Alpha Subunit Research Articles

Weighted Gene Co-expression Network Analysis of the Inflammatory Wnt Signaling Reveals Biomarkers Related to Bone Formation.

Background and aim Osteocytes regulate bone metabolism and balance through various mechanisms, including the Wnt (Wingless-related integration site signal transduction) signaling pathway. Weighted gene co-expression network analysis (WGCNA) is a computational method to identify functionally related genes based on expression patterns, especially in the Wnt-beta-catenin and osteo-regenerative pathways. This study aims to analyze gene modules of the Wnt signaling pathway from WGCNA analysis. Methods The study used a microarray dataset from the GEO (GSE228306) to analyze differential gene expression in human primary monocytes. The study standardized datasets usingRobust Multi-Array Average (RMA)expression measure and Integrated Differential Expression and Pathway (IDEP) analysistool, building a co-expression network for group-specific component (GC) genes. Results The study uses WGCNA to identify co-expression modules with dysregulated mRNAs, revealing enrichment in Wnt-associated pathways and top hub-enriched genes like colony-stimulating factor 3 (CSF3), interleukin-6 (IL-6), IL-23 subunit alpha (IL23A), suppressor of cytokine signaling 1 (SOCS1), and C-C motif chemokine ligand 19 (CCL19). Conclusion WGCNA analysis of the Wnt signaling pathway will involve functional annotation, network visualization, validation, integration with other omics data, and addressing method limitations for better understanding.

Association between PIK3CA mutations and disease-free survival in patients with HR+, HER2- tumors treated with adjuvant hormonal therapy: A real-world study in Croatia.

e12512 Background: Disease recurrence of early hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast tumor subtype is particularly challenging to manage due to its complex and very heterogeneous biological nature. Namely, due to primary and secondary resistance, one-quarter of patients with early disease will present with the disease recurrence. This variability in the timing of recurrence highlights the need to better identify key biomarkers that could predict therapeutic outcomes and guide personalized treatment strategies for these patients. Mutations in the phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene have a high prevalence of 30% to 40% in HR+/HER2- advanced breast cancer. They lead to activation of the PI3K/AKT/mTOR pathway, promoting cell growth, and proliferation, and are associated with poor prognosis in advanced breast cancer. Our aim was to examine this association and impact of PIK3CA mutation status on disease free survival (DFS) in HR+/ HER2- early breast cancer patients. Methods: We conducted a multicentric, retrospective cohort study in five Croatian institutions from July 2020 to December 2021. Study included initially early HR+/HER2- breast cancer patients who have been diagnosed with disease recurrence during adjuvant hormonal treatment or within first six years of follow up. The Cobas PIK3CA Mutation Testing Kit was used for real-time PCR-based amplification and mutation detection on the Cobas z 480 analyzer. We have analyzed differences in DFS from initiation of adjuvant endocrine therapy, using Cox, proportional hazard regression. Results: There was total of 186 patients, out of which 40.9% tested positive for the PIK3CA mutation. Primary and adjuvant treatment and in particular adjuvant endocrine treatment was similar between the two groups. After adjustment for 14 relevant covariates, we found that patients with positive PIK3CA status and with H1047 PIK3CA mutation had significantly lower hazard for disease recurrence in comparison to patients with no PIK3CA mutation (HR 0.65; 95% CI 0.45; 0.95; p = 0.024; HR 6.52; 95% CI 1.15; 36.95; p = 0.034 respectively; false discovery rate, FDR < 10%) and higher odds for late recurrence (p = 0.001, FDR < 5%). Conclusions: This study highlights the potential impact of PIK3CA mutations on disease recurrence during/following adjuvant endocrine treatment and by that potentially opens the door for further investigation of possibly more personalized treatment strategies.

Development of a prediction model for lung metastasis by using postoperative circulating tumor DNA binding clinicopathological risk factors in patients with colorectal cancer.

e15035 Background: Lung metastasis is common in patients with colorectal cancer (CRC). However, diagnosing lung metastasis in CRC is challenging due to slow-growing pulmonary lesions. Circulating tumor DNA (ctDNA) testing provides a direct indication of the presence of minimal residual disease (MRD) which contributing to cancer recurrence. Yet, there are no reports on the potential indications of ctDNA for the site of metastasis. The objective of this study is to develop a predictive model for lung metastasis in CRC patients who have received adjuvant therapy by integrating ctDNA with clinicopathological factors. Methods: Two independent cohorts' series were evaluated including 299 patients from China as training cohort and 134 patients from Spain as validation cohort from 2012-2020. We specifically focused on the detection of ctDNA in these patients. We employed LASSO regression for optimal feature selection and built a predictive model by multivariable logistic regression. Results: In training cohort, 59/299(19.7%) patients have detectable ctDNA. In validation cohort, 24/134 (18.6%) patients have detectable ctDNA. Patients who tested ctDNA-positive had a significantly higher incidence of lung metastasis (33.9% vs. 11.2%, P<0.001) compared to ctDNA-negative patients. A predictive model for lung metastasis was developed by multivariable logistic regression analysis. The model incorporated factors such as ctDNA status, preoperative carcinoembryonic antigen (CEA) level, presence of cancerous nodes, and the mutation status of Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) in plasma. The lung metastasis prediction nomogram demonstrated a favorable ability to distinguish with a concordance index of 0.775 (95% CI: 0.706-0.775) during internal validation. The Hosmer-Lemeshow test, with p-value of 0.967, indicated that prediction nomogram model had a good fit. In addition, the prediction model of validation cohort demonstrated a discriminative performance with a concordance index of 0.724(95% CI: 0.698-0.724). Furthermore, the DCA (Decision Curve Analysis) and CIC (Clinical Impact Curve) in both cohorts indicated that the lung metastasis nomogram exhibited a favorable clinical net benefit. Finally, through multi-model comparison, it was found that the predictive performance of the model, which includes ctDNA and blood PIK3CA mutation status, was significantly superior to all clinical prediction models. Conclusions: A lung metastasis prediction nomogram was developed by integrating postoperative ctDNA and clinicopathological risk factors. The purpose of this nomogram is to assist clinicians in predicting the probability of lung metastasis in patients who have undergone surgery for CRC, with the aim to facilitate prompt intervention.

Prevalence of somatic PIK3CA mutations in hormone receptor-positive metastatic breast cancer: The Indian experience.

e13047 Background: The phosphatidylinositol 3-kinase (PI3K) inhibitors have been found to improve survival outcomes in hormone receptor-positive metastatic breast cancer (MBC) and are FDA-approved in this setting after progression on hormonal therapy. However, real-world data from India on the prevalence of PIK3CA mutations is scarce. Methods: This is a retrospective study including patients with hormone receptor-positive breast cancer registered at our center between January 2017- and December 2023 who developed metastatic disease on follow-up after getting appropriate treatment (adjuvant /neoadjuvant) as per institutional protocol. The PIK3CA mutation testing was done on the biopsy specimen. FDA-approved therascreen PIL3CA RGQ PCR kit from Qiagen was used and the assay is a real-time qualitative PCR test for the detection of 11 mutations phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene (Exon 7: C420R; Exon 9: E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y) using genomic DNA (gDNA) extracted from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue. The clinical demographics of patients with PIK3CA mutations were extracted from the hospital database and represented descriptively. Results: A total of 80 consecutive patients with hormone-positive breast cancer at relapse were screened and 26 were found to have PIK3CA mutations (32.5%) of which 24 were female (92.3%). The median age of patients with mutated PIK3CA cases was 50 years (range: 31 to 72 years). The clinical stage of disease at presentation was: stage II in 11.5%, IIIA in 23.1%, and IIIB in 65.4% of cases. The median time of relapse was 21 months from diagnosis (range: 10 to 42 months). The disease sites at relapse were multiple in 23 patients (88.46%) [including central nervous system (CNS) relapse in 3 patients) and CNS (brain) only in 3 patients (11.54%). The mutations detected included H1047R (3140A>G at exon 20) in 8 cases, E545K (1633 G>A at exon 9) in 5 cases, Q546R (1637 A>G at exon 9) in 4 cases, E542K (1624 G>A at exon7) in 3 cases and synchronous mutations (E542K/E545K at Exon 7) in 3 cases and (H1047R/H1047L at exon 20 ) in 3 cases. Only eight patients received alpelisib with fulvestrant due to financial constraints. Conclusions: This is the first Indian study that describes the prevalence of PIK3CA mutations in hormone-positive breast cancer at relapse. Around one-third of patients harbor this mutation and 23.1% of cases have CNS involvement at relapse. The most common PIK3CA mutation detected was H1047R (exon 20) followed by E545K (exon 9).

Comprehensive Glycomic and Proteomic Analysis of Mouse Striatum and Lateral Hypothalamus Following Repeated Exposures to Cocaine or Methamphetamine

Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry–based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adeno-associated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascin-R, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.

Single-Base Gene Variants in MIR-146A and SCN1A Genes Related to the Epileptogenic Process in Drug-Responsive and Drug-Resistant Temporal Lobe Epilepsy-A Preliminary Study in a Brazilian Cohort Sample.

The drug-resistant temporal lobe epilepsy (TLE) has recently been associated with single nucleotide variants (SNVs) in microRNA(miR)-146a (MIR-146A) (rs2910164) and Sodium Voltage-Gated Channel Alpha Subunit 1 (SCN1A) (rs2298771 and rs3812718) genes. Moreover, no studies have shown an association between these SNVs and susceptibility to drug-resistant and drug-responsive TLE in Brazil. Thus, deoxyribonucleic acid (DNA) samples from 120 patients with TLE (55 drug-responsive and 65 drug-resistant) were evaluated by real-time polymerase chain reaction (RT-PCR). A total of 1171 healthy blood donor individuals from the Online Archive of Brazilian Mutations (ABraOM, from Portuguese Arquivo Brasileiro On-line de Mutações), a repository containing genomic variants of the Brazilian population, were added as a control population for the studied SNVs. MIR-146A and SCN1A relative expression was performed by quantitative RT-PCR (qRT-PCR). The statistical analysis protocol was performed using an alpha error of 0.05. TLE patient samples and ABraOM control samples were in Hardy-Weinberg equilibrium for all studied SNVs. For rs2910164, the frequencies of the homozygous genotype (CC) (15.00% vs. 9.65%) and C allele (37.80% vs. 29.97%) were superior in patients with TLE compared to controls with a higher risk for TLE disease [odds ratio (OR) = 1.89 (95% confidence interval (95%CI) = 1.06-3.37); OR = 1.38 (95%CI = 1.04-1.82), respectively]. Drug-responsive patients also presented higher frequencies of the CC genotype [21.81% vs. 9.65%; OR = 2.58 (95%CI = 1.25-5.30)] and C allele [39.09% vs. 29.97%; OR = 1.50 (95%CI = 1.01-2.22)] compared to controls. For rs2298771, the frequency of the heterozygous genotype (AG) (51.67% vs. 40.40%) was superior in patients with TLE compared to controls with a higher risk for TLE disease [OR = 2.42 (95%CI = 1.08-5.41)]. Drug-resistant patients presented a higher AG frequency [56.92% vs. 40.40%; OR = 3.36 (95%CI = 1.04-17.30)] compared to the control group. For rs3812718, the prevalence of genotypes and alleles were similar in both studied groups. The MIR-146A relative expression level was lower in drug-resistant compared to drug-responsive patients for GC (1.6 vs. 0.1, p-value = 0.049) and CC (1.8 vs. 0.6, p-value = 0.039). Also, the SCN1A relative expression levels in samples from TLE patients were significantly higher in AG [2.09 vs. 1.10, p-value = 0.038] and GG (3.19 vs. 1.10, p-value < 0.001) compared to the AA genotype. In conclusion, the rs2910164-CC and rs2298771-AG genotypes are exerting significant risk influence, respectively, on responsive disease and resistant disease, probably due to an upregulated nuclear factor kappa B (NF-kB) and SCN1A loss of function.

Glycolysis and beyond in glucose metabolism: exploring pulmonary fibrosis at the metabolic crossroads

At present, pulmonary fibrosis (PF) is a prevalent and irreversible lung disease with limited treatment options, and idiopathic pulmonary fibrosis (IPF) is one of its most common forms. Recent research has highlighted PF as a metabolic-related disease, including dysregulated iron, mitochondria, lipid, and glucose homeostasis. Systematic reports on the regulatory roles of glucose metabolism in PF are rare. This study explores the intricate relationships and signaling pathways between glucose metabolic processes and PF, delving into how key factors involved in glucose metabolism regulate PF progression, and the interplay between them. Specifically, we examined various enzymes, such as hexokinase (HK), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), pyruvate kinase (PK), and lactate dehydrogenase (LDH), illustrating their regulatory roles in PF. It highlights the significance of lactate, alongside the role of pyruvate dehydrogenase kinase (PDK) and glucose transporters (GLUTs) in modulating pulmonary fibrosis and glucose metabolism. Additionally, critical regulatory factors such as transforming growth factor-beta (TGF-β), interleukin-1 beta (IL-1β), and hypoxia-inducible factor 1 subunit alpha (HIF-1α) were discussed, demonstrating their impact on both PF and glucose metabolic pathways. It underscores the pivotal role of AMP-activated protein kinase (AMPK) in this interplay, drawing connections between diabetes mellitus, insulin, insulin-like growth factors, and peroxisome proliferator-activated receptor gamma (PPARγ) with PF. This study emphasizes the role of key enzymes, regulators, and glucose transporters in fibrogenesis, suggesting the potential of targeting glucose metabolism for the clinical diagnosis and treatment of PF, and proposing new promising avenues for future research and therapeutic development.

Enhancing intraneural revascularization following peripheral nerve injury through hypoxic Schwann-cell-derived exosomes: an insight into endothelial glycolysis

BackgroundEndothelial cell (EC)-driven intraneural revascularization (INRV) and Schwann cells-derived exosomes (SCs-Exos) both play crucial roles in peripheral nerve injury (PNI). However, the interplay between them remains unclear. We aimed to elucidate the effects and underlying mechanisms of SCs-Exos on INRV following PNI.ResultsWe found that GW4869 inhibited INRV, as well as that normoxic SCs-Exos (N-SCs-Exos) exhibited significant pro-INRV effects in vivo and in vitro that were potentiated by hypoxic SCs-Exos (H-SCs-Exos). Upregulation of glycolysis emerged as a pivotal factor for INRV after PNI, as evidenced by the observation that 3PO administration, a glycolytic inhibitor, inhibited the INRV process in vivo and in vitro. H-SCs-Exos more significantly enhanced extracellular acidification rate/oxygen consumption rate ratio, lactate production, and glycolytic gene expression while simultaneously suppressing acetyl-CoA production and pyruvate dehydrogenase E1 subunit alpha (PDH-E1α) expression than N-SCs-Exos both in vivo and in vitro. Furthermore, we determined that H-SCs-Exos were more enriched with miR-21-5p than N-SCs-Exos. Knockdown of miR-21-5p significantly attenuated the pro-glycolysis and pro-INRV effects of H-SCs-Exos. Mechanistically, miR-21-5p orchestrated EC metabolism in favor of glycolysis by targeting von Hippel-Lindau/hypoxia-inducible factor-1α and PDH-E1α, thereby enhancing hypoxia-inducible factor-1α-mediated glycolysis and inhibiting PDH-E1α-mediated oxidative phosphorylation.ConclusionThis study unveiled a novel intrinsic mechanism of pro-INRV after PNI, providing a promising therapeutic target for post-injury peripheral nerve regeneration and repair.Graphical

SIRT5-mediated PRKAA2 succinylation ameliorates apoptosis of human placental trophoblasts in hypertensive disorder complicating pregnancy

ABSTRACT Purpose Hypertensive disorder complicating pregnancy (HDCP) is a serious clinical disorder syndrome during pregnancy. This study aims at finding novel targets for HDCP therapy. Methods HDCP-related mRNAs were firstly screened out and subjected to gene enrichment analysis. We chose protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2) as the research object. Thirty-nine HDCP patients at 32 to 40 weeks of gestation were selected as the HDCP group, and 39 normal controls who received cesarean section delivery at 37–42 weeks of pregnancy were enrolled in this study. Chorionic villi samples were collected within 30 min of delivery. The apoptosis of isolated placental trophoblasts was monitored to investigate the regulatory role of PRKAA2. Results PRKAA2 expression was further proven to be enhanced in the placental tissues of HDCP patients compared with that of normal puerpera. Subsequently, the results of flow cytometry analysis and western blot indicated that PRKAA2 overexpression accelerated primary placental cell apoptosis, while its knockdown attenuated cell apoptosis. Mechanistically, we determined that the level of PRKAA2 succinylation was elevated in the placental tissue of HDCP patients. Through in vitro succinylation assay and mutagenesis, we confirmed that sirtuin 5 (SIRT5) interacts with PRKAA2 at K69 and K260 to induce PRKAA2 desuccinylation. SIRT5 regulated primary HDCP cell apoptosis through PRKAA2. Finally, the animal study revealed that PRKAA2 elevates the systolic blood pressure of HDCP rat model. Conclusion Our findings indicated that SIRT5-mediated PRKAA2 succinylation modulates placental cell apoptosis in HDCP, suggesting that PRKAA2 is a potential therapeutic target for HDCP treatment.

In vitro identification of single-stranded DNA aptamers targeting human IL-23 using the protein-SELEX strategy

Interleukin (IL)-23 inhibitor monoclonal antibodies shown significant efficacy in treating autoimmune diseases. DNA or RNA aptamers exhibit comparable specificity to antibodies, are cost-effective, non-immunogenic, and do not have batch to batch variation. This study aimed to characterize a single-stranded DNA (ssDNA) aptamer targeting human IL-23. The alpha subunit of IL-23 (P19) and intact IL-23 were cloned, expressed, and the proteins finally were purified through Ni2+-iminodiacetic acid affinity chromatography. The selection and characterization of ssDNA aptamer against P19 were conducted using the protein-systematic evolution of ligands by exponential enrichment (SELEX). Dot blot assay was carried out to monitor binding of the aptamer output of SELEX rounds, to P19 protein. The dissociation constant (Kd) of aptamers with positive results in dot blot assay, determined based on their binding to IL-23 using an ELISA method. Recombinant P19 and IL-23 proteins were 26 and 72 kDa, respectively, observed on SDS-PAGE .12 %. The aptamers output from 7, 8, 9, 10, 11, and 12 rounds of the SELEX was monitored by dot blot assay, revealing that the aptamer from the round 8 has stronger luminescent signal and was selected for TA-cloning. After analyzing the biotinylated aptamers from clones, positive clones in dot blot assay and ELISA were sequenced. Finally, the Kd calculation revealed three aptamers with high affinity, named A23P3, A23P6, and A23P15 with Kd values of 1.37, 2.139, and 2.88 nM, respectively. Results of this study introduced three specific anti-IL-23 ssDNA aptamers with high affinity, which could be utilized for therapeutic and diagnostic purposes.

Polymorphisms of ITGA9 Gene and Their Correlation with Milk Quality Traits in Yak (Bos grunniens).

A single-nucleotide polymorphism (SNP) is a genome-level trait that arises from a variation in a single nucleotide, leading to diversity in DNA sequences. SNP screening is commonly used to provide candidate genes for yak breeding efforts. Integrin Subunit Alpha 9 (ITGA9) is an integrin protein. It plays an important role in cell adhesion, signalling, and other processes. The aim of this study was to discuss the association between genetic polymorphisms in the ITGA9 gene and milk quality traits and to identify potential molecular marker loci for yak breeding quality. We genotyped 162 yaks using an Illumina Yak cGPS 7K liquid chip and identified the presence of polymorphisms at nine SNP loci in the ITGA9 gene of yaks. The results showed that the mutant genotypes in the loci g.285,808T>A, g.306,600T>C, and g.315,413C>T were positively correlated with the contents of casein, protein, total solids (TS), and solid nonfat (SNF) in yak milk. In other loci, heterozygous genotypes had a positive correlation with nutrient content in yak milk. Then, two ITGA9 haplotype blocks were constructed based on linkage disequilibrium, which facilitated a more accurate screening of ITGA9 as a candidate gene for yak milk quality improvement. In conclusion, we identified SNPs and haplotype blocks related to yak milk quality traits and provided genetic resources for marker-assisted selection in yak breeding.

Gαz-independent and -dependent Improvements With EPA Supplementation on the Early Type 1 Diabetes Phenotype of NOD Mice.

Prostaglandin E2 (PGE2) is a key mediator of inflammation and is derived from the omega-6 polyunsaturated fatty acid, arachidonic acid (AA). In the β-cell, the PGE2 receptor, Prostaglandin EP3 receptor (EP3), is coupled to the unique heterotrimeric G protein alpha subunit, Gɑz to reduce the production of cyclic adenosine monophosphate (cAMP), a key signaling molecule that activates β-cell function, proliferation, and survival pathways. Nonobese diabetic (NOD) mice are a strong model of type 1 diabetes (T1D), and NOD mice lacking Gɑz are protected from hyperglycemia. Therefore, limiting systemic PGE2 production could potentially improve both the inflammatory and β-cell dysfunction phenotype of T1D. Here, we sought to evaluate the effect of eicosapentaenoic acid (EPA) feeding, which limits PGE2 production, on the early T1D phenotype of NOD mice in the presence and absence of Gαz. Wild-type and Gαz knockout NOD mice were fed a control or EPA-enriched diet for 12 weeks, beginning at age 4 to 5 weeks. Oral glucose tolerance, splenic T-cell populations, islet cytokine/chemokine gene expression, islet insulitis, measurements of β-cell mass, and measurements of β-cell function were quantified. EPA diet feeding and Gɑz loss independently improved different aspects of the early NOD T1D phenotype and coordinated to alter the expression of certain cytokine/chemokine genes and enhance incretin-potentiated insulin secretion. Our results shed critical light on the Gαz-dependent and -independent effects of dietary EPA enrichment and provide a rationale for future research into novel pharmacological and dietary adjuvant therapies for T1D.

The saccus vasculosus of the neotropical cichlid fish Cichlasoma dimerus: characterization, developmental studies and its response to photoperiod.

The saccus vasculosus is an organ present in gnathostome fishes, located ventral to the hypothalamus and posterior to the pituitary gland, whose structure is highly variable among species. In some fishes, this organ is well-developed; however, its physiological function is still under debate. Recently, it has been proposed that this organ is a seasonal regulator of reproduction. In the present work, we examined the histology, ultrastructure, and development of the saccus vasculosus in Cichlasoma dimerus. In addition, immunohistochemical studies of proteins related to reproductive function were performed. Finally, the potential response of this organ to different photoperiods was explored. C. dimerus presented a well-developed saccus vasculosus consisting of a highly folded epithelium, composed of coronet and supporting cells, closely associated with blood vessels, and a highly branched lumen connected to the third ventricle. Coronet cells showed all the major characteristics described in other fish species. In addition, some of the vesicles of the globules were positive for thyrotropin beta subunit, while luteinizing hormone beta subunit immunostaining was observed at the edge of the apical processes of some coronet cells. Furthermore, neuropeptide Y and gonadotropin inhibitory hormone innervation in the saccus vasculosus of C. dimerus were shown. Finally, animals exposed to the long photoperiod showed lower levels of thyrotropin beta and common alpha subunits expression in the saccus compared to those of animals exposed to short photoperiod. All these results support the hypothesis that the saccus vasculosus is involved in the regulation of reproductive function in fish.

The predictive value of TNF family for pulmonary tuberculosis: a pooled causal effect analysis of multiple datasets.

Despite extensive research on the relationship between pulmonary tuberculosis (PTB) and inflammatory factors, more robust causal evidence has yet to emerge. Therefore, this study aims to screen for inflammatory proteins that may contribute to the susceptibility to PTB in different populations and to explain the diversity of inflammatory and immune mechanisms of PTB in different ethnicity. The inverse variance weighted (IVW) model of a two-sample Mendelian Randomization (MR) study was employed to conduct causal analysis on data from a genome-wide association study (GWAS). This cohort consisting PTB GWAS datasets from two European and two East Asian populations, as well as 91 human inflammatory proteins collected from 14,824 participants. Colocalization analysis aimed to determine whether the input inflammatory protein and PTB shared the same causal single nucleotide polymorphisms (SNPs) variation within the fixed region, thereby enhancing the robustness of the MR Analysis. Meta-analyses were utilized to evaluate the combined causal effects among different datasets. In this study, we observed a significant negative correlation between tumor necrosis factor-beta levels (The alternative we employ is Lymphotoxin-alpha, commonly referred to as LT) (P < 0.05) and tumor necrosis factor receptor superfamily member 9 levels (TNFRSF9) (P < 0.05). These two inflammatory proteins were crucial protective factors against PTB. Additionally, there was a significant positive correlation found between interleukin-20 receptor subunit alpha levels (IL20Ra) (P < 0.05), which may elevate the risk of PTB. Colocalization analysis revealed that there was no overlap in the causal variation between LT and PTB SNPs. A meta-analysis further confirmed the significant combined effect of LT, TNFRSF9, and IL20Ra in East Asian populations (P < 0.05). Levels of specific inflammatory proteins may play a crucial role in triggering an immune response to PTB. Altered levels of LT and TNFRSF9 have the potential to serve as predictive markers for PTB development, necessitating further clinical validation in real-world settings to ascertain the impact of these inflammatory proteins on PTB.

The Rac pathway prevents cell fragmentation in a nonprotrusively migrating leader cell during C. elegans gonad organogenesis

The C.elegans hermaphrodite distal tip cell (DTC) leads gonadogenesis. Loss-of-function mutations in a C.elegans ortholog of the Rac1 GTPase (ced-10) and its GEF complex (ced-5/DOCK180, ced-2/CrkII, ced-12/ELMO) cause gonad migration defects related to directional sensing; we discovered an additional defect class of gonad bifurcation in these mutants. Using genetic approaches, tissue-specific and whole-body RNAi, and invivo imaging of endogenously tagged proteins and marked cells, we find that loss of Rac1 or its regulators causes the DTC to fragment as it migrates. Both products of fragmentation-the now-smaller DTC and the membranous patch of cellular material-localize important stem cell niche signaling (LAG-2 ligand) and migration (INA-1/integrin subunit alpha) factors to their membranes, but only one retains the DTC nucleus and therefore the ability to maintain gene expression over time. The enucleate patch can lead a bifurcating branch off the gonad arm that grows through germ cell proliferation. Germ cells in this branch differentiate as the patch loses LAG-2 expression. While the nucleus is surprisingly dispensable for aspects of leader cell function, it is required for stem cell niche activity long term. Prior work found that Rac1-/-;Rac2-/- mouse erythrocytes fragment; in this context, our new findings support the conclusion that maintaining a cohesive but deformable cell is a conserved function of this important cytoskeletal regulator.

EGFR and PI3K Signalling Pathways as Promising Targets on Circulating Tumour Cells from Patients with Metastatic Gastric Adenocarcinoma.

The prognosis for metastatic gastric adenocarcinoma (mGAC) remains poor. Gene alterations in receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and their downstream effectors including catalytic subunit alpha of the phosphatidylinositol 3-kinase (PIK3CA) are common in mGAC. Targeted RTK and phosphatidylinositol-3-kinase (PI3K) treatments have demonstrated clinical benefits in other solid tumours and are key potential targets for clinical development against mGAC given the presence of recurrent alterations in these pathways. Furthermore, combination RTK/PI3K treatments may overcome compensatory mechanisms that arise using monotherapies, leading to improved patient outcomes. Herein, we investigated RTK/PI3K single and combination drug responses against our unique human mGAC-derived PIK3CA gain-of-function mutant, human epidermal growth factor receptor 2 (HER2)-negative, EGFR-expressing circulating tumour cell line, UWG02CTC, under two- and three-dimensional culture conditions to model different stages of metastasis. UWG02CTCs were highly responsive to the PI3K p110α-subunit targeted drugs PIK-75 (IC50 = 37.0 ± 11.1 nM) or alpelisib (7.05 ± 3.7 µM). Drug sensitivities were significantly increased in 3D conditions. Compensatory MAPK/ERK pathway upregulation by PI3K/Akt suppression was overcome by combination treatment with the EGFR inhibitor gefitinib, which was strongly synergistic. PIK-75 plus gefitinib significantly impaired UWG02CTC invasion in an organotypic assay. In conclusion, UWG02CTCs are a powerful ex vivo mGAC drug responsiveness model revealing EGFR/PI3K-targeted drugs as a promising combination treatment option for HER2-negative, RAS wild-type mGAC patients.

Expression of Recombinant Stonustoxin Alpha Subunit and Preparation of polyclonal antiserum for StonustoxinNeutralization Studies.

Stonustoxin (SNTX) is a lethal protein found in stonefish venom, responsible for many of the symptoms associated with stonefish envenomation. To counter stonefish venom challenges, antivenom is a well-established and effective solution. In this study, we aimed to produce the recombinant alpha subunit protein of Stonustoxin from Synanceia horrida and prepare antibodies against it The SNTXα gene sequence was optimized for E. coli BL21 (DE3) expression and cloned into the pET17b vector. Following purification, the recombinant protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbit´s serum using a G protein column As a result of codon optimization, the codon adaptation index for the SNTXα cassette increased to 0.94. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5kDa with a yield of 60mg/l. ELISA results demonstrated rabbits antibody titers were detectable up to a 1:256,000 dilution. The isolated antibody from rabbit´s serum exhibited a concentration of 1.5mg/ml, and its sensitivity allowed the detection of a minimum protein concentration of 9.7ng. In the neutralization assay the purified antibody against SNTXα protected mice challenged with 2 LD50. In conclusion, our study successfully expressed the alpha subunit of Stonustoxin in a prokaryotic host, enabling the production of antibodies for potential use in developing stonefish antivenom.

Sex-Dimorphic Effects of Hypoglycemia on Metabolic Sensor mRNA Expression in Ventromedial Hypothalamic Nucleus-Dorsomedial Division (VMNdm) Growth Hormone-Releasing Hormone Neurons.

Growth hormone-releasing hormone (Ghrh) neurons in the dorsomedial ventromedial hypothalamic nucleus (VMNdm) express the metabolic transcription factor steroidogenic factor-1 and hypoglycemia-sensitive neurochemicals of diverse chemical structures, transmission modes, and temporal signaling profiles. Ghrh imposes neuromodulatory control of coexpressed transmitters. Multiple metabolic sensory mechanisms are employed in the brain, including screening of the critical nutrient glucose or the energy currency ATP. Here, combinatory laser-catapult-microdissection/single-cell multiplex qPCR tools were used to investigate whether these neurons possess molecular machinery for monitoring cellular metabolic status and if these biomarkers exhibit sex-specific sensitivity to insulin-induced hypoglycemia. Data show that hypoglycemia up- (male) or downregulated (female) Ghrh neuron glucokinase (Gck) mRNA; Ghrh gene silencing decreased baseline and hypoglycemic patterns of Gck gene expression in each sex. Ghrh neuron glucokinase regulatory protein (Gckr) transcript levels were respectively diminished or augmented in hypoglycemic male vs female rats; this mRNA profile was decreased by Ghrh siRNA in both sexes. Gene transcripts encoding catalytic alpha subunits of the energy monitor 5-AMP-activated protein kinase (AMPK), i.e., Prkaa1 and 2, were increased by hypoglycemia in males, yet only the former mRNA was hypoglycemia-sensitive in females. Ghrh siRNA downregulated baseline and hypoglycemia-associated Prkaa subunit mRNAs in males but elicited divergent changes in Prkaa2 transcripts in eu- vs hypoglycemic females. Results provide unique evidence that VMNdm Ghrh neurons express the characterized metabolic sensor biomarkers glucokinase and AMPK and that the corresponding gene profiles exhibit distinctive sex-dimorphic transcriptional responses to hypoglycemia. Data further document Ghrh neuromodulation of baseline and hypoglycemic transcription patterns of these metabolic gene profiles.

Integrin subunit alpha 5 maintains mitochondrial function in ox-LDL-induced cardiac microvascular endothelial cells via activating the PI3K/AKT signaling pathway.

Cardiac microvascular endothelial cells (CMECs) assume a pivotal role in the regulation of blood flow, and their impairment precipitates a spectrum of pathological transformations. Our previous study unveiled a notable mitigation of CMECs dysfunction through the intervention of integrin subunit alpha 5 (ITGA5), a member of the integrin protein family. This study delves into the effect of ITGA5 on the mitochondrial function in CMECs and reveals the regulation pathway. CMECs were stimulated with oxidized low-density lipoprotein (ox-LDL) to mimic coronary artery disease (CAD). The effects of ITGA5 on diverse facets of CMEC behavior, encompassing viability, apoptosis, angiogenesis, oxidative stress, and mitochondrial function, was systematically ascertained. Employing the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway as a focal point of investigation, the mediation of this pathway was substantiated utilizing the PI3K inhibitor LY294002. ITGA5 overexpression exerted a mitigating influence upon the ox-LDL-induced detriment to CMECs, manifested as increased viability, angiogenesis, mitochondrial function, and diminished apoptosis and oxidative stress. The counteraction of these salubrious effects by the administration of the PI3K inhibitor attests to the engagement of the PI3K/AKT signaling pathway. Overall, this study has discerned that ITGA5 activates the PI3k/Akt signaling pathway to orchestrate mitochondrial function and diminish ox-LDL-induced CMEC dysfunction. Thus, the targeted amelioration of this cellular injury emerges as a strategically pivotal endeavor for the prevention and amelioration of this ailment.

M6A modification of lncRNA PHKA1-AS1 enhances Actinin Alpha 4 stability and promotes non-small cell lung cancer metastasis.

Cancer is a disease with molecular heterogeneity that is closely related to gene mutations and epigenetic changes. The principal histological subtype of lung cancer is non-small cell lung cancer (NSCLC). Long noncoding RNA (lncRNA) is a kind of RNA that is without protein coding function, playing a critical role in the progression of cancer. In this research, the regulatory mechanisms of lncRNA phosphorylase kinase regulatory subunit alpha 1 antisense RNA 1 (PHKA1-AS1) in the progression of NSCLC were explored. The increased level of N6-methyladenosine (m6A) modification in NSCLC caused the high expression of PHKA1-AS1. Subsequently, high-expressed PHKA1-AS1 significantly facilitated the proliferation and metastasis of NSCLC cells, and these effects could be reversed upon the inhibition of PHKA1-AS1 expression, both in vivo and in vitro. Additionally, the target protein of PHKA1-AS1 was actinin alpha 4 (ACTN4), which is known as an oncogene. Herein, PHKA1-AS1 could enhance the protein stability of ACTN4 by inhibiting its ubiquitination degradation process, thus exerting the function of ACTN4 in promoting the progress of NSCLC. In conclusion, this research provided a theoretical basis for further exploring the potential mechanism of NSCLC metastasis and searching novel biomarkers related to the pathogenesis and progression of NSCLC.