, expression in 8 was suppressed following transformation. In the presence of colistin in the culture medium, 39 new proteins were expressed following transformation with mcr-1 and mcr-2 plasmids. The proteins with altered expression play various roles. The results of this study highlight numerous protein alterations in E. coli resulting from mcr-1 and mcr-2-mediated resistance to colistin. This understanding can shed light on the resistance mechanism. Additionally, the proteomic variations observed in the presence and absence of colistin might indicate potential adverse effects of indiscriminate antibiotic exposure on treatment efficacy and heightened pathogenicity of microorganisms.

Sperm proteomic landscape is altered in breeding bulls with greater sperm DNA fragmentation index

Although, it is well understood that sperm DNA damage is associated with infertility, the molecular details of how damaged sperm DNA affects fertility are not fully elucidated. Since sperm proteins play an important role in fertilization and post-fertilization events, the present study aimed to identify the sperm proteomic alterations in bulls with high sperm DNA Fragmentation Index (DFI%). Semen from Holstein-Friesian crossbred breeding bulls (n = 50) was subjected to Sperm Chromatin Structure Assay. Based on DFI%, bulls were classified into either high- (HDFI; n = 6), or low-DFI (LDFI; n = 6) and their spermatozoa were subjected to high throughput proteomic analysis. Liquid chromatography and mass spectrometry analysis identified 4567 proteins in bull spermatozoa. A total of 2660 proteins were found common to both the groups, while 1193 and 714 proteins were unique to HDFI and LDFI group, respectively. A total of 265 proteins were up regulated and 262 proteins were down regulated in HDFI group. It was found that proteins involved in capacitation [heparin binding (molecular function), ERK1 and ERK2 cascade (biological process), PI3K-Akt signalling (pathway), Jak-STAT signalling (pathway)], spermatogenesis [TLR signalling (pathway), gamete generation (biological process)] and DNA repair mechanism (biological process) were significantly altered in the bulls with high DFI%.

Discovery of plasma proteome markers associated with clinical outcome and immunological stress after cardiac surgery.

Molecular mechanisms underlying perioperative acute phase reactions in cardiac surgery are largely unknown. We aimed to characterise perioperative alterations of the acute phase plasma proteome in a cohort of adult patients undergoing on-pump cardiac surgery using high-throughput mass spectrometry and to identify candidate proteins potentially relevant to postoperative clinical outcome through a novel, multi-step approach. This study is an analysis of the Bern Perioperative Biobank, a prospective cohort of adults who underwent cardiac surgery with the use of cardiopulmonary bypass (CPB) at Bern University Hospital between January and December 2019. Blood samples were taken before induction of anaesthesia and on postoperative day one. Proteomic analyses were performed by mass spectrometry. Through a multi-step, exploratory approach, hit-proteins were first identified according to their perioperative prevalence and dynamics. The set of hit-proteins were associated with predefined clinical outcome measures (all-cause one-year mortality, length of hospital stay, postoperative myocardial infarction and stroke until hospital discharge). 192 patients [75.5% male, median age 67.0 (IQR 60.0-73.0)] undergoing cardiac surgery with the use of CPB were included in this analysis. In total, we identified and quantified 402 proteins across all samples, whereof 30/402 (7%) proteins were identified as hit-proteins. Three hit-proteins-LDHB, VCAM1 and IGFBP2-demonstrated the strongest associations with clinical outcomes. After adjustment both for age, sex, BMI and for multiple comparisons, the scaled preoperative levels of IGFBP2 were associated with 1-year all-cause mortality (OR 10.63; 95% CI: 2.93-64.00; p = 0.046). Additionally, scaled preoperative levels of LDHB (OR 5.58; 95% CI: 2.58-8.57; p = 0.009) and VCAM1 (OR 2.32; 95% CI: 0.88-3.77; p = 0.05) were found to be associated with length of hospital stay. We identified a subset of promising candidate plasma proteins relevant to outcome after on-pump cardiac surgery. IGFBP2 showed a strong association with clinical outcome measures and a significant association of preoperative levels with 1-year all-cause mortality. Other proteins strongly associated with outcome were LDHB and VCAM1, reflecting the dynamics in the acute phase response, inflammation and myocardial injury. We recommend further investigation of these proteins as potential outcome markers after cardiac surgery. ClinicalTrials.gov; NCT04767685, data are available via ProteomeXchange with identifier PXD046496.