Proteomic Analysis Research Articles

SNX4 Is Correlated With Immune Infiltration and Prognosis in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is known as the most common and malignant histologic subtype of renal carcinoma. Sorting nexin 4 (SNX4) plays a regulatory role in recycling from endosomes to the plasma membrane and promotes autophagosome assembly and transport, which may exert the cancerous growth and progression. This study aimed to assess the biological role of SNX4 in ccRCC and their clinical association via public biological data platforms combined with experimental verification. In our study, we analyzed the mRNA and protein expression of SNX4 in ccRCC under different clinicopathological characteristics through The Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases. We used the Gene Expression Profiling Interactive Analysis (GEPIA) platform to conduct the survival analysis and figure out the immune cell infiltration level under different expression levels of SNX4 combined with Tumor Immune Estimation Resource (TIMER) database. Furthermore, we predicted competing endogenous RNA (ceRNA) regulatory network using TargetScan, miRDB, starBase and miRTarBase online databases. We totally collected six paired ccRCC tissues and adjacent tissues and applied quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) to detect the expression of SNX4 in the collected clinical specimens. The mRNA and protein expression level of SNX4 was significantly lower in ccRCC than those in normal tissues. The results proposed that lower SNX4 was expressed in patients with higher histologic grade and in male patients. Kaplan-Meier analysis demonstrated that lower mRNA expression level of SNX4 was correlated with poorer prognosis. SNX4 had positive correlation with immune cell infiltrating levels and programmed cell death-ligand 1 (PD-L1) expression. Furthermore, we constructed the SNX4/miR-221-3p/miR-222-3p/DHRS4-AS1 axis, which may be the underlying ceRNA interaction network. Finally, we verified the reduced expression of SNX4 in ccRCC by qRT-PCR and WB. The expression of SNX4 in ccRCC was lower than adjacent tissues and its downregulated expression was associated with poor prognosis of ccRCC patients. SNX4 may exert critical roles in the tumorigenesis, development and migration of ccRCC via various mechanisms.

Proteomic Analysis Unveiled Exercise-induced Changes In Extracellular Vesicles From Balf And Blood In Cigarette Smoke Exposed Mice

Proteomic Analysis Unveiled Exercise-induced Changes In Extracellular Vesicles From Balf And Blood In Cigarette Smoke Exposed Mice

Comprehensive Proteomic Analysis of Dysferlinopathy Unveiling Molecular Mechanisms and Biomarkers Linked to Pathological Progression.

Previous proteomics studies in dysferlinopathy muscle have been limited in scope, often utilizing 2D-electrophoresis and yielding only a small number of differential expression calls. To address this gap, this study aimed to employ high-resolution proteomics to explore the proteomic landscapes of dysferlinopathy and analyze the correlation between muscle pathological changes and alterations in protein expression in muscle biopsies. We conducted a comprehensive approach to investigate the proteomic profile and disease-associated changes in the muscle tissue proteome from 15 patients with dysferlinopathy, exhibiting varying degrees of dystrophic pathology, alongside age-matched controls. Our methodology encompasses tandem mass tag (TMT)-labeled liquid chromatography-mass spectrometry (LC-MS/MS)-based proteomics, protein-protein interaction (PPI) network analysis, weighted gene co-expression network analysis, and differential expression analysis. Subsequently, we examined the correlation between the expression of key proteins and the clinical characteristics of the patients to identify pathogenic targets associated with DYSF mutations in dysferlinopathy. A total of 1600 differentially expressed proteins were identified, with 1321 showing high expression levels and 279 expressed at lower levels. Our investigation yields a molecular profile delineating the altered protein networks in dysferlinopathy-afflicted skeletal muscle, uncovering dysregulation across numerous cellular pathways and molecular processes, including mRNA metabolic processes, regulated exocytosis, immune response, muscle system processes, energy metabolic processes, and calcium transmembrane transport. Moreover, we observe significant associations between the protein expression of ANXA1, ANXA2, ANXA4, ANXA5, LMNA, PYGM, and the extent of histopathologic changes in muscle biopsies from patients with dysferlinopathy, validated through immunoblotting and immunofluorescence assays. Through the aggregation of expression data from dysferlinopathy-impacted muscles exhibiting a range of pathological alterations, we identified multiple key proteins associated with the dystrophic pathology of patients with dysferlinopathy. These findings provide novel insights into the pathogenesis of dysferlinopathy and propose promising targets for future therapeutic endeavors.

Loss of Function in Protein Z (PROZ) is Associated with Increased Risk of Ischemic Stroke in the UK Biobank

BackgroundThe vitamin K-dependent coagulation factor protein Z (PZ), encoded by the PROZ gene, is canonically considered to have anticoagulant effects through negative regulation of factor Xa. Paradoxically, higher circulating PZ concentrations have repeatedly been associated with an elevated risk of acute ischemic stroke. ObjectiveWe performed a large-scale genetic association study to examine the relationship between germline genetic variants in PROZ and the risk of ischemic stroke. MethodsUsing whole exome sequencing and clinical data for 416,711 participants in the UK Biobank (UKB), we identified individuals with rare (MAF ≤ 0.1%) putatively function-altering variants in PROZ. Using Firth’s logistic regression and controlling for known stroke risk factors, we evaluated the association between variant carrier status and non-cardioembolic ischemic stroke (NCEIS). Additionally, we evaluated differences in the plasma levels of 1,472 proteins between PROZ variant carriers and non-carriers in a subset of 48,893 UKB participants. ResultsAfter accounting for missing data, qualifying variants in PROZ were identified in 414 UKB participants (99.0% heterozygous). Variant carriers had a significantly increased risk of NCEIS (OR=2.34, 95% CI: 1.15-4.13, P=0.02) but not of venous thromboembolism, myocardial infarction, or peripheral artery disease. Plasma proteomics analysis revealed that PROZ variant carriers had significantly elevated levels of two proteins related to the response to cerebral ischemia, peroxiredoxins 1 and 6 (PRDX1, fold-change=1.83, P=1.3 x 10-5 and PRDX6, fold-change=1.78, P=9.6 x 10-10). ConclusionsLifelong exposure to decreased PZ levels confers a significantly increased risk of NCEIS, consistent with the role of PZ as an anticoagulant factor.

Identification of membrane proteins targeted by small-molecule compounds using nanomagnetic beads.

In drug discovery research, it is important to identify target proteins of bioactive small-molecule compounds and analyse their functions. In this study, we examined whether target membrane proteins could be captured by compounds that bind to membrane proteins on the cell surface. For this purpose, we performed affinity purification using the compound-immobilized nanomagnetic beads. Affinity purification with nanomagnetic beads is known to be effective for determining the protein binding partners of small molecules. However, most previous studies have targeted proteins in the cytoplasm. As a model compound, we chose BMS-1166 (a representative small-molecule compound from Bristol Myers Squibb), a PD-1/PD-L1 immune checkpoint inhibitor that binds to PD- L1 and promotes PD-L1 dimerization. BMS-1166-immobilized beads were manufactured and incubated with extracts of cells with high PD-L1 protein expression. The bound protein was confirmed by western blotting and proteomic analysis to be PD-L1. BMS-1166-immobilized nano-magnetic beads were able to specifically bind and capture the membrane protein PD-L1. In addition, high-purity protein could be obtained from cell extracts in a single step. This is the first report of the purification of a membrane protein to high purity with nanobeads. Nanomagnetic beads with immobilized compounds are an effective tool for identifying the protein binding partners of small molecules, especially when the targets are membrane proteins.

Mapping the Proteomic Landscape and Anti-inflammatory Role of Streptococcus parauberis Extracellular Vesicles

Bacterial extracellular vesicles (BEVs) are nanoscale membrane-bound structures involved in intercellular communication and transport of bioactive molecules. In this study, we described the proteomic insight and anti-inflammatory activity of Streptococcus parauberis BEVs (SpEVs). Proteomics analysis of SpEVs identified 6,209 distinct peptides and 1039 proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated enrichment in pathways related to the biosynthesis of aminoacyl tRNA, amino acids, and secondary metabolites. Based on the predicted protein-protein interactions, we discovered key immunological proteins such as IL12A, IL12B, IL8, CD28, and NF-κB between SpEVs and human proteins. Functionally, SpEVs exhibit strong anti-inflammatory activity in LPS-stimulated Raw 264.7 cells by reducing the production of key inflammatory mediators. These include nitric oxide (NO), reactive oxygen species (ROS), inflammatory cytokines such as TNFα and IL6, as well as inflammation-related proteins like inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). qRT-PCR and immunoblotting results clearly indicate that SpEVs modulate the NF-κB and MAPK pathways to induce anti-inflammatory activity. Furthermore, in vivo experiments with zebrafish larvae demonstrated that SpEVs treatment reduced the NO and ROS production with minimal cell mortality. Finally, we validated the anti-inflammatory activity of SpEVs in vivo by systematically assessing the inhibition of NO production, reduction in ROS generation, prevention of cell death, and modulation of NF-κB and MAPK signaling pathways. In conclusion, SpEVs are rich in unique proteins that play crucial roles in mediating anti-inflammatory effects.

Explore the mechanism of yishenjiangya formula in the treatment of senile hypertension based on multi-omics technology

Ethnopharmacological relevanceThe Yishenjiangya formula (YSJ) is a traditional Chinese medicine (TCM) primarily composed of qi-tonifying components. This classic formula is commonly utilized to treat kidney qi deficiency in elderly patients with hypertension. According to TCM, maintaining a balance between qi and blood is crucial for stable blood pressure. Kidney qi deficiency can disrupt this balance, altering fluid shear force and, ultimately, leading to hypertension, particularly in elderly populations. Despite YSJ's efficacy in treating hypertension, its specific anti-hypertensive mechanisms remain unclear. Aim of the studyYSJ is commonly prescribed for elderly patients with hypertension. Earlier metabolomics studies demonstrated that YSJ exerts antihypertensive effects by influencing four key pathways: linoleic acid metabolism, glycerol phospholipid metabolism, arginine and proline metabolism, and steroid hormone biosynthesis. This study aims to combine metabolomic and proteomic analyses to thoroughly understand the molecular biological mechanisms responsible for YSJ's anti-hypertensive properties. MethodsUltra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) metabolomics, combined with Label-Free Quantitation (LFQ) proteomics, was employed to analyze serum samples from elderly individuals with and without hypertension pre- and post-YSJ intervention. Serum levels of candidate proteins were assessed using enzyme-linked immunosorbent assay, and receiver operating characteristic curves were used to evaluate the diagnostic performance of the target proteins. ResultsEight differentially expressed metabolites and three differentially expressed proteins were identified as potential therapeutic targets of YSJ. These substances are primarily involved in unsaturated fatty acid metabolism, fluid shear stress and atherosclerosis pathway, primary bile acid biosynthesis, proline metabolism, apoptosis, and endoplasmic reticulum stress. YSJ exerts its therapeutic effects on hypertension in the elderly by modulating these pathways. ConclusionsYSJ effectively treats senile hypertension. By analyzing the correlation between therapeutic targets and pathways, YSJ's anti-hypertensive effect was achieved by inhibiting lipid peroxidation and matrix degeneration. Combining metabolomics and proteomics provides an effective method for uncovering YSJ's anti-hypertensive mechanisms.

Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein–Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1)

As a component of circulating lipoproteins, APOE binds to cell surface receptors mediating lipoprotein metabolism and cholesterol transport. A growing body of evidence, including the identification of a broad variety of cellular proteins interacting with APOE, suggests additional independent functions. Investigating cellular localization and protein–protein interactions in cultured human hepatocytes, we aimed to contribute to the elucidation of hitherto unnoted cellular functions of APOE. We observed a strong accumulation of APOE in MAMs, equally evident for the two major isoforms APOE3 and APOE4. Using mass spectrometry proteome analyses, novel and previously noted APOE interactors were identified, including the mitochondrial proteins TOMM40, LONP1 and VDAC1. All three interactors were present in MAM fractions, which we think initially facilitates interactions with APOE. LONP1 is a protease with chaperone activity, which migrated to MAMs in response to ER stress, displaying a reinforced interaction with APOE. We therefore hypothesize that APOE may help in the unfolded protein response (UPR) by acting as a co-chaperone in cooperation with LONP1 at the interface of mitochondria and ER membranes. The interaction of APOE with the integral proteins TOMM40 and VDAC1 may point to the formation of bridging complexes connecting mitochondria with other organelles.

Dominant Circulating Cell-free Mycobacterial Proteins in In-use Machining Fluid and their Antigenicity Potential.

Occupational exposure to industrial Metalworking Fluid (MWF) colonized by Mycobacterium immunogenum (MI) has been associated with immune lung disease hypersensitivity pneumonitis (HP) in machinists. This warrants regular fluid monitoring for early detection of mycobacterial proteins, especially those with antigenic potential. To detect and identify dominant MI proteins and antigens directly from the field-drawn in-use MWF using an integrated immunoproteomic-immunoinformatic approach. An MI-positive MWF selected by DNA-based screening of several field-drawn MWF samples was cultured to isolate the colonizing strain and profiled for dominant circulating cell-free (ccf) MI proteins, including antigens using an integrated immunoproteomic (1D- and 2Dgel fractionation of seroreactive proteins combined with shotgun proteomic analysis using LC-MS/MS) and immunoinformatic strategy. A new MI strain (MJY-27) was identified. The gel fractionated MI protein bands (1Dgel) or spots (2D-gel) seroreactive with anti-MI sera probes (Rabbit and Patient sera) yielded 86 MI proteins, 29 of which showed peptide abundance. T-cell epitope analysis revealed high (90-100%) binding frequency for HLA-I & II alleles for 13 of the 29 proteins. Their antigenicity analysis revealed the presence of 6 to 37 antigenic determinants. Interestingly, one of the identified candidates corresponded to an experimentally validated strong B- and T-cell antigen (AgD) from our laboratory culture-based studies. This first report on dominant proteins, including putative antigens of M. immunogenum prevalent in field in-use MWF, is a significant step towards the overall goal of developing fluid monitoring for exposure and disease risk assessment for HP development in machining environments.

Circulating Proteins Associated with Anti-IL6 Receptor Therapeutic Resistance in the Sera of Patients with Severe COVID-19.

Circulating proteomes provide a snapshot of the physiological state of a human organism responding to pathogenic challenges and drug interventions. The outcomes of patients with COVID-19 and acute respiratory distress syndrome triggered by the SARS-CoV2 virus remain uncertain. Tocilizumab is an anti-interleukin-6 treatment that exerts encouraging clinical activity by controlling the cytokine storm and improving respiratory distress in patients with COVID-19. We investigate the biological determinants of therapeutic outcomes after tocilizumab treatment. Overall, 28 patients hospitalized due to severe COVID-19 who were treated with tocilizumab intravenously were included in this study. Sera were collected before and after tocilizumab, and the patient's outcome was evaluated until day 30 post-tocilizumab infusion for favorable therapeutic response to tocilizumab and mortality. Hyperreaction monitoring measurements by liquid chromatography-mass spectrometry-based proteomic analysis with data-independent acquisition quantified 510 proteins and 7019 peptides in the serum of patients. Alterations in the serum proteome reflect COVID-19 outcomes in patients treated with tocilizumab. Our results suggested that circulating proteins associated with the most significant prognostic impact belonged to the complement system, platelet degranulation, acute-phase proteins, and the Fc-epsilon receptor signaling pathway. Among these, upregulation of the complement system by activation of the classical pathway was associated with poor response to tocilizumab, and upregulation of Fc-epsilon receptor signaling was associated with lower mortality.

Integrin α6-containing extracellular vesicles promote lymphatic remodelling for pre-metastatic niche formation in lymph nodes via interplay with CD151.

Heterogeneous extracellular vesicles (EVs) from various types of tumours are acknowledged for inducing the formation of pre-metastatic "niches" in draining lymph nodes (LNs) to promote lymphatic metastasis. In order to identify the specific subpopulations of EVs involved, we performed high-resolution proteomic analysis combined with nanoflow cytometry of bladder cancer (BCa) tissue-derived EVs to identify a novel subset of tumour-derived EVs that contain integrin α6 (ITGA6+EVs) and revealed the positive correlation of ITGA6+EVs with the formation of pre-metastatic niche in draining LNs and lymphatic metastasis in multicentre clinical analysis of 820-case BCa patients. BCa-derived ITGA6+EVs induced E-selectin (SELE)-marked lymphatic remodelling pre-metastatic niche and promoted metastasis in draining LNs through delivering cargo circRNA-LIPAR to lymphatic endothelial cells in vivo and in vitro. Mechanistically, LIPAR linked ITGA6 to the switch II domain of RAB5A and sustained RAB5A GTP-bound activated state, thus maintaining the production of ITGA6+EVs loaded with LIPAR through endosomal trafficking. ITGA6+EVs targeted lymphatic vessels through ITGA6-CD151 interplay and released LIPAR to induce SELE overexpression-marked lymphatic remodelling pre-metastatic niche. Importantly, we constructed engineered-ITGA6 EVs to inhibit lymphatic pre-metastatic niche, which suppressed lymphatic metastasis and prolonged survival in preclinical models. Collectively, our study uncovers the mechanism of BCa-derived ITGA6+EVs mediating pre-metastatic niche and provides an engineered-EV-based strategy against BCa lymphatic metastasis.

Dissecting the importance and origin of circulating myokines in gastric cancer cachexia

BackgroundSome experimental data suggest that myokines may play an important role in developing cancer-associated cachexia (CAC), but their relevance in humans remains poorly explored. In our study, we tested the hypothesis that circulating myokines are associated with the pathogenesis of CAC in a model population of gastric cancer.MethodsA group of 171 treatment naïve patients with adenocarcinoma of the stomach were prospectively examined. Cachexia was defined as weight loss >5% or weight loss >2% with either BMI <20 kg/m2 or sarcopenia. A panel of 19 myokines was measured in portal and peripheral blood as well as tumour tissue and surrounding gastric mucosa. Moreover, a serum proteomic signature of cachexia was identified by a label-free quantitative proteomics with a nano LC-MS/MS system and stored in a ProteomeXchange database (PXD049334).ResultsOne hundred (58%) patients were diagnosed with CAC. The concentrations of fatty acid-binding protein 3 (FABP3), follistatin-like 1 protein (FSTL−1), interleukin 6 (IL 6), and interleukin 8 (IL 8) were significantly higher in the peripheral blood of cachectic subjects, while leptin levels were lower. Of all the evaluated myokines, tumour tissues showed higher expression levels only for IL-15 and myostatin. However, the analysis of paired samples failed to demonstrate a decreasing concentration gradient between the portal and peripheral blood for any of the myokines, evidencing against their release by the primary tumour. Proteomic analysis identified 28 proteins upregulated and 24 downregulated in the peripheral blood of patients with cachexia. Differentially expressed proteins and 5 myokines with increased serum levels generated a significant protein-protein interaction network.ConclusionsOur study provides clinical evidence that some myokines are involved in the pathogenesis of cachexia and are well integrated into the regulatory network of circulating blood proteins identified among cachectic patients with gastric cancer.

Jieduquyuziyin prescription alleviates lupus development via inhibiting neddylation pathway to promote Bim-induced apoptosis of double negative T cells

Ethnopharmacological relevanceJieduquyuziyin prescription (JP) is an empirical prescription approved for application to treat systemic lupus erythematosus (SLE) in hospital within China. Despite the prominent treatment effect of JP clinically, further investigation is imperative to explore its underlying mechanisms. Aim of the studyWe aim to investigate the impact of JP on DN T cell apoptosis in the treatment of SLE and the specific regulation mechanisms. Materials and methodsFirstly, female MRL/lpr mice were treated with JP and the therapeutic efficacy of JP was evaluated via skin lesions, lymphoid organ enlargement, accumulation of autoantibodies and renal function. Then, flow cytometer analysis was performed to evaluate the proportions and the apoptosis of T cell subpopulations. Based on the above results, double-negative (DN) T cells were subjected to proteomic with subsequent differential screening. The expression of Ube2m and Bim was further validated using real-time PCR and western blot. Subsequently, DN T cells were incubated with JP-contained serum in vitro, and cell apoptosis was quantified using flow cytometry. Additionally, the expression levels of Ube2m, Bim and other associated proteins were also assessed through western blotting. To further clarify whether Ube2m serves as the key target of JP in regulating DN T cell apoptosis, the mice that Ube2m was specific deleted in T cells with spontaneous lupus (Ube2m-/-lpr) were utilized. JP was administered to WTlpr or Ube2m-/-lpr mice, followed by assessment of the lupus condition and DN T cell apoptosis. ResultsJP administration effectively ameliorated the lupus phenotype. Then flow cytometry assay showed that JP treatment enhanced DN T cell apoptosis to reduce their accumulation and restored the immune homeostasis. Proteomic analysis revealed a significant inhibition of Ube2m for JP treatment, which is essential for maintaining homeostasis of DN T cells. Further experiments confirmed that JP treatment effectively downregulated the expression of Ube2m and subsequently upregulated the level of pro-apoptotic protein Bim with decreased Bim degradation. In vitro experiments also confirmed that JP-contained serum significantly facilitated DN T cell apoptosis and reduced DN T cell accumulation by inhibiting Ube2m expression. Furthermore, Ube2m-/-lpr mice were utilized and the impact of JP treatment on the apoptosis of DN T cells was found to be minimal in the absence of Ube2m. Mechanistic investigation reveals that JP exerts its effects by suppressing the expression of Ube2m, subsequently inhibiting CRL-dependent degradation of Bim, and ultimately promoting Bim-induced apoptosis in DN T cells. Furthermore, the blockade of Ube2m in T cells effectively prevents JP-induced apoptosis in DN T cells, underscoring Ube2m as one crucial therapeutic target of JP in mediating DN T cell apoptosis and managing SLE. ConclusionsOur findings indicate that JP treatment effectively restores the homeostasis of DN T cells in SLE by inhibiting Ube2m expression, thereby reducing Bim ubiquitination degradation. This ultimately enhanced DN T cell apoptosis and alleviates lupus phenotype.

Sex-Stratified Genome-Wide Association Study in the Spanish Population Identifies a Novel Locus for Lacunar Stroke.

Ischemic stroke (IS) represents a significant health burden globally, necessitating a better understanding of its genetic underpinnings to improve prevention and treatment strategies. Despite advances in IS genetics, studies focusing on the Spanish population and sex-stratified analyses are lacking. A case-control genome-wide association study was conducted with 9081 individuals (3493 IS cases and 5588 healthy controls). IS subtypes using Trial of ORG 10172 in Acute Stroke Treatment criteria were explored in a sex-stratified approach. Replication efforts involved the MEGASTROKE, GIGASTROKE, and the UK Biobank international cohorts. Post-genome-wide association study analysis included: in silico proteomic analysis, gene-based analysis, quantitative trait loci annotation, transcriptome-wide association analysis, and bioinformatic analysis using chromatin accessibility data. Identified as associated with IS and its subtypes were 4 significant and independent loci. Replication confirmed 5p15.2 as a new locus associated with small-vessel occlusion stroke, with rs59970332-T as the lead variant (beta [SE], 0.13 [0.02]; P=4.34×10-8). Functional analyses revealed CTNND2 given proximity and its implication in pathways involved in vascular integrity and angiogenesis. Integration of Hi-C data identified additional potentially modulated genes, and in silico proteomic analysis suggested a distinctive blood proteome profile associated with the lead variant. Gene-set enrichment analyses highlighted pathways consistent with small-vessel disease pathogenesis. Gene-based associations with known stroke-related genes such as F2 and FGG were also observed, reinforcing the relevance of our findings. We found CTNND2 as a potential key molecule in small-vessel occlusion stroke risk, and predominantly in males. This study sheds light on the genetic architecture of IS in the Spanish population, providing novel insights into sex-specific associations and potential molecular mechanisms. Further research, including replication in larger cohorts, is essential for a comprehensive understanding of these findings and for their translation to clinical practice.

LRG1 promotes metastatic colorectal cancer growth through HER3 signaling

Background and aimsTherapy failure in patients with metastatic colorectal cancer (mCRC, ∼80% occur in the liver) remains an overarching challenge. Preclinical studies demonstrated that HER3 promotes CRC cell survival, but therapies blocking the neuregulin-induced canonical HER3 signaling have made little impact in the clinic. Recent studies suggest that the liver microenvironment promotes CRC growth by activating HER3 in a neuregulin-independent fashion, thus elucidation of these mechanisms may reveal new strategies for treating patients with mCRC. MethodsPatient-derived primary liver endothelial cells (ECs) were used to interrogate EC-CRC crosstalk. We conducted proteomic analysis to identify EC-secreted factor(s) that triggers non-canonical HER3 activation in CRC, and determined the subsequent effects on mCRC using diverse murine mCRC models. In vitro studies with genetic and pharmacological interventions were used to map the non-canonical HER3 pathway. ResultsWe demonstrated that EC-secreted leucine-rich alpha-2-glycoprotein 1 (LRG1) directly binds and activates HER3 and promotes CRC growth distinct from neuregulin, the canonical HER3 ligand. Blocking host-derived LRG1 by gene knockout or a neutralizing antibody impaired mCRC outgrowth in the liver and prolonged mouse survival. We identified protein synthesis activated by the PI3K-PDK1-RSK-eIF4B axis as the biologically relevant signaling cascade downstream of the LRG1-HER3 interaction, which was not blocked by conventional HER3-specific antibodies that failed in prior clinical trials. ConclusionsLRG1 is a novel HER3 ligand and mediates liver-mCRC crosstalk. The LRG1-HER3 signaling axis is distinct from canonical HER3 signaling and represents a new therapeutic opportunity to treat patients with mCRC, and potentially other types of liver metastases.

Proteomic Analysis of Storage Proteins in Phaseolus vulgaris Associated with Resistance to Water Stress

The common bean (Phaseolus vulgaris), a widely consumed legume in Ecuador, boasts low economic value, significant nutritional contributions, and a remarkable capacity to enhance soil fertility. Despite these attributes, its field productivity often needs to improve, with the water deficit emerging as a primary hindrance. Consequently, genetic enhancements have been incorporated into select varieties, conferring tolerance to specific levels of water scarcity stress. This study aimed to elucidate the distinctions in protein expression patterns responding to water deficiency stress across nine bean varieties. Protein patterns were scrutinized through two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and selected protein spots were subjected to mass spectrometry analysis (MALDI-TOF MS/RP-LC-MS/MS). A comprehensive identification of 111 proteins was achieved and categorized based on their respective functions. Noteworthy among these were the desiccation-protectant protein (LEA14) and Desiccation-related protein PCC13-62, identified as proteins associated with the response to abiotic stress, particularly prevalent in the INIAP_473 cultivar. These findings underscore the potential for targeted genetic improvements to mitigate the impact of water deficit stress on common bean cultivation, contributing to enhanced agricultural resilience and productivity.

Long-term exposure to third-hand smoke could accelerate biological aging via mitochondrial dysfunction: Evidence from Population and Animal Studies

The relationship between third-hand smoke (THS) exposure and lifespan remains inadequately explored. Our study sought to clarify the effects of THS on aging and lifespan. In this pursuit, our cross-sectional analysis assessed hematological aging markers in 986 non-smokers and examined lifespan alterations using a Drosophila model. THS exposure levels were quantified through survey metrics consistent with the Global Adult Tobacco Survey. The findings revealed that THS exposure significantly accelerated biological aging, with exposed individuals exhibiting an average increase in biological age of 3.04 years compared to their unexposed counterparts (p < 0.05). Correspondingly, the Drosophila model reflected these outcomes, showing a reduction in lifespan by 16.07 days (p < 0.01). Proteomic analyses identified MRPL2 as a pivotal protein in THS-induced aging, linking its expression to mitochondrial dysfunction and oxidative stress. Further metabolomic profiling highlighted disruptions in energy metabolism pathways. Follow-up in vitro experiments confirmed the role of MRPL2 in the aging processes at the cellular level. Overall, our results indicate that THS exposure is a significant accelerant of aging, providing new perspectives on the health consequences of environmental smoke residues.

Characterization of decellularized porcine oviduct- and uterine-derived scaffolds evaluated by spermatozoa-based biocompatibility and biotoxicity

Extracellular matrix-derived scaffolds (dECM) are widely utilized in regenerative medicine and tissue engineering due to their ability to promote cell growth, proliferation, and differentiation. In reproduction, research is focused on using these scaffolds to treat pathologies causing reproductive dysfunction or to improve assisted reproduction technologies (ARTs). We developed an efficient protocol employing the immersion-agitation technique to decellularize porcine oviductal and uterine sections, comparing the efficacy of fresh versus frozen treatments. Both methods successfully generated acellular matrices with less than 3% residual DNA, effectively preserving structural and protein integrity. Scanning and transmission electron microscopy confirmed the ultrastructural integrity, whereas Masson's Trichrome staining highlighted better collagen preservation in frozen treatments. Proteomic analysis of decellularized scaffolds revealed collagen and key macromolecules such as laminin, filamin, dermatopontin, and fibronectin, which are essential for extracellular matrix structure and cell functions such as adhesion and migration. Innovatively, we assessed the biocompatibility and cytotoxicity of the scaffolds using spermatozoa, demonstrating that thorough washing ensures the scaffold biocompatibility without compromising sperm viability or motility. Our findings not only contribute to the standardization of decellularization protocols for female reproductive organs but also emphasize the importance of evaluating sperm biocompatibility to ensure the safety of dECM scaffolds.

HSP70 inhibits CHIP E3 ligase activity to maintain germline function in Caenorhabditis elegans

The ubiquitin-proteasome system is crucial for proteostasis, particularly during proteotoxic stress. The interaction between heat shock protein 70 (HSP70) and the ubiquitin ligase CHIP plays a key role in this process. Our study investigates the C. elegans orthologs HSP-1 and CHN-1, demonstrating that HSP-1 binding decreases CHN-1 E3 ligase activity, aligning with the inhibitory effects observed in human HSP70-CHIP interactions. To explore the physiological significance of this inhibition, we utilized the HSP-1EEYD mutant, which binds CHN-1 without reducing its activity, expressed in C. elegans. Our results reveal that the HSP-1-CHN-1 interaction is critical for maintaining germline integrity under heat stress by preventing excessive turnover of essential reproductive proteins. In HSP-1EEYD nematodes, this protective mechanism is impaired, leading to disrupted stress-induced apoptosis, which is restored by CHN-1 depletion. Additionally, proteomic analysis identified DAF-18/PTEN as a potential CHN-1 substrate, which becomes destabilized when CHN-1 activity is not downregulated by HSP-1 during stress. Depleting DAF-18 significantly compromises the reproductive benefits observed from CHN-1 knockout in HSP-1EEYD mutants, suggesting that the maintenance of DAF-18 plays a role in the observed phenotypes. These findings highlight the importance of HSP-1 in regulating CHN-1 E3 ligase activity to preserve germline function under stress conditions.

Akt Inhibitors Prevent CyHV-2 Infection In Vitro

Cyprinid herpesvirus 2 (CyHV-2) is a double-stranded DNA virus that infects goldfish (Carassius auratus) and crucian carp (C. carassius), resulting in substantial mortality rates and significant epidemiological implications. To gain deeper insights into CyHV-2-host interactions and identify potential therapeutic targets, quantitative proteomics analysis was conducted on CyHV-2-infected Ryukin goldfish fin (RyuF-2) cells. Our findings revealed significant alterations in the expression of proteins associated with the PI3K/Akt signaling pathway, which were up-regulated upon viral infection. Building on these observations, we employed LY294002, a specific inhibitor of PI3K, to investigate its impact on viral replication by inhibiting the PI3K/Akt pathway in GiCF cell line derived from the caudal fin of Carassius auratus gibelio (Bloch). Our results demonstrated the inhibition of both CyHV-2 replication and Akt phosphorylation within this pathway. Quercetin, a plant-derived analogue of LY294002, was further investigated for its anti-CyHV-2 effects in vitro as well as its underlying mechanism. The results suggested that quercetin exhibits antiviral properties against CyHV-2 and may exert its effects through mechanisms similar to those observed with LY294002. Given that aquaculture water serves as a vector for aquaculture viral diseases and the release of chemical compounds can lead to pollution of the aquatic environment, our study shifted focus to crude extracts obtained from plants. We confirmed crude quercetin extract derived from Cuminum anisum has antiviral activity against CyHV-2 in vitro. Therefore, based on our identification of the involvement of PI3K/Akt signaling pathway in CyHV-2 replication, along with verification of its antiviral mechanism using LY294002, we propose natural dietary quercetin as a promising candidate for development into a novel anti-CyHV-2 drug.