Proteomic Screen Research Articles

Repurposing neuroleptics: clozapine as a novel, adjuvant therapy for melanoma brain metastases

The blood-brain barrier and the distinct brain immunology provide challenges in translating commonly used chemotherapeutics to treat intracranial tumors. Previous reports suggest anti-tumoral effects of antipsychotics, encouraging investigations into potential treatment effects of neuroleptics on brain metastases. For the first time, the therapeutic potential of the antipsychotic drug clozapine in treating melanoma brain metastases (MBM) was investigated using three human MBM cell lines. Through in vitro cell culture and viability experiments, clozapine displayed potent anti-tumoral effects on MBM cells with an exploitable therapeutic window when compared to normal human astrocytes or rat brain organoids. Further, it was shown that clozapine inhibited migration, proliferation, and colony formation in a dose-dependent manner. Through flow cytometry and proteome screening, we found that clozapine induced apoptosis in MBM cells and potentially altered the tumor immunological environment by upregulating proteins such as macrophage inflammatory protein-1 alpha (MIP-1α) and interleukin-8 (IL-8). In conclusion, clozapine shows significant and selective anti-tumoral effects on MBM cell lines in vitro. Further in vivo experiments are warranted to translate these results into clinical use.Graphical abstract

SPT5 regulates RNA polymerase II stability via Cullin 3-ARMC5 recognition.

The stability of RNA polymerase II (Pol II) is tightly regulated during transcriptional elongation for proper control of gene expression. Our recent studies revealed that promoter-proximal Pol II is destabilized via the ubiquitin E3 ligase cullin 3 (CUL3) upon loss of transcription elongation factor SPT5. Here, we investigate how CUL3 recognizes chromatin-bound Pol II as a substrate. Using an unbiased proteomic screening approach, we identify armadillo repeat-containing 5 (ARMC5) as a CUL3 adaptor required for VCP/p97-dependent degradation of SPT5-depleted, chromatin-bound Pol II. Genome-wide analyses indicate that ARMC5 targets promoter-proximal Pol II in a BTB domain-dependent manner. Further biochemical analysis demonstrates that interaction between ARMC5 and Pol II requires the transcriptional cyclin-dependent kinase 9 (CDK9), supporting a phospho-dependent degradation model. We propose that defective, promoter-proximal Pol II that lacks SPT5 is rapidly eliminated from chromatin in a noncanonical early termination pathway that requires CDK9-dependent interaction with the CUL3-ARMC5 ubiquitin ligase complex.

Multivariable Predictive Model of Postoperative Delirium in Cardiac Surgery Patients: Proteomic and Demographic Contributions.

Delirium after cardiac surgery is common, morbid, and costly, but may be prevented with risk stratification and targeted intervention. In this study, we aimed to identify protein biomarkers and develop a predictive model for postoperative delirium in older patients undergoing cardiac surgery. SomaScan analysis of 1305 proteins in the plasma from 57 older adults undergoing cardiac surgery requiring cardiopulmonary bypass was conducted to define delirium-specific protein signatures at baseline (preoperative baseline timepoint [PREOP]) and postoperative day 2 (POD2). Selected proteins were validated in 115 patients using the Enzyme-Linked Lectin Assay (ELLA) multiplex immunoassay platform. Proteins were combined with clinical and demographic variables to build multivariable models that estimate the risk of postoperative delirium and bring light to the underlying pathophysiology. Of the 115 patients, 21 (18.3%) developed delirium after surgery. The SomaScan proteome screening evidenced differential expression of 115 and 85 proteins in delirious patients compared to nondelirious preoperatively and at POD2, respectively ( P < .05). Following biological and methodological criteria, 12 biomarker candidates (Tukey's fold change [|tFC|] >1.4, Benjamini-Hochberg [BH]- P < .01) were selected for ELLA multiplex validation. Statistical analyses of model fit resulted in the combination of age, sex, and 3 proteins (angiopoietin-2; C-C motif chemokine 5; and metalloproteinase inhibitor 1; area under the curve [AUC] = 0.829) as the best performing predictive model for delirium. Analyses of pathways showed that delirium-associated proteins are involved in inflammation, glial dysfunction, vascularization, and hemostasis. Our results support the identification of patients at higher risk of developing delirium after cardiac surgery using a multivariable model that combines demographic and physiological features, also bringing light to the role of immune and vascular dysregulation as underlying mechanisms.

Molecular analysis of acute pyelonephritis—excessive innate and attenuated adaptive immunity

This study investigated the molecular basis of disease severity in acute pyelonephritis (APN), a common and potentially life-threatening bacterial infection. Two cohorts of infants with febrile urinary tract infection were included. Renal involvement was defined by DMSA scans and molecular disease determinants by gene expression analysis and proteomic screens, at diagnosis and after 6 mo. Innate immune hyper-activation, systemically and locally in the urinary tract, was defined as a cytokine storm. Neutrophil degranulation and renal toxicity genes were strongly regulated, with overexpression in the APN group (first DMSA+). Adaptive immune attenuation in the APN group further supported the notion of an immune imbalance. DNA exome genotyping identified APN and febrile urinary tract infection as genetically distinct and scarring associated genes, but the activation of renal toxicity genes during acute infection was unrelated to the development of renal scarring. The results define APN as a hyper-inflammatory disorder with the characteristics of a cytokine storm combined with adaptive immune attenuation. The findings are consistent with innate immune dysfunctions and neutrophil disorders identified as determinants of APN susceptibility in genetic models.

SENP1 promotes deacetylation of isocitrate dehydrogenase 2 to inhibit ferroptosis of breast cancer via enhancing SIRT3 stability.

Breast cancer, one of the most prevalent malignant tumors in women worldwide, is characterized by a poor prognosis and high susceptibility to recurrence and metastasis. Ferroptosis, a lipid peroxide-dependent programed cell death pathway, holds significant potential for breast cancer treatment. Therefore, investigating the regulatory targets and associated mechanisms of ferroptosis is crucial. In this study, we conducted proteomic screening and identified isocitrate dehydrogenase 2 (IDH2) as an important player in breast cancer progression. Our findings were further supported by CCK-8 assays, transwell experiments, and scratch assays, which demonstrated that the elevated expression of IDH2 promotes breast cancer progression. Through both in vitro and in vivo experiments along with the erastin treatment, we discovered that increased expression of IDH2 confers resistance to ferroptosis in breast cancer cells. By employing Western blot analysis, Co-IP techniques, and immunofluorescence staining methods, we elucidated the upstream molecular mechanism involving SENP1-mediated SIRT3 de-SUMOylatase, which enhances IDH2 enzyme activity through deacetylation, thereby regulating cell ferroptosis. In conclusion, our study highlights the role of the SENP1-SIRT3 axis in modulating ferroptosis via IDH2 in breast cancer cells, providing valuable insights for developing targeted therapies aimed at enhancing ferroptosis for improved management of breast cancer.

Exploration of Novel Biomarkers for Neurodegenerative Diseases Using Proteomic Analysis and Ligand-Binding Assays.

Neurodegenerative diseases are a major cause of morbidity and mortality worldwide, and their public health burden continues to increase. There is an urgent need to develop reliable and sensitive biomarkers to aid the timely diagnosis, disease progression monitoring, and therapeutic development for neurodegenerative disorders. Proteomic screening strategies, including antibody microarrays, are a powerful tool for biomarker discovery, but their findings should be confirmed using quantitative assays. The current study explored the feasibility of combining an exploratory proteomic strategy and confirmatory ligand-binding assays to screen for and validate biomarker candidates for neurodegenerative disorders. It analyzed cerebrospinal fluid (CSF) and plasma samples from patients with Alzheimer's disease, Parkinson's disease, and multiple sclerosis and healthy controls using an exploratory antibody microarray and validatory ligand-binding assays. The screening antibody microarray identified differentially expressed proteins between patients with neurodegenerative diseases and healthy controls, including cluster of differentiation 14 (CD14), osteopontin, and vascular endothelial growth factor 165b. Quantitative ligand-binding assays confirmed that CD14 levels were elevated in CSF of patients with Alzheimer's disease (p = 0.0177), whereas osteopontin levels were increased in CSF of patients with Parkinson's disease (p = 0.0346). The current study demonstrated the potential utility of combining an exploratory proteomic approach and quantitative ligand-binding assays to identify biomarker candidates for neurodegenerative disorders. To further validate and expand these findings, large-scale analyses using well-characterized samples should be conducted.

Development of Dual Aurora‐A and Aurora‐B Degrading PROTACs for MYCN‐Amplified Neuroblastoma

In neuroblastoma, MYCN amplification is associated with survival rates of &lt;50%. Overexpression of the mitotic kinases Aurora‐A and Aurora‐B are also associated with low survival and exacerbate the oncogenic effects of N‐Myc. As N‐Myc is stabilized by Aurora‐A, Aurora‐A targeting proteolysis targeting chimeras (PROTACs) have been developed that reduce Aurora‐A and N‐Myc levels. However, simultaneous degradation of N‐Myc, Aurora‐A, and Aurora‐B has not been previously achieved. Given the contributions of both Aurora kinases to MYCN‐amplified neuroblastoma, we designed PROTACs capable of degrading both Aurora‐A and Aurora‐B. Dual‐degrading PROTACs dAurAB2 and dAurAB5 potently degraded Aurora‐A (DC50 = 59 nM and 8.8 nM, respectively) and Aurora‐B (DC50 = 39 nM and 6.1 nM), eliminated 89% ‐ 97% of Aurora‐A and Aurora‐B, and reduced N‐Myc levels by 38% and 45% in MYCN‐amplified IMR32 neuroblastoma cells. Global proteomics screening revealed that while dAurAB2 demonstrated good selectivity, dAurAB5 downregulated additional targets including threonine tyrosine kinase (TTK). Interestingly, TKK is also associated with MYCN‐amplified neuroblastoma, and multi‐target PROTAC dAurAB5 reduced the viability of neuroblastoma IMR32 cells by 55% at 24 hours. The development of dAurAB2 and dAurAB5 generates new modalities for inhibiting the oncogenic activities of Aurora‐A, Aurora‐B, N‐Myc, and TTK in neuroblastoma and other cancers.

Immunoinformatics investigation on pathogenic Escherichia coli proteome to develop an epitope-based peptide vaccine candidate.

Escherichia coli (E. coli), a gram-negative bacterium, quickly colonizes in the human gastrointestinal tract after birth and typically sustains a long-term, symbiotic relationship with the host. However, certain virulent strains of E. coli can cause diseases such as urinary tract infections, meningitis, and enteric disorders. The rising antibiotic resistance among these strains has heightened the urgency for an effective vaccine. This study employs immunoinformatics and a reverse vaccinology technique to identify prospective antigens and create an efficient vaccine construct. In this study, we reported the "Attaching and Effacing Protein" a novel outer-membrane protein conserved in all pathogenic E. coli strains, based on proteome screening. We developed an in silico multi-epitope vaccine that includes helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL), B cell lymphocyte (BCL), and pan HLA DR-binding reactive epitope (PADRE) sequences, along with appropriate linkers and adjuvants. Machine Learning algorithms were used to evaluate antigenicity, solubility, stability, and non-allergenicity of the vaccine construct. Additionally, molecular docking analysis revealed that vaccine construct has a strong predicted binding affinity for human toll-like receptors on the cell surface. In this context, laboratory validations are necessary to demonstrate the effectiveness of the possible vaccine design that showed encouraging findings through computational validation.

Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions.

The network of proteins at the interface between cell-cell adherens junctions and the actomyosin cytoskeleton provides robust yet dynamic connections that facilitate cell shape change and motility. While this was initially thought to be a simple linear connection via classic cadherins and their associated catenins, we now have come to appreciate that many more proteins are involved, providing robustness and mechanosensitivity. Defining the full network of proteins in this network remains a key objective in our field. Proximity proteomics provides a means to define these networks. Mammalian Afadin and its Drosophila homolog Canoe are key parts of this protein network, facilitating diverse cell shape changes during gastrulation and other events of embryonic morphogenesis. Here we report results of several proximity proteomics screens, defining proteins in the neighborhood of both the N- and C-termini of mammalian Afadin in the premier epithelial model, MDCK cells. We compare our results with previous screens done in other cell types, and with proximity proteomics efforts with other junctional proteins. These reveal the value of multiple screens in defining the full network of neighbors and offer interesting insights into the overlap in protein composition between different epithelial cell junctions.

Low toxicity ginsenoside Rg1-carbon nanodots as a potential therapeutic agent for human non-small cell lung cancer

Here ginsenoside Rg1 was used to synthesise Rg1 carbon nanodots via a one-step hydrothermal method. The surface of the Rg1 carbon nanodots is rich in hydrophilic functional groups with good water solubility and biocompatibility. The Rg1 carbon nanodots exhibited a high inhibitory effect on the proliferation, migration, and proapoptotic ability of non-small cell lung cancer A549 cells. The changes in the levels of ROS, Ca2+, and MMP in A549 cells after the administration of Rg1 carbon nanodots were evaluated and further correlated with relevant proteins in the caspase apoptotic pathway. Proteomic screening revealed that the Rg1 carbon nanodots could regulate A549 cell apoptosis by activating the expression of MAPK pathway-related proteins. In the in vivo experiment, the therapeutic efficacy of the Rg1 carbon nanodots in inhibiting tumour growth was much higher than that of commonly used chemotherapy drugs, with negligible toxicity and side effects. Immunohistochemical staining showed that the expression of caspase- and MAPK pathway-related proteins in mouse tumour tissues was consistent with that at the cellular level. The results suggest that Rg1 carbon nanodots can promote tumour apoptosis and represent a potential therapeutic agent for human non-small-cell lung cancer.

Increased expression of mesencephalic astrocyte-derived neurotrophic factor (MANF) contributes to synapse loss in Alzheimer’s disease

BackgroundThe activation of endoplasmic reticulum (ER) stress is an early pathological hallmark of Alzheimer’s disease (AD) brain, but how ER stress contributes to the onset and development of AD remains poorly characterized. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a non-canonical neurotrophic factor and an ER stress inducible protein. Previous studies reported that MANF is increased in the brains of both pre-symptomatic and symptomatic AD patients, but the consequence of the early rise in MANF protein is unknown.MethodsWe examined the expression of MANF in the brain of AD mouse models at different pathological stages. Through behavioral, electrophysiological, and neuropathological analyses, we assessed the level of synaptic dysfunctions in the MANF transgenic mouse model which overexpresses MANF in the brain and in wild type (WT) mice with MANF overexpression in the hippocampus. Using proteomic and transcriptomic screening, we identified and validated the molecular mechanism underlying the effects of MANF on synaptic function.ResultsWe found that increased expression of MANF correlates with synapse loss in the hippocampus of AD mice. The ectopic expression of MANF in mice via transgenic or viral approaches causes synapse loss and defects in learning and memory. We also identified that MANF interacts with ELAV like RNA-binding protein 2 (ELAVL2) and affects its binding to RNA transcripts that are involved in synaptic functions. Increasing or decreasing MANF expression in the hippocampus of AD mice exacerbates or ameliorates the behavioral deficits and synaptic pathology, respectively.ConclusionsOur study established MANF as a mechanistic link between ER stress and synapse loss in AD and hinted at MANF as a therapeutic target in AD treatment.

7000-Plex Proteomic Screen of Urine for Flare Monitoring and Treatment Response Biomarkers in Active Lupus Nephritis

7000-Plex Proteomic Screen of Urine for Flare Monitoring and Treatment Response Biomarkers in Active Lupus Nephritis

Tapasin assembly surveillance by the RNF185/Membralin ubiquitin ligase complex regulates MHC-I surface expression

Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here, we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD), a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin, essential for peptide loading onto MHC-I, as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance.

Exploration of potential drug targets for Glaucoma by plasma proteome screening

BackgroundGlaucoma is the leading cause of irreversible blindness. However, the current available treatment methods are still unsatisfactory. Therefore, the exploration of new drug targets for the treatment of glaucoma is of paramount importance. MethodsWe conducted two-sample Mendelian randomization (MR) using plasma protein quantitative trait loci (pQTL) data from two datasets (n = 734, n = 4907) and their instrumental variables to investigate the causal relationship between plasma proteins and glaucoma. The analysis was validated by replacing the exposure and outcome cohorts. Additionally, we utilized protein-protein interaction networks to assess the associations between these potential drug targets and existing drug targets. ResultsThrough two-sample Mendelian randomization analysis, we identified causal relationships between Glaucoma and the following proteins: AZU1, OBP2B, ENPP5, INPP5B, KREMEN1, LYPLAL1, and PTPRJ. External validation confirmed the protective effect of LYPLAL1 on Glaucoma, while ENPP5, KREMEN1, and PTPRJ increased the risk of Glaucoma. Reverse MR and Steiger filtering did not indicate any reverse causal associations of the aforementioned proteins with Glaucoma. ConclusionOur study demonstrates a causal impact of ENPP5, KREMEN1, PTPRJ, and LYPLAL1 on the risk of Glaucoma. These findings suggest that these four proteins may serve as promising drug targets for Glaucoma treatment. SignificanceCurrently, the pharmacological treatment of glaucoma primarily focuses on lowering intraocular pressure, which has its limitations. Targeted therapy is a personalized treatment approach that aims to inhibit or block the development and progression of diseases such as cancer and inflammation by selectively acting on specific biomolecules or signaling pathways. Our research employs a two-sample Mendelian randomization (MR) method, integrating a large amount of GWAS and pQTL data to perform MR analysis. This has enabled us to explore several plasma proteins as potential drug targets for glaucoma, providing direction and a research foundation for future investigations into glaucoma drug targets.

AAMP and MTSS1 Are Novel Negative Regulators of Endothelial Barrier Function Identified in a Proteomics Screen.

Cell-cell adhesion in endothelial monolayers is tightly controlled and crucial for vascular integrity. Recently, we reported on the importance of fast protein turnover for maintenance of endothelial barrier function. Specifically, continuous ubiquitination and degradation of the Rho GTPase RhoB is crucial to preserve quiescent endothelial integrity. Here, we sought to identify other barrier regulators, which are characterized by a short half-life, using a proteomics approach. Following short-term inhibition of ubiquitination with E1 ligase inhibitor MLN7243 or Cullin E3 ligase inhibitor MLN4924 in primary human endothelial cells, we identified sixty significantly differentially expressed proteins. Intriguingly, our data showed that AAMP and MTSS1 are novel negative regulators of endothelial barrier function and that their turnover is tightly controlled by ubiquitination. Mechanistically, AAMP regulates the stability and activity of RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, possibly regulating F-actin dynamics. Taken together, these findings demonstrate the critical role of protein turnover of specific proteins in the regulation of endothelial barrier function, contributing to our options to target dysregulation of vascular permeability.

Influence of RNA Methylation on Cancerous Cells: A Prospective Approach for Alteration of In Vivo Cellular Composition.

RNA methylation is a dynamic and ubiquitous post-transcriptional modification that plays a pivotal role in regulating gene expression in various conditions like cancer, neurological disorders, cardiovascular diseases, viral infections, metabolic disorders, and autoimmune diseases. RNA methylation manifests across diverse RNA species including messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA), exerting pivotal roles in gene expression regulation and various biological phenomena. Aberrant activity of writer, eraser, and reader proteins enables dysregulated methylation landscape across diverse malignancy transcriptomes, frequently promoting cancer pathogenesis. Numerous oncogenic drivers, tumour suppressors, invasion/metastasis factors, and signalling cascade components undergo methylation changes that modulate respective mRNA stability, translation, splicing, transport, and protein-RNA interactions accordingly. Functional studies confirm methylation-dependent alterations drive proliferation, survival, motility, angiogenesis, stemness, metabolism, and therapeutic evasion programs systemically. Methyltransferase overexpression typifies certain breast, liver, gastric, and other carcinomas correlating with adverse clinical outcomes like diminished overall survival. Mapping efforts uncover nodal transcripts for targeted drug development against hyperactivated regulators including METTL3. Some erasers and readers also suitable lead candidates based on apparent synthetic lethality. Proteomic screens additionally highlight relevant methylation-sensitive effector pathways amenable to combinatorial blockade, reversing compensatory signalling mechanisms that facilitate solid tumour progression. Quantifying global methylation burdens and responsible enzymes clinically predicts patient prognosis, risk stratification for adjuvant therapy, and overall therapeutic responsiveness.

Impaired degradation of PLCG1 by chaperone-mediated autophagy promotes cellular senescence and intervertebral disc degeneration

ABSTRACT Defects in chaperone-mediated autophagy (CMA) are associated with cellular senescence, but the mechanism remains poorly understood. Here, we found that CMA inhibition induced cellular senescence in a calcium-dependent manner and identified its role in TNF-induced senescence of nucleus pulposus cells (NPC) and intervertebral disc degeneration. Based on structural and functional proteomic screens, PLCG1 (phospholipase C gamma 1) was predicted as a potential substrate for CMA deficiency to affect calcium homeostasis. We further confirmed that PLCG1 was a key mediator of CMA in the regulation of intracellular calcium flux. Aberrant accumulation of PLCG1 caused by CMA blockage resulted in calcium overload, thereby inducing NPC senescence. Immunoassays on human specimens showed that reduced LAMP2A, the rate-limiting protein of CMA, or increased PLCG1 was associated with disc senescence, and the TNF-induced disc degeneration in rats was inhibited by overexpression of Lamp2a or knockdown of Plcg1. Because CMA dysregulation, calcium overload, and cellular senescence are common features of disc degeneration and other age-related degenerative diseases, the discovery of actionable molecular targets that can link these perturbations may have therapeutic value. Abbreviation: ATRA: all-trans-retinoic acid; BrdU: bromodeoxyuridine; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16-INK4A: cyclin dependent kinase inhibitor 2A; CMA: chaperone-mediated autophagy; DHI: disc height index; ER: endoplasmic reticulum; IP: immunoprecipitation; IP3: inositol 1,4,5-trisphosphate; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; IVD: intervertebral disc; IVDD: intervertebral disc degeneration; KD: knockdown; KO: knockout; Leu: leupeptin; MRI: magnetic resonance imaging; MS: mass spectrometry; N/L: NH4Cl and leupeptin; NP: nucleus pulposus; NPC: nucleus pulposus cells; PI: protease inhibitors; PLC: phospholipase C; PLCG1: phospholipase C gamma 1; ROS: reactive oxygen species; RT-qPCR: real-time quantitative reverse transcription PCR; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SASP: senescence-associated secretory phenotype; STV: starvation; TMT: tandem mass tag; TNF: tumor necrosis factor; TP53: tumor protein p53; UPS: ubiquitin-proteasome system.

Multi-omics landscapes reveal heterogeneity in long COVID patients characterized with enhanced neutrophil activity

BackgroundOmicron variant impacts populations with its rapid contagiousness, and part of patients suffered from persistent symptoms termed as long COVID. The molecular and immune mechanisms of this currently dominant global variant leading to long COVID remain unclear, due to long COVID heterogeneity across populations.MethodsWe recruited 66 participants in total, 22 out of 66 were healthy control without COVID-19 infection history, and 22 complaining about long COVID symptoms 6 months after first infection of Omicron, referred as long COVID (LC) Group. The left ones were defined as non-long COVID (NLC) Group. We profiled them via plasma neutralizing antibody titer, SARS-CoV-2 viral load, transcriptomic and proteomics screening, and machine learning.ResultsNo serum residual SARS-CoV-2 was observed in the participants 6 months post COVID-19 infection. No significant difference in neutralizing antibody titers was found between the long COVID (LC) Group and the non-long COVID (NLC) Group. Transcriptomic and proteomic profiling allow the stratification of long COVID into neutrophil function upregulated (NU-LC) and downregulated types (ND-LC). The NU-LC, identifiable through a refined set of 5 blood gene markers (ABCA13, CEACAM6, CRISP3, CTSG and BPI), displays evidence of relatively higher neutrophil counts and function of degranulation than the ND-LC at 6 months after infection, while recovered at 12 months post COVID-19.ConclusionThe transcriptomic and proteomic profiling revealed heterogeneity among long COVID patients. We discovered a subgroup of long COVID population characterized by neutrophil activation, which might associate with the development of psychiatric symptoms and indicate a higher inflammatory state. Meanwhile, a cluster of 5 genes was manually curated as the most potent discriminators of NU-LC from long COVID population. This study can serve as a foundational exploration of the heterogeneity in the pathogenesis of long COVID and assist in therapeutic targeting and detailed epidemiological investigation of long COVID.

Abstract Mo068: The role of FGF13 in modulating gap junction protein Connexin-43

Background: Fibroblast growth factor homologous factors (FHFs) are associated with arrhythmias like Brugada syndrome and atrial fibrillation, but their pathological mechanisms remain poorly understood. FHFs are best characterized as regulators of voltage gated sodium channels (VGSCs), however recent studies suggest broader, non-VGSC-related functions, including regulation of microtubules. Whether and how non-VGSC regulatory functions modulate cardiac rhythm is not known. Hypothesis: FGF13 regulates Cx43 trafficking and expression in ventricular cardiomyocytes via destabilizing microtubules. Aims: Building on an unbiased proteomic screen, we investigated if FGF13, the predominant FHF in rodent heart, affects the expression and trafficking of Connexin-43 (Cx43), the predominant gap junction protein in ventricular myocytes. Methods: To identify additional FGF13-dependent functional roles, we employed proximity labeling proteomics in mouse heart with the TurboID biotin ligase fused to FGF13. We used immunoblots, immunohistochemistry, and immunocytochemistry to further characterize the effects of FGF13 ablation on Cx43 pattern distribution and expression. We also leveraged electrophysiology techniques using gap junction inhibitors to detect any Cx43-dependent functional changes in FGF13-ablated cardiomyocytes. Results: The proteomic data revealed that cell junction proteins associated with the intercalated disc (ID) were among the top FGF13 near neighbors, of which Cx43 was heavily enriched, together with microtubule associated proteins (MAPs) known to be critical for cardiomyocyte function and ion channel trafficking. In ventricular cardiomyocytes from constitutive cardiomyocyte-restricted Fgf13 knockout mice, we found markedly perturbed trafficking of Cx43 and reduced colocalization with the ID marker N-Cadherin. Separately, MAP4 abundance was markedly reduced. The addition of colchicine, a microtubule destabilizing drug, to WT cells altered Cx43 distribution pattern similar to that observed in FGF13-ablated cardiomyocytes. Re-expression of FGF13 restored Cx43 trafficking and ID targeting. Electrophysiology experiments using a Cx43 hemichannel inhibitor, Gap19, showed a perturbation of the action potential duration in knockout cardiomyocytes. Isoproterenol-induced action potential prolongation in knockout myocytes was decreased by the addition of Gap19. Conclusions: FGF13 regulates microtubule-based trafficking and targeting of Cx43.

Differential miRNA and Protein Expression Reveals miR-1285, Its Targets TGM2 and CDH-1, as Well as CD166 and S100A13 as Potential New Biomarkers in Patients with Diabetes Mellitus and Pancreatic Adenocarcinoma.

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.