Protein Alpha Research Articles

Identification of Goat Supernumerary Teat Phenotype Using Wide-Genomic Copy Number Variants

Supernumerary teats (SNTs) or nipples often emerge around the mammary line. This study performed a genome-wide selective sweep analysis (GWS) at the copy number variant (CNV) level using two selected signal calculation methods (VST and FST) to identify candidate genes associated with SNTs in goats. A total of 12,310 CNVs were identified from 37 animals and 123 CNVs, with the top 1% VST values including 84 candidate genes (CDGs). Of these CDGs, minichromosome maintenance complex component 3, ectodysplasin A receptor associated via death domain, and cullin 5 demonstrated functions closely related to mammary gland development. In addition, 123 CNVs with the top 1% FST values were annotated to 97 CDGs. 5-Hydroxytryptamine receptor 2A, CCAAT/enhancer-binding protein alpha, and the polymeric immunoglobulin receptor affect colostrum secretion through multiple signaling pathways. Two genes, namely, RNA-binding motif protein 46 and β-1,3-galactosyltransferase 5, showed a close relation to mammary gland development. Six CNVs were identified and annotated to five genes by intersecting the top 1% of candidate CNVs with both parameters. These genes include LOC102185621, LOC102190481, and UDP-glucose pyrophosphorylase 2, which potentially affect the occurrence of BC through multiple biological processes, such as cell detoxification, glycogen synthesis, and phospholipid metabolism. In conclusion, we discovered numerous genes related to mammary development and breast cancer (BC) through a GWS, which suggests the mechanism of SNTs in goats and a certain association between mammary cancer and SNTs.

Early-Life Stress Influences the Transcriptional Activation of Alpha-2A Adrenergic Receptor and Associated Protein Kinase A Signaling Molecules in the Frontal Cortex of Rats

AbstractEarly life is a highly sensitive period associated with profound changes in brain structure and function. Adverse experiences of early-life stress (ELS) are prominent risk factors for the precipitation of major depressive disorder (MDD). In recent years, dysfunction of the central noradrenergic (NA) system and subsequent deficits in norepinephrine (NE) signaling have gained increasing attention in the pathophysiology of MDD. However, the role of the α-2A adrenergic receptor and its downstream second messenger signaling system has not been investigated in connection to early-life stress-induced depression, limiting valuable insights into neurobiological mechanisms underlying this disorder. In this study, we used maternal separation (MS) as a rodent model of ELS to investigate whether ELS-induced depressive behavior is related to the α-2A adrenergic receptor and its associated second messenger signaling cascade. To do so, we studied expression levels of the α-2A adrenergic receptor (Adra2a), G alpha proteins (stimulatory subunit-Gαs [Gnas] and inhibitory subunit-Gαi [Gnai1 and Gnai2]), and downstream protein kinase A (PKA) catalytic [Prkarcα and Prkarcβ] and regulatory subunits [Prkar1α, Prkar1β, Prkar2α, and Prkar2β]) in the frontal cortex (FC) of MS rats. We found reduced sucrose preference in MS animals, along with reduced transcript levels of Adra2a, Gnai2, Prkar1β, and Prkarcβ. These findings suggest that ELS exposure may contribute to depression symptomatology via alterations in the expression of key genes involved in the NA system, highlighting potential mechanisms underlying ELS-induced depressive behavior.

Hydrogen peroxide disrupts the regulatory pathway of saliva secretion in two salivary acinar rat cell lines

BackgroundSecretion of saliva is controlled by autonomic nerve signals via regulation of Ca2+-dependent ion transport across acinar cell membranes. Oxidative stress may affect this process, leading to a decrease in saliva production. This study investigates elements of the Ca2+ regulatory pathway and their vulnerability to hydrogen peroxide-induced oxidative stress.MethodsRat parotid and submandibular salivary gland acinar cell lines were exposed to different hydrogen peroxide concentrations to simulate oxidative stress. Cell viability and intracellular reactive oxygen species were measured, mRNA levels were assessed via RT-qPCR, and protein expression was studied using western blot and immunofluorescence microscopy.ResultsElevated concentrations of hydrogen peroxide reduced cell viability and increased intracellular levels of reactive oxygen species and led to a decrease in cholinergic receptor muscarinic 3 and adrenoreceptor alpha 1A mRNA and protein levels in both cell lines. In parotid gland cells, both mRNA and protein levels of stromal interaction molecule 1 and Orai1 decreased with increasing concentrations of hydrogen peroxide. In contrast, in submandibular gland cells stromal interaction molecule 1 and Orai1 displayed differential mRNA and protein expression levels.ConclusionOur study revealed that hydrogen peroxide exposure alters rat parotid and submandibular acinar cells, increasing reactive oxygen species and reducing autonomic receptor expression. Differential mRNA and protein expression of stromal interaction molecule 1 and Orai1 highlight complex oxidative stress effects on Ca2⁺ signaling. Most likely these effects will be deleterious to salivary secretion, but some effects may be protective.

Investigating the Effects of Gossypetin on Cardiovascular Function in Diet-Induced Pre-Diabetic Male Sprague Dawley Rats

Gossypetin (GTIN) is a naturally occurring flavonoid recognised for its pharmacological properties. This study examined the effects of GTIN on cardiovascular function in a diet-induced pre-diabetic rat model, which has not been previously studied. Pre-diabetes was induced using a high-fat high-carbohydrate (HFHC) diet supplemented with 15% fructose water for 20 weeks. Thereafter, the pre-diabetic animals were sub-divided into five groups (n = 6), where they were either orally treated with GTIN (15 mg/kg) or metformin (MET) (500 mg/kg), both in the presence and absence of dietary intervention for 12 weeks. The results demonstrated that the pre-diabetic (PD) control group exhibited significantly higher plasma triglyceride, total cholesterol, low-density lipoprotein and very low-density lipoprotein levels, along with decreased high-density lipoprotein (HDL) levels in comparison to the non-pre-diabetic (NPD) group. This was accompanied by significantly higher mean arterial pressure (MAP), body mass index (BMI), waist circumference (WC) and plasma endothelial nitric oxide (eNOS) levels in PD control. Additionally, there were increased heart malondialdehyde levels, reduced heart superoxide dismutase and glutathione peroxidase activity as well as increased plasma interleukin-6, tumour necrosis factor alpha and c-reactive protein levels present in the PD control group. Notably, both GTIN-treated groups showed significantly reduced plasma lipid levels and increased HDL, as well as decreases in MAP, BMI, WC and eNOS levels in comparison to PD control. Additionally, GTIN significantly decreased heart lipid peroxidation, enhanced antioxidant activity and decreased plasma inflammation markers. These findings may suggest that GTIN administration in both the presence and absence of dietary intervention may offer therapeutic potential in ameliorating cardiovascular disturbances associated with the PD state. However, future studies are needed to determine the physiological mechanisms by which GTIN improves cardiovascular function in the PD state.

Enhancing anti-EGFRvIII CAR T cell therapy against glioblastoma with a paracrine SIRPγ-derived CD47 blocker

A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive microenvironment, which is densely populated by protumoral glioma-associated microglia and macrophages (GAMs). Myeloid immune checkpoint therapy targeting the CD47-signal regulatory protein alpha (SIRPα) axis induces GAM phagocytic function, but CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. In this work, we engineer a CAR T cell against epidermal growth factor receptor variant III (EGFRvIII), constitutively secreting a signal regulatory protein gamma-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eradicate orthotopic EGFRvIII-mosaic GBM in vivo, promoting GAM-mediated tumor cell phagocytosis. In a subcutaneous CD19+ lymphoma mouse model, anti-CD19-SGRP CAR T cell therapy is superior to conventional anti-CD19 CAR T. Thus, combination of CAR and SGRP eliminates bystander tumor cells in a manner that could overcome main mechanisms of CAR T cell therapy resistance, including immune suppression and antigen escape.

Mendelian Randomization Estimates the Effects of Plasma and Cerebrospinal Fluid Proteins on Intelligence, Fluid Intelligence Score, and Cognitive Performance.

Observational studies have revealed associations between levels of plasma and cerebrospinal fluid (CSF) proteins and cognition-related traits. However, these associations may be influenced by confounding factors inherent in observational research. This study aims to identify plasma and CSF proteins associated with intelligence, fluid intelligence score, and cognitive performance through the application of Mendelian randomization (MR). Proteomic quantitative trait locus (pQTL) data for plasma and CSF proteins were sourced from existing genome-wide association study (GWAS). Intelligence, fluid intelligence score, and cognitive performance GWAS summary statistics provided comprehensive data for two-sample MR analysis. Extensive sensitivity analyses, including Steiger testing, reverse MR analysis, and Bayesian co-localization, were conducted to validate associations and identify shared genetic variants. Phenotype scanning explored potential pleiotropic effects. MR analysis identified several proteins in plasma and CSF significantly associated with intelligence, fluid intelligence scores, and cognitive performance. For intelligence, negatively associated proteins in plasma include endoplasmic reticulum aminopeptidase 2 (ERAP2) and secretogranin III (SCG3), while positively associated proteins are myeloperoxidase (MPO), signal regulatory protein alpha (SIRPA), regulator of microtubule dynamics 1 (RMDN1), and endoplasmic reticulum lectin 1 (ERLEC1). In CSF, C1-esterase inhibitor and carboxypeptidase E (CBPE) both exhibited positive associations with intelligence. For fluid intelligence scores, negatively associated proteins in plasma are copine 1 (CPNE1) and SCG3, while positively associated proteins are nudix hydrolase 12 (NUDT12) and RMDN1.In CSF, Macrophage Stimulating Protein (MSP) demonstrated a significant negative impact. For cognitive performance, negatively associated proteins in plasma include ERAP2, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), and SCG3, while positively associated proteins are NUDT12, RMDN1, ERLEC1, and ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (ENPP5). In CSF, C1-esterase inhibitor was positively associated, while MSP and soluble tyrosine kinase with immunoglobulin-like and EGF-like domains 1(sTie-1) showed a negative association. Bayesian co-localization analysis revealed significant genetic overlaps between SIRPA, RMDN1, and ERLEC1 in plasma with intelligence; NUDT12 and SCG3 in plasma with fluid intelligence scores; and TIE1, NUDT12, RMDN1, ERLEC1, and ENPP5 in plasma with cognitive performance. Additionally, significant co-localization was identified between C1-esterase inhibitor and CBPE in CSF with intelligence, as well as between C1-esterase inhibitor and sTie-1 in CSF with cognitive performance. Reverse causality analysis confirmed the causal direction from proteins to cognitive traits. This study identifies specific plasma and CSF proteins that significantly impact intelligence, fluid intelligence scores, and cognitive performance. These proteins could serve as biomarkers and targets for future research and therapeutic interventions aimed at sustaining cognitive abilities and reducing impairment risks.

Hepatoprotective effects of the xanthine oxidase inhibitor Febuxostat against thioacetamide-induced liver injury in rats: The role of the Nrf2/ HO-1 and TLR4/ NF-κB pathways

Experimental models of liver injury have been established utilizing thioacetamide (TAA), a classic liver toxic chemical that causes organ damage via oxidative stress and inflammatory induction. This study examined the impact of Febuxostat (a xanthine oxidase inhibitor; Febu, 10–15 mg/kg, orally) against TAA (500 mg/kg, i.p.) -induced liver injury in rats.Febu significantly attenuated TAA-induced alterations in liver function parameters, in addition to promoting hepatic antioxidant effects through a significant elevation of Heme-oxygenase-1(HO-1), nuclear factor erythroid 2-related factor2 (Nrf2), reduced glutathione (GSH) and superoxide dismutase (SOD) levels and reduction in hepatic malondialdehyde (MDA) content. Moreover, Febu improved the hepatic anti-inflammatory status by increasing the anti-inflammatory cytokine Interleukin (IL-10) level and reducing the levels of the pro-inflammatory cytokines (Nuclear factor kappa B (NF-κB), IL-1β, high-mobility group box1 (HMGB1), receptor for advanced glycation end products (RAGE), and toll-like receptor4 (TLR4) levels, in addition to suppressing the increased protein and mRNA expression levels of tumor necrosis factor alpha (TNF-α) and IL-6, hepatic expression of TNF-α and activated mitogen-activated protein kinases (p-JNK/p-p38 MAPK). Histopathologically, Febu markedly normalized TAA-induced alteration in liver sections. In conclusion, Febu, in a dose-dependent fashion, refines TAA-induced hepatotoxicity by enhancing antioxidant capabilities and decreasing inflammatory signals.

Cebpa is required for haematopoietic stem and progenitor cell generation and maintenance in zebrafish.

The CCAAT enhancer binding protein alpha (CEBPA) is crucial for myeloid differentiation and the balance of haematopoietic stem and progenitor cell (HSPC) quiescence and self-renewal, and its dysfunction can drive leukemogenesis. However, its role in HSPC generation has not been fully elucidated. Here, we utilized various zebrafish cebpa mutants to investigate the function of Cebpa in the HSPC compartment. Co-localization analysis showed that cebpa expression is enriched in nascent HSPCs. Complete loss of Cebpa function resulted in a significant reduction in early HSPC generation and the overall HSPC pool during embryonic haematopoiesis. Interestingly, while myeloid differentiation was impaired in cebpa N-terminal mutants expressing the truncated zP30 protein, the number of HSPCs was not affected, indicating a redundant role of Cebpa P42 and P30 isoforms in HSPC development. Additionally, epistasis experiments confirmed that Cebpa functions downstream of Runx1 to regulate HSPC emergence. Our findings uncover a novel role of Cebpa isoforms in HSPC generation and maintenance, and provide new insights into HSPC development.

Integrated omics profiling reveals systemic dysregulation and potential biomarkers in the blood of patients with neuromyelitis optica spectrum disorders

BackgroundNeuromyelitis optica spectrum disorders (NMOSD) are autoimmune conditions that affect the central nervous system. The contribution of peripheral abnormalities to the disease's pathogenesis is not well understood.MethodsTo investigate this, we employed a multi-omics approach analyzing blood samples from 52 NMOSD patients and 46 healthy controls (HC). This included mass cytometry, cytokine arrays, and targeted metabolomics. We then analyzed the peripheral changes of NMOSD, and features related to NMOSD's disease severity. Furthermore, an integrative analysis was conducted to identify the distinguishing characteristics of NMOSD from HC. Additionally, we unveiled the variations in peripheral features among different clinical subgroups within NMOSD. An independent cohort of 40 individuals with NMOSD was utilized to assess the serum levels of fibroblast activation protein alpha (FAP).ResultsOur analysis revealed a distinct peripheral immune and metabolic signature in NMOSD patients. This signature is characterized by an increase in monocytes and a decrease in regulatory T cells, dendritic cells, natural killer cells, and various T cell subsets. Additionally, we found elevated levels of inflammatory cytokines and reduced levels of tissue-repair cytokines. Metabolic changes were also evident, with higher levels of bile acids, lactates, triglycerides, and lower levels of dehydroepiandrosterone sulfate, homoarginine, octadecadienoic acid (FA[18:2]), and sphingolipids. We identified distinctive biomarkers differentiating NMOSD from HC and found blood factors correlating with disease severity. Among these, fibroblast activation protein alpha (FAP) was a notable marker of disease progression.ConclusionsOur comprehensive blood profile analysis offers new insights into NMOSD pathophysiology, revealing significant peripheral immune and metabolic alterations. This work lays the groundwork for future biomarker identification and mechanistic studies in NMOSD, highlighting the potential of FAP as a marker of disease progression.

C/EBPα-mediated ACSL4-dependent ferroptosis exacerbates tubular injury in diabetic kidney disease

Diabetic kidney disease (DKD) is a prevalent and debilitating complication of diabetes characterized by progressive renal function decline and a lack of effective treatment options. Here, we investigated the role of the transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) in DKD pathogenesis. Analysis of renal biopsy samples revealed increased C/EBPα expression in patients with DKD. Using RNA sequencing and proteomics, we explored the mechanisms through which the C/EBPα contributes to DKD. Our findings demonstrated that C/EBPα exacerbated tubular injury by promoting acyl-CoA synthetase long-chain family member 4 (ACSL4)-dependent ferroptosis. We identified that C/EBPα upregulated ACSL4 expression by binding to its transcription regulatory sequence (TRS), leading to elevated lipid peroxidation and ferroptosis. Furthermore, inhibition or genetic ablation of C/EBPα attenuated ferroptosis and mitigated tubular injury in DKD. These results highlighted the C/EBPα-ACSL4-ferroptosis pathway as a promising therapeutic target for DKD treatment.

A Study of JUN’s Promoter Region and Its Regulators in Chickens

Background: The Jun proto-oncogene (JUN), also referred to as C-JUN, is an integral component of the JNK signaling pathway, which is crucial for the formation and differentiation of spermatogonial stem cells (SSCs). Investigations into the transcriptional regulation of chicken JUN can offer a molecular foundation for elucidating its mechanistic role in SSCs. Methods: In this study, we successfully cloned a 2000 bp upstream sequence of the JUN transcription start site and constructed a series of pGL3 recombinant vectors containing JUN promoters of varying lengths. Results: We verified the promoter activity of the 2000 bp upstream sequence by assessing the fluorescence intensity of DF-1 and identified the promoter activities of different regions via dual-luciferase assays. The transcription of JUN and its promoter region spanning −700 to 0 bp was modulated by an activator of the JNK signaling pathway. Bioinformatics analysis revealed that this −700 to 0 bp region was highly conserved among avian species and predicted the presence of binding sites for Wilms tumor 1 (WT1) and CCAAT/enhancer binding protein alpha (CEBPA). The JNK signaling pathway activator was found to upregulate the expression of these transcription factors in DF-1 cells. Through the deletion of binding sites and the overexpression of WT1 and CEBPA, we demonstrated that WT1 inhibited the transcription of JUN, while CEBPA promoted it. Conclusions: In conclusion, the −700 to 0 bp region is the key region of the JUN promoter, with WT1 inhibiting JUN transcription. The results of the study not only provide ideas for exploring the regulatory mechanism of JUN in chicken SSCs, but also lay an important foundation for the study of avian SSCs.

Cryo-electron microscopy reveals a single domain antibody with a unique binding epitope on fibroblast activation protein alpha.

Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues but is overexpressed in a variety of pathologies, including cancer. Despite this unique expression profile, designing effective diagnostic and therapeutic agents that effectively target this biomarker remain elusive. Here we report the structural characterization of the interaction between a novel single domain antibody (sdAbs), I3, and FAP using cryo-electron microscopy. The reconstructions were determined to a resolution of 2.7 Å and contained two distinct populations; one I3 bound and two I3 molecules bound to the FAP dimer. In both cases, the sdAbs bound a unique epitope that was distinct from the active site of the enzyme. Furthermore, this report describes the rational mutation of specific residues within the complementarity determining region 3 (CDR3) loop to enhance affinity and selectivity of the I3 molecule for FAP. This report represents the first sdAb-FAP structure to be described in the literature.

Werner syndrome RECQ helicase participates in and directs maintenance of the protein complexes of constitutive heterochromatin in proliferating human cells.

Werner syndrome of premature aging is caused by mutations in the WRN RECQ helicase/exonuclease, which functions in DNA replication, repair, transcription, and telomere maintenance. How the loss of WRN accelerates aging is not understood in full. Here we show that WRN is necessary for optimal constitutive heterochromatin levels in proliferating human fibroblasts. Locally, WRN deficiency derepresses SATII pericentromeric satellite repeats but does not reduce replication fork progression on SATII repeats. Globally, WRN loss reduces a subset of protein-protein interactions responsible for the organization of constitutive heterochromatin in the nucleus, namely, the interactions involving Lamin B1 and Lamin B receptor, LBR. Both the mRNA level and subcellular distribution of LBR are affected by WRN deficiency, and unlike the former, the latter phenotype does not require WRN catalytic activities. The phenotypes of heterochromatin disruption seen in WRN-deficient proliferating fibroblasts are also observed in WRN-proficient fibroblasts undergoing replicative or oncogene-induced senescence. WRN interacts with histone deacetylase 2, HDAC2; WRN/HDAC2 association is mediated by heterochromatin protein alpha, HP1α, and WRN complexes with HP1α and HDAC2 are downregulated in senescing cells. The data suggest that the effect of WRN loss on heterochromatin is separable from senescence program, but mimics at least some of the heterochromatin changes associated with it.

Proteomics Reveals Divergent Cardiac Inflammatory and Metabolic Responses After Inhalation of Ambient Particulate Matter With or Without Ozone.

Inhalation of ambient particulate matter (PM) and ozone (O3) has been associated with increased cardiovascular morbidity and mortality. However, the interactive effects of PM and O3 on cardiac dysfunction and disease have not been thoroughly examined, especially at a proteomic level. The purpose of this study was to identify and compare proteome changes in spontaneously hypertensive (SH) rats co-exposed to concentrated ambient particulates (CAPs) and O3, with a focus on investigating inflammatory and metabolic pathways, which are the two major ones implicated in the pathophysiology of cardiac dysfunction. For this, we measured and compared changes in expression status of 9 critical pro- and anti-inflammatory cytokines using multiplexed ELISA and 450 metabolic proteins involved in ATP production, oxidative phosphorylation, cytoskeletal organization, and stress response using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) in cardiac tissue of SH rats exposed to CAPs alone, O3 alone, and CAPs + O3. Proteomic expression profiling revealed that CAPs alone, O3 alone, and CAPs + O3 differentially altered protein expression patterns, and utilized divergent mechanisms to affect inflammatory and metabolic pathways and responses. Ingenuity Pathway Analysis (IPA) of the proteomic data demonstrated that the metabolic protein network centered by gap junction alpha-1 protein (GJA 1) was interconnected with the inflammatory cytokine network centered by nuclear factor kappa beta (NF-kB) potentially suggesting inflammation-induced alterations in metabolic pathways, or vice versa, collectively contributing to the development of cardiac dysfunction in response to CAPs and O3 exposure. These findings may enhance understanding of the pathophysiology of cardiac dysfunction induced by air pollution and provide testable hypotheses regarding mechanisms of action.

Searching for New Biomarkers of Neuroendocrine Tumors: A Comparative Analysis of Chromogranin A and Inflammatory Cytokines in Patients with Neuroendocrine Tumors.

Neuroendocrine neoplasms (NENs) present a diagnostic challenge due to their heterogeneous nature and non-specific clinical manifestations. This study aimed to explore novel biomarkers for NENs. Serum chromogranin A (CgA) levels and a panel of 48 inflammatory cytokines were analyzed in a cohort of 84 NEN patients and 40 healthy controls using enzyme-linked immunosorbent assay (ELISA) and multiplex ELISA. Significant alterations in cytokine levels were observed in the NEN patients compared to the controls, including elevated levels of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha (TNF-α), and reduced levels of angiogenic factors like platelet-derived growth factor-BB (PDGF-BB) and tumor necrosis factor beta (TNF-β). Notably, cytokines such as growth-regulated alpha protein (GRO-α) and TNF-β demonstrated strong potential as diagnostic markers, with receiver operating characteristic (ROC) curve analyses showing high sensitivity and specificity. Additionally, a positive correlation was found between CgA levels and several inflammatory cytokines, suggesting their synergistic role in tumor progression. These findings highlight the limited reliability of CgA alone as a diagnostic marker and underscore the importance of a multi-marker approach in diagnosing and monitoring NENs. Further research on a larger cohort is necessary to validate these biomarkers and their potential clinical applications.

A comprehensive analysis of CEBPA on prognosis and function in uterine corpus endometrial carcinoma

Uterine corpus endometrial carcinoma (UCEC) is one of the most common tumours of the female reproductive system. CCAAT enhancer-binding protein alpha (CEBPA) is a member of the transcription factor family involved in regulating processes such as cell proliferation, differentiation, metabolism, and the immune response. However, the role of CEBPA in UCEC has not been clarified. Here, we performed a comprehensive analysis to explore the expression level, prognostic value, immune infiltration and biological function of CEBPA in UCEC. In this study, we found that CEBPA expression was upregulated and associated with poor prognosis in UCEC patients. KEGG and GO analyses revealed that the genes positively correlated with CEBPA were enriched primarily in immune regulation and oxidative phosphorylation. Immune infiltration analysis revealed that CEBPA is strongly correlated with immune cell infiltration in UCEC. RT-qPCR indicated that CEBPA may regulate the OXPHOS level in Ishikawa cells. CCK-8, cell cycle, Transwell and scratch wound healing assays revealed that CEBPA promoted Ishikawa cell proliferation, invasion and migration. In addition, PPI and survival analyses suggested that CEBPG may be a potential target of CEBPA in UCEC. These results demonstrated that CEBPA may be a potential therapeutic target in UCEC.

Combining mathematical modeling, in vitro data and clinical target expression to support bispecific antibody binding affinity selection: a case example with FAP-4-1BBL.

The majority of bispecific costimulatory antibodies in cancer immunotherapy are capable of exerting tumor-specific T-cell activation by simultaneously engaging both tumor-associated targets and costimulatory receptors expressed by T cells. The amount of trimeric complex formed when the bispecific antibody is bound simultaneously to the T cell receptor and the tumor-associated target follows a bell-shaped curve with increasing bispecific antibody exposure/dose. The shape of the curve is determined by the binding affinities of the bispecific antibody to its two targets and target expression. Here, using the case example of FAP-4-1BBL, a fibroblast activation protein alpha (FAP)-directed 4-1BB (CD137) costimulator, the impact of FAP-binding affinity on trimeric complex formation and pharmacology was explored using mathematical modeling and simulation. We quantified (1) the minimum number of target receptors per cell required to achieve pharmacological effect, (2) the expected coverage of the patient population for 19 different solid tumor indications, and (3) the range of pharmacologically active exposures as a function of FAP-binding affinity. A 10-fold increase in FAP-binding affinity (from a dissociation constant [KD] of 0.7 nM-0.07nM) was predicted to reduce the number of FAP receptors needed to achieve 90% of the maximum pharmacological effect from 13,400 to 4,000. Also, the number of patients with colon cancer that would achieve 90% of the maximum effect would increase from 6% to 39%. In this work, a workflow to select binding affinities for bispecific antibodies that integrates preclinical in vitro data, mathematical modeling and simulation, and knowledge on target expression in the patient population, is provided. The early implementation of this approach can increase the probability of success with cancer immunotherapy in clinical development.

Coffee Bioactive N-Methylpyridinium: Unveiling Its Antilipogenic Effects by Targeting De Novo Lipogenesis in Human Hepatocytes.

Type 2 diabetes and nonalcoholic fatty liver diseases (NAFLDs) are promoted by insulin resistance (IR), which alters lipid homeostasis in the liver. This study aims to investigate the effect of N-methylpyridinium (NMP), a bioactive alkaloid of coffee brew, on lipid metabolism in hepatocytes. The effect of NMP in modulating lipid metabolism is evaluated at physiological concentrations in a diabetes cell model represented by HepG2 cells cultured in a high-glucose medium. Hyperglycemia triggers lipid droplet accumulation in cells and enhances the lipogenic gene expression, which is transactivated by sterol regulatory element binding protein-1 (SREBP-1). Lipid droplet accumulation alters the redox status and endoplasmic reticulum (ER) stress, leading to the activation of the unfolded protein response and antioxidative pathways by X-Box Binding Protein 1(XBP-1)/eukaryotic Initiation Factor 2 alpha (eIF2α) Protein Kinase RNA-Like ER Kinase and nuclear factor erythroid 2-related factor 2 (NRF2), respectively. NMP induces the phosphorylation of AMP-dependent protein kinase (AMPK) and acetyl-CoA carboxylase α (ACACA), and improves the redox status and ER homeostasis, essential steps to reduce lipogenesis and lipid droplet accumulation. These results suggest that NMP may be beneficial for the management of T2D and NAFLD by ameliorating the cell oxidative and ER homeostasis and lipid metabolism.

The role of S-palmitoylation of C4BPA in regulating murine sperm motility and complement resistance

The epididymis and epididymosomes are crucial for regulating sperm motility, a key factor in male fertility. Palmitoylation, a lipid modification involving the attachment of palmitic acid to cysteine residues, is essential for protein function and localization. Additionally, this modification plays a vital role in the sorting of proteins into exosomes. This study investigates the role of S-palmitoylation at the Cys15 residue of the C4b binding protein alpha chain (C4BPA) in murine sperm motility. Our findings revealed high expression of C4BPA mRNA in the caput epididymis, with the protein present across all regions of the epididymis. Palmitoylation of C4BPA in epididymal epithelial cells was essential for its enrichment in epididymosomes and on sperm, thereby maintaining sperm motility. Inhibition of palmitoylation significantly reduced sperm motility and the localization of C4BPA on sperm. Additionally, palmitoylated C4BPA in exosomes resisted complement C4 attacks, preserving motility, unlike mutated C4BPA (C15S). These results highlight the critical role of palmitoylated C4BPA in protecting sperm from complement attacks and maintaining motility, suggesting that reversible palmitoylation of epididymal proteins could be explored as a therapeutic strategy for male contraception. Our study underscores the importance of post-translational modifications in sperm function and presents new insights into potential male contraceptive methods.

Combined PET Radiotracer Approach Reveals Insights into Stromal Cell-Induced Metabolic Changes in Pancreatic Cancer In Vitro and In Vivo.

Background/Objective Pancreatic stellate cells (PSCs) in pancreatic adenocarcinoma (PDAC) are producing extracellular matrix, which promotes the formation of a dense fibrotic microenvironment. This makes PDAC a highly heterogeneous tumor-stroma-driven entity, associated with reduced perfusion, limited oxygen supply, high interstitial fluid pressure, and limited bioavailability of therapeutic agents. Methods In this study, spheroid and tumor xenograft models of human PSCs and PanC-1 cells were characterized radiopharmacologically using a combined positron emission tomography (PET) radiotracer approach. [18F]FDG, [18F]FMISO, and [18F]FAPI-74 were employed to monitor metabolic activity, hypoxic metabolic state, and functional expression of fibroblast activation protein alpha (FAPα), a marker of activated PSCs. Results In vitro, PanC-1 and multi-cellular tumor spheroids demonstrated comparable glucose uptake and hypoxia, whereas FAPα expression was significantly higher in PSC spheroids. In vivo, glucose uptake as well as the transition to hypoxia were comparable in PanC-1 and multi-cellular xenograft models. In mice injected with PSCs, FAPα expression decreased over a period of four weeks post-injection, which was attributed to the successive death of PSCs. In contrast, FAPα expression increased in both PanC-1 and multi-cellular xenograft models over time due to invasion of mouse fibroblasts. Conclusion The presented models are suitable for subsequently characterizing stromal cell-induced metabolic changes in tumors using noninvasive molecular imaging techniques.