Protein Array Research Articles

Parallel Analyses by Mass Spectrometry (MS) and Reverse Phase Protein Array (RPPA) Reveal Complementary Proteomic Profiles in Triple-Negative Breast Cancer (TNBC) Patient Tissues and Cell Cultures.

High-plex proteomic technologies have made substantial contributions to mechanism studies and biomarker discovery in complex diseases, particularly cancer. Despite technological advancements, inherent limitations in individual proteomic approaches persist, impeding the achievement of comprehensive quantitative insights into the proteome. In this study, we employed two widely used proteomic technologies, mass spectrometry (MS) and reverse phase protein array (RPPA) to analyze identical samples, aiming to systematically assess the outcomes and performance of the different technologies. Additionally, we sought to establish an integrated workflow by combining these two proteomic approaches to augment the coverage of protein targets for discovery purposes. We used 14 fresh frozen tissue samples from triple-negative breast cancer (TNBC: seven tumors versus seven adjacent non-cancerous tissues) and cell line samples to evaluate both technologies and implement this dual-proteomic strategy. Using a single-step protein denaturation and extraction protocol, protein samples were subjected to reverse-phase liquid chromatography (LC) followed by electrospray ionization (ESI)-mediated MS/MS for proteomic profiling. Concurrently, identical sample aliquots were analyzed by RPPA for profiling of over 300 proteins and phosphoproteins that are in key signaling pathways or druggable targets in cancer. Both proteomic methods demonstrated the expected ability to differentiate samples by groups, revealing distinct proteomic patterns under various experimental conditions, albeit with minimal overlap in identified targets. Mechanism-based analysis uncovered divergent biological processes identified with the two proteomic technologies, capitalizing on their complementary exploratory potential.

Tackling immunosuppression by Neisseria gonorrhoeae to facilitate vaccine design.

Gonorrhoea, caused by Neisseria gonorrhoeae, is a common sexually transmitted infection. Increasing multi-drug resistance and the impact of asymptomatic infections on sexual and reproductive health underline the need for an effective gonococcal vaccine. Outer membrane vesicles (OMVs) from Neisseria meningitidis induce modest cross-protection against gonococcal infection. However, the presence of proteins in OMVs derived from N. gonorrhoeae that manipulate immune responses could hamper their success as a vaccine. Here we modified two key immunomodulatory proteins of the gonococcus; RmpM, which can elicit 'blocking antibodies', and PorB, an outer membrane porin which contributes to immunosuppression. As meningococcal PorB has adjuvant properties, we replaced gonococcal PorB with a meningococcal PorB. Immunisation with OMVs from N. gonorrhoeae lacking rmpM and expressing meningococcal porB elicited higher antibody titres against model antigens in mice compared to OMVs with native PorB. Further, a gonococcal protein microarray revealed stronger IgG antibody responses to a more diverse range of antigens in the Nm PorB OMV immunised group. Finally, meningococcal PorB OMVs resulted in a Th1-skewed response, exemplified by increased serum IgG2a antibody responses and increased IFNɣ production by splenocytes from immunised mice. In summary, we demonstrate that the replacement of PorB in gonococcal OMVs enhances immune responses and offers a strategy for gonococcal vaccine development.

EXTH-14. IRREVERSIBLE TARGETING OF THE C-MYC/TOPOISOMERASE I AXIS IN GLIOBLASTOMA: DUAL INHIBITION USING A NOVEL INDENOISOQUINOLINE INHIBITOR

Abstract Glioblastoma–the most common primary CNS malignancy–is notorious for its cellular and molecular complexity and uniform fatality. Overexpression of the c-MYC and TOPOISOMERASE 1 (TOP1) oncogenes is implicated in more than 50% of human cancers. They are involved in the pathogenesis of glioblastomas with overexpression correlating with decreased overall median survival in glioblastoma patients. LMP744 is a non-camptothecin indenoisoquinoline small molecule inhibitor designed to target the C-MYC/TOP1 axis. The compound is inherently stable, has high brain penetrance, and irreversibly binds to its target providing several advantages over the camptothecin class of drugs. Human glioblastoma cells treated with LMP744 show a dose-dependent decrease in viability with LD50 ranging between 50 and 100 nM compared to 100-200µM following treatment with temozolomide in the same cell lines. Treatment results in a pro-apoptotic effect in exposed cells as well as S-phase arrest. Protein arrays and confirmatory western blots demonstrate increased CHK2 and decreased p53 and GSK-a/ß in treated cells. Orthotopic xenotransplantation via stereotactic injection of human glioblastoma cells (7×105 cells/4 µL) in nude mice showed exquisite sensitivity to LMP744 without adverse events. Administration of 20 mg/kg of LMP744 via tail vein injection resulted in total eradication of tumor signal on bioluminescent imaging. These preliminary findings suggest that LMP744–and perhaps the class of non-camptothecin indenoisoquinolones–is a promising novel candidate for glioblastoma pharmacotherapy.

EXTH-83. CHARACTERIZATION OF SPLICING ABERRATIONS INDUCED BY PRMT5 INHIBITION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) are lethal tumors with limited treatment options, with a high unmet need for novel therapies. PRMT5, an arginine methyltransferase that regulates several histone and non-histone targets, is overexpressed in cancers including GBM and is a promising therapeutic target given its role in promoting oncogenic processes. Here, we characterized the effects of pharmacological inhibition of PRMT5 by the brain-penetrant, orally bioavailable inhibitors, PRT811 and PRT808 on treatment-induced splicing aberrations using a panel of patient-derived glioma stem cells (GSC) and organotypic human glioma slice cultures. Changes in protein expression upon PRMT5 inhibition were assessed using reverse phase protein array (RPPA) analysis. Additionally, splicing aberrations due to PRMT5 inhibition in were tested in several GSCs using RNA-seq. We observed a potent reduction of symmetrical dimethylation of arginine (SDMA protein marks by both PRMT5 inhibitors across various GSC lines and ex-vivo brain tumor tissue slices. Notably, GSCs with both mutant and wild-type p53 displayed similar sensitivity to PRMT5 inhibition, independent of MTAP status. Moreover, PRMT5 inhibition elicited reduced cell proliferation, altered cell survival pathways, and apoptosis. RNA-seq analysis revealed that splicing alterations affecting numerous oncogenes and regulatory factors, encompassing 42 cancer driver genes, 80 splicing factors, and 42 transcriptional factors across these GSC lines, with specific splicing events observed in nuclear and cytoplasmic subcellular compartments. Additional analysis is ongoing to determine which of the aberrant transcripts are translated to yield tumor specific neoantigens that can be potential targets for tumor specific immune strategies. These results underscore the efficacy of pharmacological PRMT5 inhibition in genetically and epigenetically diverse GSCs, and highlight its potential for developing novel immune and non-immune therapeutic strategies against GBM.

TMIC-40. TARGETING GLIOMA TUMOR MICROENVIRONMENT BY SPECIFIC NEUTRALIZATION OF EXTRACELLULAR NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (ENAMPT) USING ALT-100, A NOVEL HUMANIZED MONOCLONAL ANTIBODY

Abstract BACKGROUND Intracellular nicotinamide phosphoribosyltransferase (iNAMPT is the rate-limiting enzyme of NAD salvage pathway and is preferentially used by glioma cells for regulating energy metabolism and homeostasis. When secreted, extracellular NAMPT (eNAMPT) exerts cytokine-like functions and is associated with poor prognosis in cancer patients. We have previously reported an essential role of iNAMPT for glioma cell proliferation and survival. Here, we examined whether eNAMPT influences glioma biology and tumor microenvironment by neutralizing eNAMPT using ALT-100, a novel humanized monoclonal antibody. METHODS Native levels of eNAMPT in blood samples of patients with primary/recurrent gliomas were determined using ELISA under an IRB approved protocol. Next, the effects of ALT-100 on glioma microenvironment were determined by treating viable human glioma tissue slices with ALT-100 and assessing downstream effects on RNA and protein using RNASeq, reverse phase protein array and spatial transcriptomics. Lastly, the in vivo effect of ALT-100-mediated eNAMPT neutralization on survival was determined in a mouse intracranial glioma model. RESULTS Measurable levels of eNAMPT were noted in all patient samples with a range of (3.7-71.2 ng/ml). Preliminary results showed a correlation of eNAMPT levels with grade of tumor. Human organotypic glioma slice cultures treated with ALT-100, showed a profound alteration of gene and protein expression particularly related to key energy metabolism and proliferation pathways and the PD-1/PD-L1 axis. Additionally, results of spatial profiling of ALT-100 treated human organotypic glioma slices will be presented. Survival and response data from ALT-100 treatment in ongoing in vivo experiment will also be reported. CONCLUSIONS This is the first report of ALT-100-mediated eNAMPT neutralization in gliomas and demonstrates profound effects on the tumor and its microenvironment. Given that ALT-100 is in phase 2 trials (NCT05938036) against ARDS, data from our studies can potentially accelerate translating ALT-100-mediated eNAMPT neutralization to treat gliomas.

Activation of MEK‐ERK‐c‐MYC signaling pathway promotes splenic M2-like macrophage polarization to inhibit PHcH-liver cirrhosis

IntroductionPortal hypertension combined with hypersplenism (PHcH) is the main cause of hypocytosis and esophagogastric variceal hemorrhage in patients with liver cirrhosis. Activated macrophages that destroy excess blood cells are the main cause of hypersplenism, but the activating pathway is not very clear. This study aims to investigate the activation types of splenic macrophages and their activation mechanisms, to provide experimental evidence for the biological treatment of splenomegaly, and to find a strategy to improve liver fibrosis and inflammation by intervening in splenic immune cells. This study revealed the occurrence of M2-like polarization of macrophages and upregulation of c-Myc gene expression in the PH spleen.MethodsRNAseq, protein chip, western blot, and chip-seq were performed on macrophages and the in vitro MEK inhibitor rafametinib was used. Carbon tetrachloride and thioacetamide induced mouse cirrhosis models were separately constructed.Resultsc-Myc gene knockout in splenic macrophages reduced M2-like polarization and exacerbated liver fibrosis inflammation. c-Myc activated the MAPK signaling pathway and upregulated the expression of IL-4 and M2-like related genes in PH hypersplenism through the MEK-ERK-c-Myc axis. In addition, the c-Myc gene exerted anti-inflammatory effects by upregulating IL-4-mediated signal transduction to promote M2-like differentiation and anti-inflammatory cytokine secretion.ConclusionsActivation of MEK‐ERK‐c‐MYC signaling pathway promotes splenic M2-like macrophage polarization to inhibit PHcH-liver cirrhosis. Therefore, the induction of macrophage depolarization might represent a new therapeutic approach in the cure of PH hypersplenism, making c-Myc a potential candidate for macrophage polarization therapy.

FGF6 inhibits oral squamous cell carcinoma progression by regulating PI3K/AKT and MAPK pathways

To explore diagnostic and prognostic biomarkers in the progression of oral squamous cell carcinoma (OSCC) and to reveal their regulatory mechanisms in key pathways. A RayBiotech protein chip was used to screen differentially expressed serum proteins in OSCC, oral leukoplakia (OLK), and healthy participants. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used to determine the pathways enriched by characteristic differential proteins. Immunohistochemical analysis and western blotting were used to verify the expression of characteristic differential proteins and key regulatory factors in human tissues and in a nude mouse model. Fibroblast growth factor 6 (FGF6) was identified as a key differential protein and was weakly expressed in OSCC tissues. The mitogen-activated protein kinases (MAPK) and PI3K-AKT pathways were identified as key signaling pathways. The results showed that pERK, Cyclin D1, pAKT, and BCL2 were highly expressed in OSCC, Caspase9 was lowly expressed in OSCC. With an increase in FGF6 expression in nude mice, the expression of FGFR4, pERK, Cyclin D1, pAKT, BCL2, GPX4, and ACSL4 increased, and the expression of Caspase9 decreased. FGF6 may change the expression of apoptosis-related proteins and proliferation factors by binding to FGFR4 in the PI3K-AKT/MAPK pathway and may inhibit the ferroptosis of OSCC, thereby possibly participating in the process of inhibiting OSCC.

Dental pulp mesenchymal stem cell (DPSCs)-derived soluble factors, produced under hypoxic conditions, support angiogenesis via endothelial cell activation and generation of M2-like macrophages

BackgroundCell therapy has emerged as a revolutionary tool to repair damaged tissues by restoration of an adequate vasculature. Dental Pulp stem cells (DPSC), due to their easy biological access, ex vivo properties, and ability to support angiogenesis have been largely explored in regenerative medicine.MethodsHere, we tested the capability of Dental Pulp Stem Cell-Conditioned medium (DPSC-CM), produced in normoxic (DPSC-CM Normox) or hypoxic (DPSC-CM Hypox) conditions, to support angiogenesis via their soluble factors. CMs were characterized by a secretome protein array, then used for in vivo and in vitro experiments. In in vivo experiments, DPSC-CMs were associated to an Ultimatrix sponge and injected in nude mice. After excision, Ultimatrix were assayed by immunohistochemistry, electron microscopy and flow cytometry, to evaluate the presence of endothelial, stromal, and immune cells.For in vitro procedures, DPSC-CMs were used on human umbilical-vein endothelial cells (HUVECs), to test their effects on cell adhesion, migration, tube formation, and on their capability to recruit human CD14+ monocytes.ResultsWe found that DPSC-CM Hypox exert stronger pro-angiogenic activities, compared with DPSC-CM Normox, by increasing the frequency of CD31+ endothelial cells, the number of vessels and hemoglobin content in the Ultimatrix sponges. We observed that Utimatrix sponges associated with DPSC-CM Hypox or DPSC-CM Normox shared similar capability to recruit CD45− stromal cells, CD45+ leukocytes, F4/80+ macrophages, CD80+ M1-macrophages and CD206+ M2-macropages. We also observed that DPSC-CM Hypox and DPSC-CM Normox have similar capabilities to support HUVEC adhesion, migration, induction of a pro-angiogenic gene signature and the generation of capillary-like structures, together with the ability to recruit human CD14+ monocytes.ConclusionsOur results provide evidence that DPSCs-CM, produced under hypoxic conditions, can be proposed as a tool able to support angiogenesis via macrophage polarization, suggesting its use to overcome the issues and restrictions associated with the use of staminal cells.

Blood-brain barrier permeability increases with the differentiation of glioblastoma cells in vitro

BackgroundGlioblastoma multiforme (GBM) is an aggressive tumor, difficult to treat pharmacologically because of the blood-brain barrier (BBB), which is rich in ATP-binding cassette (ABC) transporters and tight junction (TJ) proteins. The BBB is disrupted within GBM bulk, but it is competent in brain-adjacent-to-tumor areas, where eventual GBM foci can trigger tumor relapse. How GBM cells influence the permeability of BBB is poorly investigated.MethodsTo clarify this point, we co-cultured human BBB models with 3 patient-derived GBM cells, after separating from each tumor the stem cell/neurosphere (SC/NS) and the differentiated/adherent cell (AC) components. Also, we set up cultures of BBB cells with the conditioned medium of NS or AC, enriched or depleted of IL-6. Extracellular cytokines were measured by protein arrays and ELISA. The intracellular signaling in BBB cells was measured by immunoblotting, in the presence of STAT3 pharmacological inhibitor or specific PROTAC. The competence of BBB was evaluated by permeability assays and TEER measurement.ResultsThe presence of GBM cells or their conditioned medium increased the permeability to doxorubicin, mitoxantrone and dextran-70, decreased TEER, down-regulated ABC transporters and TJ proteins at the transcriptional level. These effects were higher with AC or their medium than with NS. The secretome analysis identified IL-6 as significantly more produced by AC than by NS. Notably, AC-conditioned medium treated with an IL-6 neutralizing antibody reduced the BBB permeability to NS levels, while NS-conditioned medium enriched with IL-6 increased BBB permeability to AC levels. Mechanistically, IL-6 released by AC GBM cells activated STAT3 in BBB cells. In turn, STAT3 down-regulated ABC transporter and TJ expression, increased permeability and decreased TEER. The same effects were obtained in BBB cells treated with STA-21, a pharmacological inhibitor of STAT3, or with a PROTAC targeting STAT3.ConclusionsOur work demonstrates for the first time that the degree of GBM differentiation influences BBB permeability. The crosstalk between GBM cells that release IL-6 and BBB cells that respond by activating STAT3, controls the expression of ABC transporters and TJ proteins on BBB. These results may pave the way for novel therapeutic tools to tune BBB permeability and improve drug delivery to GBM.

Anti-interferon gamma-inducible protein 16 antibodies: Identification of a novel autoantigen in idiopathic interstitial pneumonia and its clinical characteristics based on a multicenter cohort study

Autoantibodies are detected in idiopathic interstitial pneumonias (IIPs) without a clear connective tissue disease diagnosis, and their clinical significance is unclear. This study aimed to identify a novel autoantibody in IIPs. We screened 295 IIP patients using a 35S-methionine labeled protein immunoprecipitation assay. Candidate autoantigens were identified via protein array and confirmed by immunoprecipitation. Six sera from 295 IIP patients immunoprecipitated common tetrameric proteins (100 kDa). The protein array identified interferon gamma-inducible protein 16 (IFI16) as the candidate autoantigen. Patients with anti-IFI16 antibodies received immunosuppressants less frequently. Five-year survival rates were 50 %, 69 %, and 63 % (P = 0.60), and acute exacerbation-free rates were 50 %, 96 %, and 84 % (P = 0.15) for patients with anti-IFI16, anti-aminoacyl tRNA antibodies, and others. Anti-IFI16 is a novel autoantibody in IIPs. Patients with this antibody often receive less immunosuppressive therapy and could have a poor prognosis. Further research is needed to refine patient stratification and management.

Screening of cancer-specific biomarkers for hepatitis B-related hepatocellular carcinoma based on a proteome microarray

Hepatocellular carcinoma (HCC) is associated with one of the highest mortality rates among cancers, rendering its early diagnosis clinically invaluable. Serum biomarkers, specifically alpha-fetoprotein (AFP), represent the most promising and widely used diagnostic biomarkers for HCC. However, its detection rate is low in the early stages of HCC progression, and distinguishing specific false positives for other liver-related diseases, such as cirrhosis and acute hepatitis, remains challenging. Therefore, this study was conducted to identify biomarkers for hepatitis B (HBV)-related liver diseases by screening differentially expressed autoantibodies against tumor-associated antigens (TAAbs). We designed a large-scale multistage investigation, encompassing initial screening, HCC-focused, and ELISA validation cohorts to identify potential TAAbs in HBV-related liver diseases, spanning from healthy control (HC) individuals to patients with chronic hepatitis B (CHB), hepatitis B-related cirrhosis (HBC), and HCC, using protein microarray technology. The differential biological characteristics of TAAbs were analyzed using bioinformatics analysis. Validation of tumor-specific biomarkers for HCC was performed using ELISA. In the screening cohort, 547 candidate TAAbs were identified in the HCC group compared to those in the HC group. In the HCC-focused cohort, 64, 61, and 65 candidate TAAbs were identified in the CHB, HBC, and HCC groups, respectively, compared to those in the HC group. Thirty-four proteins exhibited continuously elevated expression from HCs to patients with CHB, HBC, and HCC. Among these, nine were identified as cancer-specific proteins. In the validation cohort, UBE2Z, CNOT3, and EID3 were correlated with liver function indicators in patients with hepatitis B-related HCC. Overall, UBE2Z, CNOT3, and EID3 emerged as cancer-specific biomarkers for HBV-related liver disease, providing a scientific basis for clinical application.

Impact of Green-synthesized Copper Silver Nanoparticles Against Human Colon Cancer Cells Utilizing Sargassum muticum Extract in Terms of Cytotoxicity and Apoptosis

Background and Objectives Brown seaweed, Sargassum muticum, can grow up to a maximum length of 10 m. This study aimed to create novel bimetallic nanoparticles (NPs) by utilizing the entire plant extract. The bimetallic green-Ag-CuNPs was synthesized by using these substances extract of Sargassum muticum algae, copper oxide (CuO), silver (AgCl) by a reduction procedure and studied its toxic action against human colon cancer (HCT-116) cells. Methods SEM and TEM were used to measure the NPs’ size prior to the treatment of g-Ag-Cu NPs. The g-Ag-Cu nanoparticles had a circular shape and measured 46 ± 1 nm in size. MTT and NRU tests were used to assess the cytotoxicity of g-Ag-Cu NPs on HCT-116 cells. Results The results showed that the cytotoxicity of NPs increased in a concentration-dependent manner. The median inhibitory concentration (IC50) for HCT-116 cells at 24 h was determined to be 66.4 μg/ml based on the MTT result. At 50 µg/ml of g-Ag-Cu NPs, HCT-116 cells generated large amounts of intracellular ROS, induced caspase 3/7. Using Rhodamine123 labeling, the impact of NPs on mitochondrial membrane potential in HCT-116 cells was evaluated and it was highly compromised at 50 µg/ml of g-Ag-Cu NPs. Expression of apoptotic protein was assessed by using protein array methods and it was down and up regulated as per concentration of exposure of g-Ag-Cu NPs. Conclusion Our findings support the g-Ag-Cu NPs’ lethal effects on human colon cancer cells. We believe that because this nanoparticle increases cytotoxicity and triggers apoptosis, it may be useful as a supplement in the treatment of colon cancer.

Study on the changes and significance of CXCL10 level in serum of isolated polymyalgia rheumatica.

To investigate the significance of CXC chemokine ligand 10 (CXCL10) in the pathogenesis of isolated polymyalgia rheumatica (PMR). The serum of six PMR patients diagnosed and treated at the First Affiliated Hospital of Wannan Medical College from September 2019 to December 2020 before treatment and after remission was collected, and the serum of six active rheumatoid arthritis (RA) patients and six healthy medical checkups were also collected, and protein microarray technology was used to detect 24 cytokines, including IL-6, IL-4, CXCL10, CXCL8, and CXCL2. Subsequently, serum was collected from other 28 patients with active PMR, 26 patients with PMR in remission, 24 patients with active RA, and 24 healthy medical checkups who were diagnosed and treated at the First Affiliated Hospital of Wannan Medical College from January 2021 to July 2023, and the enzyme-linked immunosorbent assay (ELISA) was used to validate and compare the levels of CXCL10 in each group and analyze the correlation between the levels of serum CXCL10 and the parameters of the clinical activities of PMR. Protein microarray screening revealed significant differences in CXCL10 before and after PMR treatment, and ELISA validation revealed that peripheral serum CXCL10 levels were significantly higher in the PMR-active group than in the remission group (P < 0.001), and also significantly higher than in the RA-active group (P = 0.003) and in the healthy control group (P < 0.001); correlation analysis showed a significant positive correlation between serum CXCL10 levels and serum ferritin in PMR patients (r = 0.450, P = 0.024). In the ROC curve for distinguishing PMR and RA, the area under the curve is 0.741, sensitivity = 0.643, and specificity = 0.792. CXCL10 may play a role in the pathogenesis of isolated PMR and its level might contribute to the differential diagnosis of PMR and RA. Key Points •The concentration of CXCL10 was higher in peripheral blood of isolated PMR patients. •CXCL10 is a potential diagnostic biomarker for isolated PMR patients. •The level of CXCL10 might contribute to the differential diagnosis of PMR and RA.

Molecular mechanisms underlying amyloid beta peptide mediated upregulation of vascular cell adhesion molecule-1 in Alzheimer's disease.

Amyloid beta (Aβ) deposition and neurofibrillary tangles are widely considered as the primary pathological hallmarks of familial and sporadic forms of Alzheimer's disease (AD). However, cerebrovascular inflammation, which is prevalent in 70% of AD patients is emerging as another core feature of AD pathology. In addition, activation of inflammatory signaling pathways have been observed in AD patients; specifically, cerebrovascular inflammation was found to be augmented in Alzheimer's patients. Our studies have demonstrated that the inflammation signaling pathway is upregulated in AD patient brains. Moreover, vascular cell adhesion molecule-1 (VCAM-1), a cerebrovascular inflammatory marker expressed on the blood-brain barrier (BBB) endothelium, was observed to be upregulated in APP,PS1 mice (a mouse model that overexpresses Ab42), as detected by dynamic SPEC/CT imaging. While there is a strong association between Aβ42 exposure and an increase in VCAM-1 expression, the mechanisms underlying the influence of Aβ42 on VCAM-1 expression remain understudied. Therefore, we investigated the hypothesis that Ab42 exposure increases VCAM-1 expression in human cerebral microvascular endothelial cell (hCMEC/D3) monolayers. In addition, reverse phase protein array assays (RPPA) and immunocytochemistry demonstrated that Ab42 increases VCAM-1 expression through the Src/p38/MEK signaling pathway specifically within the blood-brain barrier (BBB) endothelium. In summary, these results demonstrate that Ab42 augments cerebrovascular inflammation by elevating VCAM-1 expression via Src/MEK/p38 pathway. Hence, targeting VCAM-1 at the BBB as a diagnostic and therapeutic marker may hold potential for detecting and mitigating cerebrovascular inflammation in Alzheimer's disease. Significance Statement While considered a core pathological feature of Alzheimer's disease, molecular pathways leading to cerebrovascular inflammation remain partially understood. Moreover, clinical diagnostic methods for detecting cerebrovascular inflammation are underdeveloped. In this study, we demonstrated VCAM-1 detection using radio-iodinated VCAM-1 antibody and SPECT/CT imaging. The study demonstrated that the exposure to Aβ42 increases VCAM-1 expression on the BBB endothelium via Src/p38/MEK pathway. These findings are expected to aid in the development of diagnostic and therapeutic approaches for addressing cerebrovascular inflammation in AD.

Unfractionated Heparin Enhances Sepsis Prognosis Through Inhibiting Drp1‐Mediated Mitochondrial Quality Imbalance

AbstractUnfractionated heparin (UFH) is commonly used as an anticoagulant in sepsis treatment and has recently been found to have non‐anticoagulant effects, but underlying mechanisms remain unclear. This retrospective clinical data showed that UFH has significant protective effects in sepsis compared to low‐molecular‐weight heparin and enoxaparin, indicating potential benefits of its non‐anticoagulant properties. Recombinant protein chip screening, surface plasmon resonance, and molecular docking data demonstrated that UFH specifically bound to the cytoplasmic Drp1 protein through its zone 2 non‐anticoagulant segment. In‐vitro experiments verified that UFH's specific binding to Drp1 suppressed Drp1 translocation to mitochondria following “sepsis” challenge, thereby improving mitochondrial morphology, function and metabolism in vascular endothelial cells. Consequently, UHF comprehensively protected mitochondrial quality, thus reducing vascular leakage and improving prognosis in a sepsis rat model. These findings highlight the potential of UFH as a sepsis treatment strategy targeting non‐anticoagulation mechanism.

Prevalent and persistent new-onset autoantibodies in mild to severe COVID-19

Autoantibodies have been shown to be implied in COVID-19 but the emerging autoantibody repertoire remains largely unexplored. We investigated the new-onset autoantibody repertoire in 525 healthcare workers and hospitalized COVID-19 patients at five time points over a 16-month period in 2020 and 2021 using proteome-wide and targeted protein and peptide arrays. Our results show that prevalent new-onset autoantibodies against a wide range of antigens emerged following SARS-CoV-2 infection in relation to pre-infectious baseline samples and remained elevated for at least 12 months. We found an increased prevalence of new-onset autoantibodies after severe COVID-19 and demonstrated associations between distinct new-onset autoantibodies and neuropsychiatric symptoms post-COVID-19. Using epitope mapping, we determined the main epitopes of selected new-onset autoantibodies, validated them in independent cohorts of neuro-COVID and pre-pandemic healthy controls, and identified sequence similarities suggestive of molecular mimicry between main epitopes and the conserved fusion peptide of the SARS-CoV-2 Spike glycoprotein. Our work describes the complexity and dynamics of the autoantibody repertoire emerging with COVID-19 and supports the need for continued analysis of the new-onset autoantibody repertoire to elucidate the mechanisms of the post-COVID-19 condition.

A proteome-wide analysis unveils a core Epstein-Barr virus antibody signature of classic Hodgkin lymphoma across ethnically diverse populations.

Epstein-Barr virus (EBV) is an oncogenic virus associated with various malignancies, including classical Hodgkin lymphoma (cHL). Despite its known association, the specific role of humoral immune response to EBV remains poorly characterized in cHL. To address this, we conducted a study using a custom protein microarray to measure the antibody responses in cHL patients and matched healthy controls recruited from an East-Asian hospital-based case-control study. We identified 16 IgG antibodies significantly elevated in EBV-positive cHL compared with controls, defining an "East-Asian antibody signature of EBV-positive cHL." We evaluated responses against these 16 antibodies in a distinct European population, leveraging data from our previous European cHL case-control study from the UK, Denmark, and Sweden. A subset of antibodies (14/16, 87.5%) from the "East-Asian antibody signature of EBV-positive cHL" exhibited significant associations with cHL in the European population. Conversely, we assessed the "European antibody signature of EBV-positive cHL" identified in our prior study which consisted of 18 EBV antibodies (2 IgA, 16 IgG), in the East-Asian population. A subset of these antibodies (15/18, 83.3%) maintained significant associations with cHL in the East-Asian population. This cross-comparison of antibody signatures underscores the robust generalizability of EBV antibodies across populations. Five anti-EBV IgG antibodies (LMP-1, TK, BALF2, BDLF3, and BBLF1), found in both population-specific antibody signatures, represent a "core signature of EBV-positive cHL." Our findings suggest that the antibody responses targeting these core EBV proteins reflect a specific EBV gene expression pattern, serving as potential biomarkers for EBV-positive cHL independent of population-specific factors.

Understanding the relationship between inflammation, apoptosis, and diabetes osteoporosis: A bioinformatics approach and experimental verification.

Diabetes osteoporosis (DOP) is a chronic metabolic bone disease. This study aimed to identify potential biomarkers of DOP and explore their underlying mechanisms through bioinformatics methods and experimental verification. Bioinformatics methods were used to identify differentially expressed genes (DEGs) for DOP based on GEO data and the GeneCards database. GO and KEGG enrichment analyses were used to search the key pathways. The STRING website was used to construct a protein-protein interaction (PPI) network and identify key genes. Then, 50 mg/mL glucose was used to interveneosteoblasts (OBs).CCK-8 and Alizarin Red staining were used to investigate the proliferation and differentiation changes in OBs. Flowcytometry was used to investigate apoptosis. The membrane protein chip, WB, and RT-PCR were used to verify the expression of key targets or pathways about DOP. Forty-two common genes were screened between DOP-related targets and DEGs. GO and KEGG enrichment analysis showed that DOP was mainly associated with cytokine-cytokine receptor interactions, and apoptosis. PPI network analysis showed that TNF, IL1A, IL6, IL1B, IL2RA, Fas ligand (FASLG), and Fas cell surface death receptor (FAS) were key up-regulated genes in the occurrence of DOP. The experiment results show that 50 mg/mL glucose significantly inhibited OBs proliferation but presented an increase in apoptosis. Membrane protein chip, WB, and RT-PCR-verified a significantly active in the expression of TNF/FASLG/FAS pathway. High glucose activated the TNF-α/FAS/FASLG pathway and induced the inflammatory microenvironment and apoptosis, then impaired osteogenic differentiation of OBs. These may be an important mechanism for the occurrence and development of DOP.

Bacterial Lysate-Based Bifunctional mRNA Nanoformulation for Efficient Colon Cancer Immunogene Therapy.

mRNA-based nonviral gene therapy has played an important role in cancer therapy, however, the limited delivery efficiency and therapeutic capacity still require further exploration and enhancement. Immunogene therapy provides a strategy for cancer treatment. Bacteria are tiny single-celled living organisms, many of which can be found in and on the human body and are beneficial to humans. Lactobacillus reuteri is a bacterial member of the gut flora, and recent research has shown that it can reduce intestinal inflammation by stimulating an immunomodulatory response. L. reuteri lysate represents an ideal resource for constructing advanced mRNA delivery systems with immune stimulation potential. Here, we prepared a bifunctional mRNA delivery system DMP-Lac (DOTAP-mPEG-PCL-L. reuteri lysate), which successfully codelivered L. reuteri lysate and IL-23A mRNA, exhibited a high mRNA delivery efficiency of 75.56% ± 0.85%, and strongly promoted the maturation and activation of the immune system in vivo. Both the CT26 abdominal metastasis model and the lung metastasis model also exhibited a good therapeutic effect, and the tumor inhibition rate of DMP-Lac/IL-23A group reached 97.92%. Protein chip technology verified that DMP acted as an immune adjuvant, demonstrating that the L. reuteri lysate could regulate the related immune cells, while IL-23 mRNA caused changes in downstream factors, thus producing the corresponding tumor treatment effect. The DMP-Lac/IL-23A complex exhibited strong anticancer immunotherapeutic effects. Our results demonstrated that this bifunctional mRNA formulation served as a tumor-specific nanomedicine, providing an advanced strategy for colon cancer immunogene therapy.

Autoantibodies towards HFE and SYT5 in anti-neutrophil cytoplasm antibody-associated vasculitis relapse.

Identification of those at high and low risk of disease relapse is a major unmet need in the management of patients with ANCA-associated vasculitis (AAV). Precise stratification would allow tailoring of immunosuppressive medication. We profiled the autoantibody repertoire of AAV patients in remission to identify novel autoantibodies associated with relapse risk. Plasma samples collected from 246 AAV patients in remission were screened for novel autoantibodies using in-house generated protein arrays including 42 000 protein fragments representing 18 000 unique human proteins. Patients were categorized based on the occurrence and frequency of relapses. We modelled the association between these antibodies and relapse occurrence using descriptive and high dimensional regression approaches. We observed nine autoantibodies at higher frequency in samples from AAV patients experiencing multiple relapses compared with patients in long-term remission off therapy (LTROT). LASSO analysis identified six autoantibodies that exhibited an association with relapse occurrence after sample collection. Antibodies targeting HFE and SYT5 were identified as associated with relapse in both analyses. Through a broad protein array-based autoantibody screening, we identified two novel autoantibodies directed against HFE and SYT5 as candidate biomarkers of relapse in AAV.