Regulation Of Cytoskeleton Research Articles

Proteomics-Derived Biomarker Panel Facilitates Distinguishing Primary Lung Adenocarcinomas With Intestinal or Mucinous Differentiation From Lung Metastatic Colorectal Cancer

The diagnosis of primary lung adenocarcinomas with intestinal or mucinous differentiation (PAIM) remains challenging due to the overlapping histomorphological, immunohistochemical (IHC), and genetic characteristics with lung metastatic colorectal cancer (lmCRC). This study aimed to explore the protein biomarkers that could distinguish between PAIM and lmCRC. To uncover differences between the two diseases, we used tandem mass tagging–based shotgun proteomics to characterize proteomes of formalin-fixed, paraffin-embedded tumor samples of PAIM (n = 22) and lmCRC (n = 17).Then three machine learning algorithms, namely support vector machine (SVM), random forest, and the Least Absolute Shrinkage and Selection Operator, were utilized to select protein features with diagnostic significance. These candidate proteins were further validated in an independent cohort (PAIM, n = 11; lmCRC, n = 19) by IHC to confirm their diagnostic performance. In total, 105 proteins out of 7871 proteins were significantly dysregulated between PAIM and lmCRC samples and well-separated two groups by Uniform Manifold Approximation and Projection. The upregulated proteins in PAIM were involved in actin cytoskeleton organization, platelet degranulation, and regulation of leukocyte chemotaxis, while downregulated ones were involved in mitochondrial transmembrane transport, vasculature development, and stem cell proliferation. A set of ten candidate proteins (high-level expression in lmCRC: CDH17, ATP1B3, GLB1, OXNAD1, LYST, FABP1; high-level expression in PAIM: CK7 (an established marker), NARR, MLPH, S100A14) was ultimately selected to distinguish PAIM from lmCRC by machine learning algorithms. We further confirmed using IHC that the five protein biomarkers including CDH17, CK7, MLPH, FABP1 and NARR were effective biomarkers for distinguishing PAIM from lmCRC. Our study depicts PAIM-specific proteomic characteristics and demonstrates the potential utility of new protein biomarkers for the differential diagnosis of PAIM and lmCRC. These findings may contribute to improving the diagnostic accuracy and guide appropriate treatments for these patients.

Identification of Cellular Protein Targets of a Half-Sandwich Iridium(III) Complex Reveals Its Dual Mechanism of Action via Both Electrophilic and Oxidative Stresses.

Identification of intracellular targets of anticancer drug candidates provides key information on their mechanism of action. Exploiting the ability of the anticancer (C∧N)-chelated half-sandwich iridium(III) complexes to covalently bind proteins, click chemistry with a bioorthogonal azido probe was used to localize a phenyloxazoline-chelated iridium complex within cells and profile its interactome at the proteome-wide scale. Proteins involved in protein folding and actin cytoskeleton regulation were identified as high-affinity targets. Upon iridium complex treatment, the folding activity of Heat Shock Protein HSP90 was inhibited in vitro and major cytoskeleton disorganization was observed. A wide array of imaging and biochemical methods validated selected targets and provided a multiscale overview of the effects of this complex on live human cells. We demonstrate that it behaves as a dual agent, inducing both electrophilic and oxidative stresses in cells that account for its cytotoxicity. The proposed methodological workflow can open innovative avenues in metallodrug discovery.

Identification of the Hub Genes Involved in Chikungunya Viral Infection.

Background Chikungunya virus (CHIKV) infection poses a significant global health threat, necessitating a deeper understanding of its molecular mechanisms for effective management and treatment. This study aimed to understand the molecular and geneticmechanisms of CHIKV infection by analyzing microarray expression data. Methodology National Center for Biotechnology Information (NCBI) GEO2R with an adjusted p-value cut-off of <0.05 and |log2FC ≥ 1.5| was used to identify the differentially expressed genes involved in CHIKV infection using microarray data from the Gene Expression Omnibus (GEO) database, followed by enrichment analysis, protein-protein interaction (PPI) network construction, and, finally, hub gene identification. Results Analysis of the microarray dataset revealed 25 highly significant differentially expressed genes (DEGs), including 21 upregulated and four downregulated genes. PPI network analysis elucidated interactions among these DEGs, with hub genes such as ACTB and CTNNB1 exhibiting central roles. Enrichment analysis identified crucial pathways, including leukocyte transendothelial migration, regulation of actin cytoskeleton, and thyroid hormone signaling, implicating their involvement in CHIKV infection. Furthermore, the study highlights potential therapeutic targets such as ACTB and CTNNB1, which showed significant upregulation in infected cells. Conclusions These findings underscore the complex interplay between viral infection and host cellular processes, shedding light on novel avenues for diagnostic marker discovery and advancing antiviral strategies. In this study, we shed light on the molecular and genetic mechanisms of CHIKV infection and the potential role of ACTB and CTNNB1 genes.

Interplay of RAP2 GTPase and the cytoskeleton in Hippo pathway regulation

The Hippo signaling is instrumental in regulating organ size, regeneration, and carcinogenesis. The cytoskeleton emerges as a primary Hippo signaling modulator. Its structural alterations in response to environmental and intrinsic stimuli control Hippo signaling pathway activity. However, the precise mechanisms underlying the cytoskeleton regulation of Hippo signaling are not fully understood. RAP2 GTPase is known to mediate the mechanoresponses of Hippo signaling via activating the core Hippo kinases LATS1/2 through MAP4Ks and MST1/2. Here we show the pivotal role of the reciprocal regulation between RAP2 GTPase and the cytoskeleton in Hippo signaling. RAP2 deletion undermines the responses of the Hippo pathway to external cues tied to RhoA GTPase inhibition and actin cytoskeleton remodeling, such as energy stress and serum deprivation. Notably, RhoA inhibitors and actin disruptors fail to activate LATS1/2 effectively in RAP2-deficient cells. RNA sequencing highlighted differential regulation of both actin and microtubule networks by RAP2 gene deletion. Consistently, Taxol, a microtubule-stabilizing agent, was less effective in activating LATS1/2 and inhibiting cell growth in RAP2 and MAP4K4/6/7 knockout cells. In summary, our findings position RAP2 as a central integrator of cytoskeletal signals for Hippo signaling, which offers new avenues for understanding Hippo regulation and therapeutic interventions in Hippo-impaired cancers.

Integrated Analysis Identified TGFBI as a Biomarker of Disease Severity and Prognosis Correlated with Immune Infiltrates in Patients with Sepsis.

Sepsis is a major contributor to morbidity and mortality among hospitalized patients. This study aims to identify markers associated with the severity and prognosis of sepsis, providing new approaches for its management and treatment. Data were mined from the Gene Expression Omnibus (GEO) databases and were analyzed by multiple statistical methods like the Spearman correlation coefficient, Kaplan-Meier analysis, Cox regression analysis, and functional enrichment analysis. Candidate indicator' associations with immune infiltration and roles in sepsis development were evaluated. Additionally, we employed techniques such as flow cytometry and neutral red staining to evaluate its impact on macrophage functions like polarization and phagocytosis. Twenty-eight genes were identified as being closely linked to the severity of sepsis, among which transforming growth factor beta induced (TGFBI) emerged as a distinct marker for predicting clinical outcomes. Notably, reductions in TGFBI expression during sepsis correlate with poor prognosis and rapid disease progression. Elevated expression of TGFBI has been observed to mitigate abnormalities in sepsis-related immune cell infiltration that are critical to the pathogenesis and prognosis of the disease, including but not limited to type 17 T helper cells and activated CD8 T cells. Moreover, the protein-protein interaction network revealed the top ten genes that interact with TGFBI, showing significant involvement in the regulation of the actin cytoskeleton, extracellular matrix-receptor interactions, and phagosomes. These are pivotal elements in the formation of phagocytic cups by macrophages, squaring the findings of the Human Protein Atlas. Additionally, we discovered that TGFBI expression was significantly higher in M2-like macrophages, and its upregulation was found to inhibit lipopolysaccharide-induced polarization and phagocytosis in M1-like macrophages, thereby playing a role in preventing the onset of inflammation. TGFBI warrants additional exploration as a promising biomarker for assessing illness severity and prognosis in patients with sepsis, considering its significant association with immunological and inflammatory responses in this condition.

PlexinA1 promotes gastric cancer migration through preventing MICAL1 protein ubiquitin/proteasome-mediated degradation in a Rac1-dependent manner

Metastasis promotes the development of tumors and is a significant cause of gastric cancer death. For metastasis to proceed, tumor cells must become mobile by modulating their cytoskeleton. MICAL1 (Molecule Interacting with CasL1) is known as an actin cytoskeleton regulator, but the mechanisms by which it drives gastric cancer cell migration are still unclear. Analysis of gastric cancer tissues revealed that MICAL1 expression is dramatically upregulated in stomach adenocarcinoma (STAD) samples as compared to noncancerous stomach tissues. Patients with high MICAL1 expression had shorter overall survival (OS), post-progression survival (PPS) and first-progression survival (FPS) compared with patients with low MICAL1 expression. RNAi-mediated silencing of MICAL1 inhibited the expression of Vimentin, a protein involved in epithelial-mesenchymal transition. This effect correlates with a significant reduction in gastric cancer cell migration. MICAL1 overexpression reversed these preventive effects. Immunoprecipitation experiments and immunofluorescence assays revealed that PlexinA1 forms a complex with MICAL1. Importantly, specific inhibition of PlexinA1 blocked the Rac1 activation and ROS production, which, in turn, impaired MICAL1 protein stability by accelerating MICAL1 ubiquitin/proteasome-dependent degradation. Overexpression of PlexinA1 enhanced Rac1 activation, ROS production, MICAL1 and Vimentin expressions, and favored cell migration. In conclusion, this study identified MICAL1 as an important facilitator of gastric cancer cell migration, at least in part, by affecting Vimentin expression and PlexinA1 promotes gastric cancer cell migration by binding to and suppressing MICAL1 degradation in a Rac1/ROS-dependent manner.

A Role for Aquaporin-5 Variants in Regulation of the Actin Cytoskeleton in Non-Epidermolytic Palmoplantar Keratoderma

A Role for Aquaporin-5 Variants in Regulation of the Actin Cytoskeleton in Non-Epidermolytic Palmoplantar Keratoderma

Hepatocyte-derived exosomes deliver the lncRNA CYTOR to hepatic stellate cells and promote liver fibrosis.

Liver fibrosis is characterized by the activation and transformation of hepatic stellate cells (HSCs) induced by various injury factors. The degree of liver fibrosis can be significantly improved, but persistent injury factors present a significant therapeutic challenge. Hepatocytes are the most important parenchymal cell type in the liver. In this study, we explored the molecular mechanisms by which damaged liver cells activate HSCs through extracellular vesicles. We established a coculture model of LO2 and LX2 and validated its exosomal transmission activity. Subsequently, differentially expressed long noncoding RNAs (lncRNAs) were screened through RNA sequencing and their mechanisms of action as competing endogenous RNAs (ceRNAs) further confirmed using biological methods, such as FISH and luciferase assays. Damaged liver cells induced activation of LX2 and upregulation of liver fibrosis-related markers. Exosomes extracted and identified from the supernatant fraction contained differentially expressed lncRNA cytoskeleton regulator RNA (CYTOR) that competed with microRNA-125 (miR-125) for binding to glial cell line-derived neurotrophic factor (GDNF) in HSCs, in turn, promoting LX2 activation. MiR-125 could target and regulate both CYTOR and GDNF and vice versa, as verified using the luciferase assay. In an invivo model, damaged liver extracellular vesicles induced the formation of liver fibrosis. Notably, downregulation of CYTOR within extracellular vesicles effectively inhibited liver fibrosis. The lncRNA CYTOR in exosomes of damaged liver cells is upregulated and modulates the expression of downstream GDNF through activity as a ceRNA, providing an effective mechanism for activation of HSCs.

Abstract 1701: ABI1 regulates prostate tumor plasticity through chromatin remodeling and transcriptional regulation

Abstract Background: Prostate cancer remains a challenging issue affecting men worldwide. In contrast to localized disease, metastatic prostate cancer tumor is incurable. Progression to metastatic disease is characterized by tumor heterogeneity and plasticity regulated by epigenetic changes of tumor cells. While tumor heterogeneity is often associated with gene mutations, tumor plasticity is ascribed to transcriptional regulation and chromatin activity. The latter is modulated by treatment of prostate cancer leading to treatment resistance. Here, we identified a novel mechanism of epigenetic regulation in prostate cancer by an actin regulatory and signaling protein, Abelson Interactor 1 (ABI1). Methods: To demonstrate the ABI1-DNA binding, we employed in vitro binding assays, NMR spectroscopy, and ChIP-seq. from cells expressing alternatively spliced isoforms of ABI1 that contain or lack the DNA binding HHR domain. To determine the ABI1 interactions with several transcriptional factors, we used immunoprecipitation and/or proximity ligation assay (PLA). We used ATAC sequencing to determine the chromatin accessibility in the presence or absence of ABI1 gene. RNA-seq established the activity of transcriptional factors and signaling pathways regulated by ABI1. Results: Using NMR spectroscopy, we established that ABI1 demonstrated DNA binding dependent on the presence of alternatively spliced Exon 4. Exon 4 is located within the homeobox homology region (HHR). The DNA binding activity was confirmed using in vitro binding assays; the assay results confirmed the role of ABI1 HHR. Moreover, cells lacking ABI1 or expressing ABI1 lacking Exon 4 show low accessibility and activity of chromatin. In contrast, cells expressing ABI1 containing Exon 4 show active chromatin. RNA-seq analysis demonstrated that ABI1 regulated transcription in an Exon 4-dependent manner. Conclusions: We uncovered a role of ABI1 in chromatin stability, that is dependent on the presence of the DNA binding region. Notably, ABI1's contribution to transcriptional activity occurs via a transient yet precisely defined interaction between its intrinsically disordered region and DNA. This regulatory control plays a pivotal role in managing tumor plasticity by establishing connections between the actin cytoskeleton, cellular signaling, and transcriptional regulation. Based on these findings, we propose that ABI1 functions as an epigenetic regulator, maintaining transcriptional homeostasis in prostate cancer. Citation Format: Leszek Kotula. ABI1 regulates prostate tumor plasticity through chromatin remodeling and transcriptional regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1701.

Circadian disruption dysregulates lung gene expression associated with inflammatory lung injury.

Circadian systems drive the expression of multiple genes in nearly all cells and coordinate cellular-, tissue-, and system-level processes that are critical to innate immunity regulation. We examined the effects of circadian rhythm disorganization, produced by light shift exposure, on innate immunity-mediated inflammatory lung responses including vascular permeability and gene expression in a C57BL/6J murine model of inflammatory lung injury. A total of 32 C57BL/6J mice were assigned to circadian phase shifting (CPS) with intratracheal phosphate-buffered saline (PBS), CPS with intratracheal lipopolysaccharide (LPS), control (normal lighting) condition with intratracheal PBS, and control condition with intratracheal LPS. Bronchoalveolar lavage (BAL) protein, cell counts, tissue immunostaining, and differentially expressed genes (DEGs) were measured in lung tissues at 2 and 10 weeks. In mice exposed to both CPS and intratracheal LPS, both BAL protein and cell counts were increased at both 2 and 10 weeks compared to mice exposed to LPS alone. Multiple DEGs were identified in CPS-LPS-exposed lung tissues compared to LPS alone and were involved in transcriptional pathways associated with circadian rhythm disruption, regulation of lung permeability, inflammation with Rap1 signaling, and regulation of actin cytoskeleton. The most dysregulated pathways included myosin light chain kinase, MAP kinase, profilin 2, fibroblast growth factor receptor, integrin b4, and p21-activated kinase. Circadian rhythm disruption results in exacerbated immune response and dysregulated expression of cytoskeletal genes involved in the regulation of epithelial and vascular barrier integrity-the mechanistic underpinnings of acute lung injury. Further studies need to explore circadian disorganization as a druggable target.

The Specific Molecular Changes Induced by Diabetic Conditions in Valvular Endothelial Cells and upon Their Interactions with Monocytes Contribute to Endothelial Dysfunction.

Aortic valve disease (AVD) represents a global public health challenge. Research indicates a higher prevalence of diabetes in AVD patients, accelerating disease advancement. Although the specific mechanisms linking diabetes to valve dysfunction remain unclear, alterations of valvular endothelial cells (VECs) homeostasis due to high glucose (HG) or their crosstalk with monocytes play pivotal roles. The aim of this study was to determine the molecular signatures of VECs in HG and upon their interaction with monocytes in normal (NG) or high glucose conditions and to propose novel mechanisms underlying valvular dysfunction in diabetes. VECs and THP-1 monocytes cultured in NG/HG conditions were used. The RNAseq analysis revealed transcriptomic changes in VECs, in processes related to cytoskeleton regulation, focal adhesions, cellular junctions, and cell adhesion. Key molecules were validated by qPCR, Western blot, and immunofluorescence assays. The alterations in cytoskeleton and intercellular junctions impacted VEC function, leading to changes in VECs adherence to extracellular matrix, endothelial permeability, monocyte adhesion, and transmigration. The findings uncover new molecular mechanisms of VEC dysfunction in HG conditions and upon their interaction with monocytes in NG/HG conditions and may help to understand mechanisms of valvular dysfunction in diabetes and to develop novel therapeutic strategies in AVD.

Chlorella alleviates the intestinal damage of tilapia caused by microplastics

Polyethylene microplastics (MPs) of the different sizes may result in different response in fish. Studies showed microorganisms adhered to the surface of MPs have toxicological effect. Juveniles tilapia (Oreochromis niloticus, n = 600, 26.5 ± 0.6 g) were dispersed into six groups: the control group (A), 75 nm MP exposed group (B), 7.5 μm group (C) and 750 (D) μm group, 75 nm + 7.5 μm+750 μm group (E) and 75 nm + Chlorella vulgaris group (F), and exposed for 10 and 14 days. The intestinal histopathological change, enzymic activities, and the integrated “omics” workflows containing transcriptomics, proteomics, microbiota and metabolomes, have been performed in tilapia. Results showed that MPs were distributed on the surface of goblet cells, Chlorella group had severe villi fusion without something like intestinal damage, as in other MPs groups. The intestinal Total Cholesterol (TC, together with group E) and Tumor Necrosis Factor α (TNFα, except for group B) contents in group F were significantly increased, cytochrome p450 1a1 (EROD, group B and E) significantly increased, adenosine triphosphate (ATP), lipoprotein lipase (LPL) and caspase 3 (except group B) also significantly increased at 14 d. At 14 days, group E saw considerably higher regulation of the actin cytoskeleton, focal adhesion, insulin signaling pathway, and AGE-RAGE signaling pathway in diabetes complications. Whereas, chlorella enhanced the focal adhesion, cytokine-cytokine receptor interaction, and MAPK signaling pathways. PPAR signaling pathway has been extremely significantly enriched via the proteomics method. Candidatus latescibacteria, C. uhrbacteria, C. abyssubacteria, C. cryosericota significantly decreased caused by MPs of different particle sizes. Carboxylic acids and derivatives, indoles and derivatives, organooxygen compounds, fatty acyls and organooxygen compounds significantly increased with long-term duration, especially PPAR signaling pathway. MPs had a size-dependent long-term effect on histopathological change, gene and protein expression, and gut microbial metabolites, while chlorella alleviates the intestinal histopathological damage via the integrated “omics” workflows.

Comprehensive analysis of exosome gene LYPD3 and prognosis/immune cell infiltration in lung cancer.

Lung cancer (LC) is a leading cause of cancer-associated mortality worldwide, with high incidence and mortality rates. Ly6/PLAUR domain containing 3 (LYPD3) is a tumorigenic and highly glycosylated cell surface protein that has been rarely reported in LC. This study aimed to explore the prognostic role and immune cell infiltration of LYPD3 in LC. We used ExoCarta, a database of exosomal proteins and RNA, to select exosomes in LC. The Tumor Immune Estimation Resource (TIMER) and Human Protein Atlas (HPA) databases were utilized to compare the expression of LYPD3 in LC. We applied Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and Kaplan-Meier (KM) plotter to evaluate the prognostic prediction performance of LYPD3. Biological processes (BPs), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and gene set enrichment analysis (GSEA) analyses were performed to illustrate the possible role of LYPD3 in LC. The correlations between LYPD3 and immune cell infiltration were explored using Tumor and Immune System Interaction Database (TISIDB), GEPIA2, and TIMER. R software was used for statistical analysis and mapping. A total of 904 exosome molecules were screened in LC. Further analysis showed that the up-regulation of LYPD3 in these 904 exosome molecules was associated with poor prognosis in LC. Pan-cancer analyses revealed that the expression of LYPD3 varied in many cancers, particularly in LC. Clinical correlation analysis indicated that LYPD3 was associated with stage and T classification in LC. We observed that LYPD3 co-expression genes were associated with cell cycle, DNA replication, proteasome, and regulation of the actin cytoskeleton by GSEA. Moreover, LYPD3 was associated with immune modulators. Immunophenoscores (IPS) and IPS-CTLA4 were significantly different between the high LYPD3 group and low LYPD3 group. Additionally, the median half maximal inhibitory concentration (IC50) of bexarotene, cyclopamine, etoposide, and paclitaxel in LYPD3 high group was significantly lower than that in LYPD3 low group. LYPD3 is involved in many BPs of LC, such as regulating immune cell infiltration and affecting prognosis. Therefore, LYPD3 may have potential value as a biomarker for prognosis and immunotherapy in LC.

Discrete-state models identify pathway specific B cell states across diseases and infections at single-cell resolution

Oxygen (O2) regulated pathways modulate B cell activation, migration and proliferation during infection, vaccination, and other diseases. Modeling these pathways in health and disease is critical to understand B cell states and ways to mediate them. To characterize B cells by their activation of O2 regulated pathways we develop pathway specific discrete state models using previously published single-cell RNA-sequencing (scRNA-seq) datasets from isolated B cells. Specifically, Single Cell Boolean Omics Network Invariant-Time Analysis (scBONITA) was used to infer logic gates for known pathway topologies. The simplest inferred set of logic gates that maximized the number of “OR” interactions between genes was used to simulate B cell networks involved in oxygen sensing until they reached steady network states (attractors). By focusing on the attractors that best represented sequenced cells, we identified genes critical in determining pathway specific cellular states that corresponded to diseased and healthy B cell phenotypes. Specifically, we investigate the transendothelial migration, regulation of actin cytoskeleton, HIF1A, and Citrate Cycle pathways. Our analysis revealed attractors that resembled the state of B cell exhaustion in HIV+ patients as well as attractors that promoted anerobic metabolism, angiogenesis, and tumorigenesis in breast cancer patients, which were eliminated after neoadjuvant chemotherapy (NACT). Finally, we investigated the attractors to which the Azimuth-annotated B cells mapped and found that attractors resembling B cells from HIV+ patients encompassed a significantly larger number of atypical memory B cells than HIV− attractors. Meanwhile, attractors resembling B cells from breast cancer patients post NACT encompassed a reduced number of atypical memory B cells compared to pre-NACT attractors.

Complex changes in serum protein levels in COVID-19 convalescents

The COVID-19 pandemic, triggered by severe acute respiratory syndrome coronavirus 2, has affected millions of people worldwide. Much research has been dedicated to our understanding of COVID-19 disease heterogeneity and severity, but less is known about recovery associated changes. To address this gap in knowledge, we quantified the proteome from serum samples from 29 COVID-19 convalescents and 29 age-, race-, and sex-matched healthy controls. Samples were acquired within the first months of the pandemic. Many proteins from pathways known to change during acute COVID-19 illness, such as from the complement cascade, coagulation system, inflammation and adaptive immune system, had returned to levels seen in healthy controls. In comparison, we identified 22 and 15 proteins with significantly elevated and lowered levels, respectively, amongst COVID-19 convalescents compared to healthy controls. Some of the changes were similar to those observed for the acute phase of the disease, i.e. elevated levels of proteins from hemolysis, the adaptive immune systems, and inflammation. In contrast, some alterations opposed those in the acute phase, e.g. elevated levels of CETP and APOA1 which function in lipid/cholesterol metabolism, and decreased levels of proteins from the complement cascade (e.g. C1R, C1S, and VWF), the coagulation system (e.g. THBS1 and VWF), and the regulation of the actin cytoskeleton (e.g. PFN1 and CFL1) amongst COVID-19 convalescents. We speculate that some of these shifts might originate from a transient decrease in platelet counts upon recovery from the disease. Finally, we observed race-specific changes, e.g. with respect to immunoglobulins and proteins related to cholesterol metabolism.

Conditional Deletion of β-Catenin in the Mediobasal Hypothalamus Impairs Adaptive Energy Expenditure in Response to High-Fat Diet and Exacerbates Diet-Induced Obesity.

β-Catenin is a bifunctional molecule that is an effector of the wingless-related integration site (Wnt) signaling to control gene expression and contributes to the regulation of cytoskeleton and neurotransmitter vesicle trafficking. In its former role, β-catenin binds transcription factor 7-like 2 (TCF7L2), which shows strong genetic associations with the pathogenesis of obesity and type-2 diabetes. Here, we sought to determine whether β-catenin plays a role in the neuroendocrine regulation of body weight and glucose homeostasis. Bilateral injections of adeno-associated virus type-2 (AAV2)-mCherry-Cre were placed into the arcuate nucleus of adult male and female β-catenin flox mice, to specifically delete β-catenin expression in the mediobasal hypothalamus (MBH-β-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-β-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-β-cat KO mice were significantly heavier than the control mice in both sexes (p < 0.05). This deficit seemed to be due to a failure to show an adaptive increase in energy expenditure seen in controls, which served to offset the increased calories by HFD. Both male and female MBH-β-cat KO mice were highly glucose intolerant when on HFD and displayed a significant reduction in both leptin and insulin sensitivity compared with controls. This study highlights a critical role for β-catenin in the hypothalamic circuits regulating body weight and glucose homeostasis and reveals potential mechanisms by which genetic variation in this pathway could impact on development of metabolic disease.

Characterization of runs of homozygosity islands in American mink using whole-genome sequencing data.

The genome-wide analysis of runs of homozygosity (ROH) islands can be an effective strategy for identifying shared variants within a population and uncovering important genomic regions related to complex traits. The current study performed ROH analysis to characterize the genome-wide patterns of homozygosity, identify ROH islands and annotated genes within these candidate regions using whole-genome sequencing data from 100 American mink (Neogale vison). After sequence processing, variants were called using GATK and Samtools pipelines. Subsequent to quality control, 8,373,854 bi-allelic variants identified by both pipelines remained for further analysis. A total of 34,652 ROH segments were identified in all individuals, among which shorter segments (0.3-1 Mb) were abundant throughout the genome, approximately accounting for 84.39% of all ROH. Within these segments, we identified 63 ROH islands housing 156 annotated genes. The genes located in ROH islands were associated with fur quality (EDNRA, FGF2, FOXA2 and SLC24A4), body size/weight (MYLK4, PRIM2, FABP2, EYS and PHF3), immune capacity (IL2, IL21, PTP4A1, SEMA4C, JAK2, CCNA2 and TNIP3) and reproduction (ADAD1, KHDRBS2, INSL6, PGRMC2 and HSPA4L). Furthermore, Gene Ontology and KEGG pathway enrichment analyses revealed 56 and 9 significant terms (FDR-corrected p-value < 0.05), respectively, among which cGMP-PKG signalling pathway, regulation of actin cytoskeleton, and calcium signalling pathway were highlighted due to their functional roles in growth and fur characteristics. This is the first study to present ROH islands in American mink. The candidate genes from ROH islands and functional enrichment analysis suggest possible signatures of selection in response to the mink breeding targets, such as increased body length, reproductive performance and fur quality. These findings contribute to our understanding of genetic characteristics, and provide complementary information to assist with implementation of breeding strategies for genetic improvement in American mink.

Comparative transcriptomics reveals common and strain-specific responses of human macrophages to infection with Mycobacterium tuberculosis and Mycobacterium bovis BCG

Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (M. bovis) are closely related pathogenic mycobacteria known to cause chronic pulmonary infections in both humans and animals. Despite sharing nearly identical genomes and virulence factors, these two bacteria display variations in host tropism, epidemiology, and clinical presentations. M. bovis Bacillus Calmette–Guérin (BCG) is an attenuated strain of M. bovis commonly utilized as a vaccine for tuberculosis (TB). Nevertheless, the molecular underpinnings of these distinctions and the intricacies of host-pathogen interactions remain areas of ongoing research. In this study, a comparative transcriptomic analysis was conducted on human leukemia macrophages (THP-1) infected with either MTB H37Rv or M. bovis BCG (Tokyo strain) to elucidate common and strain-specific responses at the transcriptional level. RNA sequencing was utilized to characterize the transcriptomes of human primary macrophages infected with MTB or BCG at 6 and 24 h post-infection. The findings indicate that both MTB and BCG induce substantial and dynamic alterations in the transcriptomes of THP-1, with a notable overlap in the quantity and extent of differentially expressed genes (DEGs). Moreover, gene ontology (GO) enrichment analysis unveiled shared pathways related to immune response, cytokine signaling, and apoptosis. The immune response of macrophages to bacterial infections at 6 h exhibited significantly greater intensity compared to that at 24 h. Furthermore, distinct gene sets displaying notable variances between MTB and BCG infections were identified. The profound impact of MTB infection on macrophage gene expression, particularly within the initial 6 h, was evident. Additionally, downregulation of pathways such as Focal adhesion, Rap1 signaling pathway, and Regulation of actin cytoskeleton was observed. The pathways associated with inflammation reactions and cell apoptosis exhibited significant differences, with BCG triggering macrophage apoptosis and MTB enhancing the survival of intracellular bacteria. Our findings reveal that MTB and BCG provoke similar yet distinct transcriptional responses in human macrophages, indicating variations in their pathogenesis and ability to adapt to host environments. These results offer novel insights into the molecular mechanisms governing host-pathogen interactions and may contribute to a deeper understanding of TB pathogenesis.

Identification of hub genes and transcription factors in patients with primary gout complicated with atherosclerosis

Evidence shows that primary gout is prone to develop to atherosclerosis, but the mechanism of its occurrence is still not fully clarified. The aim of this study was to explore the molecular mechanism of the occurrence of this complication in gout. The gene expression profiles of primary gout and atherosclerosis were downloaded from the gene expression omnibus database. Overlapping differentially expressed genes (DEGs) between gout and atherosclerosis were identified. The biological roles of common DEGs were explored through enrichment analyses. Hub genes were identified using protein–protein interaction networks. The immune infiltrations of 28 types of immune cells in gout and control samples from GSE160170 were evaluated by the ssGSEA method. Transcription factors (TFs) were predicted using Transcriptional Regulatory Relationships Unraveled by Sentence Based Text Mining (TRRUST) database. A total of 168 overlapping DEGs were identified. Functional enrichment analyses indicated that DEGs were mostly enriched in chemokine signaling pathway, regulation of actin cytoskeleton, and TNF signaling pathway. CytoScape demonstrated 11 hub genes and two gene cluster modules. The immune infiltration analysis showed that the expression of DEGs in gout was significantly upregulated in activated CD4 T cells, gamma delta T cells, T follicular helper cell, CD56dim natural killer cells, and eosinophil. TRRUST predicted one TF, RUNX family transcription factor 1. Our study explored the pathogenesis of gout with atherosclerosis and discovered the immune infiltration of gout. These results may guide future experimental research and clinical transformation.

A mutation in F-actin polymerization factor suppresses the distal arthrogryposis type 5 PIEZO2 pathogenic variant in Caenorhabditis elegans.

The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole-genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]), significantly increased brood size and ovulation rate, as well as alleviating the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Expression of GEX-3 in the soma is required to rescue the brood size defects in pezo-1(R2405P) animals. Actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the PIEZO coordinates with the cytoskeleton regulator to maintain the F-actin network and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.