Analysis Of Protein Expression Research Articles

Microfibril-Associated Protein-3-Like Regulates TGFβ-Induced EMT Process via TNFR2/p38 MAPK Pathway in Endothelial Cells

Abdominal aortic inflammation (AAI) is a major arterial vasculitis characterized by chronic inflammation and fibrosis. Endothelial cells transform into mesenchymal cells (EMT) is one of the significant mechanisms of vasculitis fibrosis. Despite its importance, the molecular mechanism of EMT in AAI remains poorly understood. In this study, we induced AAI in mice through intraperitoneal injection of tumor necrosis factor-alpha (TNFα). To analyze protein expression, we performed Western blotting. Additionally, we extracted RNA using the nanomagnetic bead method to investigate the expression of functionally related genes. We conducted cell migration and invasion assays using scratch and Transwell techniques. Western blot analysis revealed the upregulation of microfibril-associated protein-3-like (MFAP3L) and tumor necrosis factor (TNF) receptor 2 (TNFR2), along with p38 signaling pathway activation. Notably, MFAP3L expression played a crucial role in the transforming growth factor-beta (TGFβ)-induced EMT process in endothelial cells. Furthermore, we identified that MFAP3L-mediated EMT relied on both TNFR2 expression and the activity of the TNFR2/p38 signaling pathway.

Effects of voluntary and forced physical exercise on the retinal health of aging Wistar rats.

Aging is accompanied by an increased prevalence of degenerative conditions, including those affecting ocular health, which significantly impact quality of life and increase the burden on healthcare systems. Among these, retinal aging is of particular concern due to its direct link to vision impairment, a leading cause of disability in the elderly. Vision loss in the aging population is associated with heightened risks of cognitive decline, social isolation, and morbidity. This study addresses the critical gap in our understanding of modifiable lifestyle factors, such as physical exercise, that may mitigate retinal aging and its related pathologies. We investigated the effects of different exercise regimens-voluntary (recreational-type) and forced (high-intensity)-on the retinal health of aging Wistar rats (18-month-old), serving as a model for studying the translational potential of exercise interventions in humans. Male Wistar rats were divided into four groups: a young control (3-month-old) for baseline comparison, an aged sedentary control, an aged group engaging in voluntary exercise via a running wheel in their cage, and an aged group subjected to forced exercise on a treadmill for six sessions of 20 min each per week. After a 6-month experimental period, we assessed retinal function via electroretinography (ERG), measured retinal thickness histologically, and analyzed protein expression changes relevant to oxidative stress, inflammation, and anti-aging mechanisms. Our findings reveal that voluntary exercise positively impacts retinal function and morphology, reducing oxidative stress and inflammation markers while enhancing anti-aging protein expression. In contrast, forced exercise showed diminished benefits. These insights underscore the importance of exercise intensity and preference in preserving retinal health during aging. The study highlights the potential of recreational physical activity as a non-invasive strategy to counteract retinal aging, advocating for further research into exercise regimens as preventative therapies for age-related ocular degenerations.

Genome-wide identification and expression analysis of the universal stress protein (USP) gene family in Arabidopsis thaliana, Zea mays, and Oryza sativa.

The Universal Stress Protein (USP) primarily participates in cellular responses to biotic and abiotic stressors, playing a pivotal role in plant growth, development, and Stress responses to adverse environmental conditions. Totals of 23, 26 and 26 USP genes were recognized in Arabidopsis thaliana, Zea mays, and Oryza sativa, respectively. According to USP genes physicochemical properties, proteins from USP I class were identified as hydrophilic proteins with high stability. Based on phylogenetic analysis, USP genes family were classified into nine groups, USP II were rich in motifs. Additionally, members of the same subgroup exhibited similar numbers of introns/exons, and shared conserved domains, indicating close evolutionary relationships. Motif analysis results demonstrated a high degree of conservation among USP genes. Chromosomal distribution suggested that USP genes might have undergone gene expansion through segmental duplication in Arabidopsis thaliana, Zea mays, and Oryza sativa. Most Ka/Ks ratios were found to be less than 1, suggesting that USP genes in Arabidopsis thaliana, Zea mays, and Oryza sativa have experienced purifying selection. Expression profile analysis revealed that USP genes primarily respond to drought stress in Oryza sativa, temperature, and drought stress in Zea mays, and cold stress in Arabidopsis thaliana. Gene collinearity analysis can reveal correlations between genes, aiding subsequent in-depth investigations. This study sheds new light on the evolution of USP genes in monocots and dicots and lays the foundation for a better understanding of the biological functions of the USP genes family.

Involvement of N4BP2L1, PLEKHA4, and BEGAIN genes in breast cancer and muscle cell development

Patients with breast cancer show altered expression of genes within the pectoralis major skeletal muscle cells of the breast. Through analyses of The Cancer Genome Atlas (TCGA)-breast cancer (BRCA), we identified three previously uncharacterized putative novel tumor suppressor genes expressed in normal muscle cells, whose expression was downregulated in breast tumors. We found that NEDD4 binding protein 2-like 1 (N4BP2L1), pleckstrin homology domain-containing family A member 4 (PLEKHA4), and brain-enriched guanylate kinase-associated protein (BEGAIN) that are normally highly expressed in breast myoepithelial cells and smooth muscle cells were significantly downregulated in breast tumor tissues of a cohort of 50 patients with this cancer. Our data revealed that the low expression of PLEKHA4 in patients with menopause below 50 years correlated with a higher risk of breast cancer. Moreover, we identified N4BP2L1 and BEGAIN as potential biomarkers of HER2-positive breast cancer. Furthermore, low BEGAIN expression in breast cancer patients with blood fat, heart problems, and diabetes correlated with a higher risk of this cancer. In addition, protein and RNA expression analysis of TCGA-BRCA revealed N4BP2L1 as a promising diagnostic protein biomarker in breast cancer. In addition, the in silico data of scRNA-seq showed high expression of these genes in several cell types of normal breast tissue, including breast myoepithelial cells and smooth muscle cells. Thus, our results suggest their possible tumor-suppressive function in breast cancer and muscle development.

MRGPRX2 antagonist GE1111 attenuated DNFB-induced atopic dermatitis in mice by reducing inflammatory cytokines and restoring skin integrity

IntroductionAtopic dermatitis (AD) is a chronic inflammatory skin disorder characterised by itching, erythema, and epidermal barrier dysfunction. The pathogenesis of AD is complex and multifactorial; however,mast cell (MC) activation has been reported to be one of the crucial mechanisms in the pathogenesis of AD. The MC receptor Mas related G protein-coupled receptor-X2 (MRGPRX2) has been identified as a prominent alternative receptor to the IgE receptor in causing MC activation and the subsequent release of inflammatory mediators. The current study aimed to evaluate the therapeutic effect of a novel small molecule MRGPRX2 antagonist GE1111 in AD using in vitro and in vivo approaches. MethodsWe developed an in vitro cell culture disease model by using LAD-2 MC, HaCaT keratinocytes and RAW 264.7 macrophage cell lines. We challenged keratinocytes and macrophage cells with CST-14 treated MC supernatant in the presence and absence of GE1111 and measured the expression of tight junction protein claudin 1, inflammatory cytokines and macrophage phagocytosis activity through immunohistochemistry, western blotting, RT-qPCR and fluorescence imaging techniques. In addition to this, we developed a DFNB-induced AD model in mice and evaluated the protective effect and underlying mechanism of GE1111.Results and DiscussionOur in vitro findings demonstrated a potential therapeutic effect of GE1111, which inhibits the expression of TSLP, IL-13, MCP-1, TNF-a, and IL-1ß in MC and keratinocytes. In addition to this, GE1111 was able to preserve the expression of claudin 1 in keratinocytes and the phagocytotic activity of macrophage cells. The in vivo results demonstrated that GE1111 treatment significantly reduced phenotypic changes associated with AD (skin thickening, scaling, erythema and epidermal thickness). Furthermore, immunohistochemical analysis demonstrated that GE1111 treatment preserved the expression of the tight junction protein Involucrin and reduced the expression of the inflammatory mediator periostin in the mouse model of AD. These findings were supported by gene and protein expression analysis, where GE1111 treatment reduced the expression of TSLP, IL-13, and IL-1ß, as well as downstream signalling pathways of MRGPRX2 in AD skin lesions. In conclusion, our findings provide compelling in vitro and in vivo evidence supporting the contribution of MRGPRX2-MC interaction with keratinocytes and macrophages in the pathogenesis of AD.

Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage.

Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary sgRNA validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

Bcl-2 dependent modulation of Hippo pathway in cancer cells

IntroductionBcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2.MethodsWe performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts.ResultsWe demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation.ConclusionsWe discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

Glutathione S-transferase omega class 1 (GSTO1)-associated large extracellular vesicles are involved in tumor-associated macrophage-mediated cisplatin resistance in bladder cancer.

Bladder cancer poses a significant challenge to chemotherapy due to its resistance to cisplatin, especially at advanced stages. Understanding the mechanisms behind cisplatin resistance is crucial for improving cancer therapy. The enzyme glutathione S-transferase omega class 1 (GSTO1) is known to be involved in cisplatin resistance in colon cancer. This study focused on its role in cisplatin resistance in bladder cancer. Our analysis of protein expression in bladder cancer cells stimulated by secretions from tumor-associated macrophages (TAMs) showed a significant increase in GSTO1. This prompted further investigation into the role of GSTO1 in bladder cancer. We found a strong correlation between GSTO1 expression and cisplatin resistance. Mechanistically, GSTO1 triggered the release of large extracellular vesicles (EVs) that promoted cisplatin efflux, thereby reducing cisplatin-DNA adduct formation and enhancing cisplatin resistance. Inhibition of EV release effectively counteracted the cisplatin resistance associated with GSTO1. In conclusion, GSTO1-mediated EV release may contribute to cisplatin resistance caused by TAMs in bladder cancer. Strategies to target GSTO1 could potentially improve the efficacy of cisplatin in treating bladder cancer.

Mechanistic Characterization of Cancer Associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein.

Cancer-associated fibroblasts (CAFs) are a prominent cell type within the tumor microenvironment where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease Fibroblast Activation Protein (FAP) is expressed on the surface of highly pro-tumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating pro-tumorigenic CAFs will disrupt the crosstalk between components of TME resulting in cancer cell death and immune infiltration. This hypothesis, however, has never been directly proven. To eliminate FAP-expressing CAFs, we developed an antibody-drug conjugate (ADC) using our anti-FAP antibody, huB12, coupled to a monomethyl auristatin E (huB12-MMAE) payload. After determining that huB12 was an effective targeting vector, we found that huB12-MMAE potently eliminated FAP-expressing cells as monocultures in vitro and significantly prolonged survival in vivo using a xenograft engineered to overexpress FAP. We investigated the effects of selectively eliminating CAFs using a layered, open microfluidic cell co-culture platform, known as the Stacks. Analysis of mRNA and protein expression found that treatment with huB12-MMAE resulted in the increased secretion of the pro-inflammatory cytokines IL6 and IL8 by CAFs and an associated increase in expression of pro-inflammatory genes in cancer cells. We also detected increased secretion of CSF1, a cytokine involved in myeloid recruitment and differentiation. Our findings suggest that the mechanism of FAP-targeted therapies are through effects on the immune microenvironment and anti-tumor immune response.

Combined transcriptome and proteome analysis reveals MSN and ARFIP2 as biomarkers for trastuzumab resistance of breast cancer.

HER2-positive breast cancer (BC) accounts for 20-30% of all BC subtypes and is linked to poor prognosis. Trastuzumab (Tz), a humanized anti-HER2 monoclonal antibody, is a first-line treatment for HER2-positive breast cancer which faces resistance challenges. This study aimed to identify the biomarkers driving trastuzumab resistance. Differential expression analysis of genes and proteins between trastuzumab-sensitive (TS) and trastuzumab-resistant (TR) cells was conducted using RNA-seq and iTRAQ. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) were used to study their functions. The prognostic significance and protein levels of ARFIP2 and MSN were evaluated using online tools and immunohistochemistry. Sensitivity of MSN and ARFIP2 to other therapies was assessed using public pharmacogenomics databases and the R language. Five genes were up-regulated, and nine genes were down-regulated in TR cells at both transcriptional and protein levels. Low ARFIP2 and high MSN expression linked to poor BC prognosis. MSN increased and ARFIP2 decreased in TR patients, correlating with shorter OS. MSN negatively impacted fulvestrant and immunotherapy sensitivity, while ARFIP2 had a positive impact. Our findings suggest that MSN and ARFIP2 could serve as promising biomarkers for predicting response to Tz, offering valuable insights for future research in the identification of diagnostic and therapeutic targets for BC patients with Tz resistance.

Heterogeneous Profile of ROR1 Protein Expression across Tumor Types.

The Wnt receptor ROR1 has generated increased interest as a cancer therapeutic target. Research on several therapeutic approaches involving this receptor is ongoing; however, ROR1 tissue expression remains understudied. We performed an immunohistochemistry analysis of ROR1 protein expression in a large cohort of multiple tumor and histologic types. We analyzed 12 anonymized multi-tumor tissue microarrays (TMAs), including mesothelioma, esophageal and upper gastrointestinal carcinomas, and uterine endometrioid carcinoma, among other tumor types. Additionally, we studied 5 different sarcoma types of TMAs and 6 patient-derived xenografts (PDX) TMAs developed from 19 different anatomic sites and tumor histologic types. A total of 1142 patient cases from different histologic types and 140 PDXs placed in TMAs were evaluated. Pathologists assessed the percentage of tumor cells in each case that were positive for ROR1 and the intensity of staining. For determining the prevalence of staining for each tumor type, a case was considered positive if >1% of its tumor cells showed ROR1 staining. Our immunohistochemistry assays revealed a heterogeneous ROR1 expression profile. A high prevalence of ROR1 expression was found in mesothelioma (84.6%), liposarcoma (36.1%), gastrointestinal stromal tumors (33.3%), and uterine endometrioid carcinoma (28.9%). Other histologic types such as breast, lung, renal cell, hepatocellular, urothelial carcinoma, and colon carcinomas; glioblastoma; cholangiocarcinoma; and leiomyosarcoma showed less ROR1 overall expression, ranging between 0.9 and 13%. No ROR1 expression was seen in mesenchymal chondrosarcoma, rhabdomyosarcoma, or gastric adenocarcinoma cases. Overall, ROR1 expression was relatively infrequent and low in most tumor types investigated; however, ROR1 expression was infrequent but high in selected tumor types, such as gastroesophageal GIST, suggesting that ROR1 prescreening may be preferable for those indications. Further, mesothelioma exhibited frequent and high levels of ROR1 expression, which represents a previously unrecognized therapeutic opportunity. These findings can contribute to the development of ROR1-targeted therapies.

Oxidative Stress, Lipid Peroxidation and Ferroptosis Are Major Pathophysiological Signatures in the Placental Tissue of Women with Late-Onset Preeclampsia.

Preeclampsia, a serious and potentially life-threatening medical complication occurring during pregnancy, is characterized by hypertension and often accompanied by proteinuria and multiorgan dysfunction. It is classified into two subtypes based on the timing of diagnosis: early-onset (EO-PE) and late-onset preeclampsia (LO-PE). Despite being less severe and exhibiting distinct pathophysiological characteristics, LO-PE is more prevalent than EO-PE, although both conditions have a significant impact on placental health. Previous research indicates that different pathophysiological events within the placenta may contribute to the development of preeclampsia across multiple pathways. In our experimental study, we investigated markers of oxidative stress, ferroptosis, and lipid peroxidation pathways in placental tissue samples obtained from women with LO-PE (n = 68) compared to healthy control pregnant women (HC, n = 43). Through a comprehensive analysis, we observed an upregulation of specific molecules associated with these pathways, including NADPH oxidase 1 (NOX-1), NADPH oxidase 2 (NOX-2), transferrin receptor protein 1 (TFRC), arachidonate 5-lipoxygenase (ALOX-5), acyl-CoA synthetase long-chain family member 4 (ACSL-4), glutathione peroxidase 4 (GPX4) and malondialdehyde (MDA) in women with LO-PE. Furthermore, increased ferric tissue deposition (Fe3+) was observed in placenta samples stained with Perls' Prussian blue. The assessment involved gene and protein expression analyses conducted through RT-qPCR experiments and immunohistochemistry assays. Our findings underscore the heightened activation of inflammatory pathways in LO-PE compared to HC, highlighting the pathological mechanisms underlying this pregnancy disorder.

Expression of CD39 is associated with T cell exhaustion in ovarian cancer and its blockade reverts T cell dysfunction

ABSTRACT Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the “don’t eat me” molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

Integrated Proteomics Analysis of Baseline Protein Expression in Pig Tissues.

The availability of an increasingly large amount of public proteomics data sets presents an opportunity for performing combined analyses to generate comprehensive organism-wide protein expression maps across different organisms and biological conditions. Sus scrofa, a domestic pig, is a model organism relevant for food production and for human biomedical research. Here, we reanalyzed 14 public proteomics data sets from the PRIDE database coming from pig tissues to assess baseline (without any biological perturbation) protein abundance in 14 organs, encompassing a total of 20 healthy tissues from 128 samples. The analysis involved the quantification of protein abundance in 599 mass spectrometry runs. We compared protein expression patterns among different pig organs and examined the distribution of proteins across these organs. Then, we studied how protein abundances were compared across different data sets and studied the tissue specificity of the detected proteins. Of particular interest, we conducted a comparative analysis of protein expression between pig and human tissues, revealing a high degree of correlation in protein expression among orthologs, particularly in brain, kidney, heart, and liver samples. We have integrated the protein expression results into the Expression Atlas resource for easy access and visualization of the protein expression data individually or alongside gene expression data.

Cannabidiol’s cytotoxicity in pancreatic cancer is induced via an upregulation of ceramide synthase 1 and ER stress

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a median 5 year-survival rate of 12%. Cannabidiol (CBD) has been found to exhibit antineoplastic potential and may potentiate the anticancer effects of cytotoxic’s such as gemcitabine. CBD therapy has been linked to de novo synthesis of ceramide. The sphingolipid ceramide is a potent tumour suppressor lipid with roles in apoptosis and autophagy. One of the key players involved is ceramide synthase, an enzyme with six isoforms (CerS1-CerS6), reported to have disease prognostic value. Quantitative real time PCR was used to determine mRNA expression levels of ceramide synthase isoforms, GRP78, ATF4 and CHOP. Western blotting was used to analyze protein expression of these markers and knockdown of CerS1 and GRP78 were applied via an siRNA and confirmed by the two mentioned methods. Mice with PDAC xenografts were injected via intraperitoneal method with drugs and tumours were analysed with flow cytometry and processed using H&E and IHC staining. siRNA knockdown of ceramide synthase 1 (CerS1) and analysis point to evidence of a putative CerS1 dependent pathway driven by CBD in activating endoplasmic reticulum (ER) stress target; GRP78. Upon CBD treatment, CerS1 was upregulated and downstream this led to the GRP78/ATF4/CHOP arm of the unfolded protein response (UPR) pathway being activated. In an in vivo model of PDAC in which CerS1 was not upregulated on IHC, there was no observed improvement in survival of animals, however a reduction in tumour growth was observed in combination chemotherapy and CBD group, indicating further investigations in vivo. These findings provide evidence of a potential ceramide induced cytotoxic mechanism of action of CBD in pancreatic ductal adenocarcinoma.

Study of the potential role of CASPASE-10 mutations in the development of autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is a primary disorder of lymphocyte homeostasis, leading to chronic lymphoproliferation, autoimmune cytopenia, and increased risk of lymphoma. The genetic landscape of ALPS includes mutations in FAS, FASLG, and FADD, all associated with apoptosis deficiency, while the role of CASP10 defect in the disease remains debated. In this study, we aimed to assess the impact of CASP10 variants on ALPS pathogenesis. We benefit from thousands of genetic analysis datasets performed in our Institute’s genetic platform to identify individuals carrying CASP10 variants previously suspected to be involved in ALPS outcome: p.C401LfsX15, p.V410I and p.Y446C, both at heterozygous and homozygous state. Clinical and laboratory features of the six included subjects were variable but not consistent with ALPS. Two individuals were healthy. Comprehensive analyses of CASP10 protein expression and FAS-mediated apoptosis were conducted and compared to healthy controls and ALPS patients with FAS mutations. Missense CASP10 variants (p.V410I and p.Y446C), which are common in the general population, did not disrupt CASP10 expression, nor FAS-mediated apoptosis. In contrast, homozygous p.C401LfsX15 CASP10 variant lead to a complete abolished CASP10 expression but had no impact on FAS-mediated apoptosis function. At heterozygous state, this p.C401LfsX15 variant lead to a reduced CASP10 protein levels but remained associated with a normal FAS-mediated apoptosis function. These findings demonstrate that CASPASE 10 is dispensable for FAS-mediated apoptosis. In consequences, CASP10 defect unlikely contribute to ALPS pathogenesis, since they did not result in an impairment of FAS-mediated apoptosis nor in clinical features of ALPS in human. Moreover, the absence of FAS expression up-regulation in subjects with CASP10 variants rule out any compensatory mechanisms possibly involved in the normal apoptosis function observed. In conclusion, this study challenges the notion that CASP10 variants contribute to the development of ALPS.

SDPR expression in human trabecular meshwork and its potential role in racial disparities of glaucoma

In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients’ surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.

Abstract PO1-13-11: ZNF689 promotes homologous recombination via NBS1 ubiquitination and its loss confers sensitivity to PARP inhibition in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) poses significant clinical challenges due to the lack of targeted therapies and poor prognosis. Homologous recombination (HR) deficiency is a defining characteristic of TNBC and renders tumors susceptible to Poly (ADP-ribose) polymerase (PARP) inhibitors. However, not all TNBC patients respond to PARP inhibitors, highlighting the need to identify novel biomarkers to predict therapeutic response and improve treatment strategies. Methods: In this study, we investigated the role of zinc finger protein 689 (ZNF689) in DNA damage response (DDR) and its impact on PARP inhibitor sensitivity in TNBC. Using a comprehensive set of molecular and cellular techniques, including immunoprecipitation, in vitro and in vivo ubiquitination assays, protein expression analysis, and functional assays using TNBC cell lines, patient-derived organoids (PDOs), patient-derived xenograft (PDX) models, and cell line xenograft models, we elucidated the mechanistic role of ZNF689 in HR and PARP inhibitor response. Results: Our findings revealed ZNF689 as a novel player in DDR. ZNF689 is activated by ATM-mediated phosphorylation and recruited to DNA damage sites in an ATM-dependent manner. At the DNA double-strand break sites, ZNF689 collaborates directly with NBS1 to facilitate K63-linked ubiquitination of NBS1 via the E3 ligase SKP2. Furthermore, ZNF689-mediated NBS1 ubiquitination stabilizes the MRE11-RAD50-NBS1 (MRN) complex and feedbacks activation of ATM, therefore accelerating HR repair. Loss of ZNF689 results in HR deficiency, leading to increased sensitivity of TNBC cells to PARP inhibitors in both in vitro and in vivo models. Additionally, the combination of PARP inhibitors and paclitaxel demonstrated potential for improved efficacy in treating ZNF689-deficient TNBC. In our clinical analysis, we observed a positive correlation between ZNF689 expression and the levels of NBS1 and phosphorylated ATM in TNBC patient samples. Importantly, low ZNF689 expression was associated with a favorable response to PARP inhibitors in TNBC patients. Conclusions: Our study highlights the significant role of ZNF689 in HR and its impact on the response to PARP inhibitors in TNBC. These findings suggest ZNF689 as a promising therapeutic target for enhancing the effectiveness of PARP inhibitors in TNBC patients. Working Modle Citation Format: Li-Ping Ge, Zi-Yu Wang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. ZNF689 promotes homologous recombination via NBS1 ubiquitination and its loss confers sensitivity to PARP inhibition in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-11.

Acetic acid affects porcine oocyte metabolism and improves oocyte developmental ability

Improvement in in vitro maturation culture conditions has been achieved by mimicking in vivo culture environments such as the follicular fluid. Acetic acid is an energy substrate that is abundantly present in the follicular fluid but has not been considered in in vitro maturation. This study examined the effects of acetic acid on oocyte quality during nuclear maturation. Cumulus cells and oocyte complexes were collected from the porcine antral follicles of gilt ovaries and matured with 0, 0.1 or 1 mmol/L of acetic acid. After 44 h of in vitro maturation, the energy status, mitochondrial quality and function and embryonic developmental rate following parthenogenetic activation were determined. RNA-sequencing and protein expression analyses were conducted to predict the effects of acetic acid. Supplementation of the in vitro maturation medium with acetic acid (1 mmol/L) improved embryonic development. Oocytes matured with acetic acid had low adenosine triphosphate and lipid contents, mitochondrial membrane potential and reactive oxygen species levels. RNA-sequencing revealed differential expression of genes associated with the adenosine monophosphate-activated protein kinase signalling pathway. Immunostaining revealed that acetic acid increased the levels of phospho-adenosine monophosphate-activated protein kinase, phospho-acetyl-coenzyme A carboxylase, and sirtuin 1 and decreased those of fatty acid synthase and acetyl-coenzyme A synthetase 1. In summary, the use of acetic acid during oocyte maturation improved oocyte developmental ability and metabolism by altering mitochondrial activity and lipid metabolism.

HIV-derived Proteins induce Hypertension, Sympatho-activation, and neuroinflammation via T cell-dependent mechanisms

Combination antiretroviral therapy (cART) has markedly increased life expectancy in people living with HIV (PLWH) and induced a switch in the cause of death from opportunistic diseases to cardiovascular disease (CVD). CVD is currently the leading cause of death in PLWH and hypertension, the primary risk factor for CVD, presents in more than 65% of them. However, the mechanisms whereby hypertension develops in PLWH remain unknown. In PLWH on cART, T lymphocytes (T cells) remain a viral reservoir for HIV and continue to secrete viral proteins. Viral proteins remain in circulation and are expressed in the central nervous system. Herein, we tested the hypothesis that expression of HIV-derived proteins increases blood pressure (BP), induces sympatho-activation and inflammation of the central nervous system. We utilized the Tg26 mouse model which expresses 7/9 viral proteins. Quantitative PCR analysis of viral protein expression showed that Tat, gp120, Nef, Vif, and Vpr are primarily expressed in spleen, lung, and brain tissues in Tg26 mice. Morphometric analysis revealed slight but significant increases and decreases in lean mass and fat weights respectively in Tg26. Flow cytometry analysis showed no reduction in CD4+ T cells in Tg26 mice indicating that this mouse model does not progress to AIDS and reproduce the conditions of virally suppressed PLWH. Blood pressure (BP) measurement via telemetry revealed a hypertensive phenotype in male and female Tg26 mice. Hypertension was associated with a larger drop in BP in response to ganglionic blockade but no alterations in heart rate responses to muscarinic and β-adrenergic receptor blockade in Tg26 mice, indicating that viral proteins expression increases neurogenic control of BP but did not alter cardiac autonomic control. β-adrenergic receptor blockade did not reduce BP in Tg26 mice. Tg26 mice also display increased aortic vasoconstriction to phenylephrine. To test the contribution of T cells in BP regulation and sympatho-activation, mice were subjected to a CTLA-4 inhibitor, abatacept, which inhibits T cell activation. Abatacept treatment significantly reduced BP, sympatho-activation, and phenylephrine constriction in Tg26 mice with no effects on wild-type mice. To determine whether the increase in sympatho-activation involves central inflammation, inflammatory cytokines and T cell markers levels were measured in the brain. Levels of pro-inflammatory cytokines including IL-1α, TNFα, IL-6, and CCL2 were increased in the brain of Tg26 mice as well as levels of the T-cell marker GATA3. Altogether, our data shows that the expression of HIV-derived proteins increases BP, sympatho-activation, and vascular adrenergic contractility via T cell-dependent mechanisms likely through central mechanisms involving neuro-inflammation. R01HL147639-01A1, R01HL15526501 and 19EIA34760167 (E.J.B). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.