Target Of Pathway Research Articles

Epigenetic trajectory predicts development of clinical rheumatoid arthritis in ACPA+ individuals: Targeting Immune Responses for Prevention of Rheumatoid Arthritis (TIP-RA).

The presence of autoantibodies to citrullinated protein antigens (ACPAs) in the absence of clinically-apparent inflammatory arthritis (IA) identifies individuals "at-risk" for developing future clinical rheumatoid arthritis (RA). However, it is unclear why some ACPA+ individuals convert to clinical RA while others do not. We explored the possibility in the Targeting Immune Responses for Prevention of Rheumatoid Arthritis (TIP-RA) study that epigenetic remodeling is part of the trajectory from an at-risk state to clinical disease and identifies novel biomarkers associated with conversion to clinical RA. ACPA-Controls, ACPA+ At-Risk, and Early RA individuals were followed for up to 5 years, including obtaining blood samples annually and at RA diagnosis. Peripheral blood mononuclear cells (PBMCs) were separated into CD19+ B cells, memory CD4+ T cells, and naïve CD4+ T cells using antibodies and magnetic beads. Genome-wide methylation within each cell lineage was assayed using the Illumina MethylationEPIC v1.0 beadchip. ACPA+ At-Risk participants who did or did not develop RA were designated "Pre-RA" or "Non-converters", respectively.Differentially methylated loci (DML) were selected using the Limma software package. Using the Caret package, we constructed machine learning models in test and validation cohorts and identified the most predictive loci of clinical RA conversion. Cross-sectional differential methylation analysis at baseline revealed DMLs that distinguish the Pre-RA methylome from ACPA+ Non-converters, the latter which closely resembled ACPA-Controls. Genes overlapping these DMLs correspond to aberrant NOTCH signaling and DNA repair pathways in B cells. Longitudinal analysis showed that ACPA-Control and ACPA+ Non-converter methylomes are relatively constant. In contrast, the Pre-RA methylome remodeled along a dynamic "RA methylome trajectory" characterized by epigenetic changes in active regulatory elements. Clinical conversion to RA, defined based on diagnosis, marked an epigenetic inflection point for cell cycle pathways in B cells and adaptive immunity pathways in naïve T cells. Machine learning revealed individual loci associated with RA conversion. This model significantly outperformed autoantibodies plus acute phase reactants as predictors of RA conversion. DNA methylation is a dynamic process in ACPA+ individuals at-risk for developing RA that eventually transition to clinical disease. In contrast, non-converters and controls have stable methylomes. The accumulation of epigenetic marks over time prior to conversion to clinical RA conforms to pathways that are associated with immunity and can be used to identify potential pathogenic pathways for therapeutic targeting and/or use as prognostic biomarkers.

Phylogenetic Relations and High-Altitude Adaptation in Wild Boar (Sus scrofa), Identified Using Genome-Wide Data.

The Qinghai-Tibet Plateau (QTP) wild boar is an excellent model for investigating high-altitude adaptation. In this study, we analyzed genome-wide data from 93 wild boars compiled from various studies worldwide, including the QTP, southern and northern regions of China, Europe, Northeast Asia, and Southeast Asia, to explore their phylogenetic patterns and high-altitude adaptation based on genome-wide selection signal analysis and run of homozygosity (ROH) estimation. The findings demonstrate the alignment between the phylogenetic associations among wild boars and their geographical location. An ADMIXTURE analysis indicated a relatively close genetic relationship between QTP and southern Chinese wild boars. Analyses of the fixation index and cross-population extended haplotype homozygosity between populations revealed 295 candidate genes (CDGs) associated with high-altitude adaptation, such as TSC2, TELO2, SLC5A1, and SLC5A4. These CDGs were significantly overrepresented in pathways such as the mammalian target of rapamycin signaling and Fanconi anemia pathways. In addition, 39 ROH islands and numerous selective CDGs (e.g., SLC5A1, SLC5A4, and VCP), which are implicated in glucose metabolism and mitochondrial function, were discovered in QTP wild boars. This study not only assessed the phylogenetic history of QTP wild boars but also advanced our comprehension of the genetic mechanisms underlying the adaptation of wild boars to high altitudes.

Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR: ABL1 signaling

BackgroundThe inability of biologics to pass the plasma membrane prevents their development as therapeutics for intracellular targets. To address the lack of methods for cytosolic protein delivery, we used the type III secretion system (T3SS) of Y. enterocolitica, which naturally injects bacterial proteins into eukaryotic host cells, to deliver monobody proteins into cancer cells. Monobodies are small synthetic binding proteins that can inhibit oncogene signaling in cancer cells with high selectivity upon intracellular expression. Here, we engineered monobodies targeting the BCR::ABL1 tyrosine kinase for efficient delivery by the T3SS, quantified cytosolic delivery and target engagement in cancer cells and monitored inhibition of BCR::ABL1 signaling.MethodsIn vitro assays were performed to characterize destabilized monobodies (thermal shift assay and isothermal titration calorimetry) and to assess their secretion by the T3SS. Immunoblot assays were used to study the translocation of monobodies into different cell lines and to determine the intracellular concentration after translocation. Split-Nanoluc assays were performed to understand translocation and degradation kinetics and to evaluate target engagement after translocation. Phospho flow cytometry and apoptosis assays were performed to assess the functional effects of monobody translocation into BCR:ABL1-expressing leukemia cells.ResultsTo enable efficient translocation of the stable monobody proteins by the T3SS, we engineered destabilized mutant monobodies that retained high affinity target binding and were efficiently injected into different cell lines. After injection, the cytosolic monobody concentrations reached mid-micromolar concentrations considerably exceeding their binding affinity. We found that injected monobodies targeting the BCR::ABL1 tyrosine kinase selectively engaged their target in the cytosol. The translocation resulted in inhibition of oncogenic signaling and specifically induced apoptosis in BCR::ABL1-dependent cells, consistent with the phenotype when the same monobody was intracellularly expressed.ConclusionHence, we establish the T3SS of Y. enterocolitica as a highly efficient protein translocation method for monobody delivery, enabling the selective targeting of different oncogenic signaling pathways and providing a foundation for future therapeutic application against intracellular targets.

Improvement effect and mechanism of XuanFuDaiZhe tang on rats with diarrheal irritable bowel syndrome induced by colorectal dilation

Ethnopharmacological relevanceXuanFuDaiZhe Tang (XFDZT) is used in traditional Chinese medicine (TCM) to treat diarrhoea-predominant irritable bowel syndrome (IBS-D). Our laboratory has demonstrated that XFDZT remarkably improves various gastrointestinal motility disorders in animal models. However, previous studies have only focused on one or several protein targets without systematically investigating dynamic changes and protein interrelations. Aim of the studyTo explore the mechanisms underlying the therapeutic action of XFDZT in IBS-D using a network pharmacology approach and in vivo experiments. Materials and methodsThe active compounds of XFDZT were selected from TCM Systems Pharmacology and TCM Integrated databases, and potential targets were identified using the Swiss Target Prediction databases. Targets related to IBS-D were mined from the DisGeNet, Drug Bank, and Therapeutic Target databases. The intersecting protein–protein interactions (PPIs) of the drug–disease crossover genes were analysed, and a central PPI network was constructed using the STRING database and Cytoscape 3.7.2. Following Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, a gene pathway network was constructed to identify key target genes and pathways. Using haematoxylin and eosin staining and western blotting, we validated how XFDZT controls water expression in the body to treat IBS-D infection. ResultsFirst, the results showed that XFDZT contained 1037 active ingredients and 1458 corresponding targets. After intersecting the 252 IBS targets, 108 targets were identified. The main targets of XFDZT were albumin, aquaporins such as AQP1 and AQP3, calmodulin, and the cellular enzyme CYP2C9. GO and KEGG enrichment predicted that the action pathways were the neuroactive ligand–receptor interaction, calcium signalling pathway, serotonergic synapse signalling pathway, cGMP–PKG signalling pathway, cAMP signalling pathway, and MLCK–MLC signalling pathway. Second, an IBS-D rat model was constructed using colorectal dilation (CRD). CRD can significantly induce IBS-D symptoms such as diarrhoea, abdominal pain, and anxiety and depression-like behaviour in rats. XFDZT (10, 20, and 40 g/kg) administered for 14, 21, and 28 days significantly reversed these changes in IBS-D rats in a time- and dose-dependent manner, suggesting that XFDZT significantly improved IBS-D. Finally, the mechanism by which XFDZT improves IBS-D was explored from the perspective of AQPs, tight junction proteins, and motility-related proteins in colon tissue. Compared with the control group, the protein expression of AQP1, AQP3, and AQP8 in the colon tissue of IBS-D rats was significantly downregulated, whereas the protein expression of AQP7 was significantly upregulated. The expression of tight junction-related proteins claudin-1, occludin, and ZO-1 in colon tissue was significantly downregulated, whereas the expression of motility-related proteins p-MLC, MLC, MLCK, and CaM in colon tissue was significantly upregulated, suggesting that IBS-D rats had AQP disorders, epithelial intercellular connections, and motility in colon tissue. The above changes were significantly reversed by XFDZT administration (5, 10, and 20 g/kg) for 14 days. ConclusionXFDZT significantly improved diarrhoea, abdominal pain, anxiety, and depression in IBS-D rats, and its mechanism of action may be related to the regulation of AQPs, tight junction proteins, and the MLCK–MLC pathway. This study provided a pharmacological experimental basis for the development of XFDZT as a novel drug for the treatment of IBS-D.

Cepharanthine sensitizes gastric cancer cells to chemotherapy by targeting TRIB3-FOXO3-FOXM1 axis to inhibit autophagy

BackgroundGastric cancer is among the common solid tumors. Chemotherapy resistance is the most common issue in gastric cancer treatment. Inhibiting intracellular autophagy may be a feasible method for overcoming chemotherapy resistance. Cepharanthine (CEP), a natural small molecule extracted from the stephania cephalantha Hayata plant, has been demonstrated to significantly inhibit cancer growth and can regulate autophagy. Although CEP can significantly inhibit cancer growth, it remains unclear whether CEP can regulate autophagy in gastric cancer. This study aimed to investigate whether CEP can enhance the sensitivity of gastric cancer to chemotherapy and elucidate its molecular mechanism. MethodsThree gastric cancer cell lines (AGS, SGC7901, and MFC) and one normal gastric mucosal epithelial cell line (GES-1) were used for in vitro experiments. The characterization of autophagy in gastric cancer cells included the detection of autophagy markers and autophagy flux through immunofluorescence staining and Western blotting, as well as the assessment of lysosomal function using fluorescence staining (LysoTracker Red DND-99, Acridine Orange staining) and Western blotting. The cytotoxicity of CEP, autophagy inhibitors (chloroquine [CQ] and 3-methyladenine [3MA]), and chemotherapy drugs (doxorubicin [DOX] and cisplatin [CIS]) was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell colony formation, and fluorescence staining techniques (H2DCFDA, Dihydroethidium, and JC-1 staining). The interaction between CEP and autophagy inhibitors was tested in a 615 mice model, and changes in the gut microbiota were determined through accurate 16S absolute quantification sequencing. The signaling pathway and autophagy regulatory target TRIB3-FOXO3-FOXM1 were confirmed through molecular docking, RNA sequencing, bioinformatic analysis, transfection techniques, and Western blotting. ResultsCEP blocked autophagic flux in gastric cancer cells without affecting lysosomal function. As a novel autophagy inhibitor, CEP could combine with conventional autophagy inhibitors (CQ and 3MA) to block intracellular autophagy, thereby inhibiting gastric cancer growth. During this process, changes in the gut microbiota were observed, including low-level changes in Odoribacterium, Erysipelatoclostridium, and ParaPrevotella and high-level changes in Ileibacterium, Enterorhabdus, and Bifidobacterium. Additionally, CEP synergistically inhibited the growth of gastric cancer when combined with chemotherapy drugs. Mechanistically, the TRIB3-FOXO3-FOXM1 signaling axis was found to be involved in the inhibition of gastric cancer by CEP combined with autophagy inhibitors and chemotherapy drugs, thereby mediating cell apoptosis. ConclusionThis study links the TRIB3-FOXO3-FOXM1 axis with chemotherapy efficacy. Our findings demonstrated that CEP inhibits autophagy by modulating the FOXO3-FOXM1 axis. When combined with chemotherapy drugs (DOX and CIS), CEP, as an autophagy inhibitor, can limit TRIB3 protein expression, thereby regulating the FOXO3-FOXM1 axis and enhancing its ability to prevent gastric cancer growth. These findings may contribute to improving the prognosis of patients with gastric cancer. Furthermore, these results enrich the fundamental understanding of how autophagy inhibition can enhance clinical cancer treatment efficacy and provide insights into the potential mechanisms by which CEP functions as an anti-tumor drug, thereby exploring its value for clinical application.

Sex-specific proximal tubular cell differentiation pathways identified by single-nucleus RNA sequencing

Postnatal kidney growth is substantial and involves expansion in kidney tubules without growth of new nephrons, which are the functional units of the kidney. Proliferation and differentiation pathways underpinning nephron elongation are not well defined. To address this, we performed sequential characterization of mouse kidney transcriptomics at the single cell level. Single nuclear RNA sequencing (snRNA-seq) was performed on kidney tissue from male and female mice at 1, 2, 4 and 12 weeks of age using the 10x Chromium platform. Unbiased clustering was performed on 68,775 nuclei from 16 animals. 31 discrete cellular clusters were seen, which were identified through comparison of their gene expression profiles to canonical markers of kidney cell populations. High levels of proliferation were evident at early time points in some cell types, especially tubular cells, but not in other cell types, for example podocytes. Proliferation was especially evident in Proximal Tubular Cells (PTCs) which are the most abundant cell type in the adult kidney. Uniquely when compared to other kidney cell types, PTCs demonstrated sex-specific expression profiles at late, but not early, time points. Mapping of PTC differentiation pathways using techniques including trajectory and RNA Velocity analyses delineated increasing PTC specialization and sex-specific phenotype specification. Our single-cell transcriptomics data characterise cellular states observed during kidney growth. We have identified PTC differentiation pathways that lead to sex-specific tubular cell phenotypes. Tubular proliferative responses are of central importance in postnatal kidney growth and have also been linked to kidney recovery versus fibrosis following injury. Our unbiased and comprehensive dataset of tubular cell development can be used to identify candidate pathways for therapeutic targeting.

Asiatic acid impedes NSCLC progression by inhibiting COX-2 and modulating PI3K signaling.

Non-small cell lung cancer comprises up to 85% of lung cancer cases and has a poor prognosis. At present, there are still no effective treatments for this illness. Evidence suggests that the prostaglandin [cyclooxygenase-2 (COX-2)] and leukotriene [lipoxygenase-5 (5-LOX)] pathways are involved in lung cancer carcinogenesis. Therefore, novel agents that target COX-2 and 5-LOX may have therapeutic potential. In the present study, we examined the role of asiatic acid (AA), a triterpenoid saponin, in targeting the protein kinases responsible for lung cancer proliferation and mobility. The experimental data revealed that AA inhibited the growth of lung cancer cells (> 50%) and it significantly impeded the proliferation of lung cancer cells by inhibiting COX-2, which results in downregulation of the phosphotidyl inositol-3 kinase/protein kinase B/mammalian target of rapamycin signaling pathway, leading to an induction of cytotoxic autophagy-mediated apoptosis. Mechanistically, the expression of mitogen-activated protein kinase/extracellular signal-regulated kinase, hypoxia-inducible factor-1 and vascular endothelial growth factor is downregulated by AA, thereby reducing cell mobility and invasion. It also shows negative osmotic fragility on healthy human erythrocytes. It is concluded that AA may be a viable therapeutic drug for non-small cell lung cancer treatment, which opens new opportunities for synthesizing analogues.

Rhein induces apoptosis of AGS and MGC803 cells by regulating the Ras/PI3K/AKT and p38/MAPK signaling pathway.

Rhein is one of the main bioactive compounds in the Polygonaceae plant, and has been proven to have anti-cancer activity in some reports. But the mechanism of Rhein in the treatment of gastric cancer (GC) is limited reported. In this research, network pharmacology combined with in vitro experiments was used for systematically studying the mechanism of Rhein. Network pharmacology confirmed the major effect signaling pathway and key targets of Rhein in the treatment of GC. Cell viability assay, colony formation assay, fluorescence probe assay, apoptosis assay, western blot and qRT-PCR verified the mechanism of Rhein in the treatment of GC cells (AGS and MGC803 cells). The results showed that Rhein significantly induced the apoptosis process of AGS and MGC803 cells by regulating the Ras/phosphoinositide-3 kinase (PI3K)/protein kinase B (AKT) and the p38/mitogen-activated protein kinase signaling pathways. The AKT activator (SC79) and p38 inhibitor (SB202190) inhibited Rhein-induced apoptosis. All results proved that Rhein could be recognized as a potential natural drug for the treatment of GC.

Metabolic effects of quercetin on inflammatory and autoimmune responses in rheumatoid arthritis are mediated through the inhibition of JAK1/STAT3/HIF-1α signaling

BackgroundRheumatoid arthritis, a chronic autoimmune disease, is characterized by synovial hyperplasia and cartilage erosion. Here, we investigated the potential mechanism of action of quercetin, the main component of flavonoids, in treating rheumatoid arthritis.ObjectTo examine the anti-arthritic effects of quercetin and elucidate the specific mechanisms that differentiate its metabolic effects on autoimmune and inflammatory responses at the synovial cell level.MethodsWe created a collagen-induced arthritis (CIA) model in Wistar rats, which were administered quercetin (50 or 100 mg/kg) continuously for four weeks via stomach perfusion. The arthritis score, histopathological staining, radiological assessment, and serum biochemical parameters were used to study the impact of quercetin on disease improvement. Additionally, immunofluorescence was employed to detect JAK1/STAT3/HIF-1α expression in rat joints. Moreover, the effects of quercetin (20, 40, and 80 µmol/L) on the properties and behavior of synovial fibroblasts were evaluated in an in vitro MH7A cell model using flow cytometry, CCK8, and transwell assays. Further, the mRNA expression levels of inflammatory cytokines IL1β, IL6, IL17, and TNFα were assessed by quantitative real-time PCR. Glucose, lactate, lactate dehydrogenase, pyruvate, pyruvate dehydrogenase, and adenosine triphosphate assay kits were employed to measure the metabolic effects of quercetin on synovial fibroblasts. Finally, immunoblotting was used to examine the impact of quercetin on the JAK1/STAT3/HIF-1α signaling pathway in synovial fibroblasts.ResultsIn vivo experiments confirmed the favorable effects of quercetin in CIA rats, including an improved arthritis score and reduced ankle bone destruction, in addition to a decrease in the pro-inflammatory cytokines IL-1β, IL-6, IL-17, and TNF-α in serum. Immunofluorescence verified that quercetin may ameliorate joint injury in rats with CIA by inhibiting JAK1/STAT3/HIF-1α signaling. Various in vitro experiments demonstrated that quercetin effectively inhibits IL-6-induced proliferation of MH7A cells and reduces their migratory and invasive behavior, while inducing apoptosis and reducing the expression of the pro-inflammatory cytokines IL1β, IL6, IL17, and TNFα at the mRNA level. Quercetin caused inhibition of glucose, lactate, lactate dehydrogenase, pyruvate, and adenosine triphosphate and increased pyruvate dehydrogenase expression in MH7A cells. It was further confirmed that quercetin may inhibit energy metabolism and inflammatory factor secretion in MH7A cells through JAK1/STAT3/HIF-1α signaling.ConclusionsQuercetin’s action on multiple target molecules and pathways makes it a promising treatment for cartilage injury in rheumatoid arthritis. By reducing joint inflammation, improving joint metabolic homeostasis, and decreasing immune system activation energy, quercetin inhibits the JAK1/STAT3/HIF-1α signaling pathway to improve disease status.

Yiqi Yangxue formula inhibits cartilage degeneration in knee osteoarthritis by regulating LncRNA-UFC1/miR-34a/MMP-13 axis

Ethnopharmacological relevanceKnee osteoarthritis (KOA) is a prevalent and disabling clinical condition affecting joint structures worldwide. The Yiqi Yangxue formula (YQYXF) is frequently prescribed in clinical settings for the treatment of KOA. Existing research has primarily focused on alterations in drug metabolism, with limited investigation into the epigenetic effects of YQYXF, particularly in relation to non-coding RNA. Aim of the studyExploring the effects of YQYXF on critical factors of long chain non-coding RNA UFC1/miR-34a/matrix metalloproteinase-13 (MMP-13) axis and their interrelationships. MethodsUHPLC-QE-MS technology was used to identify the YQYXF ingredients in rat serum. KEGG and GO analysis were performed on the targets of blood components acting on KOA using a database. Simultaneously, a protein interaction network was constructed using target proteins and metabolites to identify the core components and key pathways of YQYXF. The KOA rat model was established using an improved Hulth method. SPF SD rats were randomly divided into normal group, sham surgery group, model group, celecoxib capsules group (18 mg/kg), YQYXF low, medium and high dose groups (4.6 g/kg, 9.2 g/kg, 18.4 g/kg). Observe the synovial and cartilage tissues of rats using pathological methods. RT-PCR was used to detect the levels of UFC1, miR-34a, and MMP-13 in cartilage. Immunohistochemistry was used to detect the levels of MMP-13 and ADAMTS-5 in cartilage. ELISA method was used to detect the levels of MMP-13 and ADAMTS-5 in serum. In addition, we further validated the regulation of crucial factor expression levels of UFC1/miR-34a/MMP-13 axis in rat chondrocytes and degenerative chondrocytes of KOA patients by YQYXF, providing a basis for its treatment of KOA. ResultsThe compounds that YQYXF enters the bloodstream mainly contain flavonoids and phenylpropanoid compounds. The core components that act on OA include quercetin, fisetin, and demethylweldelolactone. The main target pathways are the IL-17 signaling pathway, lipid and atherosclerosis, cellular sensitivity, inflammatory mediator regulation of TRP channels, TNF signaling pathway, relaxin signaling pathway and C-type lectin receptor signaling pathway. YQYXF inhibited the expression of miR-34a and MMP-13 mRNA, and reduced the protein levels of MMP-13 and ADAMTS-5. In vitro studies have confirmed that 20% YQYXF serum promoted UFC1 and reduce miR-34a levels. In addition, miR-34a in sh-UFC1+10% YQYXF serum and sh-UFC1+20% YQYXF serum groups significantly decreased compared to the sh-UFC1 group. ConclusionThe anti-KOA cartilage degeneration effect of YQYXF might be related to inhibiting cell apoptosis and promoting cell proliferation, which regulated the lncRNA-UFC1/miR-34a/MMP-13 axis.

One path, two solutions: Network-based analysis identifies targetable pathways for the treatment of comorbid type II diabetes and neuropsychiatric disorders

Comorbid diseases complicate patient outcomes and escalate healthcare costs, necessitating the need for a deeper mechanistic understanding. Neuropsychiatric disorders (NPDs) such as Neurotic Disorder, Major Depression, Bipolar Disorder, Anxiety Disorder, and Schizophrenia significantly exacerbate Type 2 Diabetes Mellitus (DM2), often leading to suboptimal treatment outcomes. The neurobiological mechanisms underlying this comorbidity remain poorly understood. To address this gap, we developed a novel pathway-based network computational framework to identify critical shared disease mechanisms between DM2 and these five prevalent comorbid NPDs. Our approach involves reconstructing an integrated DM2 ∩ NPDs KEGG pathway-pathway network and employs two complementary analytical methods, including the "minimum path to comorbidity" method to identify the shortest path fostering comorbid development. This analysis uncovered shared pathways like the PI3K-Akt signaling pathway and highlighted key nodes such as calcium signaling, MAPK, estrogen signaling, and apoptosis pathways. Dysregulation of these pathways likely contributes to the development of DM2-NPDs comorbidity. These findings have significant clinical implications, as they identify promising therapeutic targets that could lead to more effective treatments addressing both DM2 and NPDs simultaneously. Our model not only elucidates the intricate molecular interactions driving this comorbidity but also identifies promising therapeutic targets, paving the way for innovative treatment strategies. Additionally, the framework developed in this study can be adapted to study other complex comorbid conditions, advancing personalized medicine for comorbidities and improving patient care.

NEK10 kinase ablation affects mitochondrial morphology, function and protein phosphorylation status

BackgroundNEK10, a serine/threonine/tyrosine kinase belonging to the NEK (NIMA-related kinases) family, has been associated with diverse cellular processes. However, no specific target pathways have been identified. Our previous work knocking down NEK10 in HeLa cells suggested a functional association with mitochondria, as we observed altered mitochondrial morphology, mitochondrial oxygen consumption, mtDNA integrity, and reactive oxygen species levels.MethodsTo better understand this association, we studied human HAP1 cells fully knockout for NEK10 and confirmed that NEK10 has an important role in mitochondrial homeostasis.We performed the study of mitochondrial respiration, mitochondrial morphology, mitochondrial mass, and mtDNA analysis. Additionally, we showed proteome and phosphoproteome data of crude mitochondrial fraction of Parental and NEK10 KO cells using liquid chromatography-mass spectrometry (LC–MS/MS).ResultsIn the absence of NEK10 several mitochondrial functions were disturbed. Moreover, proteome and phosphoproteome analyses of mitochondrial fractions showed that NEK10 alters the threonine phosphorylation status of several mitochondrial/endoplasmic reticulum components, including HSP60, NDUFB4, and TOM20. These changes impacted the steady-state levels of a larger group of proteins, preferentially involving respiratory complexes and autophagy pathways.ConclusionWe concluded that NEK10 plays a key role in mitochondrial function, possibly by modulating the phosphorylation status of mitochondrial proteins.

Heading Date 3a Stimulates Tiller Bud Outgrowth in Oryza sativa L. through Strigolactone Signaling Pathway.

Heading date 3a (Hd3a, a FLOWERING LOCUS T (FT) ortholog from rice) is well known for its important role in rice (Oryza sativa L.), controlling floral transition under short-day (SD) conditions. Although the effect of Hd3a on promoting branching has been found, the underlying mechanism remains largely unknown. In this report, we overexpressed an Hd3a and BirAG (encoding a biotin ligase) fusion gene in rice, and found that early flowering and tiller bud outgrowth was promoted in BHd3aOE transgenic plants. On the contrary, knockout of Hd3a delayed flowering and tiller bud outgrowth. By using the BioID method, we identified multiple Hd3a proximal proteins. Among them, D14, D53, TPR1, TPR2, and TPRs are central components of the strigolactone signaling pathway, which has an inhibitory effect on rice tillering. The interaction between Hd3a, on the one hand, and D14 and D53 was further confirmed by the bimolecular fluorescence complementation (BiFC), yeast two-hybrid (Y2H), and co-immunoprecipitation (Co-IP) methods. We also found that Hd3a prevented the degradation of D53 induced by rac-GR24 (a strigolactone analog) in rice protoplasts. RT-qPCR assay showed that the expression levels of genes involved in strigolactone biosynthesis and signal transduction were altered significantly between WT and Hd3a overexpression (Hd3aOE) or mutant (hd3a) plants. OsFC1, a downstream target of the strigolactone signaling transduction pathway in controlling rice tillering, was downregulated significantly in Hd3aOE plants, whereas it was upregulated in hd3a lines. Collectively, these results indicate that Hd3a promotes tiller bud outgrowth in rice by attenuating the negative effect of strigolactone signaling on tillering and highlight a novel molecular network regulating rice tiller outgrowth by Hd3a.

Data mining of PubChem bioassay records reveals diverse OXPHOS inhibitory chemotypes as potential therapeutic agents against ovarian cancer

Focused screening on target-prioritized compound sets can be an efficient alternative to high throughput screening (HTS). For most biomolecular targets, compound prioritization models depend on prior screening data or a target structure. For phenotypic or multi-protein pathway targets, it may not be clear which public assay records provide relevant data. The question also arises as to whether data collected from disparate assays might be usefully consolidated. Here, we report on the development and application of a data mining pipeline to examine these issues. To illustrate, we focus on identifying inhibitors of oxidative phosphorylation, a druggable metabolic process in epithelial ovarian tumors. The pipeline compiled 8415 available OXPHOS-related bioassays in the PubChem data repository involving 312,093 unique compound records. Application of PubChem assay activity annotations, PAINS (Pan Assay Interference Compounds), and Lipinski-like bioavailability filters yields 1852 putative OXPHOS-active compounds that fall into 464 clusters. These chemotypes are diverse but have relatively high hydrophobicity and molecular weight but lower complexity and drug-likeness. These chemotypes show a high abundance of bicyclic ring systems and oxygen containing functional groups including ketones, allylic oxides (alpha/beta unsaturated carbonyls), hydroxyl groups, and ethers. In contrast, amide and primary amine functional groups have a notably lower than random prevalence. UMAP representation of the chemical space shows strong divergence in the regions occupied by OXPHOS-inactive and -active compounds. Of the six compounds selected for biological testing, 4 showed statistically significant inhibition of electron transport in bioenergetics assays. Two of these four compounds, lacidipine and esbiothrin, increased in intracellular oxygen radicals (a major hallmark of most OXPHOS inhibitors) and decreased the viability of two ovarian cancer cell lines, ID8 and OVCAR5. Finally, data from the pipeline were used to train random forest and support vector classifiers that effectively prioritized OXPHOS inhibitory compounds within a held-out test set (ROCAUC 0.962 and 0.927, respectively) and on another set containing 44 documented OXPHOS inhibitors outside of the training set (ROCAUC 0.900 and 0.823). This prototype pipeline is extensible and could be adapted for focus screening on other phenotypic targets for which sufficient public data are available.Scientific contributionHere, we describe and apply an assay data mining pipeline to compile, process, filter, and mine public bioassay data. We believe the procedure may be more broadly applied to guide compound selection in early-stage hit finding on novel multi-protein mechanistic or phenotypic targets. To demonstrate the utility of our approach, we apply a data mining strategy on a large set of public assay data to find drug-like molecules that inhibit oxidative phosphorylation (OXPHOS) as candidates for ovarian cancer therapies.Graphical

To boldly go where no microRNAs have gone before: spaceflight impact on risk for small-for-gestational-age infants

In the era of renewed space exploration, comprehending the effects of the space environment on human health, particularly for deep space missions, is crucial. While extensive research exists on the impacts of spaceflight, there is a gap regarding female reproductive risks. We hypothesize that space stressors could have enduring effects on female health, potentially increasing risks for future pregnancies upon return to Earth, particularly related to small-for-gestational-age (SGA) fetuses. To address this, we identify a shared microRNA (miRNA) signature between SGA and the space environment, conserved across humans and mice. These miRNAs target genes and pathways relevant to diseases and development. Employing a machine learning approach, we identify potential FDA-approved drugs to mitigate these risks, including estrogen and progesterone receptor antagonists, vitamin D receptor antagonists, and DNA polymerase inhibitors. This study underscores potential pregnancy-related health risks for female astronauts and proposes pharmaceutical interventions to counteract the impact of space travel on female health.

8738 Unveiling Potential Therapeutic Candidates for ACTH-Silent Pituitary Neuroendocrine Tumors Through High Throughput Drug Screening

Abstract Disclosure: J.M. Marques: None. N.D. D' Alessandre: None. G.D. Guardia: None. F.L. Craveiro: None. C. Grutzmacher: None. G.O. Silva: None. B.B. Mendonca: None. L.R. Carvalho: None. F. Fattahi: None. P.A. Galante: None. R.L. Batista: None. Introduction: ACTH-silent pituitary neuroendocrine tumours (PitNETs) are a subtype of PitNETs that arise from corticotroph cells of the anterior pituitary gland. They can show positive immunoreactivity for adrenocorticotropic hormone (ACTH) but with no clinical or laboratory evidence of hypercortisolism. ACTH-silent PitNETs display notably more aggressive behavior and treatment resistance. Currently, there is no established pharmacological therapy for this specific tumor subtype. Aim: To use high throughput drug screening to identify compounds that modulate ACTH secretion and tumor progression in ACTH-silent PitNETs. Methods: WTC11 cells (GM25256) were differentiated into pituitary using E6 with SB431542 10µM, SHH 200ng/mL, FGF8 100ng/mL and FGF10 50ng/mL until day 30. Pituitary cells were passaged in 384 well plates and treated with FDA-approved compound library with 3113 drugs at 1μM for 72h (#L1300, Selleckchem). Cells were stained with ACTH and imaged on ImageXpress® HCS. ACTH intensity was normalized to total cell number, measured by DAPI, using MetaXpress Software and compounds z-score was calculated, considering -2.5 for analysis. Gene and pathway enrichment analysis were performed and PitNET RNAseq data, publicly available (GSE207937), was used to determine target drugs based on similar target pathways and gene expression. To validate hits, we collected tumor samples from a patient with an aggressive silent ACTH PitNET. PitNET was histologically confirmed and hypercortisolism was excluded clinically and laboratory. Samples were cultured on DMEN/F12 with 10% FBS and 1% antibiotic and pituitary hormones were measured at days 3, 6 and 8. Cells were treated with 1uM of Letrozole, Linsitinib and Raloxifene for 48h and sample for RNA and hormone dosage were collected. Results: We identified 8 drugs that decreased ACTH in induced pituitary cells and selected 3 of them for validation. PitNET was successfully cultured and treated with selected drugs and ACTH secretion was detected at all periods (972.1 pg/mL to 10490 pg/mL), gonadotropins were below the detected level. Letrozole significantly reduced ACTH in the PitNET culture at day 8 (600.9 ± 25.8pg/mL, p<0,001). ACTH was also decreased in Linsitinib treated cells (932 ± 12.7pg/mL, p<0.05), while Raloxifene did not change ACTH secretion (980 ± 95.1pg/mL, p=0.061). Microscopically, PitNET cells aggregate in spheres and this seems to be affected by letrozole treatment, not influencing cell viability. Conclusion: We performed a high throughput drug screen using induced pituitary cells that led to the discovery of 3 compounds that might be used as an alternative for PitNET treatment. Pathway and gene expression analysis from target drugs and PitNET RNAseq may help in the understanding of drug effect in the PitNET context and improve the understanding of tumorigenesis and pharmacological treatment. Presentation: 6/1/2024

From apoptosis to pyroptosis: A two-decade analysis of spinal cord injury systematic review.

Spinal cord injury has a significant impact on patients' physical and mental health, with cell death playing a key role in the pathology. The Web of Science Core Collection database was searched and screened according to inclusion criteria for publications between January 1, 2000 and December 31, 2023. Bibliometric analysis was conducted using bibliomatrix R-package, CiteSpace, and VOSviewer software. A total of 2309 publications were identified, with a stable growth in the first 13 years and accelerated growth in the past decade, indicating an increasing trend in this field. These publications on cell death after spinal cord injury originated from 251 countries/regions. China contributed the most publications, with funding support primarily from China, and Wenzhou Medical University in China being the leading institution with the highest number of publications. Salvatore Cuzzocrea was the most influential author. The research direction in this field after 2000 mainly focused on topics such as apoptosis, autophagy, mesenchymal stem cells, mammalian target of rapamycin pathway, ferroptosis, functional recovery, neuronal regeneration, and NOD-like receptor protein 3.Current hot topics and future research trends revolve around extracellular vesicles including exosomes, extracellular vesicle-mediated mesenchymal stem cells, microRNA, autophagy, NOD-like receptor protein 3 inflammasome-mediated cell pyroptosis, and mechanisms of glial scar formation. Publications in this field have grown rapidly in the past 2 decades and are expected to keep increasing. Current hot topics include mesenchymal stem cells, apoptosis, autophagy, and ferroptosis. Future research will focus on extracellular vesicle-mediated mesenchymal stem cells, autophagy, inflammasome-mediated cell pyroptosis, and glial scar formation.

Characterization of aberrant splicing in pediatric central nervous system tumors reveals CLK1 as a candidate oncogenic dependency.

Pediatric brain cancer is the leading cause of disease-related mortality in children, and many aggressive tumors still lack effective treatment strategies. We characterized aberrant alternative splicing across pediatric brain tumors, identifying pediatric high-grade gliomas (HGGs) among the most heterogeneous. Annotating these events with UniProt, we identified 11,940 splice events in 5,368 genes leading to potential protein function changes. We discovered CDC-like kinase 1 (CLK1) is aberrantly spliced to include exon 4, resulting in a gain of two phosphorylation sites and subsequent activation. Inhibition of CLK1 with Cirtuvivint significantly decreased both cell viability and proliferation in the pediatric HGG KNS-42 cell line. Morpholino-mediated depletion of CLK1 exon 4 splicing reduced RNA expression, protein abundance, and cell viability with concurrent differential expression of 78 cancer genes and differential splicing at functional sites in 193 cancer genes. Our findings highlight a dependency of pediatric HGGs on CLK1 and represent a promising therapeutic strategy.

Rnf111 has a pivotal role in regulating development of definitive hematopoietic stem and progenitor cells through the Smad2/3-Gcsfr/NO axis in zebrafish.

The ubiquitination or SUMOylation of hematopoietic related factors plays pivotal roles in hematopoiesis. RNF111, known as a ubiquitin ligase (Ubl), is a newly discovered SUMO-targeted ubiquitin ligase (STUbl) involved in multiple signaling pathways mediated by TGF-β family members. However, its role in hematopoiesis remains unclear. Herein, a heritable Rnf111 mutant zebrafish line was generated by CRISPR/Cas9-mediated genome editing. Impaired hematopoietic stem and progenitor cells (HSPC) of definitive hematopoiesis was found in Rnf111 deficient mutants. Ablation of Rnf111 resulted in decreased phosphorylation of Smad2/3 in HSPC. Definitive endoderm 2 inducer (IDE2), which specifically activates TGF-β signaling and downstream Smad2 phosphorylation, can restore the definitive hematopoiesis in Rnf111-deficient embryos. Further molecular mechanism studies revealed that Gcsfr/NO signaling was an important target pathway of Smad2/3 involved in Rnf111-mediated HSPC development. In conclusion, our study demonstrated that Rnf111 contributes to the development of HSPC by maintaining Smad2/3 phosphorylation and the Gcsfr/NO signaling pathway activation. Keywords: Rnf111, Ubiquitin ligase (UbL), HSPC, Smad2/3, Gcsfr/NO.

Single-cell transcriptional atlas of human breast cancers and model systems.

Breast cancer's complex transcriptional landscape requires an improved understanding of cellular diversity to identify effective treatments. The study of genetic variations among breast cancer subtypes at single-cell resolution has potential to deepen our insights into cancer progression. In this study, we amalgamate single-cell RNA sequencing data from patient tumours and matched lymph metastasis, reduction mammoplasties, breast cancer patient-derived xenografts (PDXs), PDX-derived organoids (PDXOs), and cell lines resulting in a diverse dataset of 117 samples with 506719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 positive (HER2+), and triple-negative breast cancer (TNBC) subtypes, including isogenic model pairs. Herein, we delineated similarities and distinctions across models and patient samples and explore therapeutic drug efficacy based on subtype proportions. PDX models more closely resemble patient samples in terms of tumour heterogeneity and cell cycle characteristics when compared with TNBC cell lines. Acquired drug resistance was associated with an increase in basal-like cell proportions within TNBC PDX tumours as defined with SCSubtype and TNBCtype cell typing predictors. All patient samples contained a mixture of subtypes; compared to primary tumours HR+ lymph node metastases had lower proportions of HER2-Enriched cells. PDXOs exhibited differences in metabolic-related transcripts compared to PDX tumours. Correlative analyses of cytotoxic drugs on PDX cells identified therapeutic efficacy was based on subtype proportion. We present a substantial multimodel dataset, a dynamic approach to cell-wise sample annotation, and a comprehensive interrogation of models within systems of human breast cancer. This analysis and reference will facilitate informed decision-making in preclinical research and therapeutic development through its elucidation of model limitations, subtype-specific insights and novel targetable pathways. Patient-derived xenografts models more closely resemble patient samples in tumour heterogeneity and cell cycle characteristics when compared with cell lines. 3D organoid models exhibit differences in metabolic profiles compared to their in vivo counterparts. A valuable multimodel reference dataset that can be useful in elucidating model differences and novel targetable pathways.