Search Results (165)

Background Plasma membrane proteins (PMPs) play key roles in cell signalling, adhesion, and trafficking, and are attractive therapeutic targets in cancer due to their surface accessibility. However, their typically low abundance limits detection by conventional proteomic approaches. Methods: To improve PMP detection, we employed a surface proteomics workflow combining cell surface biotinylation and affinity purification prior to LC-MS/MS analysis in cervical (SiHa) and bladder (UMUC3) cancer cell lines cultured under normoxic (21% O₂) or hypoxic (0.1% O₂) conditions. Results: In SiHa cells, 43 hypoxia-upregulated proteins were identified exclusively in the biotin-enriched fraction, including ITGB2, ITGA7, AXL, MET, JAG2, and CAV1/CAV2. In UMUC3 cells, 32 unique upregulated PMPs were detected, including CD55, ADGRB1, SLC9A1, NECTIN3, and ACTG1. These proteins were not observed in corresponding whole-cell lysates and are associated with extracellular matrix remodelling, immune modulation, and ion transport. Biotinylation enhanced the detection of membrane-associated pathways such as ECM organisation, integrin signalling, and PI3K–Akt activation. Protein–protein interaction analysis revealed links between membrane receptors and intracellular stress regulators, including mitochondrial proteins. Conclusions: These findings demonstrate that surface biotinylation improves the sensitivity and selectivity of plasma membrane proteomics under hypoxia, revealing hypoxia-responsive proteins and pathways not captured by standard whole-cell analysis. Full article

Cancer is a systemic disease rather than a localized pathology and is characterized by widespread effects, including whole-body exhaustion and chronic inflammation. A thorough understanding of cancer pathophysiology requires a systemic approach that accounts for the complex interactions between cancer cells and host tissues. To explore these dynamics, we employed a comprehensive metabolomic analysis of plasma samples from patients with either esophageal or head and neck squamous cell carcinoma (SCC). Plasma samples from 149 patients were metabolically profiled and correlated with clinical data. Among the metabolites identified, lysophosphatidylcholine (LPC) emerged as the sole biomarker strongly correlated with prognosis. A significant reduction in plasma LPC levels was linked to poorer overall survival. Plasma LPC levels demonstrated minimal correlation with patient-specific factors, such as tumor size and general condition, but showed significant association with the response to immune checkpoint inhibitor therapy. Proteomic and cytokine analyses revealed that low plasma LPC levels reflected systemic chronic inflammation, characterized by high levels of inflammatory proteins, the cytokines interleukin-6 and tumor necrosis factor-α, and coagulation-related proteins. These findings indicate that plasma LPC levels may be used as reliable biomarkers for predicting prognosis and evaluating the efficacy of immunotherapy in patients with SCC. Full article

Background/Objectives: Guillain–Barré syndrome (GBS) is a rare autoimmune peripheral neuropathy that affects both the myelin sheaths and axons of the peripheral nervous system. It is the leading cause of acute neuromuscular paralysis worldwide, with an annual incidence of less than two cases per 100,000 people. Although most patients recover, a small proportion do not regain mobility and even remain dependent on mechanical ventilation. In this study, we refer to the analysis of samples collected from GBS patients at different defined time points during hospital recovery and performed by a medical or research group. Methods: The conditions for whole blood collection, peripheral blood mononuclear cell isolation, and serum collection from GBS patients and volunteer donors are explained. Aliquots of these human samples have been used for red blood cell phenotyping, transcriptomic and proteomic analyses, and serum biochemical parameter studies. Results: The initial sporadic preservation of human samples from GBS patients and control volunteers enabled the creation of a biobank collection for current and future studies related to the diagnosis and treatment of GBS. Conclusions: In this article, we describe the laboratory procedures and the integration of a GBS biobank collection, local medical services, and academic institutions collaborating in its respective field. The report establishes the intra-disciplinary and inter-institutional network to conduct long-term longitudinal studies on GBS. Full article

Precision medicine is transforming hematologic cancer care by tailoring treatments to individual patient profiles and moving beyond the traditional “one-size-fits-all” model. This review outlines foundational technologies, disease-specific advances, and emerging directions in precision hematology. The field is enabled by molecular profiling techniques, including next-generation sequencing (NGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), as well as epigenomic and proteomic analyses. Complementary tools such as liquid biopsy and minimal residual disease (MRD) monitoring have improved diagnosis, risk stratification, and therapeutic decision making. We discuss major molecular targets and personalized strategies across hematologic malignancies: FLT3 and IDH1/2 in acute myeloid leukemia (AML); Philadelphia chromosome–positive and Ph-like subtypes in acute lymphoblastic leukemia (ALL); BCR-ABL1 in chronic myeloid leukemia (CML); TP53 and IGHV mutations in chronic lymphocytic leukemia (CLL); molecular subtypes and immune targets in diffuse large B-cell lymphoma (DLBCL) and other lymphomas; and B-cell maturation antigen (BCMA) in multiple myeloma. Despite significant progress, challenges remain, including high costs, disparities in access, a lack of standardization, and integration barriers in clinical practice. However, advances in single-cell sequencing, spatial transcriptomics, drug repurposing, immunotherapies, pan-cancer trials, precision prevention, and AI-guided algorithms offer promising avenues to refine treatment and improve outcomes. Overcoming these barriers will be critical for ensuring the equitable and widespread implementation of precision medicine in routine hematologic oncology care. Full article

Sickle cell disease (SCD) is the most common hemoglobinopathy, caused by a mutation in the β-globin gene of hemoglobin. It predisposes patients to painful Vaso-occlusive crises (VOC) and multi-organ dysfunctions. The disease exhibits significant phenotypic variability, making it challenging to predict severity and outcomes. This study aimed to characterize the whole blood gene expression profile of Bahraini SCD patients, identifying differentially expressed genes during steady-state (n = 10) and VOC (n = 10) compared to healthy controls (n = 8). Analysis revealed 2073 and 3363 dysregulated genes during steady-state and VOC, respectively, compared to controls, with 1078 genes differentially expressed during VOC versus steady-state. Gene Ontology (GO) enrichment analysis highlighted significant deregulation in immune and hematopoietic pathways, including down-regulation of critical genes for immune modulation and hematopoietic balance. Notably, the transcription factor RUNX3, involved in immune cell differentiation and inflammation, was among the 668 down-regulated genes. RUNX3 was four-fold down-regulated in microarray analysis, three-fold in PCR, and showed a mean protein concentration of 11.13 pg/mL during VOC compared to 457.93 pg/mL during steady-state (p < 0.01). These findings suggest that RUNX3 may serve as a potential biomarker for VOC. Future large-scale validation, additional proteomic studies, and functional investigations are recommended to confirm its clinical utility and significance. Full article

Background: Cancer vaccines represent a groundbreaking advancement in cancer immunotherapy, utilizing tumor antigens to induce tumor-specific immune responses. However, challenges like tumor-induced immune resistance and technical barriers limit the widespread application of predefined antigen vaccines. Here, we investigated the potential of viral protein R (Vpr) peptides as effective candidates for constructing anonymous antigen vaccines in situ by directly injecting at the tumor site and releasing whole-tumor antigens, inducing robust anti-tumor immune responses to overcome the limitations of predefined antigen vaccines. Methods: The cytotoxic effects of Vpr peptides were evaluated using the CCK8 reagent kit. Membrane penetration ability of Vpr peptides was observed using a confocal laser scanning microscope and quantitatively analyzed using flow cytometry. EGFR levels in the cell culture supernatants of cells treated with Vpr peptides were evaluated using an ELISA. Surface exposure of CRT on the tumor cell surface was observed using a confocal laser scanning microscope and quantitatively analyzed using flow cytometry. The secretion levels of ATP from tumor cells were evaluated using an ATP assay kit. HMGB1 release was evaluated using an ELISA. Mouse (Male C57BL/6 mice aged 4 weeks) MC38 and LLC bilateral subcutaneous tumor models were established to evaluate the therapeutic effects of Vpr peptides through in situ vaccination. Proteomic analysis was performed to explore the mechanism of anti-tumor activity of Vpr peptides. Results: Four Vpr peptides were designed and synthesized, with P1 and P4 exhibiting cytotoxic effects on tumor cells, inducing apoptosis and immunogenic cell death. In mouse tumor models, in situ vaccination with Vpr peptide significantly inhibited tumor growth and activated various immune cells. High-dose P1 monotherapy demonstrated potent anti-tumor effects, activating DCs, T cells, and macrophages. Combining ISV of P1 with a CD47 inhibitor SIRPαFc fusion protein showed potent distant tumor suppression effects. Proteomic analysis suggested that Vpr peptides exerted anti-tumor effects by disrupting tumor cell morphology, movement, and adhesion, and promoting immune cell infiltration. Conclusions: The designed Vpr peptides show promise as candidates for in situ vaccination, with significant anti-tumor effects, immune activation, and favorable safety profiles observed in mouse models. In situ vaccination with Vpr-derived peptides represents a potential approach for cancer immunotherapy. Full article

Hypoxia leads to endothelial dysfunction and increased blood–brain barrier (BBB) permeability, promoting the incidence of diseases such as stroke and acute high-altitude illness. Brain microvascular endothelial cells (BMECs) are important structural and functional components of the BBB; however, the molecular changes that occur in BMECs during hypoxia remain unknown. We reported the molecular and functional changes in BMECs under hypoxia through whole-transcriptome sequencing, small RNA microarray, TMT quantitative proteomic, and untargeted metabolomic analyses. We found that hypoxia affected pathways such as ncRNA processing, the HIF-1 signaling pathway, the cell cycle, DNA replication, glucose metabolism, protein synthesis, and inflammation pathways. ncRNA processing was significantly downregulated. However, the levels of some miRNAs, tRNAs, tsRNAs, snoRNAs, lncRNAs, and circRNAs were significantly upregulated under hypoxia. These results suggest that ncRNAs may play an important role in oxidative stress and cellular adaptation to hypoxia, helping us understand the pathological process of BBB injury and providing potential targets for the treatment of BBB-related cerebrovascular diseases. Full article

Escherichia coli and Klebsiella pneumoniae are major contributors to the global challenge of antimicrobial resistance, posing serious threats to public health. Among current preventive strategies, conjugate vaccines that utilize bacterial surface polysaccharides have emerged as a promising and effective approach to counter multidrug-resistant strains. In this study, both the Wzy/Wzx-dependent and ABC transporter-dependent biosynthetic pathways for antigenic polysaccharides were introduced into E. coli W3110 cells. This dual-pathway engineering enabled the simultaneous biosynthesis of two structurally distinct polysaccharides within a single host, offering a streamlined and potentially scalable strategy for vaccine development. Experimental findings confirmed that both polysaccharide types were successfully produced in the engineered strains, although co-expression levels were moderately reduced. A weak competitive interaction was noted during the initial phase of induction, which may be attributed to competition for membrane space or the shared use of activated monosaccharide precursors. Interestingly, despite a reduction in plasmid copy number and transcriptional activity of the biosynthetic gene clusters over time, the overall polysaccharide yield remained stable with prolonged induction. This suggests that extended induction does not adversely affect final product output. Additionally, two glycoproteins were efficiently generated through in vivo bioconjugation of the synthesized polysaccharides with carrier proteins, all within the same cellular environment. This one-cell production system simplifies the workflow and enhances the feasibility of generating complex glycoprotein vaccines. Whole-cell proteomic profiling followed by MFUZZ clustering and Gene Ontology analysis revealed that core biosynthetic genes were grouped into two functional clusters. These genes were predominantly localized to the cytoplasm and were enriched in pathways related to translation and protein binding. Such insights not only validate the engineered biosynthetic routes but also provide a molecular basis for optimizing future constructs. Collectively, this study presents a robust synthetic biology platform for the co-expression of multiple polysaccharides in a single bacterial host. The approach holds significant promise for the rational design and production of multivalent conjugate vaccines targeting drug-resistant pathogens. Full article

In this study, a global response analysis was performed to explore the mechanism of action of Usnic acid and its synergy with Norfloxacin, a well-known quinolone antibiotic to which MRSA clinical isolates showed resistance (MIC, 500 µg/mL). A microdilution assay, a growth kinetics analysis, a microscopic analysis, and cell-based assays consistently showed that Usnic acid possesses strong anti-staphylococcal activity (MIC, 7.8 µg/mL), causes cell leakage, modulates efflux pump activity, and synergizes with Norfloxacin against the multi-drug-resistant clinical isolate MRSA 2071. Whole-cell proteome profiling using gel-free proteomics-based nano-LC-ESI-QTOF-MS/MS revealed several proteins whose expression was significantly modulated by Usnic acid and Norfloxacin alone or in combination. Usnic acid downregulated the abundance of RNA polymerase subunits (RpoB and RpoC), carbamoyl phosphate synthase large subunit (PyrAB), chaperone (GroEL), and adenylosuccinate synthetase (PurA). Interestingly, proteins found to be upregulated in the presence of Usnic acid and Norfloxacin included oxidative-stress-related proteins such as peroxidase (Tpx), alkyl hydroperoxide reductase (AphC), and general stress protein (UspA). This study clearly shows that Usnic acid affects numerous cellular targets and can potentiate the action of Norfloxacin. Furthermore, an in vivo study showed that UA at low concentrations prevents body weight gain, but changes in other tested toxicological parameters were found to be within normal limits. Thus, UA at low doses appears to be a promising candidate for repurposing old antibiotics through combination therapy against MRSA infections. Full article

Mucolipidosis type II is a very rare lysosomal disease affecting the UDP-GlcNAc N-acetylglucosamine-1-phosphotransferase enzyme, which catalyzes the synthesis of the targeting signal mannose 6-phosphate in lysosomal acid hydrolases. Its deficiency hinders the arrival of lysosomal enzymes to the lysosome, diminishing the multiple degradations of components that cells need to perform. Due to the low prevalence of this condition, available information is scarce. This article aims to deepen the understanding of the disease; clinical, biochemical, and proteomic data are analyzed. Three patients have been identified presenting GNPTAB pathogenic variants using whole exome sequencing. A biochemical profile for these patients has been carried out through quantification of glycosaminoglycans in urine samples and enzymatic analysis in dried blood spot (DBS) samples. Quantitative proteomic studies were performed. Results show how enzymatic assays in DBS can be used to diagnose this disease both during the neonatal period or in patients of more advanced age. Increased levels of acid sphingomyelinase, alpha-iduronidase, iduronidate 2-sulfatase, alpha-N-acetyl glucosaminidase, and beta-glucuronidase are found. Conclusion: this biochemical method could potentially improve early diagnosis. Proteomic data supporting these results reveal disrupted biochemical pathways, including the degradation of dermatan sulfate, heparan sulfate, and cellular cholesterol trafficking. Full article

Purpose: Horse racing may cause stress-induced physiological changes and tissue damage in horses, but the changes in miRNA expression, protein expression, and metabolic substances in the plasma exosomes of the Yili horse after racing are still unclear. This study detected miRNA, protein expression, and metabolic substances in the plasma exosomes of Yili horses before and after competition, providing new insights for post-race recovery and care of Yili horses. Method: Eight three-year-old Yili horses that had undergone training were selected as the research subjects, with four horses that had not competed as the control group and four horses that had participated in the competition for half an hour as the training group. Extract whole blood and separate plasma from two groups of horses, and then extract plasma exosomes; MiRNAs, proteins, and metabolites in extracellular vesicles were detected and analyzed using miRNAomics, proteomics, and metabolomics. P Result: After the competition, the levels of miRNAs related to the cytoplasm and nucleus in Yili horse plasma exosomes increased, and miRNAs related to the transcription and transcriptional regulation of biological processes significantly increased. The levels of proteins related to the cytoplasm and nucleus also increased, and the levels of proteins related to cell signaling function increased, carbohydrates and their metabolites were significantly reduced. Conclusions: The competition process causes significant changes in the miRNA, proteomics, and metabolomics of plasma exosomes in the Yili horses, which are mainly related to metabolic regulation. Full article

(1) Background: Single-cell multi-omics is a powerful method for the dissection and detection of complicated immunologic functions and synapses. However, most currently available technologies merge datasets of different omics from separate portions of the same sample to generate combined multi-omics. This process is a source of bias, mainly in the field of immunology on cells originating from pluripotent hematopoietic stem cells with high flexibility during maturation. (2) Methods: Although new multi-omics approaches have been developed to use the advantages of cellular and molecular barcoding and next-generation sequencing to solve this issue, one of the main current challenges is intracellular proteomics, which should be combined with other omics data with high importance for immune system studies. We designed this study to evaluate previously recommended minimal permeabilization and fixation methods on the quality and quantity of transcriptomics and proteomics data generated by the BD Rhapsody™ Single-Cell Analysis System. (3) Results: Our findings showed that high-throughput sequencing with advanced quality and read-out is required for the combination of multi-omics outcomes from a permeabilized single cell. Therefore, the HiseqX platform was selected for further analysis. The effect of immune stimulation was observed clearly as the separated clusters of helper and cytotoxic T cells using unsupervised clustering. Importantly, fixation and permeabilization did not affect the general expression profile of unstimulated cells. However, fixation and permeabilization were proved to negatively impact the detection of the whole transcriptome for single-cell assay. Nevertheless, about 60% of the transcriptomic signature of the stimulation was detected. If the measurement of combined surface and intracellular markers is required to be achieved, the modified fixation and permeabilization method is recommended because of a lower transcriptomic loss and more precise proteomic fingerprint detected. (4) Conclusions: The findings of this study support the potential possibility for integrating intracellular proteomics, which needs to be optimized and tested with newly designed oligonucleotide-tagged antibodies targeting intracellular proteins. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the large coronavirus family with high infectivity and pathogenicity and is the primary pathogen causing the global pandemic of coronavirus disease 2019 (COVID-19). Phosphorylation is a major type of protein post-translational modification that plays an essential role in the process of SARS-CoV-2–host interactions. The precise identification of phosphorylation sites in host cells infected with SARS-CoV-2 will be of great importance to investigate potential antiviral responses and mechanisms and exploit novel targets for therapeutic development. Numerous computational tools have been developed on the basis of phosphoproteomic data generated by mass spectrometry-based experimental techniques, with which phosphorylation sites can be accurately ascertained across the whole SARS-CoV-2-infected proteomes. In this work, we have comprehensively reviewed several major aspects of the construction strategies and availability of these predictors, including benchmark dataset preparation, feature extraction and refinement methods, machine learning algorithms and deep learning architectures, model evaluation approaches and metrics, and publicly available web servers and packages. We have highlighted and compared the prediction performance of each tool on the independent serine/threonine (S/T) and tyrosine (Y) phosphorylation datasets and discussed the overall limitations of current existing predictors. In summary, this review would provide pertinent insights into the exploitation of new powerful phosphorylation site identification tools, facilitate the localization of more suitable target molecules for experimental verification, and contribute to the development of antiviral therapies. Full article

Importance: Although prolonged fasting has become increasingly popular, the favourable biological adaptations and possible adverse effects in humans have yet to be fully elucidated. Objective: To investigate the effects of a three-day water-only fasting, with or without exercise-induced glycogen depletion, on autophagy activation and the molecular pathways involved in cellular damage accumulation and repair in healthy humans. Design: A randomised, single-centre, two-period, two-sequence crossover trial. The primary outcome is autophagic activity, assessed as flux in peripheral blood mononuclear cells (PBMCs) measured in the context of whole blood. Secondary outcomes include changes in body composition, heart rate variability, endothelial function, and genomic, epigenomic, metabolomic, proteomic, and metagenomic adaptations to fasting in plasma, platelets, urine, stools, and PBMCs. Detailed profiling of circulating immune cell populations and their functional states will be assessed by flow cytometry. Setting: All clinical investigations will be undertaken at the Charles Perkins Centre Royal Prince Alfred Hospital clinic, University of Sydney, Australia. Participants: Twenty-four individuals aged 18 to 70 years, with a BMI of 20–40 kg/m², free of major health conditions other than obesity. Discussion: While autophagic flux induction through fasting has garnered interest, there is a notable lack of human studies on this topic. This trial aims to provide the most detailed and integrated analysis of how three days of prolonged water-only fasting, combined with glycogen-depleting exercise, affects autophagy activation and other crucial metabolic and molecular pathways linked to cellular, metabolic, and immune health. Insights from this study may pave the way for safe and effective strategies to induce autophagy, offering potential preventive interventions for a range of chronic conditions. Full article

Differences in cell count and growth factor expression between first- and second-generation autologous platelet concentrates (APCs) have been well described. The debate over which formula best supports wound healing in various surgical procedures is still ongoing. This study aims to assess the whole proteome assembly, cell content, immunological potential and pro-angiogenic potential of second-generation APC, Platelet-Rich Fibrin (PRF) vs. first-generation APC, Platelet-Rich Plasma (PRP). The global proteome of the APCs was analyzed using nano-liquid chromatography mass spectrometry. Blood cell concentrations were determined by an automated cell counter. The effect of APCs on macrophage polarization was analyzed by flow cytometry. A yolk sac membrane (YSM) assay was used to monitor the neo-vessel formation and capillary branching in vivo. Cell count analysis revealed a higher number/concentration of leukocytes in PRF vs. PRP. Incubation of macrophages with PRP or platelet-free plasma (PFP) did not induce a significant pro-inflammatory state but led to a shift to the M0/M2 phenotype as seen in wound healing for all tested formulas. Label-free proteomics analysis identified a total of 387 proteins from three biological replicates of the respective designated groups. PRF induced increased formation of neo-vessels and branching points in vivo in comparison to PRP and PFP (each p < 0.001), indicating the enhanced pro-angiogenic potential of PRF. Overall, PRF seems superior to PRP, an important representative of first-generation formulas. Inclusion of leucocytes in PRF compared to PRP suggested rather an anti-inflammatory effect on macrophages. These results are important to support the versatile clinical applications in regenerative medicine for second-generation autologous platelet concentrates to optimize wound healing. Full article