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Excessive alarmins S100A8/A9 escalate the inflammation and even exacerbate immune-driven thrombosis and multi-organ damage. However, the regulatory mechanisms of S100A8/A9 expression in infectious diseases remain unclear. In this study, high-dimensional transcriptomic data analyses revealed a high proportion of CD14+FCN1hi macrophages within the pulmonary niche post-severe SARS-CoV-2 infection. By constructing the S100-coexpression gene list and supervised module scoring, we found that CD14+FCN1hi macrophages presented the highest scores of alarmin S100, and possibly served as the trigger and amplifier of inflammation in severe COVID-19. These CD14+FCN1hi cells lacked the positive regulatory activity of transcription factor PPARγ, and lost their differentiation ability towards mature macrophages. Ex vivo experiments further validated that the epithelial cells with high ORF-3a expression promoted the expression and secretion of S100A8/A9 through ANXA1/SAA1-FPR1 signaling. S100A8/A9 heterodimers, as well as the co-localization of S100A8/A9 with microtubules, were both diminished by the FPR1 inhibitor. Phospho-kinase protein array indicated that STAT3 promoted transcription, and PLC-γ and ERK1/2 pathways were involved in the hetero-dimerization and unconventional secretion of S100A8/A9. Our study highlights the pivotal role of FPR1 signaling in the excessive production of S100A8/A9 and provides a promising target for the prevention and control of severe COVID-19 and post-acute COVID-19 sequelae. CD14+FCN1hi macrophages highly express S100A8/S100A9, which is initiated by FPR1 signaling in severe COVID-19. An FPR1 inhibitor prevents the transcription, hetero-dimerization, and secretion of S100A8/A9 through STAT3/PLC-γ/ERK1/2 pathways. [ABSTRACT FROM AUTHOR]

Objective: The biochip method allows microscale multiparametric analysis of macromolecular samples using a matrix of immobilized molecular probes. Selection of materials for biochip fabrication, functionalization of the carrier surface, and the method of immobilization of molecular probes are the key tasks of biochip technology. Methods: Methods of obtaining polymer coating from polyvinyl acetate on the surface of polyethylene terephthalate polymer substrates and subsequent production of brush polymers by photoinduced radical copolymerization of acrylate monomers have been studied. Cell matrices with numerous reactive chemical groups for subsequent immobilization of proteins were formed by photolithography method. Methods of activation of carboxyl groups on brush polymers attached to the surface of polyethylene terephthalate were tested. Immobilization of model protein streptavidin labeled with fluorescent dye Su3 was performed to test the method of activation of carboxyl groups. Results and Discussion: A variant of the immunofluorescence assay in a biological microarray format was tested on the model "streptavidin–biotinylated immunoglobulin." Conclusions: Streptavidin immobilized in brush polymer cells retains functionality and spatial accessibility for binding to biotinylated immunoglobulin and subsequent manifestation by antibodies fluorescently labeled with Cy5 dye, which opens prospects for the use of biological microarrays with brush polymer cells on polyethylene terephthalate substrates for immunofluorescence analysis of various protein targets. [ABSTRACT FROM AUTHOR]

Background information: Arpin, an Arp2/3 inhibitory protein, inhibits lamellipodial protrusions and cell migration. Arpin expression is lost in tumor cells of several cancer types. Results: Here we analyzed expression levels of Arpin and various markers using Reverse Phase Protein Array (RPPA) in human mammary carcinomas. We found that Arpin protein levels were correlated with those of several DNA damage response markers. Arpin‐null cells display enhanced clustering of double stand breaks (DSBs) when cells are treated with a DNA damaging agent, in line with a previously described role of the Arp2/3 complex in promoting DSB clustering for homologous DNA repair (HDR) in the nucleus. Using a specific HDR assay, we further showed that Arpin depletion increased HDR efficiency two‐fold through its ability to inactivate the Arp2/3 complex. Conclusions: Arpin regulates both cell migration in the cytosol and HDR in the nucleus. Significance: Loss of Arpin expression coordinates enhanced cell migration with up‐regulated DNA repair, which is required when DNA damage is induced by active cell migration. [ABSTRACT FROM AUTHOR]

Objectives To identify and characterize undescribed systemic sclerosis (SSc)-specific autoantibodies targeting nucleolar antigens and to assess their clinical significance. Methods We conducted proteome-wide autoantibody screening (PWAS) against serum samples from SSc patients with nucleolar patterned anti-nuclear antibodies (NUC-ANAs) of specific antibodies (Abs) unknown, utilizing wet protein arrays fabricated from in vitro human proteome. Controls included SSc patients with already-known SSc-specific autoantibodies, patients with other connective tissue diseases and healthy subjects. The selection of nucleolar antigens was performed by database search in the Human Protein Atlas. The presence of autoantibodies was certified by immunoblots and immunoprecipitations. Indirect immunofluorescence assays on HEp-2 cells were also conducted. Clinical assessment was conducted by retrospective review of electronic medical records. Results PWAS identified three candidate autoantibodies, including anti-nuclear valosin-containing protein-like (NVL) Ab. Additional measurements in disease controls revealed that only anti-NVL Abs are exclusively detected in SSc. Detection of anti-NVL Abs was reproduced by conventional assays such as immunoblotting and immunoprecipitation. Indirect immunofluorescence assays demonstrated homogeneous nucleolar patterns. Anti-NVL Ab-positive cases were characterized by significantly low prevalence of diffuse skin sclerosis and interstitial lung disease, compared with SSc cases with NUC-ANAs other than anti-NVL Abs, such as anti-U3-RNP and anti-Th/To Abs. Conclusion Anti-NVL Ab is an SSc-specific autoantibody associated with a unique combination of clinical features, including limited skin sclerosis and lack of lung involvement. [ABSTRACT FROM AUTHOR]

Background: Personalized medicine remains an unmet need in ovarian cancer due to its heterogeneous nature and complex immune microenvironments, which has gained increasing attention in the era of immunotherapy. A key obstacle is the lack of reliable biomarkers to identify patients who would benefit significantly from the therapy. While conventional clinicopathological factors have exhibited limited efficacy as prognostic indicators in ovarian cancer, multi-omics profiling presents a promising avenue for comprehending the interplay between the tumor and immune components. Here we aimed to leverage the individual proteomic and transcriptomic profiles of ovarian cancer patients to develop an effective protein-based signature capable of prognostication and distinguishing responses to immunotherapy. Methods: The workflow was demonstrated based on the Reverse Phase Protein Array (RPPA) and RNA-sequencing profiles of ovarian cancer patients from The Cancer Genome Atlas (TCGA). The algorithm began by clustering patients using immune-related gene sets, which allowed us to identify immune-related proteins of interest. Next, a multi-stage process involving LASSO and Cox regression was employed to distill a prognostic signature encompassing five immune-related proteins. Based on the signature, we subsequently calculated the risk score for each patient and evaluated its prognostic performance by comparing this model with conventional clinicopathological characteristics. Results: We developed and validated a protein-based prognostic signature in a cohort of 377 ovarian cancer patients. The risk signature outperformed conventional clinicopathological factors, such as age, grade, stage, microsatellite instability (MSI), and homologous recombination deficiency (HRD) status, in terms of prognoses. Patients in the high-risk group had significantly unfavorable overall survival (p < 0.001). Moreover, our signature effectively stratified patients into subgroups with distinct immune landscapes. The high-risk group exhibited higher levels of CD8 T-cell infiltration and a potentially greater proportion of immunotherapy responders. The co-activation of the TGF-β pathway and cancer-associated fibroblasts could impair the ability of cytotoxic T cells to eliminate cancer cells, leading to poor outcomes in the high-risk group. Conclusions: The protein-based signature not only aids in evaluating the prognosis but also provides valuable insights into the tumor immune microenvironments in ovarian cancer. Together our findings highlight the importance of a thorough understanding of the immunosuppressive tumor microenvironment in ovarian cancer to guide the development of more effective immunotherapies. [ABSTRACT FROM AUTHOR]

Background: Osteoporosis (OP) is a systemic bone disease characterized by reduced bone mass and deterioration of bone microstructure, leading to increased bone fragility. Platelets can take up and release cytokines, and a high platelet count has been associated with low bone density. Obesity is strongly associated with OP, and adipose tissue can influence platelet function by secreting adipokines. However, the biological relationship between these factors remains unclear. Methods: We conducted differential analysis to identify OP platelet-related plasma proteins. And, making comprehensive analysis, including functional enrichment, protein-protein interaction network analysis, and Friends analysis. The key protein, Tetranectin (TNA/CLEC3B), was identified through screening. Then, we analyzed TNA's potential roles in osteogenic and adipogenic differentiation using multiple RNA-seq data sets and validated its effect on osteoclast differentiation and bone resorption function through in vitro experiments. Results: Six OP-platelet-related proteins were identified via differential analysis. Then, we screened the key protein TNA, which was found to be highly expressed in adipose tissue. RNA-seq data suggested that TNA may promote early osteoblast differentiation. In vitro experiments showed that knockdown of TNA expression significantly increased the expression of osteoclast markers, thereby promoting osteoclast differentiation and bone resorption. Conclusions: We identified TNA as a secreted protein that inhibits osteoclast differentiation and bone resorption. While, it potentially promoted early osteoblast differentiation from bioinformatic results. TNA may play a role in bone metabolism through the adipose-bone axis. [ABSTRACT FROM AUTHOR]

Proteins form the fastest-growing therapeutic class. Due to their intrinsic instability, loss of native structure is common. Structure alteration must be carefully evaluated as structural changes may jeopardize the efficiency and safety of the protein-based drugs. Hydrogen deuterium exchange (HDX) has long been used to evaluate protein structure and dynamics. The rate of exchange constitutes a sensitive marker of the conformational state of the protein and of its stability. It is often monitored by mass spectrometry. Fourier transform infrared (FTIR) spectroscopy is another method with very promising capabilities. Combining protein microarrays with FTIR imaging resulted in high throughput HDX FTIR measurements. BaF2 slides bearing the protein microarrays were covered by another slide separated by a spacer, allowing us to flush the cell continuously with a flow of N2 gas saturated with 2H2O. Exchange occurred simultaneously for all proteins and single images covering ca. 96 spots of proteins that could be recorded on-line at selected time points. Each protein spot contained ca. 5 ng protein, and the entire array covered 2.5 × 2.5 mm2. Furthermore, HDX could be monitored in real time, and the experiment was therefore not subject to back-exchange problems. Analysis of HDX curves by inverse Laplace transform and by fitting exponential curves indicated that quantitative comparison of the samples is feasible. The paper also demonstrates how the whole process of analysis can be automatized to yield fast analyses. [ABSTRACT FROM AUTHOR]

Background: Multiple sclerosis (MS), a chronic autoimmune disorder marked by demyelination in the central nervous system, is exceptionally uncommon in China, and remains poorly understood in terms of its peripheral blood manifestations. Methods: We conducted a cohort study comprising 39 MS patients and 40 normal controls (NC). High-dimensional mass cytometry, protein arrays, and targeted metabolomics were utilized to profile immune subsets, proteins, and metabolites in blood. Differences in multi-omics signatures were scrutinized across varying MS subtypes. Results: Immune profiling demonstrated an elevation in various B cell subsets and monocytes, alongside a reduction in dendritic cells among MS patients. Proteomic data revealed a downregulation in neurotrophic and tissue repair proteins. Metabolomic assessment showed a noted decrease in antiinflammatory molecules and sphingolipids. Integrated analysis identified distinct molecular patterns distinguishing MS from controls. Additionally, multi-omics differences among different MS subtypes were uncovered. Notably, hippuric acid levels was consistently lower in MS subgroups with greater disease severity. Conclusion: This study represents the pioneering exploration of multi-omics in Chinese MS patients, presenting a comprehensive view of the peripheral blood changes in MS. Our study underscores the robust capability of multi-omics assessments in identifying peripheral blood biomarkers that delineate the varied clinical presentation, and facilitates future development of biomarkers and targeted therapeutic interventions in MS. [ABSTRACT FROM AUTHOR]

Most secreted proteins are transported through the "conventional" endoplasmic reticulum-Golgi apparatus exocytic route for their delivery to the cell surface and release into the extracellular space. Nonetheless, formative discoveries have underscored the existence of alternative or "unconventional" secretory routes, which play a crucial role in exporting a diverse array of cytosolic proteins outside the cell in response to intrinsic demands, external cues, and environmental changes. In this context, lysosomes emerge as dynamic organelles positioned at the crossroads of multiple intracellular trafficking pathways, endowed with the capacity to fuse with the plasma membrane and recognized for their key role in both conventional and unconventional protein secretion. The recent recognition of lysosomal transport and exocytosis in the unconventional secretion of cargo proteins provides new and promising insights into our understanding of numerous physiological processes. [ABSTRACT FROM AUTHOR]

Blends of germinated maize and germinated lentil (100:0, 85:15, 70:30, 55:45, and 40:60) were utilised for preparation of chips by air frying. Higher contents of protein, ash, total phenols, flavonoids and antioxidant activity were confirmed in germinated flours through analysis of FTIR spectra. Doughs from blends containing higher amounts of germinated lentil flour showed improved viscoelastic behaviour indicating better protein network as compared to maize dough. Significant (P ≤ 0.05) variation was observed in colour and hardness of air fried chips prepared from various blends. The 70:30 blend was found most acceptable based on sensory evaluation. Chips from this formulation were found superior to ungerminated maize chips in terms of protein digestibility and mineral composition. [ABSTRACT FROM AUTHOR]

Detect the expression of Farnesoid X Receptor(FXR), Multiple Drug Resistance Associated Protein-1(MRP-1) and Solute Carrier Family 7, Member 5 (SLC7A5) in hepatocellular carcinoma(HCC) of rat model, so as to provide new therapeutic targets for gene therapy of HCC. Sixty male Wistar rats were randomly divided into three groups. The rats in experimental group were given 0.2% diethylnitrosamine (DEN) by gavage with a dose of 10 mg/kg, 3 times a week, and it stopped at 12 weeks. The control group rats were given physiological saline by gavage, while the sham operation group did not receive anything by gavage. At 10 weeks, one rat in the experimental group was euthanized, and the changes of livers were recorded. The procedure was repeated at 12 weeks. After 12 weeks, HCC only occurred in the experimental group. After confirming the formation of the tumor through pathological examination, liver tissues and tumor tissues were taken from the three groups. FXR, MRP-1 and SLC7A5 expression in liver tissues and tumor tissues was detected. After 7 weeks the rats in experimental group ate less, and their weight was significantly reduced. Three months later, HCC was detected in 15 rats in the experimental group. The ratio of FXR/GAPDH mRNA, MRP-1/GAPDH mRNA, SLC7A5/GAPDH mRNA were significantly different among the three groups. Under the light microscope the FXR protein, MRP-1 protein, and SLC7A5 protein react with their respective antibodies, and they showed granular expression. Every pathological section included different numbers of positive cells in each group. FXR expression in HCC of rats was significantly lower than that in normal liver tissues, but MRP-1 and SLC7A5 expression in HCC were significantly higher than that in normal liver tissues, suggesting that drugs targeting FXR, MRP-1 and SLC7A5 may be new strategies for the treatment of HCC. [ABSTRACT FROM AUTHOR]

Background: During pseudoglandular stage of the human lung development the primitive bronchial buds are initially conformed by simple tubules lined by endoderm-derived epithelium surrounded by mesenchyme, which will progressively branch into airways and start to form distal epithelial saculles. For first time alveolar type II (AT2) pneumocytes appears. This study aims to characterize the genes and microRNAs involved in this differentiation process and decipher its role in the starting alveolar differentiation. Methods: Gene and microRNA profiling was performed in human embryonic lungs from 7 to 12 post conception weeks (pcw). Protein expression location of candidate genes were analyzed by immunofluorescense in embryonic lung tissue sections. mRNA/miRNA target pairs were identified using computational approaches and their expression was studied in purified epithelial/mesenchymal cell populations and in isolated tips and stalks from the bronchial tree. Additionally, silencing experiments in human embryonic lung mesenchymal cells and in human embryonic tip-derived lung organoids were performed, as well as organoid differentiation studies. AT2 cell markers were studied by qRT-PCR and by immunofluorescence. The TGFB-β phosphorylated pathways was analyzed with membrane protein arrays. Lung explants were cultured in air/liquid interface with/without peptides. Results: We identified 88 differentially expressed genes, including IGFBP3. Although IGFBP3 mRNA was detected in both epithelial and mesenchymal populations, the protein was restricted to the epithelium, indicating post-transcriptional regulation preventing IGFBP3 protein expression in the mesenchyme. MicroRNA profiling identified miR-34a as an IGFBP3 regulator. miR-34a was up-regulated in mesenchymal cells, and its silencing in human embryonic lung mesenchymal cells increased IGFBP3 levels. Additionally, IGFBP3 expression showed a marked downregulation from 7 to 12 pcw, suggesting its involvement in the differentiation process. The differentiation of human tip-derived lung embryonic organoids showed a drastic reduction in IGFBP3, supported by the scRNAseq data. IGFBP3 silencing in organoids activated an alveolar-like differentiation process characterized by stem cell markers downregulation and upregulation of AT2 markers. This process was mediated by TGFβ signalling inhibition and BMP pathway activation. Conclusions: The IGFBP3/miR-34a axis restricts IGFBP3 expression in the embryonic undifferentiated lung epithelium, and the progressive downregulation of IGFBP3 during the pseudoglandular stage is required for alveolar differentiation. [ABSTRACT FROM AUTHOR]

Background Objective Methods Results Conclusions There are more than 30 immunoglobulin G (IgG) autoantibody biomarkers of autoimmune cerebellar ataxia reported.To describe a novel IgG biomarker targeting neurobeachin, a scaffolding protein integral to synapse formation and function.Clinical and laboratory‐based assessment was used for a novel diagnostic biomarker of autoimmune ataxia.A 46‐year‐old woman had ataxia onset followed by bilateral knee pain, attributable to rheumatoid arthritis. She was negative for standard neural antibodies. Her ataxia stabilized and tremor initially improved after corticosteroid treatment, but she progressed to wheelchair‐dependence (on methotrexate and then rituximab) over 4 years, in keeping with the trajectory encountered by many with autoimmune ataxia. In contrast, her inflammatory arthritis remitted with immunotherapy. Neurobeachin‐IgG was unmasked in serum and cerebrospinal fluid using protein arrays and confirmed using neurobeachin‐specific confocal immunofluorescence and Western blot.Neurobeachin‐IgG is a new neural antibody biomarker of cerebellar ataxia among a growing antibody repertoire, collectively common but often individually rare. © 2024 International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Invasive lobular carcinoma exhibits unique morphological features frequently associated with alterations in CDH1. Although some studies have identified abnormalities in adhesion factors other than E-cadherin, the molecular mechanisms underlying E-cadherin abnormalities in CDH1-unaltered invasive lobular carcinoma remain poorly understood. In this study, we investigated the molecular underpinnings of E-cadherin dysregulation in invasive lobular carcinoma in the absence of CDH1 gene alterations, using comprehensive bioinformatic analyses. We conducted a comparative study of CDH1-mutated and non-mutated invasive lobular carcinoma and evaluated the differences in mRNA levels, reverse-phase protein array, methylation, and miRNAs. We observed that invasive lobular carcinoma cases without CDH1 alterations exhibited a significantly higher incidence of the Claudin-low subtype (p < 0.01). The results of the reverse-phase protein array indicate no significant difference in E-cadherin expression between CDH1-mutated and non-mutated cases. Therefore, abnormalities in E-cadherin production also exist in CDH1 non-mutated invasive lobular carcinoma. Considering that there are no differences in mRNA levels and methylation status, post-translational modifications are the most plausible explanation for the same. Hence, future studies should focus on elucidating the mechanism underlying E-cadherin inactivation via post-translational modifications in CDH1 non-mutated invasive lobular carcinoma. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease caused by autoantibodies. Serum samples from patients with SLE (n = 10) were compared with those from normal controls (NCs, n = 5) using 21K protein chip analysis to identify a biomarker for SLE, revealing 63 SLE-specific autoantibodies. The anti-chaperonin-containing t-complex polypeptide-1 (TCP1) antibody exhibited higher expression in patients with SLE than in NCs. To validate the specificity of the anti-TCP1 antibody in SLE, dot blot analysis was conducted using sera from patients with SLE (n = 100), rheumatoid arthritis (RA; n = 25), Behçet's disease (BD; n = 28), and systemic sclerosis (SSc; n = 30) and NCs (n = 50). The results confirmed the detection of anti-TCP1 antibodies in 79 of 100 patients with SLE, with substantially elevated expression compared to both NCs and patients with other autoimmune diseases. We performed an enzyme-linked immunosorbent assay to determine the relative amounts of anti-TCP1 antibodies; markedly elevated anti-TCP1 antibody levels were detected in the sera of patients with SLE (50.1 ± 17.3 arbitrary unit (AU), n = 251) compared to those in NCs (33.9 ± 9.3 AU), RA (35 ± 8.7 AU), BD (37.5 ± 11.6 AU), and SSc (43 ± 11.9 AU). These data suggest that the anti-TCP1 antibody is a potential diagnostic biomarker for SLE. [ABSTRACT FROM AUTHOR]

Background: To analyze the concentration of aqueous humor (AH) cytokines in eyes with idiopathic epiretinal membrane (iERM) and to investigate their potential correlation with disease severity. Methods: Retrospective cross-sectional case-control institutional study. A total of 16 eyes of 16 iERM patients and 14 eyes of 14 age-matched healthy controls were enrolled. AH samples were analyzed for various biomarkers using a glass-chip protein array. Cytokines associated with inflammation, fibrosis, angiogenesis, and glial signal transduction were quantified. Results: Significant differences in cytokine concentration were observed between the iERM group and controls, with 19 cytokines elevated in the iERM group (among them IL-6, IL-8, PDGF-AB, PDGF-BB, TGFB-1, TGFB-2, TGFB-3, VEGF A, VEGF C, VEGF D, p < 0,05, 95% confidence interval). Correlation analysis revealed associations between cytokine levels and iERM severity. Notably, stages 2, 3, and 4 of iERM demonstrated increased levels of various biomarkers. Conclusions: This study provides insights into the complex molecular interactions underlying iERM pathogenesis, describing a correlation between neuroinflammation and iERM severity. [ABSTRACT FROM AUTHOR]

Detection of important genes affecting lung adenocarcinoma (LUAD) is critical to finding effective therapeutic targets for this highly lethal cancer. However, many existing approaches have focused on single outcomes or phenotypic associations, which may not be as thorough as investigating molecular transcript levels within cells. In this article, we apply a novel multivariate rank-distance correlation-based gene selection procedure (MrDcGene) to LUAD multi-omics data downloaded from The Cancer Genome Atlas (TCGA). MrDcGene provides additional opportunities for detecting novel susceptibility genes as it leverages information from multiple platforms, while efficiently handling challenges such as high dimensionality, low signal-to-noise ratio, unknown distributions, and non-linear structures, etc. Notably, the MrDcGene method is able to detect two different scenarios, i.e., strong association strength with a few gene expressions and weak association strength with several gene expressions. After thoroughly exploring the association between gene expression (GE) and multiple other platforms, including reverse phase protein array (RPPA), miRNA, copy number variation (CNV) and DNA methylation (ME), we detect several novel genes that may play an important role in LUAD (ZNF133, CCDC159, YWHAZ, HNRNPR. ITPR2, PTHLH, and WIPI2). In addition, we quantitatively validate several other susceptibility genes that were reported in the literature using different methods and studies. The accuracy of the MrDcGene approach is theoretically assured and empirically demonstrated by the simulation studies. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic ductal adenocarcinoma is a very lethal tumor entity with the late appearance of clinical signs and the absence of biomarkers for early diagnosis. Diabetes mellitus can increase the risk of pancreatic cancer, as well as the effect of treatment modalities, so patients with PDAC and DM represent a special study group. We validated miRNA1285-3p, TGM2, CDH-1, CD166, and S100A13 as potential meaningful biomarkers to characterize patients with PDAC + DM. The present study represents a pilot approach to address this question. The potential biomarkers presented in the current study could contribute to the search for biomarkers for the diagnosis of PDAC, enabling early detection of PDAC in DM patients in the future. Early detection of PDAC remains challenging due to the lack of early symptoms and the absence of reliable biomarkers. The aim of the present project was to identify miRNA and proteomics signatures discriminating PDAC patients with DM from nondiabetic PDAC patients. Proteomics analysis and miRNA array were used for protein and miRNA screening. We used Western blotting and Real-Time Quantitative Reverse Transcription polymerase chain reaction (qRT-PCR) for protein and miRNA validation. Comparisons between experimental groups with normal distributions were performed using one-way ANOVA followed by Tukey's post hoc test, and pairwise tests were performed using t-tests. p ≤ 0.05 was considered statistically significant. Protein clusters of differentiation 166 (CD166), glycoprotein CD63 (CD63), S100 calcium-binding protein A13 (S100A13), and tumor necrosis factor-β (TNF-β) were detected in the proteomics screening. The miRNA assay revealed a differential miRNA 1285 regulation. Previously described target proteins of miR-1285 cadherin-1 (CDH-1), cellular Jun (c-Jun), p53, mothers against decapentaplegic homolog 4 (Smad4), human transglutaminase 2 (TGM2) and yes-associated protein (YAP), were validated via Western blotting. miR-1285-3p was successfully validated as differentially regulated in PDAC + DM via qRT-PCR. Overall, our data suggest miRNA1285-3p, TGM2, CDH-1, CD166, and S100A13 as potential meaningful biomarker candidates to characterize patients with PDAC + DM. Data are available via ProteomeXchange with the identifier PXD053169. [ABSTRACT FROM AUTHOR]

Primary focal segmental glomerulosclerosis (FSGS) is a disease of the podocytes and glomerulus, leading to nephrotic syndrome and progressive loss of renal function. One of the most serious aspects is its recurrence of disease in over 30% of patients following allogeneic kidney transplantation, leading to early graft loss. This research investigates the individual genetic predispositions and differences in the immune responses leading to recurrence of FSGS after transplantation. We performed exome sequencing on six patients with recurrent FSGS to identify variants in fifty-one genes and found significant variations in the alpha-2-macroglobulin (A2M). Immunoblotting was used to investigate effects of specific gene variants at the protein level. Further expression analysis identified A2M, exophilin 5 (EXPH5) and plectin (PLEC) as specific proteins linked to podocytes, endothelial cells, and the glomerulus. Subsequent protein array screening revealed the presence of non-HLA-specific antibodies, including TRIM21, after transplantation. Using Metascape for pathway and process enrichment analysis, we focused on the IL-17 signaling and chemotaxis pathways. ELISA measurements showed significantly elevated IL-17 levels in patients with recurrent FSGS (32.30 ± 9.12 pg/mL) compared to individuals with other glomerular diseases (23.16 ± 2.49 pg/mL; p < 0.01) and healthy subjects (22.28 ± 0.94 pg/mL; p < 0.01), with no significant difference in plasma CCL2/MCP-1 levels between groups. This study explores the molecular dynamics underlying recurrence of FSGS after transplantation, offering insights into potential biomarkers and therapeutic targets for the future development of individualized treatments for transplant patients. [ABSTRACT FROM AUTHOR]

Context: The removal or supplementation of ejaculates with seminal plasma (SP) can affect cryotolerance and post-thaw survival of spermatozoa in many species. In the Asian elephant (Elephas maximus), elucidation of the SP proteome and investigation of how it affects spermatozoa may enable improvement of cryopreservation protocols. Aims: Herein, we characterise the Asian elephant SP proteome and investigate the impacts of SP on sperm cryotolerance in the presence of conspecific or heterospecific SP. Methods: Proteomic analysis of Asian elephant SP was performed using mass spectrometry on nine samples from three individuals. In a separate study, SP was removed from six ejaculates and spermatozoa were resuspended in Tris extender supplemented with: no seminal plasma (NOSP), conspecific SP from ejaculates exhibiting 'good' (GSP, >60%) or mixed sperm total motility (MSP), or horse SP (HSP). Samples underwent cryopreservation, and sperm parameters were compared prior to cryopreservation and after thawing (0 and 2 h). Key results: Mass spectrometry identified 155 proteins from an array of families. Significant differences were observed in post-thaw sperm quality between SP treatments: high concentrations of MSP (25%, v/v) displayed greater average path and straight-line velocity immediately after thawing (P < 0.05) and greater sperm motility index and beat cross frequency than NOSP after 2 h post-thaw incubation (P < 0.05). The addition of HSP improved sperm kinematic parameters compared to NOSP and GSP treatments (P < 0.05). Conclusions and implications: These preliminary findings suggest the potential of SP to enhance the cryosurvival of Asian elephant spermatozoa, with HSP showing particularly promising results compared to conspecific SP (GSP). Further research into the specific effects of Asian elephant SP proteins is warranted. There is an urgent need to develop a means of freezing Asian elephant semen, to enable captive breeding conservation programs to maintain genetic diversity. This study revealed important information about the proteins present in the seminal plasma of these animals and how this may contribute to the successful frozen storage of their spermatozoa. These findings will contribute to our understanding of differences in sperm quality between elephants and aid in developing sperm freezing protocols that will lead to the long-awaited birth of an Asian elephant calf from frozen spermatozoa. Photograph by Cameron Negus. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

Background: GP‐2250, a novel analog of taurultam (TRLT), has emerged as a potent anti‐neoplastic drug; however, the mechanisms underlying its effects are not well understood. Here, we investigated the mechanism of action and the biological effects of GP‐2250 using in vitro and in vivo models. Methods: We carried out a series of in vitro (MTT assay, Annexin V/PI assay, colony formation assay, reverse‐phase protein array [RPPA], and HRLC/IC analysis) to determine the biological activity of GP‐2250 and investigate the mechanism of action. In vivo experiments were carried out to determine the therapeutic efficacy of GP‐2250 alone and in combination with standard‐of‐care drugs (e.g., paclitaxel, cisplatin, topotecan, and poly ADP‐ribose polymerase [PARP] inhibitors). Results: We investigated the cytotoxic effect of GP‐2250 in 10 ovarian cancer cell lines and found GP‐2250 combined with a PARP inhibitor had the greatest synergy. RPPA revealed that GP‐2250 inhibited hypoxia‐inducible factor‐1α, AKT, and mammalian target of rapamycin (mTOR) activation and expression. High‐resolution mass spectrometry revealed that hexokinase2 activity and protein expression were significantly reduced by GP‐2250 exposure. Furthermore, GP‐2250 reduced glycolysis and ATP synthesis in cancer cells. An in vivo pharmacodynamic experiment using the OVCAR8 mouse model demonstrated that 500 mg/kg GP‐2250 was effective in downregulating AKT and mTOR activation and expression. In the in vivo therapy experiment using an orthotopic mouse model, a combination of GP‐2250 with either PARP inhibitors or bevacizumab showed a significant reduction of tumor weights and nodules compared to those treated with a vehicle, control IgG groups, or monotherapy groups. Conclusions: Taken together, our data indicate that GP‐2250 exerts profound effects on tumor metabolism and, in combination with PARP inhibitors or bevacizumab, showed promising anti‐tumor efficacy. These findings could have implications for the clinical development of GP‐2250. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to explore the potential to profile and distinguish varying clinical severity grades of MIH, compared to normal enamel, using proteomics. Methods: Liquid chromatography–mass spectrometry analyses were conducted on enamel samples of extracted teeth, from 11 children and adolescents, spanning an age range of 6–18 years. Enamel powder samples were collected from extracted, third molars (n = 3) and first permanent molars diagnosed with MIH (n = 8). The MIH tooth samples were categorized into subgroups based on clinical severity grade. The data were statistically analyzed using ANOVA and Welch's t test. Results: Teeth affected by MIH exhibited a diverse array of proteins, each with different functions related to dental enamel, distinguishing them from their normal enamel counterparts. The application of microdissection combined with LC–MS techniques has revealed the potential to discern unique proteomic profiles among MIH-affected teeth, characterized by varying clinical severity grades. Both analyzed MIH groups displayed consistent trends in the presentation of biological processes, including underabundance of proteins primarily associated with cell organization and biogenesis. Furthermore, proteins linked to cell death were overabundant in both MIH groups. Conclusion: Proteomics enabled the detection and differentiation of various proteins across different clinical severity grades of MIH. [ABSTRACT FROM AUTHOR]

Precise diagnostic biomarkers of anticitrullination protein antibody (ACPA)‐negative and early‐stage RA are still to be improved. We aimed to screen autoantibodies in ACPA‐negative patients and evaluated their diagnostic performance. The human genome‐wide protein arrays (HuProt arrays) were used to define specific autoantibodies from the sera of 182 RA patients and 261 disease and healthy controls. Statistical analysis was performed with SPSS 17.0. In Phase I study, 51 out of 19,275 recombinant proteins covering the whole human genome were selected. In Phase II validation study, anti‐ANAPC15 (anaphase promoting complex subunit 15) exhibited 41.8% sensitivity and 91.5% specificity among total RA patients. There were five autoantibodies increased in ACPA‐negative RA, including anti‐ANAPC15, anti‐LSP1, anti‐APBB1, anti‐parathymosin, and anti‐UBL7. Anti‐parathymosin showed the highest prevalence of 46.2% (p = 0.016) in ACPA‐negative early stage (<2 years) RA. To further improve the diagnostic efficacy, a prediction model was constructed with 44 autoantibodies. With increased threshold for RA calling, the specificity of the model is 90.8%, while the sensitivity is 66.1% (87.8% in ACPA‐positive RA and 23.8% in ACPA‐negative RA) in independent testing patients. Therefore, HuProt arrays identified RA‐associated autoantibodies that might become possible diagnostic markers, especially in early stage ACPA‐negative RA. [ABSTRACT FROM AUTHOR]

The word "secretome" was first used to describe the proteins that cells secrete under different circumstances; however, recent studies have proven the existence of other molecules such as RNA and chemical compounds in the secretome. The study of secretome has significance for the diagnosis and treatment of disease as it provides insight into cellular functions, including immune responses, development, and homeostasis. By halting cell division, cellular senescence plays a role in both cancer defense and aging by secreting substances known as senescence-associated secretory phenotypes (SASP). A variety of techniques could be used to analyze the secretome: protein-based approaches like mass spectrometry and protein microarrays, nucleic acid-based methods like RNA sequencing, microarrays, and in silico prediction. Each method offers unique advantages and limitations in characterizing secreted molecules. Top-down and bottom-up strategies for thorough secretome analysis are became possible by mass spectrometry. Understanding cellular function, disease causes, and proper treatment targets is aided by these methodologies. Their approaches, benefits, and drawbacks will all be discussed in this review. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is often diagnosed at an advanced stage when treatment options are limited. Being one of the deadliest cancers that mandates longer medication and treatment phases, there is an inevitable need to have the knowledge of drug response of anti-pancreatic cancer drugs before it is recommended for a patient. AI-driven drug response prediction has proven potential to personalize treatment strategies, improve therapeutic outcomes, and reduce adverse effects and treatment costs for cancer patients. In this research work, we have accounted for the use of different drug descriptors and their core structures known as scaffolds along with three cell line features, chromatin profiling, reverse phase protein array, and metabolomics data to build a feature engineered dataset for drug response prediction tested on various computational learning models. The 53 unique drugs against 18 unique pancreatic cancer cell lines were taken as the raw dataset. The initial dataset having a large dimension was feature selected using an ensemble method derived from five different techniques. The dataset was evaluated on various computational methods and an accuracy of 89% was achieved using the TabNet architecture. Furthermore, the common scaffolds that were persistently found among the drugs that possess high IC50-valued drug clusters were also recorded. [ABSTRACT FROM AUTHOR]

Context: tabersonine has been investigated for its role in modulating inflammation-associated pathways in various diseases. however, its regulatory effects on triple-negative breast cancer (TNBC) have not yet been fully elucidated. Objective: this study uncovers the anticancer properties of tabersonine in TNBC cells, elucidating its role in enhancing chemosensitivity to cisplatin (CDDP). Materials and methods: after tabersonine (10 μM) and/or CDDP (10 μM) treatment for 48 h in BT549 and MDA-MB-231 cells, cell proliferation was evaluated using the cell counting kit-8 and colony formation assays. Quantitative proteomics, online prediction tools and molecular docking analyses were used to identify potential downstream targets of tabersonine. transwell and wound-healing assays and Western blot analysis were used to assess epithelial--mesenchymal transition (EMT) phenotypes. Results: tabersonine demonstrated inhibitory effects on tNBc cells, with IC50 values at 48 h being 18.1 μM for BT549 and 27.0 μM for MDA-MB-231. the combined treatment of CDDP and tabersonine synergistically suppressed cell proliferation in BT549 and MDA-MB-231 cells. enrichment analysis revealed that the proteins differentially regulated by tabersonine were involved in eMt-related signalling pathways. this combination treatment also effectively restricted EMT-related phenotypes. through the integration of online target prediction and proteomic analysis, aurora kinase a (AURKA) was identified as a potential downstream target of tabersonine. aURKa expression was reduced in tNBc cells post-treatment with tabersonine. Discussion and conclusions: tabersonine significantly enhances the chemosensitivity of CDDP in tNBc cells, underscoring its potential as a promising therapeutic agent for TNBC treatment. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is caused by an expansion of CAG trinucleotide repeat in the HTT gene; the exact pathogenesis of HD currently remains unclear. One of the promising directions in the study of HDs is to determine the molecular mechanism underlying the development and role of microRNAs (miRNAs). This study aimed to identify the profile of miRNAs in an HD human cell line model as diagnostic biomarkers for HD. To study HD, the human SH-SY5Y HD cell model is based on the expression of two different forms: pEGFP-Q23 and pEGFP-Q74 of HTT. The expression of Htt protein was confirmed using aggregation assays combined with immunofluorescence and Western blotting methods. miRNA levels were measured in SH-SY5Y neuronal cell model samples stably expressing Q23 and Q74 using the extraction-free HTG EdgeSeq protocol. A total of 2083 miRNAs were detected, and 354 (top 18 miRNAs) miRNAs were significantly differentially expressed (DE) (p < 0.05) in Q23 and Q74 cell lines. A majority of the miRNAs were downregulated in the HD cell model. Moreover, we revealed that six DE miRNAs target seven genes (ATN1, GEMIN4, EFNA5, CSMD2, CREBBP, ATXN1, and B3GNT) that play important roles in neurodegenerative disorders and showed significant expression differences in mutant Htt (Q74) when compared to wild-type Htt (Q23) using RT-qPCR (p < 0.05 and 0.01). We demonstrated the most important DE miRNA-mRNA profiles, interaction binding sites, and their related pathways in HD using experimental and bioinformatics methods. This will allow the development of novel diagnostic strategies and provide alternative therapeutic routes for treating HD. [ABSTRACT FROM AUTHOR]

The prefusion conformation of human metapneumovirus fusion protein (hMPV Pre-F) is critical for eliciting the most potent neutralizing antibodies and is the preferred immunogen for an efficacious vaccine against hMPV respiratory infections. Here we show that an additional cleavage event in the F protein allows closure and correct folding of the trimer. We therefore engineered the F protein to undergo double cleavage, which enabled screening for Pre-F stabilizing substitutions at the natively folded protomer interfaces. To identify these substitutions, we developed an AI convolutional classifier that successfully predicts complex polar interactions often overlooked by physics-based methods and visual inspection. The combination of additional processing, stabilization of interface regions and stabilization of the membrane-proximal stem, resulted in a Pre-F protein vaccine candidate without the need for a heterologous trimerization domain that exhibited high expression yields and thermostability. Cryo-EM analysis shows the complete ectodomain structure, including the stem, and a specific interaction of the newly identified cleaved C-terminus with the adjacent protomer. Importantly, the protein induces high and cross-neutralizing antibody responses resulting in near complete protection against hMPV challenge in cotton rats, making the highly stable, double-cleaved hMPV Pre-F trimer an attractive vaccine candidate. The authors report an efficacious vaccine for human metapneumovirus based on a prefusion-stabilized fusion protein trimer that does not require a trimerization domain, designed using an AI convolutional classifier trained to predict optimized complex polar interactions. [ABSTRACT FROM AUTHOR]

The chemical modification of proteins serves as a fundamental tool for understanding biological processes and enables the design of biofunctional materials. Among the available methodologies, photochemical strategies have garnered significant attention because of their remarkable biocompatibility and precise spatiotemporal reaction control. Developing novel reactions tailored to specific applications necessitates a comprehensive understanding of photoreactive properties, including catalyst structures, appropriate modifiers, and reaction conditions. This review discusses chemical modifications of proteins using an array of catalysts, including photoredox catalysts for single‐electron transfer (SET), catalysts for energy transfer, long‐wavelength excitable photocatalysts, genetically encoded photocatalysts, and artificial metalloenzymes. The discussion covers the unique attributes, mechanisms, practical applications, and future prospects of each catalyst‐driven reaction, shedding light on the evolving landscape of protein chemical modifications. [ABSTRACT FROM AUTHOR]

Background: In the fight against GBM, drug repurposing emerges as a viable and time-saving approach to explore new treatment options. Chlorpromazine, an old antipsychotic medication, has recently arisen as a promising candidate for repositioning in GBM therapy in addition to temozolomide, the first-line standard of care. We previously demonstrated the antitumor efficacy of chlorpromazine and its synergistic effects with temozolomide in suppressing GBM cell malignant features in vitro. This prompted us to accomplish a Phase II clinical trial to evaluate the efficacy and safety of adding chlorpromazine to temozolomide in GBM patients with unmethylated MGMT gene promoter. In this in vitro study, we investigate the potential role of chlorpromazine in overcoming temozolomide resistance. Methods: In our experimental set, we analyzed Connexin-43 expression at both the transcriptional and protein levels in control- and chlorpromazine-treated GBM cells. DNA damage and subsequent repair were assessed by immunofluorescence of γ-H2AX and Reverse-Phase Protein microArrays in chlorpromazine treated GBM cell lines. To elucidate the relationship between DNA repair systems and chemoresistance, we analyzed a signature of DNA repair genes in GBM cells after treatment with chlorpromazine, temozolomide and Connexin-43 downregulation. Results: Chlorpromazine treatment significantly downregulated connexin-43 expression in GBM cells, consequently compromising connexin-dependent cellular resilience, and ultimately contributing to cell death. In line with this, we observed concordant post-translational modifications of molecular determinants involved in DNA damage and repair pathways. Our evaluation of DNA repair genes revealed that temozolomide elicited an increase, while chlorpromazine, as well as connexin-43 silencing, a decrease in DNA repair gene expression in GBM cells. Conclusions: Chlorpromazine potentiates the cytotoxic effects of the alkylating agent temozolomide through a mechanism involving downregulation of Cx43 expression and disruption of the cell cycle arrest essential for DNA repair processes. This finding suggests that chlorpromazine may be a potential therapeutic strategy to overcome TMZ resistance in GBM cells by inhibiting their DNA repair mechanisms. [ABSTRACT FROM AUTHOR]

Background: Expanding the number of biomarkers is imperative for studying the etiology and improving venous thromboembolism prediction. In this study, we aimed to identify promising biomarkers or targeted therapies to improve the detection accuracy of early-stage deep vein thrombosis (DVT) or reduce complications. Methods: Quantibody Human Cytokine Antibody Array 440 (QAH-CAA-440) was used to screen novel serum-based biomarkers for DVT/non-lower extremity DVT (NDVT). Differentially expressed proteins in DVT were analyzed using bioinformatics methods and validated using a customized array. Diagnostic accuracy was calculated using receiver operating characteristics, and machine learning was applied to establish a biomarker model for evaluating the identified targets. Twelve targets were selected for validation. Results: Cytokine profiling was conducted using a QAH-CAA-440 (RayBiotech, USA) quantimeter array. Cross-tabulation analysis with Venn diagrams identified common differential factors, leading to the selection of 12 cytokines for validation based on their clinical significance. These 12 biomarkers were consistent with the results of previous array analysis: FGF-6 (AUC = 0.956), Galectin-3 (AUC = 0.942), EDA-A2 (AUC = 0.933), CHI3L1 (AUC = 0.911), IL-1 F9 (AUC = 0.898), Dkk-4 (AUC = 0.88), IG-H3 (AUC = 0.876), IGFBP (AUC = 0.858), Gas-1 (AUC = 0.858), Layilin (AUC = 0.849), ULBP-2 (AUC = 0.813)and FGF-9 (AUC = 0.773). These cytokines are expected to serve as biomarkers, targets, or therapeutic targets to differentiate DVT from NDVT. Conclusions: EDA-A2, FGF-6, Dkk-4, IL-1 F9, Galentin-3, Layilin, Big-h3, CHI3L1, ULBP-2, Gas-1, IGFBP-5, and FGF-9 are promising targets for DVT diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Background: Recent studies have proved the role of autophagy in mesenchymal stem cell (MSCs) function and regenerative properties. How and by which mechanism autophagy modulation can affect the juxtacrine interaction of MSCs should be addressed. Here, the role of autophagy was investigated in the formation of tunneling nanotubes (TNTs) and homotypic mitochondrial donation. Methods: MSCs were incubated with 15 µM Metformin (Met) and/or 3 µM 3-methyladenine (3-MA) for 48 h. The formation of TNTs was assessed using bright-field and SEM images. The mitochondria density and ΔΨ values were monitored using flow cytometry analysis. Using RT-PCR and protein array, the close interaction and shared mediators between autophagy, apoptosis, and Wnt signaling pathways were also monitored. The total fatty acid profile was assessed using gas chromatography. Result: Data indicated the increase of TNT length and number, along with other cell projections after the induction of autophagy while these features were blunted in 3-MA-treated MSCs (p < 0.05). Western blotting revealed the significant reduction of Rab8 and p-FAK in 3-MA-treated MSCs (p < 0.05), indicating the inhibition of TNT assembly and vesicle transport. Likewise, the stimulation of autophagy increased autophagic flux and mitochondrial membrane integrity compared to 3-MA-treated MSCs. Despite these findings, protein levels of mitochondrial membrane Miro1 and 2 were unchanged after autophagy inhibition/stimulation (p > 0.05). We found that the inhibition/stimulation of autophagy can affect the protein, and transcription levels of several mediators related to Wnt and apoptosis signaling pathways involved in different cell bioactivities. Data confirmed the profound increase of mono and polyunsaturated/saturated fatty acid ratio in MSCs exposed to autophagy stimulator. Conclusions: In summary, autophagy modulation could affect TNT formation which is required for homotypic mitochondrial donation. Thus, the modulation of autophagy creates a promising perspective to increase the efficiency of cell-based therapies. [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer (CRC) cells are characterized by high levels of SMAD7, a protein involved in the positive control of growth and survival of cancer cells. This study aims to examine whether SMAD7 is a regulator of CRC cell migration and evaluate the underlying mechanisms. By using specific antisense oligonucleotides, we here show that knockdown of SMAD7 reduces the formation of F-ACTIN filaments and impairs CRC cell migration. Analysis of molecules involved in the control of F-ACTIN formation shows that SMAD7-deficient cells have reduced content of XIAP and this is probably related to the ability of SMAD7 to control the expression of STAT3, a transcription factor that positively regulates XIAP. Finally, we document that, in human CRC samples, there is a positive correlation between SMAD7 expression and XIAP content. Overall, these findings support the role of SMAD7 in the control of F-ACTIN filament formation and migration of CRC cells. The reorganization of the cell cytoskeleton and changes in the content of cell adhesion molecules are crucial during the metastatic spread of tumor cells. Colorectal cancer (CRC) cells express high SMAD7, a protein involved in the control of CRC cell growth. In the present study, we evaluated whether SMAD7 regulates the cytoskeleton reorganization and dynamics in CRC. Knockdown of SMAD7 with a specific antisense oligonucleotide (AS) in HCT116 and DLD1, two human CRC cell lines, reduced the migration rate and the content of F-ACTIN filaments. A gene array, real-time PCR, and Western blotting of SMAD7 AS-treated cells showed a marked down-regulation of the X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis family, which has been implicated in cancer cell migration. IL-6 and IL-22, two cytokines that activate STAT3, enhanced XIAP in cancer cells, and such induction was attenuated in SMAD7-deficient cells. Finally, in human CRC, SMAD7 mRNA correlated with XIAP expression. Our data show that SMAD7 positively regulates XIAP expression and migration of CRC cells, and suggest a mechanism by which SMAD7 controls the architecture components of the CRC cell cytoskeleton. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR) has been widely characterized as a defense system against phages and other invading elements in bacteria and archaea. A low percentage of Ralstonia lanacearum species complex (RSSC) strains possess the CRISPR array and the CRISPR-associated proteins (Cas) that would confer immunity against various phages. To provide a wide-range screen of the CRISPR presence in the RSSC, weanalyzed 378 genomes of RSSC strains to find the CRISPR locus. We found that 20.1,14.3, and 54.5% of the R. solanacearum, pseudosolanacearum, and R. syzygii strains, respectively, possess the CRISPR locus. In addition, we performed further analysis to identify the respective phages that are restricted by the CRISPR arrays. We found 252 different phages infecting different strains of the RSSC, by means of the identification of similarities between the protospacers in phages and spacers in bacteria. We compiled this information in a database with web access called CRISPRals (https: #crisprals, yachaytech, edu. ec/). Additionally, we made available a number of tools to detect and identify CRI SPR array and Cas genes in genomic sequences that could be uploaded by users. Finally, a matching tool to relate bacteria spacer with phage protospacer sequences is available. CRISPRals is a valuable resource for the scientific community that contributes to the study of bacteria-phage interaction and a starti ng point that will help to design efficient phage therapy strategies. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single‐EV resolution. To address these needs, we developed a HaloTag‐based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag‐mediated quantification is generally robust across EV analysis methods. We compared HaloTag‐labelling to antibody‐labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods. [ABSTRACT FROM AUTHOR]

Heart failure (HF) is a chronic condition that progressively worsens and continues to be a major financial burden and public health concern. The "gut-heart" axis provides an innovative perspective and therapeutic strategy for preventing and treating heart failure. Shenfu injection (SFI) is a Traditional Chinese Medicine-based treatment demonstrating potential as a therapeutic strategy for heart failure. However, the precise therapeutic mechanisms of SFI in heart failure are not completely characterized. In this study, HF models were established utilizing subcutaneous multipoint injection of isoproterenol (ISO) at a dosage of 5mg kg-1⋅d-1 for 7 days. Serum levels of inflammatory biomarkers were quantified using protein microarrays. Rat feces were analyzed using untargeted metabolomics research and 16S rRNA sequencing. The link between gut microbiota and metabolites was examined using a MetOrigin and Spearman correlation analysis. Our results show that Shenfu injection effectively enhances cardiac function in rats with ISO-induced heart failure by potentially modulating pro-/anti-inflammatory imbalance and reducing serum and urine Trimethylamine-N-oxide (TMAO) levels. Moreover, SFI significantly increases the abundance of Bacteroidota at the phylum level, thereby improving disrupted gut microbiota composition. Additionally, SFI supplementation enriches specific genera known for their capacity to produce short-chain fatty acids. SFI was found to be associated with three key metabolic pathways, as revealed by fecal metabonomics analysis, including the pentose phosphate pathway, pyrimidine metabolism, and purine metabolism. Metabolite tracing analysis revealed that Taurine and hypotaurine metabolism was found to be specific to the microbial community. The biosynthesis of Pyrimidine metabolism, Purine metabolism, beta-alanine metabolism, Naphthalene degradation, Pantothenate, and CoA biosynthesis were identified as co-metabolic pathways between microbes and host. The Spearman correlation analysis was also significantly correlated to differentially expressed metabolites regulated by SFI and the gut microbiota. These results suggest that SFI improves ISO-induced heart failure by modulating cometabolism and regulating the TMAO-inflammation axis. [ABSTRACT FROM AUTHOR]

The prevalence of allergic diseases has increased tremendously in recent decades, which can be attributed to growing exposure to environmental triggers, changes in dietary habits, comorbidity, and the increased use of medications. In this context, the multiplexed diagnosis of sensitization to various allergens and the monitoring of the effectiveness of treatments for allergic diseases become particularly urgent issues. The detection of allergen-specific antibodies, in particular, sIgE and sIgG, is a modern alternative to skin tests due to the safety and efficiency of this method. The use of allergen microarrays to detect tens to hundreds of allergen-specific antibodies in less than 0.1 mL of blood serum enables the transition to a deeply personalized approach in the diagnosis of these diseases while reducing the invasiveness and increasing the informativeness of analysis. This review discusses the technological approaches underlying the development of allergen microarrays and other protein microarrays, including the methods of selection of the microarray substrates and matrices for protein molecule immobilization, the obtainment of allergens, and the use of different types of optical labels for increasing the sensitivity and specificity of the detection of allergen-specific antibodies. [ABSTRACT FROM AUTHOR]

The demand for accurate and efficient immunoassays calls for the development of precise, high-throughput analysis methods. This paper introduces a novel approach utilizing a weak measurement interface sensor for immunoassays, offering a solution for high throughput analysis. Weak measurement is a precise quantum measurement method that amplifies the weak value of a system in the weak interaction through appropriate pre- and post-selection states. To facilitate the simultaneous analysis of multiple samples, we have developed a chip with six flow channels capable of conducting six immunoassays concurrently. We can perform real-time immunoassay to determine the binding characteristics of spike protein and antibody through real-time analysis of the flow channel images and calculating the relative intensity. The proposed method boasts a simple structure, eliminating the need for intricate nano processes. The spike protein concentration and relative intensity curve were fitted using the Log-Log fitting regression equation, and R2 was 0.91. Utilizing a pre-transformation approach to account for slight variations in detection sensitivity across different flow channels, the present method achieves an impressive limit of detection(LOD) of 0.85 ng/mL for the SARS-CoV-2 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, with a system standard deviation of 5.61. Furthermore, this method has been successfully verified for monitoring molecular-specific binding processes and differentiating binding capacities. [ABSTRACT FROM AUTHOR]

Plasmodium vivax serological exposure markers (SEMs) have emerged as promising tools for the actionable surveillance and implementation of targeted interventions to accelerate malaria elimination. To determine the dynamic profiles of SEMs in current and past P. vivax infections, we screened and selected 11 P. vivax proteins from 210 putative proteins using protein arrays, with a set of serum samples obtained from patients with acute P. vivax and documented past P. vivax infections. Then we used a murine protein immune model to initially investigate the humoral and memory B cell response involved in the generation of long-lived antibodies. We show that of the 11 proteins, especially C-terminal 42-kDa region of P. vivax merozoite surface protein 1 (PvMSP1-42) induced longer-lasting long-lived antibodies, as these antibodies were detected in individuals infected with P. vivax in the 1960-1970s who were not re-infected until 2012. In addition, we provide a potential mechanism for the maintenance of long-lived antibodies after the induction of PvMSP1-42. The results indicate that PvMSP1-42 induces more CD73+CD80+ memory B cells (MBCs) compared to P. vivax GPI-anchored micronemal antigen (PvGAMA), allowing IgG anti-PvMSP1-42 antibodies to be maintained for a long time. Author summary: P. vivax serological exposure markers (SEMs) that reflect exposure to blood-stage P. vivax parasites, could play an important role in assessing progress towards malaria elimination. Serological surveillance can be a useful tool for identifying areas of high transmission intensity or hidden asymptomatic reservoirs, especially as malaria transmission declines. These immune responses can persist long after the clearance of blood-stage infection, making antibodies valuable markers for both past exposure and current infection status. Furthermore, gaining a better understanding of the host immune response to malaria, particularly regarding the induction and maintenance of high levels of circulating antibodies, is crucial for effective serological surveillance efforts. Using serum obtained in 2007 or 2012 from patients with acute P. vivax infection, 5-year, 12-year and 30-year recovery of P. vivax-infected patients we provide evidence that PvMSP1-42 induces long-lived anti-PvMSP1-42 antibodies. Then we used a murine protein immune model, show that more CD73+CD80+ memory B cells, allowing IgG anti-PvMSP1-42 antibodies to be maintained for a long time (Fig 1). The results of this study represent an important step forward in our understanding of the host immune response to P. vivax blood stages, including the induction and maintenance of high levels of circulating antibodies. These findings may have important implications for the development of effective serological surveillance tools. [ABSTRACT FROM AUTHOR]

Introduction: Protein kinases are indispensable reversible molecular switches that adapt and control protein functions during cellular processes requiring rapid responses to internal and external events. Bacterial infections can affect kinasemediated phosphorylation events, with consequences for both innate and adaptive immunity, through regulation of antigen presentation, pathogen recognition, cell invasiveness and phagocytosis. Streptococcus pneumoniae (Spn), a human respiratory tract pathogen and a major cause of communityacquired pneumoniae, affects phosphorylation-based signalling of several kinases, but the pneumococcal mediator(s) involved in this process remain elusive. In this study, we investigated the influence of pneumococcal H2O2 on the protein kinase activity of the human lung epithelial H441 cell line, a generally accepted model of alveolar epithelial cells. Methods: We performed kinome analysis using PamGene microarray chips and protein analysis in Western blotting in H441 lung cells infected with Spn wild type (SpnWT) or with SpnDlctODspxB -a deletion mutant strongly attenuated in H2O2 production- to assess the impact of pneumococcal hydrogen peroxide (H2O2) on global protein kinase activity profiles. Results: Our kinome analysis provides direct evidence that kinase activity profiles in infected H441 cells significantly vary according to the levels of pneumococcal H2O2. A large number of kinases in H441 cells infected with SpnWT are significantly downregulated, whereas this no longer occurs in cells infected with the mutant SpnDlctODspxB strain, which lacks H2O2. In particular, we describe for the first time H2O2-mediated downregulation of Protein kinase B (Akt1) and activation of lymphocyte-specific tyrosine protein kinase (Lck) via H2O2-mediated phosphorylation. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is one of the most common mechanisms regulating cellular signaling pathways, and many kinases and phosphatases are proven drug targets. Upon phosphorylation, protein functions can be further regulated by the distinct isomerase Pin1 through cis-trans isomerization. Numerous protein targets and many important roles have now been elucidated for Pin1. However, no tools are available to detect or target cis and trans conformation events in cells. The development of Pin1 inhibitors and stereo- and phospho-specific antibodies has revealed that cis and trans conformations have distinct and often opposing cellular functions. Aberrant conformational changes due to the dysregulation of Pin1 can drive pathogenesis but can be effectively targeted in age-related diseases, including cancers and neurodegenerative disorders. Here, we review advances in understanding the roles of Pin1 signaling in health and disease and highlight conformational regulation as a distinct signal transduction checkpoint in disease development and treatment. [ABSTRACT FROM AUTHOR]

Wild‐type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria. This protein display relies on the transpeptidase activity of sortase A, which is widely engineered to allow protein ligation and protein engineering based on the interaction between sortase A and peptides. Here an unknown interaction is found between sortase A from Staphylococcus aureus and nucleic acids, in which exogenously expressed engineered sortase A binds oligonucleotides in vitro and is independent of its canonical transpeptidase activity. When incubated with mammalian cells, engineered sortase A further mediates oligonucleotide labeling to the cell surface, where sortase A attaches itself and is part of the labeled moiety. The labeling reaction can also be mediated by many classes of wild‐type sortases as well. Cell surface GAG appears involved in sortase‐mediated oligonucleotide cell labeling, as demonstrated by CRISPR screening. This interaction property is utilized to develop a technique called CellID to facilitate sample multiplexing for scRNA‐seq and shows the potential of using sortases to label cells with diverse oligonucleotides. Together, the binding between sortase A and nucleic acids opens a new avenue to understanding the virulence of wild‐type sortases and exploring the application of sortases in biotechnology. [ABSTRACT FROM AUTHOR]

Motivation Multiplexed immunofluorescence (mIF) is an emerging assay for multichannel protein imaging that can decipher cell-level spatial features in tissues. However, existing automated cell phenotyping methods, such as clustering, face challenges in achieving consistency across experiments and often require subjective evaluation. As a result, mIF analyses often revert to marker gating based on manual thresholding of raw imaging data. Results To address the need for an evaluable semi-automated algorithm, we developed GammaGateR, an R package for interactive marker gating designed specifically for segmented cell-level data from mIF images. Based on a novel closed-form gamma mixture model, GammaGateR provides estimates of marker-positive cell proportions and soft clustering of marker-positive cells. The model incorporates user-specified constraints that provide a consistent but slide-specific model fit. We compared GammaGateR against the newest unsupervised approach for annotating mIF data, employing two colon datasets and one ovarian cancer dataset for the evaluation. We showed that GammaGateR produces highly similar results to a silver standard established through manual annotation. Furthermore, we demonstrated its effectiveness in identifying biological signals, achieved by mapping known spatial interactions between CD68 and MUC5AC cells in the colon and by accurately predicting survival in ovarian cancer patients using the phenotype probabilities as input for machine learning methods. GammaGateR is a highly efficient tool that can improve the replicability of marker gating results, while reducing the time of manual segmentation. Availability and implementation The R package is available at https://github.com/JiangmeiRubyXiong/GammaGateR. [ABSTRACT FROM AUTHOR]

This article provides an overview of the epidemiology and evolution of dengue outbreaks in Bangladesh from 2020 to 2023. The study found that dengue fever cases peaked earlier than usual in 2023, resulting in the highest annual death toll ever. A novel genotype of the dengue virus was identified, and the virus strains were found to be rapidly evolving. The study suggests that this information can help in developing strategies to control future outbreaks. In 2023, there were a total of 1,705 deaths recorded, with the highest number of deaths occurring in August, September, and October. The Dhaka division had the most severe outbreak, and the majority of cases were male. Amino acid substitutions were observed in the virus, indicating the emergence of a new genotype. The study also found that the evolutionary rate of the virus was higher for one strain compared to another. The incidence rate of dengue has been increasing globally, and Bangladesh experienced a severe outbreak in 2023. The outbreak may be attributed to climate change and increased rainfall. Dengue surpassed COVID-19 as the leading cause of mortality in Bangladesh during this period. The majority of cases occurred in young adults, and there was a male predominance. The study highlights the importance of continuously monitoring the virus and its variants to prepare for future outbreaks. The article acknowledges the limitations of the study and declares no conflict of interest. The data supporting the findings are available upon request. [Extracted from the article]

Colorectal cancer is the third leading cause of cancer-related death worldwide. Hence, there is a need to identify new therapeutic agents to improve the current repertoire of therapeutic drugs. Wogonin, a flavonoid from the herbal medicine Scutellaria baicalensis, has unique antitumor activity. Our study aimed to further explore the inhibitory effects of wogonin on colorectal cancer and its specific mechanism. The results showed that wogonin significantly inhibited the proliferation of colorectal cancer cells as well as their ability to invade and metastasize. We detected phosphorylation of tumor-associated signaling pathways using a phosphorylated protein microarray and found that wogonin intervention significantly inhibited the phosphorylation level of the AKT protein in colorectal cancer cells. Through in vitro and in vivo experiments, it was confirmed that wogonin exerted its antitumor effects against colorectal cancer by inhibiting phosphorylation in the AKT pathway. Our discovery of wogonin as an inhibitor of AKT phosphorylation provides new opportunities for the pharmacological treatment of colorectal cancer. [ABSTRACT FROM AUTHOR]

Previous studies from our laboratory and others have established the dendritic cell (DC) as a key target of RSV that drives infection-induced pathology. Analysis of RSV-induced transcriptomic changes in RSV-infected DC revealed metabolic gene signatures suggestive of altered cellular metabolism. Reverse phase protein array (RPPA) data showed significantly increased PARP1 phosphorylation in RSV-infected DC. Real-time cell metabolic analysis demonstrated increased glycolysis in PARP1-/- DC after RSV infection, confirming a role for PARP1 in regulating DC metabolism. Our data show that enzymatic inhibition or genomic ablation of PARP1 resulted in increased ifnb1, il12, and il27 in RSV-infected DC which, together, promote a more appropriate anti-viral environment. PARP1-/- mice and PARP1-inhibitor-treated mice were protected against RSV-induced immunopathology including airway inflammation, Th2 cytokine production, and mucus hypersecretion. However, delayed treatment with PARP1 inhibitor in RSV-infected mice provided only partial protection, suggesting that PARP1 is most important during the earlier innate immune stage of RSV infection. [ABSTRACT FROM AUTHOR]

The recently discovered Type 9 Secretion System (T9SS) is present in bacteria of the Fibrobacteres–Bacteroidetes–Chlorobi superphylum, which are key constituents of diverse microbiomes. T9SS is instrumental in the extracellular secretion of over 270,000 proteins, including peptidases, sugar hydrolases, metal ion‐binding proteins, and metalloenzymes. These proteins are essential for the interaction of bacteria with their environment. This mini‐review explores the extensive array of proteins secreted by the T9SS. It highlights the diverse functions of these proteins, emphasizing their roles in pathogenesis, bacterial interactions, host colonization, and the overall health of the ecosystems inhabited by T9SS‐containing bacteria. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are highly expressed in preimplantation development. Preimplantation development is the phase when the cells of the early embryo undergo the first cell fate choice and change from being totipotent to pluripotent. A range of studies have advanced our understanding of TEs in preimplantation, as well as their epigenetic regulation and functional roles. However, many questions remain about the implications of TE expression during early development. Challenges originate first due to the abundance of TEs in the genome, and second because of the limited cell numbers in preimplantation. Here we review the most recent technological advancements promising to shed light onto the role of TEs in preimplantation development. We explore novel avenues to identify genomic TE insertions and improve our understanding of the regulatory mechanisms and roles of TEs and their RNA and protein products during early development. [ABSTRACT FROM AUTHOR]

In recent yearsjajajj, peptide-based therapeutics have attracted increasing interest as a potential approach to cancer treatment. Peptides are characterized by high specificity and low cytotoxicity, but they cannot be considered universal drugs for all types of cancer. Of the numerous anticancer-reported peptides, both natural and synthetic, only a few have reached clinical applications. However, in most cases, the mechanism behind the anticancer activity of the peptide is not fully understood. For this reason, in this work, we investigated the effect of the novel peptide ∆M4, which has documented anticancer activity, on two human skin cancer cell lines. A novel approach to studying the potential induction of apoptosis by anticancer peptides is the use of protein microarrays. The results of the apoptosis protein study demonstrated that both cell types, skin malignant melanoma (A375) and epidermoid carcinoma (A431), exhibited markers associated with apoptosis and cellular response to oxidative stress. Additionally, ∆M4 induced concentration- and time-dependent moderate ROS production, triggering a defensive response from the cells, which showed decreased activation of cytoplasmic superoxide dismutase. However, the studied cells exhibited a differential response in catalase activity, with A375 cells showing greater resistance to the peptide action, possibly mediated by the Nrf2 pathway. Nevertheless, both cell types showed moderate activity of caspases 3/7, suggesting that they may undergo partial apoptosis, although another pathway of programmed death cannot be excluded. Extended analysis of the mechanisms of action of anticancer peptides may help determine their effectiveness in overcoming chemoresistance in cancerous cells. [ABSTRACT FROM AUTHOR]

Migrasomes represent a recently uncovered category of extracellular microvesicles, spanning a diameter range of 500 to 3000 nm. They are emitted by migrating cells and harbour a diverse array of RNAs and proteins. Migrasomes can be readily identified in bodily fluids like serum and urine, rendering them a valuable non‐invasive source for disease diagnosis through liquid biopsy. In this investigation, we introduce a streamlined and effective approach for the capture and quantitative assessment of migrasomes, employing wheat germ agglutinin (WGA)‐coated magnetic beads and flow cytometry (referred to as WBFC). Subsequently, we examined the levels of migrasomes in the urine of kidney disease (KD) patients with podocyte injury and healthy volunteers using WBFC. The outcomes unveiled a substantial increase in urinary podocyte‐derived migrasome concentrations among individuals with KD with podocyte injury compared to the healthy counterparts. Notably, the urinary podocyte‐derived migrasomes were found to express an abundant quantity of phospholipase A2 receptor (PLA2R) proteins. The presence of PLA2R proteins in these migrasomes holds promise for serving as a natural antigen for the quantification of autoantibodies against PLA2R in the serum of patients afflicted by membranous nephropathy. Consequently, our study not only pioneers a novel technique for the isolation and quantification of migrasomes but also underscores the potential of urinary migrasomes as a promising biomarker for the early diagnosis of KD with podocyte injury. [ABSTRACT FROM AUTHOR]