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D-site binding protein, DBP, is a clock-controlled transcription factor and drives daily rhythms of physiological processes through the regulation of an array of genes harboring a DNA binding motif, D-box. DBP protein levels show a circadian oscillation with an extremely robust peak/trough ratio, but it is elusive how the temporal pattern is regulated by post-translational regulation. In this study, we show that DBP protein levels are down-regulated by the ubiquitin-proteasome pathway. Analysis using 19 dominant-negative forms of E2 enzymes have revealed that UBE2G1 and UBE2T mediate the degradation of DBP. A proteomic analysis of DBP-interacting proteins and database screening have identified Tumor necrosis factor Receptor-Associated Factor 7 (TRAF7), a RING-type E3 ligase, that forms a complex with UBE2G1 and/or UBE2T. Ubiquitination analysis have revealed that TRAF7 enhances K48-linked polyubiquitination of DBP in cultured cells. Overexpression of TRAF7 down-regulates DBP protein level, while knockdown of TRAF7 up-regulates DBP in cultured cells. Knockout of TRAF7 in NIH3T3 cells have revealed that TRAF7 mediates the time-of-the-day-dependent regulation of DBP levels. Furthermore, TRAF7 has a period-shortening effect on the cellular clock. Together, TRAF7 plays an important role in circadian clock oscillation through destabilization of DBP. An E3 ligase TRAF7 regulates time-of-the-day-dependent expression of DBP proteins via the ubiquitin-proteasome system, providing insight into the posttranslational regulation of clock proteins in the mammalian circadian clockwork. [ABSTRACT FROM AUTHOR]

Intraepithelial lymphocytes (IELs) reside in the epithelial layer and protect against foreign pathogens, maintaining the epithelial barrier function in the intestine. Interactions between IEL and epithelial cells are required for IELs to function effectively; however, the underlying molecular machinery remains to be elucidated. In this study, we found that intestinal epithelium-specific deficiency of the clathrin adaptor protein (AP)-1B, which regulates basolateral protein sorting, led to a massive reduction in IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins (E-cadherin, butyrophilin-like 2, and plexin B2), were decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among these proteins, CD166 interacted with CD6 on the surface of induced IEL. CD166 knockdown, using shRNA in intestinal organoid cultures, significantly inhibited IEL recruitment to the epithelial layer. These findings highlight the essential role of AP-1B-mediated basolateral sorting in IEL maintenance and survival within the epithelial layer. This study reveals a novel function of AP-1B in the intestinal immune system. [ABSTRACT FROM AUTHOR]

Background: Acute Kidney Injury (AKI), a prevalent complication of Liver Transplantation (LT) that occurs during the perioperative period has been established to profoundly impact the prognosis of transplant recipients. This study aimed to investigate the mechanism of the hepatic IRI-induced AKI and to identify potential therapeutic targets for treating this condition and improving the prognosis of LT patients. Methods: An integrated transcriptomics and proteomics approach was employed to investigate transcriptional and proteomic alterations in hepatic IRI-induced AKI and the hypoxia-reoxygenation (H/R) model using TCMK-1 cells and the hepatic IRI-induced AKI mouse model using male C57BL/6 J mice were employed to elucidate the underlying mechanisms. Hematoxylin–eosin staining, reverse transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot were used to assess the effect of Rosiglitazone (RGZ) on hepatic IRI-induced AKI in vitro and in vivo. Results: According to the results, 322 genes and 128 proteins were differentially expressed between the sham and AKI groups. Furthermore, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway analyses revealed significant enrichment in pathways related to amino acid and lipid metabolism. Additionally, the Protein–Protein Interaction (PPI) network analysis of the kidney tissues obtained from a hepatic IRI-induced AKI mouse model highlighted arachidonic acid metabolism as the most prominent pathway. Animal and cellular analyses further revealed that RGZ, a PPAR-γ agonist, could inhibit the expression of the PPAR-γ/NF-κB signaling pathway-associated proteins in in vitro and in vivo. Conclusions: These findings collectively suggest that RGZ ameliorates hepatic IRI-induced AKI via PPAR-γ/NF-κB signaling pathway modulation, highlighting PPAR-γ as a crucial therapeutic target for AKI prevention post-LT. [ABSTRACT FROM AUTHOR]

In developed countries, endometrial cancer (EC) is the most prevalent gynecological cancer. ATP5F1D is a subunit of ATP synthase, as well as an important component of the mitochondrial electron transport chain (ETC). ETC plays a compelling role in carcinogenesis. To date, little is known about the role of ATP5F1D in EC. We undertook data-independent acquisition mass spectrometry (DIA-MS) of 20 EC patients, comprising 10 high-grade and 10 low-grade cancer tissues. Biological functions of differentially expressed genes (DEGs) were analyzed by GO and KEGG. The expression level, clinicopathological features, diagnostic potency, prognostic value, RNA modifications, immune characteristics, and therapy response of ATP5F1D were investigated. In total, 77 DEGs were acquired by DIA analysis, which were closely related to regulating immune response and metabolic pathways. Among the five genes (NDUFB8, SLC26A2, RAF1, ATP5F1D, and GSTM5) involving in reactive oxygen species pathway, ATP5F1D showed the most significant differential expression (2.903-fold change). We found ATP5F1D had a high diagnostic value and was associated with a favorable prognosis in EC patients. After analyzing the RNA modifications of ATP5F1D, revealing a negative regulation between them. Additionally, ATP5F1D was closely related to tumor immune infiltration. Our results suggested T-cell dysfunction and TAM-M2 polarization might be the important mechanisms of ATP5F1D to facilitate tumor immune escape. Noticeably, EC patients with ATP5F1D-high expression had better immune treatment responses and were more sensitive to chemotherapy drugs. ATP5F1D can be used as a biomarker for diagnosis, prognosis, and immune infiltration of EC, and offers a crucial reference for personalized treatment of EC patients. [ABSTRACT FROM AUTHOR]

Background: The incidence of gallstones is high in Qinghai Province. However, the molecular mechanisms underlying the development of gallstones remain unclear. Methods: In this study, we collected urine samples from 30 patients with gallstones and 30 healthy controls. The urine samples were analysed using multi-omics platforms. Proteomics analysis was conducted using data-independent acquisition, whereas metabolomics analysis was performed using liquid chromatography–mass spectrometry (LC–MS). Results: Among the patients with gallstones, we identified 49 down-regulated and 185 up-regulated differentially expressed proteins as well as 195 up-regulated and 189 down-regulated differentially expressed metabolites. Six pathways were significantly enriched: glycosaminoglycan degradation, arginine and proline metabolism, histidine metabolism, pantothenate and coenzyme A biosynthesis, drug metabolism–other enzymes, and the pentose phosphate pathway. Notably, 10 differentially expressed proteins and metabolites showed excellent predictive performance and were selected as potential biomarkers. Conclusion: The findings of our metabolomics and proteomics analyses provide new insights into novel biomarkers for patients with cholelithiasis in high-altitude areas. [ABSTRACT FROM AUTHOR]

Background: Heart failure (HF) often disrupts the protein quality control (PQC) system leading to protein aggregate accumulation. Evidence from tissue biopsies showed that exercise restores PQC system in HF; however, little is known about its effects on plasma proteostasis. Aim: To determine the effects of exercise training on the load and composition of plasma SDS-resistant protein aggregates (SRA) in patients with HF with reduced ejection fraction (HFrEF). Methods: Eighteen patients with HFrEF (age: 63.4 ± 6.5 years; LVEF: 33.4 ± 11.6%) participated in a 12-week combined (aerobic plus resistance) exercise program (60 min/session, twice per week). The load and content of circulating SRA were assessed using D2D SDS-PAGE and mass spectrometry. Cardiorespiratory fitness, quality of life, and circulating levels of high-sensitive C-reactive protein, N-terminal pro-B-type natriuretic peptide (NT-proBNP), haptoglobin and ficolin-3, were also evaluated at baseline and after the exercise program. Results: The exercise program decreased the plasma SRA load (% SRA/total protein: 38.0 ± 8.9 to 36.1 ± 9.7%, p = 0.018; % SRA/soluble fraction: 64.3 ± 27.1 to 59.8 ± 27.7%, p = 0.003). Plasma SRA of HFrEF patients comprised 31 proteins, with α-2-macroglobulin and haptoglobin as the most abundant ones. The exercise training significantly increased haptoglobin plasma levels (1.03 ± 0.40 to 1.11 ± 0.46, p = 0.031), while decreasing its abundance in SRA (1.83 ± 0.54 × 1011 to 1.51 ± 0.59 × 1011, p = 0.049). Cardiorespiratory fitness [16.4(5.9) to 19.0(5.2) ml/kg/min, p = 0.002], quality of life, and circulating NT-proBNP [720.0(850.0) to 587.0(847.3) pg/mL, p = 0.048] levels, also improved after the exercise program. Conclusion: Exercise training reduced the plasma SRA load and enhanced PQC, potentially via haptoglobin-mediated action, while improving cardiorespiratory fitness and quality of life of patients with HFrEF. [ABSTRACT FROM AUTHOR]

Physical frailty is a syndrome that typically manifests in later life, although the pathogenic process causing physical frailty likely begins decades earlier. To date, few studies have examined the biological signatures in mid-life associated with physical frailty later in life. Among 4,189 middle-aged participants (57.8 ± 5.0 years, 55.8% women) from the Atherosclerosis Risk in Community (ARIC) study, we evaluated the associations of 4,955 plasma proteins (log 2-transformed and standardized) measured using the SomaScan platform with their frailty status approximately 20 years later. Using multinomial logistic regression models adjusting for demographics, health behaviors, kidney function, total cholesterol, and comorbidities, 12 and 221 proteins were associated with prefrailty and frailty in later life, respectively (FDR p < 0.05). Top frailty-associated proteins included neurocan core protein (NCAN, OR = 0.66), fatty acid-binding protein heart (FABP3, OR = 1.62) and adipocyte (FABP4, OR = 1.65), as well proteins involved in the contactin-1 (CNTN1), toll-like receptor 5 (TLR5), and neurogenic locus notch homolog protein 1 (NOTCH1) signaling pathway relevant to skeletal muscle regeneration, myelination, and inflammation. Pathway analyses suggest midlife dysregulation of inflammation, metabolism, extracellular matrix, angiogenesis, and lysosomal autophagy among those at risk for late-life frailty. After further adjusting for midlife body mass index (BMI) – an established frailty risk factor – only CNTN1 (OR = 0.75) remained significantly associated with frailty. Post-hoc analyses demonstrated that the top 41 midlife frailty-associated proteins mediate 32% of the association between mid-life BMI and late-life frailty. Our findings provide new insights into frailty etiology earlier in the life course, enhancing the potential for prevention. [ABSTRACT FROM AUTHOR]

Background: This study investigates the role of CXXC5 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) within the bone marrow microenvironment, utilizing advanced methodologies such as single-cell RNA sequencing (scRNA-seq), CRISPR-Cas9, and proteomic analysis. Methods: We employed flow cytometry to isolate HSCs from bone marrow samples, followed by scRNA-seq analysis using the 10x Genomics platform to examine cell clustering and CXXC5 expression patterns. CRISPR-Cas9 and lentiviral vectors facilitated the knockout and overexpression of CXXC5 in HSCs. The impact on HSCs was assessed through qRT-PCR, Western blot, CCK-8, CFU, and LTC-IC assays, alongside flow cytometry to measure apoptosis and cell proportions. A mouse model was also used to evaluate the effects of CXXC5 manipulation on HSC engraftment and survival rates. Results: Our findings highlight the diversity of cell clustering and the significant role of CXXC5 in HSC regulation. Knockout experiments showed reduced proliferation and accelerated differentiation, whereas overexpression led to enhanced proliferation and delayed differentiation. Proteomic analysis identified key biological processes influenced by CXXC5, including cell proliferation, differentiation, and apoptosis. In vivo results demonstrated that CXXC5 silencing impaired HSC engraftment in a bone marrow transplantation model. Conclusion: CXXC5 is crucial for the regulation of HSC self-renewal and differentiation in the bone marrow microenvironment. Its manipulation presents a novel approach for enhancing HSC function and provides a potential therapeutic target for hematological diseases. [ABSTRACT FROM AUTHOR]

Diclofenac is a hepatotoxic non-steroidal anti-inflammatory drug (NSAID) that affects liver histology and its protein expression levels. Here, we studied the effect of diclofenac on rat liver when co-administrated with either Yersinia enterocolitica strain 8081 serotype O:8 biovar 1B (D*Y) or Lactobacillus fermentum strain 9338 (D*L). Spectroscopic analysis of stool samples showed biotransformation of diclofenac. When compared with each other, D*Y rats lack peaks at 1709 and 1198 cm−1, while D*L rats lack peaks at 1411 cm−1. However, when compared to control, both groups lack peaks at 1379 and 1170 cm−1. Assessment of serum biomarkers of hepatotoxicity indicated significantly altered activities of AST (D*Y: 185.65 ± 8.575 vs Control: 61.9 ± 2.607, D*L: 247.5 ± 5.717 vs Control: 61.9 ± 2.607), ALT (D*Y: 229.8 ± 6.920 vs Control: 70.7 ± 3.109, D*L: 123.75 ± 6.068 vs Control: 70.7 ± 3.109), and ALP (D*Y: 276.4 ± 18.154 vs Control: 320.6 ± 9.829, D*L: 298.5 ± 12.336 vs Control: 320.6 ± 9.829) in IU/L. The analysis of histological alterations showed hepatic sinusoidal dilation with vein congestion and cell infiltration exclusively in D*Y rats along with other histological changes that are common to both test groups, thereby suggesting more pronounced alterations in D*Y rats. Further, LC–MS/MS based label-free quantitation of proteins from liver tissues revealed 74.75% up-regulated, 25.25% down-regulated in D*Y rats and 51.16% up-regulated, 48.84% down-regulated in D*L experiments. The proteomics-identified proteins majorly belonged to metabolism, apoptosis, stress response and redox homeostasis, and detoxification and antioxidant defence that demonstrated the potential damage of rat liver, more pronounced in D*Y rats. Altogether the results are in favor that the administration of lactobacilli somewhat protected the rat hepatic cells against the diclofenac-induced toxicity. [ABSTRACT FROM AUTHOR]

The bacterial secretome represents a comprehensive catalog of proteins released extracellularly that have multiple important roles in virulence and intercellular communication. This study aimed to characterize the secretome of an environmental isolate Pseudomonas aeruginosa S-8 by analyzing trypsin-digested culture supernatant proteins using nano-LC–MS/MS tool. Using a combined approach of bioinformatics and mass spectrometry, 1088 proteins in the secretome were analyzed by PREDLIPO, SecretomeP 2.0, SignalP 4.1, and PSORTb tool for their subcellular localization and further categorization of secretome proteins according to signal peptides. Using the gene ontology tool, secretome proteins were categorized into different functional categories. KEGG pathway analysis identified the secreted proteins into different metabolic functional pathways. Moreover, our LC–MS/MS data revealed the secretion of various CAZymes into the extracellular milieu, which suggests its strong biotechnological applications to breakdown complex carbohydrate polymers. The identified immunodominant epitopes from the secretome of P. aeruginosa showed the characteristic of being non-allergenic, highly antigenic, nontoxic, and having a low risk of triggering autoimmune responses, which highlights their potential as successful vaccine targets. Overall, the identification of secreted proteins of P. aeruginosa could be important for both diagnostic purposes and the development of an effective candidate vaccine. [ABSTRACT FROM AUTHOR]

This paper proposes a novel multi-view ensemble predictor model that is designed to address the challenge of determining synergistic drug combinations by predicting both the synergy score value values and synergy class label of drug combinations with cancer cell lines. The proposed methodology involves representing drug features through four distinct views: Simplified Molecular-Input Line-Entry System (SMILES) features, molecular graph features, fingerprint features, and drug-target features. On the other hand, cell line features are captured through four views: gene expression features, copy number features, mutation features, and proteomics features. To prevent overfitting of the model, two techniques are employed. First, each view feature of a drug is paired with each corresponding cell line view and input into a multi-task attention deep learning model. This multi-task model is trained to simultaneously predict both the synergy score value and synergy class label. This process results in sixteen input view features being fed into the multi-task model, producing sixteen prediction values. Subsequently, these prediction values are utilized as inputs for an ensemble model, which outputs the final prediction value. The 'MVME' model is assessed using the O'Neil dataset, which includes 38 distinct drugs combined across 39 distinct cancer cell lines to output 22,737 drug combination pairs. For the synergy score value, the proposed model scores a mean square error (MSE) of 206.57, a root mean square error (RMSE) of 14.30, and a Pearson score of 0.76. For the synergy class label, the model scores 0.90 for accuracy, 0.96 for precision, 0.57 for kappa, 0.96 for the area under the ROC curve (ROC-AUC), and 0.88 for the area under the precision-recall curve (PR-AUC). Scientific contribution: This paper presents an enhanced synergistic drug combination model by utilizing four different feature views for drugs and four views for cancer cell lines. Each view is then input into a multi-task deep learning model to predict both the synergy score and class label simultaneously. To address the challenge of managing diverse views and their corresponding prediction values while avoiding overfitting, an ensemble model is applied. [ABSTRACT FROM AUTHOR]

Ricin is a toxic protein regarded as a potential chemical weapon for bioterrorism or criminal use. In the event of a ricin incident, rapid analytical methods are essential for ricin confirmation in a diversity of matrices, from environmental to human or food samples. Mass spectrometry–based methods provide specific toxin identification but require prior enrichment by antibodies to reach trace-level detection in matrices. Here, we describe a novel assay using the glycoprotein asialofetuin as an alternative to antibodies for ricin enrichment, combined with the specific detection of signature peptides by high-resolution mass spectrometry. Additionally, optimizations made to the assay reduced the sample preparation time from 5 h to 80 min only. Method evaluation confirmed the detection of ricin at trace levels over a wide range of pH and in protein-rich samples, illustrating challenging matrices. This new method constitutes a relevant antibody-free solution for the fast and specific mass spectrometry detection of ricin in the situation of a suspected toxin incident, complementary to active ricin determination by adenine release assays. [ABSTRACT FROM AUTHOR]

Proteome analysis by data-independent acquisition (DIA) has become a powerful approach to obtain deep proteome coverage, and has gained recent traction for label-free analysis of single cells. However, optimal experimental design for DIA-based single-cell proteomics has not been fully explored, and performance metrics of subsequent data analysis tools remain to be evaluated. Therefore, we here formalize and comprehensively evaluate a DIA data analysis strategy that exploits the co-analysis of low-input samples with a so-called matching enhancer (ME) of higher input, to increase sensitivity, proteome coverage, and data completeness. We assess the matching specificity of DIA-ME by a two-proteome model, and demonstrate that false discovery and false transfer are maintained at low levels when using DIA-NN software, while preserving quantification accuracy. We apply DIA-ME to investigate the proteome response of U-2 OS cells to interferon gamma (IFN-γ) in single cells, and recapitulate the time-resolved induction of IFN-γ response proteins as observed in bulk material. Moreover, we uncover co- and anti-correlating patterns of protein expression within the same cell, indicating mutually exclusive protein modules and the co-existence of different cell states. Collectively our data show that DIA-ME is a powerful, scalable, and easy-to-implement strategy for single-cell proteomics. Single-cell proteomics needs improved workflows for reliable proteome characterization in minute samples. Here, the authors demonstrate that matching data to a higher-input sample faithfully improves proteome coverage to examine proteome response and heterogeneity in single-cell populations. [ABSTRACT FROM AUTHOR]

Extracellular proteins play a significant role in shaping microbial communities which, in turn, can impact ecosystem function, human health, and biotechnological processes. Yet, for many ubiquitous microbes, there is limited knowledge regarding the identity and function of secreted proteins. Here, we introduce EXCRETE (enhanced exoproteome characterization by mass spectrometry), a workflow that enables comprehensive description of microbial exoproteomes from minimal starting material. Using cyanobacteria as a case study, we benchmark EXCRETE and show a significant increase over current methods in the identification of extracellular proteins. Subsequently, we show that EXCRETE can be miniaturized and adapted to a 96-well high-throughput format. Application of EXCRETE to cyanobacteria from different habitats (Synechocystis sp. PCC 6803, Synechococcus sp. PCC 11901, and Nostoc punctiforme PCC 73102), and in different cultivation conditions, identified up to 85% of all potentially secreted proteins. Finally, functional analysis reveals that cell envelope maintenance and nutrient acquisition are central functions of the predicted cyanobacterial secretome. Collectively, these findings challenge the general belief that cyanobacteria lack secretory proteins and suggest that multiple functions of the secretome are conserved across freshwater, marine, and terrestrial species. This study introduces EXCRETE (enhanced exoproteome characterization by mass spectrometry), a workflow that enables comprehensive description of microbial exoproteomes from minimal starting material. [ABSTRACT FROM AUTHOR]

Background: Ailanthone (Ail) extracted from medicinal plants has played an anticancer role in multiple cancers, while there is no research about Ail in renal cell carcinoma (RCC). Methods: In the present study, we performed CCK-8 and flow cytometry to assess the effect of Ail on cell viability, apoptosis and cycle. We also performed tandem mass tags (TMT)-labeled quantitative proteomic technology and bioinformatic analysis to identify the functional pathway and proteins of Ail in RCC. Results: The results showed Ail could inhibit cell viability and induce cell apoptosis. Proteomic profiling identified 1732 differentially expressed proteins in cells treated with Ail, compared to the negative control group. Gene ontology function annotation and Gene Set Enrichment Analysis (GSEA) were performed to identified the involved biological processes, molecular function and pathway. Results of GSEA proved the enrichment of Deps in EZH2 targets. The comparison between Deps and EZH2 co-expressed genes revealed 44 overlapped genes and we identified 4 hub genes (CDC20, CEP55, TOP2A, and UBE2C) associated with RCC progression. The molecular docking study revealed a moderate to tight binding potential of Ail and EZH2, and western blotting showed EZH2 was suppressed after cells treated with Ail. Conclusion: Altogether, we identified the anticancer role of Ail in RCC, including inhibition of cell proliferation and induction of apoptosis. The results also screened the key proteins mediate the function of Ail, which have laid a theoretical foundation for elucidating the applications of Ail in clinical research. [ABSTRACT FROM AUTHOR]

The defense mechanisms induced in wild Chinese pine (Pinus tabuliformis) in response to herbivores are not well characterized, especially in the field. To address this knowledge gap, we established a biological model system to evaluate proteome variations in pine needles after feeding by the Chinese pine caterpillar (Dendrolimus tabulaeformis), a major natural enemy and dominant herbivore. Quantitative tandem mass tag (TMT) proteomics and bioinformatics were utilized to systematically identify differentially abundant proteins implicated in the induced defense response of Chinese pine. We validated key protein changes using parallel reaction monitoring (PRM) technology. Pathway analysis revealed that the induced defenses involved phenylpropanoid, coumarin, and flavonoid biosynthesis, among other processes. To elucidate the regulatory patterns underlying pine resistance, we determined the activities of defense enzymes and levels of physiological and biochemical compounds. In addition, the expression of upstream genes for key proteins was validated by qRT-PCR. Our results provide new molecular insights into the induced defense mechanisms in Chinese pine against this caterpillar in the field. A better understanding of these defense strategies will inform efforts to breed more-resistant pine varieties. [ABSTRACT FROM AUTHOR]

Loss of protein homeostasis is one of the hallmarks of aging. As such, interventions that restore proteostasis should slow down the aging process and improve healthspan. Two of the most broadly used anti-aging interventions that are effective in organisms from yeast to mammals are calorie restriction (CR) and rapamycin (RM) treatment. To identify the regulatory mechanisms by which these interventions improve the protein homeostasis, we carried out ribosome footprinting in the muscle of mice aged under standard conditions, or under long-term treatment with CR or RM. We found that the treatments distinctly impact the non-canonical translation, RM primarily remodeling the translation of upstream open reading frames (uORFs), while CR restores stop codon readthrough and the translation of downstream ORFs. Proteomics analysis revealed the expression of numerous non-canonical ORFs at the protein level. The corresponding peptides may provide entry points for therapies aiming to maintain muscle function and extend health span. [ABSTRACT FROM AUTHOR]

The systemic coordination of accumulation of plasma membrane aquaporins (PIP) was investigated in this study in relation to mycorrhized maize response to a rapid development of severe drought followed by rewatering. In non-mycorrhizal roots, drought led to a drop in PIP abundance, followed by a transient increase under rewatering, whereas leaves showed an opposite pattern. In contrast, mycorrhiza contributed to maintenance of high and stable levels of PIPs in both plant organs after an initial increase, prolonged over the irrigation period. Isoelectric focusing electrophoresis resolved up to 13 aquaporin complexes with highly reproducible pl positions across leaf and root samples, symbiotic and non-symbiotic, stressed or not. Mass spectrometry recognized in leaves and roots a different ratio of PIP1 and PIP2 subunits within 2D spots that accumulated the most. Regardless of symbiotic status, drought regulation of aquaporins in roots was manifested as the prevalence of complexes that comprise almost exclusively PIP2 monomers. In contrast, the leaf response involved enrichment in PIP1s. PIP1s are thought to enhance water transport, facilitate CO2 diffusion but also affect stomatal movements. These features, together with elevated aquaporin levels, might explain a stress tolerance mechanism observed in mycorrhizal plants, resulting in faster recovery of stomatal water conductance and CO2 assimilation rate after drought. [ABSTRACT FROM AUTHOR]

Shortening of telomere length (TL) is correlated with many age-related disorders and is a hallmark of biological aging. This study used proteome-wide Mendelian randomization to identify the protein biomarkers associated with telomere length. Protein quantitative trait loci (pQTL) were derived from two studies, the deCODE Health study (4907 plasma proteins) and the UK Biobank Pharma Proteomics Project (2923 plasma proteins). Summary data from genome-wide association studies (GWAS) for TL were obtained from the UK Biobank (472,174 cases) and GWAS Catalog (418,401 cases). The association between proteins and TL was further assessed using colocalization and summary data-based Mendelian randomization (SMR) analyses. The protein–protein network, druggability assessment, and phenome-wide MR were used to further evaluate the potential biological effects, druggability, and safety of the target proteins. Proteome-wide MR analysis identified 22 plasma proteins that were causally associated with telomere length. Five of these proteins (APOE, SPRED2, MAX, RALY, and PSMB1) had the highest evidence of association with TL and should be prioritized. This study revealed telomere length-related protein biomarkers, providing new insights into the development of new treatment targets for chronic diseases and anti-aging intervention strategies. [ABSTRACT FROM AUTHOR]

Background: Migraine is a highly prevalent and complex neurovascular disease. However, the currently available therapeutic drugs often fall to adequately meet clinical needs due to limited effectiveness and numerous undesirable side effects. This study aims to identify putative novel targets for migraine treatment through proteome-wide Mendelian randomization (MR). Methods: We utilized MR to estimate the causal effects of plasma proteins on migraine and its two subtypes, migraine with aura (MA) and without aura (MO). This analysis integrated plasma protein quantitative trait loci (pQTL) data with genome-wide association studies (GWAS) findings for these migraine phenotypes. Moreover, we conducted a phenome-wide MR assessment, enrichment analysis, protein–protein interaction networks construction, and mediation MR analysis to further validate the pharmaceutical potential of the identified protein targets. Results: We identified 35 protein targets for migraine and its subtypes (p < 8.04 × 10–6), with prioritized targets showing minimal side effects. Phenome-wide MR identified novel protein targets—FCAR, UBE2L6, LATS1, PDCD1LG2, and MMP3—that have no major disease side effects and interacted with current acute migraine medication targets. Additionally, MMP3, PDCD1LG2, and HBQ1 interacted with current preventive migraine medication targets. The causal effects of plasma protein on migraine were partly mediated by plasma metabolites (proportion of mediation from 3.8% to 21.0%). Conclusions: A set of potential protein targets for migraine and its subtypes were identified. These proteins showed rare side effects and were responsible for biological mechanisms involved in migraine pathogenesis, indicating priority for the development of migraine treatments. [ABSTRACT FROM AUTHOR]

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen posing a significant global public health threat. There is a pressing need for dependable and effective treatments, alongside an urgency for further research into the molecular characterization of its genome. Within the genomic landscape of Coxiella burnetii, numerous hypothetical proteins remain unidentified, underscoring the necessity for in-depth study. In this study, we conducted comprehensive in silico analyses to identify and prioritize potential hypothetical protein of Coxiella burnetii, aiming to elucidate the structure and function of uncharacterized protein. Furthermore, we delved into the physicochemical properties, localization, and molecular dynamics and simulations, and assessed the primary, secondary, and tertiary structures employing a variety of bioinformatics tools. The in-silico analysis revealed that the uncharacterized protein contains a conserved Mth938-like domain, suggesting a role in preadipocyte differentiation and adipogenesis. Subcellular localization predictions indicated its presence in the cytoplasm, implicating a significant role in cellular processes. Virtual screening identified ligands with high binding affinities, suggesting the protein's potential as a drug target against Q fever. Molecular dynamics simulations confirmed the stability of these complexes, indicating their therapeutic relevance. The findings provide a structural and functional overview of an uncharacterized protein from C. burnetii, implicating it in adipogenesis. This study underscores the power of in-silico approaches in uncovering the biological roles of uncharacterized proteins and facilitating the discovery of new therapeutic strategies. The findings provide valuable preliminary data for further investigation into the protein's role in adipogenesis. [ABSTRACT FROM AUTHOR]

Aim: The availability of disease-modifying therapies and newborn screening programs for spinal muscular atrophy (SMA) has generated an urgent need for reliable prognostic biomarkers to classify patients according to disease severity. We aim to identify cerebrospinal fluid (CSF) prognostic protein biomarkers in CSF samples of SMA patients collected at baseline (T0), and to describe proteomic profile changes and biological pathways influenced by nusinersen before the sixth nusinersen infusion (T302). Methods: In this multicenter retrospective longitudinal study, we employed an untargeted liquid chromatography mass spectrometry (LC-MS)-based proteomic approach on CSF samples collected from 61 SMA patients treated with nusinersen (SMA1 n=19, SMA2 n=19, SMA3 n=23) at T0 at T302. The Random Forest (RF) machine learning algorithm and pathway enrichment analysis were applied for analysis. Results: The RF algorithm, applied to the protein expression profile of naïve patients, revealed several proteins that could classify the different types of SMA according to their differential abundance at T0. Analysis of changes in proteomic profiles identified a total of 147 differentially expressed proteins after nusinersen treatment in SMA1, 135 in SMA2, and 289 in SMA3. Overall, nusinersen-induced changes on proteomic profile were consistent with i) common effects observed in allSMA types (i.e. regulation of axonogenesis), and ii) disease severity-specific changes, namely regulation of glucose metabolism in SMA1, of coagulation processes in SMA2, and of complement cascade in SMA3. Conclusions: This untargeted LC-MS proteomic profiling in the CSF of SMA patients revealed differences in protein expression in naïve patients and showed nusinersen-related modulation in several biological processes after 10 months of treatment. Further confirmatory studies are needed to validate these results in larger number of patients and over abroader timeframe. [ABSTRACT FROM AUTHOR]

Recent trials have shown the efficacy of trastuzumab deruxtecan (T-DXd) in HER2-negative patients, but there is not yet a way to identify which patients will best respond, especially with the inability of current HER2 IHC and FISH assays to accurately determine HER2 expression in the unamplified setting. Here, we present a heavily pre-treated patient with triple-negative breast cancer (HER2 IHC 0 who had a complete response to T-DXd. In this case, we used a CLIA-certified reverse-phase protein array-based proteomic assay (RPPA) to determine that the patient had moderate HER2 protein expression (HER2Total 2+, 42%) and activation (HER2Y1248 1+, 23%). Using these results, we determined that the patient may benefit from T-Dxd despite being traditionally qualified as HER2 IHC 0. These findings highlight the potential for proteomics-based assays that may more accurately quantitate HER2 and (its activation) in the HER2 unamplified/IHC 0 setting to better select patients whose tumors are classically molecularly defined as HER2 IHC 0, but still could respond to HER2-directed therapy, and give patients access to therapies which for which they otherwise would not be eligible. [ABSTRACT FROM AUTHOR]

Background: The effects of blue light on photosynthetic organs have been studied. However, its effects on non-photosynthetic organs, in particular, on the early stages of rice caryopsis development, are unclear. Thus, we aimed to determine the metabolic characteristics of caryopsis development under blue light to improve the metabolic quality of crop kernels. Results: We conducted a multi-omics analysis of each of the three periods from the beginning of cellular differentiation to the end of morphogenesis in post-pollination seeds of a japonica rice variety to explore the effect of blue light on metabolic levels during these metabolic changes and its genetic basis. It was found that blue light caused a gradual decrease in auxin content, a significant increase in the accumulation of JA and flavonoids, and a downregulation of the expression of many starch-related genes and proteins, leads to reduced starch synthesis and smaller starch granules. In addition, the gene co-expression network identified three transcription factors that may regulate starch and two that may regulate flavonoids. Conclusions: It was found through multi-omics testing that hormones such as jasmonic acid and auxins, and metabolites including alkaloids, flavonoids, lipids, organic acids, phenolic acids, and terpenoids altered significantly. Transcriptome and proteome analyses showed that blue light affected the seed nutrient repository activity. Specifically, starch- and gluten-related genes and proteins were significantly downregulated. Co- and WGCNA analyses identified several transcription factors that were regulated under blue light and identified key regulators of starch. Our study provides an understanding of the effects of blue light on post-flowering development in Gramineae and provides a framework for blue light-induced synthesis of secondary metabolites. Highlights: Evaluate the impact of blue light on the secondary metabolite accumulation. Confirm the accumulation of hormones and flavonoids in different blue light. Identify key candidate genes related to starch and flavonoid. Blue light leads to reduced starch synthesis and smaller starch granules. [ABSTRACT FROM AUTHOR]

Analyses of mitochondrial adaptations in human skeletal muscle have mostly used whole-muscle samples, where results may be confounded by the presence of a mixture of type I and II muscle fibres. Using our adapted mass spectrometry-based proteomics workflow, we provide insights into fibre-specific mitochondrial differences in the human skeletal muscle of men before and after training. Our findings challenge previous conclusions regarding the extent of fibre-type-specific remodelling of the mitochondrial proteome and suggest that most baseline differences in mitochondrial protein abundances between fibre types reported by us, and others, might be due to differences in total mitochondrial content or a consequence of adaptations to habitual physical activity (or inactivity). Most training-induced changes in different mitochondrial functional groups, in both fibre types, were no longer significant in our study when normalised to changes in markers of mitochondrial content. Exercise alters the mitochondrial proteome, but little is known about changes in different fibre types. Here, the authors provide insight into fibre-specific mitochondrial differences before and after training using proteomics in human skeletal muscle. [ABSTRACT FROM AUTHOR]

Understanding the endocannabinoid system in C. elegans may offer insights into basic biological processes and potential therapeutic targets for managing pain and inflammation in human. It is well established that anandamide modulates pain perception by binding to cannabinoid and vanilloid receptors, regulating neurotransmitter release and neuronal activity. One objective of this study was to demonstrate the suitability of C. elegans as a model organism for assessing the antinociceptive properties of bioactive compounds and learning about the role of endocannabinoid system in C. elegans. The evaluation of the compound anandamide (AEA) revealed antinociceptive activity by impeding C. elegans nocifensive response to noxious heat. Proteomic and bioinformatic investigations uncovered several pathways activated by AEA. Enrichment analysis unveiled significant involvement of ion homeostasis pathways, which are crucial for maintaining neuronal function and synaptic transmission, suggesting AEA's impact on neurotransmitter release and synaptic plasticity. Additionally, pathways related to translation, protein synthesis, and mTORC1 signaling were enriched, highlighting potential mechanisms underlying AEA's antinociceptive effects. Thermal proteome profiling identified NPR-32 and NPR-19 as primary targets of AEA, along with OCR-2, Cathepsin B, Progranulin, Transthyretin, and ribosomal proteins. These findings suggest a complex interplay between AEA and various cellular processes implicated in nociceptive pathways and inflammation modulation. Further investigation into these interactions could provide valuable insights into the therapeutic potential of AEA and its targets for the management of pain-related conditions. [ABSTRACT FROM AUTHOR]

Amyloids interact with plasma membranes. Extracellular amyloids cross the plasma membrane barrier. Internalized extracellular amyloids are reported to trigger amyloidogenesis of endogenous proteins in recipient cells. To what extent these extracellular and intracellular amyloids perturb the plasma membrane proteome is not investigated. Using α-synuclein as a model amyloid protein, we performed membrane shaving followed by mass spectrometry experiments to identify the conformational changes in cell surface proteins after extracellular amyloid challenge. We also performed membrane proteomics after the biogenesis of intracellular α-synuclein amyloids. Our results suggest that promiscuous interactions with extracellular amyloids stochastically alter the conformation of plasma membrane proteins. This affects the biological processes through the plasma membrane and results in loss of cell viability. Cells that survive the extracellular amyloid shock can grow normally and gradually develop intracellular amyloids which do not directly impact the plasma membrane proteome and associated biological processes. Thus, our results suggest that α-synuclein amyloids can damage the plasma membrane and related processes during cell-to-cell transfer and not during their intracellular biogenesis. [ABSTRACT FROM AUTHOR]

Heavy metals are extremely hazardous for human health due to their toxic effects. They are non-biodegradable in nature, thus remain in the environment and enter and accumulate in the human body through biomagnification; hence, there is a serious need of their remediation. Phytoremediation has emerged as a green, sustainable, and effective solution for heavy metal removal and many plant species could be employed for this purpose. Plants are able to sequester substantial quantity of heavy metals, in some cases thousands of ppm, due to their robust physiology enabling high metal tolerance and anatomy supporting metal ion accumulation. Identification and modification of potential target genes involved in heavy metal accumulation have led to improved phytoremediation capacity of plants at the molecular level. The introduction of foreign genes through genetic engineering approaches has further enhanced phytoremediation capacity manifolds. This review gives an insight towards improving the phytoremediation efficiency through a better understanding of molecular mechanisms involved, expression of different proteins, genetic engineering approaches for transgenic production, and genetic modifications. It also comprehends novel omics tools such as genomics, metabolomics, proteomics, transcriptomics, and genome editing technologies for improvement of phytoremediation ability of plants. [ABSTRACT FROM AUTHOR]

Azadirachta indica or neem is recognized for its unique properties, both against insects and in improving human health. However, proteomics studies of this plant's response to abiotic stresses have yet to be reported. Application of this technique highly depends on efficient and optimized sample preparation, in deriving highly reproducible proteomics data. The present study highlights the comparison of protein extraction method which effectively recovers proteins from recalcitrant phenolic-rich tissues of neem callus. Four protocols based on TCA-acetone or/and phenol extraction methods were compared. The TCA-acetone complemented with PVPP method was the most effective approach and generated the greatest total protein yields, spot number, good resolution, and spot intensities. Subsequently, the differential proteomics performed revealed that proteins associated with oxidoreduction, energy, transcriptional, stress response, respiration, and cell division were differentially regulated in salinity stress treatment. Meanwhile, the expression of proteins classified in the group of energy, photosynthesis, protein synthesis, oxidoreduction, and post-transcriptional regulation was altered in the JA-stressed callus. The accumulation of AZA was also noticeably reduced in salinity- and JA-treated callus, suggesting these tested stress parameters were not effective in improving AZA production in the neem callus. Overall, this analysis provides valuable information on the application of plant tissue culture as an alternative for sustainable production of major bioactive constituents in A. indica. Together, the proteomics results brought forward a possible fundamental representation of the mechanism associated with salinity and JA tolerance in the neem callus. [ABSTRACT FROM AUTHOR]

Proteome analyses of two contrasting mutant lines and their parent revealed responsive proteins in salt stress. The tandem mass spectrometry analysis resulted in identification of 67 proteins involved in various molecular processes including defense to oxidative stresses. The results demonstrated that total upregulated proteins were similar in sensitive and tolerant mutants (20), but downregulated proteins were significantly higher in sensitive line. Similarly, downregulated proteins were equal in tolerant mutant and wild type (23), though upregulated proteins were much higher than their parent. Although, tolerant, sensitive and their parent had partially the same pattern of protein regulations in protein folding and oxidative stress proteins, they were absolutely different in regulation of photosynthesis and carbohydrate proteins. Even, tolerant mutant showed lower up regulation intensity in some oxidative stress proteins than wild-type parent. Protein–protein interaction (PPI) maps revealed core protein roles in regulating salt tolerance mechanisms in mutant lines and their wild-type parent. PPI maps were created with two algorithms including K-mean and MCL clusters to discover protein–protein interactions. By employing PPI, we found 4 core proteins including Peptidyl-prolyl cis–trans isomerase, 70 kDa heat shock protein, Ferredoxin and 2-Cys peroxiredoxin that were involved in protein folding and oxidative stress pathways. These core proteins can regulate a wide range of molecular processes like carbohydrates metabolism, photosynthesis and ATP synthesis. As a result, tolerant mutant would regulate salt tolerance due to efficient and ordered protein–protein interactions. Further studies are needed to illuminate the importance of ordered protein–protein interactions leading to salt tolerance. [ABSTRACT FROM AUTHOR]

Background: Liquid biopsy represents a major development in cancer research, with significant translational potential. Similarly, it is increasingly recognized that multi-omic molecular approaches are a powerful avenue through which to understand complex and heterogeneous disease biology. We hypothesize that merging these two promising frontiers of cancer research will improve the discriminatory capacity of current models and allow for improved clinical utility. Methods: We have compiled a cohort of patients with glioblastoma, brain metastasis, and primary central nervous system lymphoma. Cell-free methylated DNA immunoprecipitation (cfMeDIP) and shotgun proteomic profiling was obtained from the cerebrospinal fluid (CSF) of each patient and used to build tumour-specific classifiers. Results: We show that the DNA methylation and protein profiles of cerebrospinal fluid can be integrated to fully discriminate lymphoma from its diagnostic counterparts with perfect AUC of 1 (95% confidence interval 1–1) and 100% specificity, significantly outperforming single-platform classifiers. Conclusions: We present the most specific and accurate CNS lymphoma classifier to date and demonstrates the synergistic capability of multi-platform liquid biopsies. This has far-reaching translational utility for patients with newly diagnosed intra-axial brain tumours. [ABSTRACT FROM AUTHOR]

Numerous studies have shown that exosomes play a regulatory role in a variety of biological processes as well as in disease development and progression. However, exosome-mediated intercellular communication between brain microvascular endothelial cells (BMECs) and astrocytes during meningitic Escherichia coli (E. coli)–induced neuroinflammation remains largely unknown. Here, by using in vivo and in vitro models, we demonstrate that exosomes derived from meningitic E. coli–infected BMECs can activate the inflammatory response of astrocytes. A label-free quantitation approach coupled with LC-MS/MS was used to compare the exosome proteomic profiles of human BMECs (hBMECs) in response to meningitic E. coli infection. A total of 57 proteins exhibited significant differences in BMEC-derived exosomes during the infection. Among these proteins, growth differentiation factor 15 (GDF15) was significantly increased in BMEC-derived exosomes during the infection, which triggered the Erk1/2 signaling pathway and promoted the activation of astrocytes. The identification and characterization of exosome protein profiles in BMECs during meningitic E. coli infection will contribute to the understanding of the underlying pathogenic mechanisms from the perspective of intercellular communication between BMECs and astrocytes, and provide new insights for future prevention and treatment of E. coli meningitis. [ABSTRACT FROM AUTHOR]

Main conclusion: This article explores possible future initiatives, such as the development of targeted breeding and integrated omics approach to boost little millet production, nutritional value, and environmental adaptation. Little millet (P. sumatrense) is a staple grain in many parts of Asia and Africa owing to its abundance in vitamins and minerals and its ability to withstand harsh agro-ecological conditions. Enhancing little millet using natural resources and novel crop improvement strategy is an effective way of boosting nutritional and food security. To understand the genetic makeup of the crop and figure out important characteristics linked to nutritional value, biotic and abiotic resistance, and production, researchers in this field are currently resorting on genomic technology. These realizations have expedited the crop's response to shifting environmental conditions by enabling the production of superior cultivars through targeted breeding. Going forward, further improvements in breeding techniques and genetics may boost the resilience, nutritional content, and production of little millet, which would benefit growers and consumers alike. The research and development on little millet improvement using novel omics platform and the integration of genetic resources are summarized in this review paper. Improved cultivars of little millet that satisfy changing farmer and consumer demands have already been developed through the use of these novel breeding strategies. This article also explores possible future initiatives, such as the development of targeted breeding, genomics, and sustainable agriculture methods. The potential for these measures to boost little millet's overall production, nutritional value, and climate adaptation will be extremely helpful in addressing nutritional security. Integrated omics for little millet improvement [ABSTRACT FROM AUTHOR]

Ant venom has several functions, including predation, communication, defense against predators, and action against pathogens. There is a scarcity of data about ant venom components that could provide support for understanding the mechanisms of action. The objective here was to identify the amino acids and proteins in the venom of the predatory ant Ectatomma brunneum and to evaluate its antimicrobial activity. The amino acids were analyzed by liquid chromatography, with diode array detection, and were identified using amino acid standards. The two-dimensional (2D) gel electrophoresis fractionation approach was used to identify the proteins, together with MALDI-TOF/TOF mass spectrometry and protein databases. The antimicrobial activity of the venom was evaluated using the minimum inhibitory and minimum microbiocidal concentrations. The venom of E. brunneum contained free amino acids, with a high amount of alanine. The 2D gel analysis showed 104 spots, of which 21 were identified and classified according to biological function, as follows: venom proteins, nontoxic reservoir protection, cellular maintenance proteins, and proteins with unknown function. The venom showed antimicrobial activity, inhibiting the growth of all the bacteria and fungi tested. The results provide new insights into ant venom components and antimicrobial activity. [ABSTRACT FROM AUTHOR]

Mass spectrometry is crucial in proteomics analysis, particularly using Data Independent Acquisition (DIA) for reliable and reproducible mass spectrometry data acquisition, enabling broad mass-to-charge ratio coverage and high throughput. DIA-NN, a prominent deep learning software in DIA proteome analysis, generates peptide results but may include low-confidence peptides. Conventionally, biologists have to manually screen peptide fragment ion chromatogram peaks (XIC) for identifying high-confidence peptides, a time-consuming and subjective process prone to variability. In this study, we introduce SeFilter-DIA, a deep learning algorithm, aiming at automating the identification of high-confidence peptides. Leveraging compressed excitation neural network and residual network models, SeFilter-DIA extracts XIC features and effectively discerns between high and low-confidence peptides. Evaluation of the benchmark datasets demonstrates SeFilter-DIA achieving 99.6% AUC on the test set and 97% for other performance indicators. Furthermore, SeFilter-DIA is applicable for screening peptides with phosphorylation modifications. These results demonstrate the potential of SeFilter-DIA to replace manual screening, providing an efficient and objective approach for high-confidence peptide identification while mitigating associated limitations. [ABSTRACT FROM AUTHOR]

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Sugarcane, commercially cultivated for the production of sucrose, is constantly confronted by Colletotrichum falcatum which is accountable for the catastrophic red rot disease. Membrane-bound sucrose transporters are the key controllers of short as well as long-distance transport of sucrose in plants. In the background of some recent reports suggesting the involvement of sucrose transporters and disease resistance proteins in plants' defense responses; we have investigated the abundance of these proteins in the stalk tissues of red rot resistant (BO91), and susceptible (CoJ64) cultivars of sugarcane using nanoLCMS/MS-based approach. The results revealed the presence of eight sucrose transporters and four disease resistance proteins cumulatively in both the cultivars of sugarcane. Further, we observed that C. falcatum inoculation influenced the peptide abundance of these proteins in both the cultivars of sugarcane. Among sucrose transporters, fungal inoculation caused a significant reduction in the peptide abundance of a large number of sucrose transporter proteins in both the cultivars of sugarcane including SUT-1, 5, 6, 7, and 8. In case of disease resistance proteins, the peptide abundance of all the four disease resistance proteins (DRP-1–4) was induced by C. falcatum inoculation in red rot susceptible cultivar. Our study indicated that sucrose transporters are repressed, whereas the disease resistance proteins are induced by C. falcatum infection in both the cultivars of sugarcane. The study might be useful in unraveling the specific roles of sucrose transporters and disease resistance proteins during stress responses in plants. [ABSTRACT FROM AUTHOR]

To facilitate our understanding of proteome dynamics during signaling events, robust workflows affording fast time resolution without confounding factors are essential. We present Surface-exposed protein Labeling using PeroxidaSe, H2O2, and Tyramide-derivative (SLAPSHOT) to label extracellularly exposed proteins in a rapid, specific, and sensitive manner. Simple and flexible SLAPSHOT utilizes recombinant soluble APEX2 protein applied to cells, thus circumventing the engineering of tools and cells, biological perturbations, and labeling biases. We applied SLAPSHOT and quantitative proteomics to examine the TMEM16F-dependent plasma membrane remodeling in WT and TMEM16F KO cells. Time-course data ranging from 1 to 30 min of calcium stimulation revealed co-regulation of known protein families, including the integrin and ICAM families, and identified proteins known to reside in intracellular organelles as occupants of the freshly deposited extracellularly exposed membrane. Our data provide the first accounts of the immediate consequences of calcium signaling on the extracellularly exposed proteome. Surface-exposed protein Labeling using PeroxidaSe, H2O2, and Tyramide-derivative (SLAPSHOT) coupled with mass spectrometry reveals the dynamics in the extracellularly exposed protein landscape in short time scales. [ABSTRACT FROM AUTHOR]

Obesity is a health problem characterized by large expansion of adipose tissue. During this expansion, genotoxic stressors can be accumulated and negatively affect the mesenchymal stem cells (MSCs) of adipose tissue. Due to the oxidative stress generated by these genotoxic stressors, senescence phenotype might be observed in adipose tissue MSCs. Senescent MSCs lose their proliferations and differentiation properties and secrete senescence-associated molecules to their niche thus triggering senescence for the rest of the tissue. Accumulation of senescent cells in adipose tissue results in decreased tissue regeneration and functional impairment not only in the close vicinity but also in the other tissues. Here we hypothesized that declined tissue regeneration might be associated with loss of stemness markers in MSCs population. We analyzed the expression of several stemness-associated genes of in vitro cultured MSCs originated from adipose tissue of high-fat diet and normal diet mice models. Since the heterogenous MSCs population covers a small percentage of the pluripotent stem cells, which have roles in proliferation and tissue regeneration, we measured the percentage of these cells via TRA-1-60 pluripotent state antigen. Additionally, by conducting a shotgun proteomic approach using LC-MS/MS, whole cell proteome of the adipose tissue MSCs of high-fat diet and normal diet mice were analyzed and identified proteins were evaluated via gene ontology and PPI network analysis. MSCs of obese mice showed senescent phenotype and altered cell cycle distribution due to a hostile environment with oxidative stress in adipose tissue where they reside. Additionally, the number of pluripotent markers expressing cells declined in the MSC population of the high-fat diet mice. Gene expression analysis evidenced the loss of stemness with a decrease in the expression of stemness-associated genes. Of the proteomic comparison of the normal and the high-fat diet group, MSCs revealed that stemness-associated molecules were decreased while inflammation and senescence-associated phenotypes emerged in obese mice MSCs. Our results showed us that the MSCs of adipose tissue may lose their stemness properties due to obesity-associated stress conditions. [ABSTRACT FROM AUTHOR]

Replacing cereals with food leftovers could reduce feed-food competition and keep nutrients and energy in the food chain. Former food products (FFPs) are industrial food leftovers no more intended for human but still suitable as alternative and sustainable feedstuffs for monogastric. In this study, omics approaches were applied to evaluate the impact of dietary FFPs on pig liver proteome and plasma peptidome. Thirty-six Swiss Large White male castrated pigs were randomly assigned to three dietary treatments [control (CTR), 30% CTR replaced with salty FFP (SA), 30% CTR replaced with sugary FFP (SU)] from the start of the growing phase (22.4 ± 1.7 kg) until slaughtering (110 ± 3 kg). The low number of differentially regulated proteins in each comparison matrix (SA/SU vs. CTR) and the lack of metabolic interaction indicated a marginal impact on hepatic lipid metabolism. The plasma peptidomics investigation showed low variability between the peptidome of the three dietary groups and identified three possible bioactive peptides in the SA group associated with anti-hypertension and vascular homeostasis regulation. To conclude, the limited modulation of liver proteome and plasma peptidome by the SA and SU diets strenghtened the idea of reusing FFPs as feed ingredients to make pig production more sustainable. [ABSTRACT FROM AUTHOR]

Primary open-angle glaucoma (POAG) is subdivided depending on eye pressure. Patients with normal-tension glaucoma (NTG) have never had high intraocular pressure (IOP) measured while patients with ocular hypertension (OHT) have high eye pressure but no signs of glaucoma. Although IOP is considered to be a risk factor for all glaucoma patients, it is reasonable to assume that other risk factors such as inflammation play a role. We aimed to characterize the proteome and cytokine profile during hypoxia in plasma from patients with NTG (n = 10), OHT (n = 10), and controls (n = 10). Participants were exposed to hypoxia for two hours, followed by 30 min of normoxia. Samples were taken before ("baseline"), during ("hypoxia"), and after hypoxia ("recovery"). Proteomics based on liquid chromatography coupled with mass spectrometry (LC–MS) was performed. Cytokines were measured by Luminex assays. Bioinformatic analyses indicated the involvement of complement and coagulation cascades in NTG and OHT. Regulation of high-density lipoprotein 3 (HDL3) apolipoproteins suggested that changes in cholesterol metabolism are related to OHT. Hypoxia decreased the level of tumor necrosis factor-α (TNF-α) in OHT patients compared to controls. Circulating levels of interleukin-1β (IL-1β) and C-reactive protein (CRP) were decreased in NTG patients compared to controls during hypoxia. After recovery, plasma interleukin-6 (IL-6) was upregulated in patients with NTG and OHT. Current results indicate an enhanced systemic immune response in patients with NTG and OHT, which correlates with pathogenic events in glaucoma. Apolipoproteins may have anti-inflammatory effects, enabling OHT patients to withstand inflammation and development of glaucoma despite high IOP. [ABSTRACT FROM AUTHOR]

Blood vessel endothelial cells (EC) display heterogeneity across vascular beds, which is anticipated to drive site-specific vascular pathology. This heterogeneity is assessed using transcriptomics in vivo, and functional assays in vitro, but how proteomes compare across human in vitro cultured ECs remains incompletely characterized. We generated an in-depth human EC proteomic landscape (>8000 proteins) across six organs and two in vitro models in steady-state and upon IFNγ-induced inflammation. EC proteomes displayed a high similarity and organ-specific proteins were limited. Variation between ECs was mainly based on proliferation and differentiation processes in which Blood outgrowth endothelial cells (BOEC) and Human umbilical vein cells (HUVEC) represented the extremes of proteomic phenotypes. The IFNγ response was highly conserved across all samples. Harnessing dynamics in protein abundances we delineated VWF and VE-Cadherin correlation networks. This EC landscape provides an extensive proteomic addition in studying EC biology and heterogeneity from an in vitro perspective. Analysis of proteomic signatures of commercially acquired endothelial cells from different organs in steady state and after IFNγ inflammation in the context of understanding vascular heterogeneity. [ABSTRACT FROM AUTHOR]

Current stage of proteomic research in the field of biology, medicine, development of new drugs, population screening, or personalized approaches to therapy dictates the need to analyze large sets of samples within the reasonable experimental time. Until recently, mass spectrometry measurements in proteomics were characterized as unique in identifying and quantifying cellular protein composition, but low throughput, requiring many hours to analyze a single sample. This was in conflict with the dynamics of changes in biological systems at the whole cellular proteome level upon the influence of external and internal factors. Thus, low speed of the whole proteome analysis has become the main factor limiting developments in functional proteomics, where it is necessary to annotate intracellular processes not only in a wide range of conditions, but also over a long period of time. Enormous level of heterogeneity of tissue cells or tumors, even of the same type, dictates the need to analyze biological systems at the level of individual cells. These studies involve obtaining molecular characteristics for tens, if not hundreds of thousands of individual cells, including their whole proteome profiles. Development of mass spectrometry technologies providing high resolution and mass measurement accuracy, predictive chromatography, new methods for peptide separation by ion mobility and processing of proteomic data based on artificial intelligence algorithms have opened a way for significant, if not radical, increase in the throughput of whole proteome analysis and led to implementation of the novel concept of ultrafast proteomics. Work done just in the last few years has demonstrated the proteome-wide analysis throughput of several hundred samples per day at a depth of several thousand proteins, levels unimaginable three or four years ago. The review examines background of these developments, as well as modern methods and approaches that implement ultrafast analysis of the entire proteome. [ABSTRACT FROM AUTHOR]

Heat stress (HS) impacts the nuclear proteome and, subsequently, protein activities in different nuclear compartments. In Arabidopsis thaliana, a short exposure to 37 °C leads to loss of the standard tripartite architecture of the nucleolus, the most prominent nuclear substructure, and, consequently, affects the assembly of ribosomes. Here, we report a quantitative label-free LC‒MS/MS (Liquid Chromatography coupled to tandem Mass Spectrometry) analysis to determine the nuclear proteome of Arabidopsis at 22 °C, HS (37 °C for 4 and 24 h), and a recovery phase. This analysis identified ten distinct groups of proteins based on relative abundance changes in the nucleus before, during and after HS: Early, Late, Transient, Early Persistent, Late Persistent, Recovery, Early-Like, Late-Like, Transient-Like and Continuous Groups (EG, LG, TG, EPG, LPG, RG, ELG, LLG, TLG and CG, respectively). Interestingly, the RNA polymerase I subunit NRPA3 and other main nucleolar proteins, including NUCLEOLIN 1 and FIBRILLARIN 1 and 2, were detected in RG and CG, suggesting that plants require increased nucleolar activity and likely ribosome assembly to restore protein synthesis after HS. [ABSTRACT FROM AUTHOR]

Knowledge of protein-protein interactions (PPI) is essential for studying protein functions and understanding the biological processes. Previously, most of the works on PPI in the BioNLP domain rely solely on textual data. With the availability of different information (structure, sequence, gene ontology) about proteins, researchers have started to use other details with textual data to predict PPI more accurately. This paper reports the first attempt in integrating gene ontology(GO)-based information with the features extracted from other two modalities of proteins namely 3D structure and existing textual information. Existing two popular text-based benchmark PPI corpora, i.e., BioInfer and HRPD50 are first extended to integrate with the structure and GO-based information. Finally, some deep learning-based techniques are employed to extract features from three modalities and those are concatenated for final prediction of protein interaction. The experimentation on generated multi-modal datasets illustrates that the proposed deep multi-modal framework outperforms the baselines (uni-modal, bi-modal and multi-modal) and state-of-the-art methods. [ABSTRACT FROM AUTHOR]

Purpose: HER2-positive breast cancer (BC) accounts for 20–30% of all BC subtypes and is linked to poor prognosis. Trastuzumab (Tz), a humanized anti-HER2 monoclonal antibody, is a first-line treatment for HER2-positive breast cancer which faces resistance challenges. This study aimed to identify the biomarkers driving trastuzumab resistance. Methods: Differential expression analysis of genes and proteins between trastuzumab-sensitive (TS) and trastuzumab-resistant (TR) cells was conducted using RNA-seq and iTRAQ. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) were used to study their functions. The prognostic significance and protein levels of ARFIP2 and MSN were evaluated using online tools and immunohistochemistry. Sensitivity of MSN and ARFIP2 to other therapies was assessed using public pharmacogenomics databases and the R language. Results: Five genes were up-regulated, and nine genes were down-regulated in TR cells at both transcriptional and protein levels. Low ARFIP2 and high MSN expression linked to poor BC prognosis. MSN increased and ARFIP2 decreased in TR patients, correlating with shorter OS. MSN negatively impacted fulvestrant and immunotherapy sensitivity, while ARFIP2 had a positive impact. Conclusion: Our findings suggest that MSN and ARFIP2 could serve as promising biomarkers for predicting response to Tz, offering valuable insights for future research in the identification of diagnostic and therapeutic targets for BC patients with Tz resistance. [ABSTRACT FROM AUTHOR]

Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 results in the mistrafficking of proteins crucial for neuronal development and survival, including FGFR3, UNC5B and SEMA4D. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 are compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome. [ABSTRACT FROM AUTHOR]

There has been a dramatic increase in the identification of non-canonical translation and a significant expansion of the protein-coding genome. Among the strategies used to identify unannotated small Open Reading Frames (smORFs) that encode microproteins, Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple genomic sites are removed since they cannot be unambiguously assigned to a specific genomic location. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of multi-mapping alignments, such that smORFs residing in these regions cannot be identified by Ribo-Seq. Moreover, it has been challenging to identify protein evidence for Ribo-Seq. To solve this, we developed Rp3, a pipeline that integrates proteogenomics and Ribosome profiling to provide unambiguous evidence for a subset of microproteins missed by current Ribo-Seq pipelines. Here, we show that Rp3 maximizes proteomics detection and confidence of microprotein-encoding smORFs. Small genes have been ignored for years in the genome of every organism due to experimental and computational limitations. Here, the authors describe a computational tool named Rp3 that integrates two high throughput approaches to identify microproteins encoded by these short genes. [ABSTRACT FROM AUTHOR]

Although proteomics is extensively used in immune research, there is currently no publicly accessible spectral assay library for the comprehensive proteome of immune cells. This study generated spectral assay libraries for five human immune cell lines and four primary immune cells: CD4 T, CD8 T, natural killer (NK) cells, and B cells. This was achieved by utilizing data-dependent acquisition (DDA) and employing fractionated samples from over 100 µg of proteins, which was applied to acquire the highest-quality MS/MS spectral data. In addition, Data-indedendent acquisition (DIA) was used to obtain sufficient data points for analyzing proteins from 10,000 primary CD4 T, CD8 T, NK, and B cells. The immune cell spectral assay library generated included 10,544 protein groups and 127,106 peptides. The proteomic profiles of 10,000 primary human immune cells obtained from 15 healthy volunteers analyzed using DIA revealed the highest heterogeneity of B cells among other immune cell types and the similarity between CD4 T and CD8 T cells. All data and spectral library are deposited in ProteomeXchange (PXD047742). [ABSTRACT FROM AUTHOR]

The paper presents an analysis of the proteomic composition in relation to both the risk of thrombosis and changes in the state of cardiomyocytes associated with the risk of cardiac fibrosis and heart failure. We examined 12 practically healthy male volunteers exposed to head-down -6° tilt bed rest (HDBR) for 21 days. The revealed decrease in the level of stimulating growth factor 2 (ST2) on days 10 and 21 relative to the initial values (background; 5 days before HDBR) indicated a decrease in the myocardial load and cardiomyocyte extensibility. The level of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) increased on day 2, decreased on days 10 and 21 of HDBR relative to the background levels, and returned to baseline values after the recovery period (5 days after HDBR). The revealed changes in the level of NT-proBNP reflected the increase in circulating blood volume corresponding to HDBR duration and the role of the gravity component in increasing the functional load on the myocardium. Unchanged blood level of D-dimer at all points of the study indicates that there is no risk of thrombosis under the conditions of this study. [ABSTRACT FROM AUTHOR]