Cell Junction Organization Research Articles

EphA2 regulates vascular permeability and prostate cancer metastasis via modulation of cell junction protein phosphorylation.

Prostate cancer morbidity and mortality demonstrate a need for more effective targeted therapies. One potential target is EphA2, although paradoxically, pro- and anti-oncogenic effects have been shown to be mediated by EphA2. We demonstrate that unique activating and blocking EphA2-targeting monoclonal antibodies display opposing tumor-suppressive and oncogenic properties in vivo. To further explore this complexity, we performed detailed phosphoproteomic analysis following ligand-induced EphA2 activation. Our analysis identified altered phosphorylation of 73 downstream proteins related to the PI3K/AKT/mTOR and ERK/MAPK pathways, with the majority implicated in cell junction and cytoskeletal organization, cell motility, and tumor metastasis. We demonstrate that the adapter protein SHB is an essential component in mediating the inhibition of the ERK/MAPK pathway in response to EphA2 receptor activation. Furthermore, we identify the adherence junction protein afadin as an EphA2-regulated phosphoprotein which is involved in prostate cancer migration and invasion.

Identification of Genes Associated with Familial Focal Segmental Glomerulosclerosis Through Transcriptomics and In Silico Analysis, Including RPL27, TUBB6, and PFDN5.

Focal segmental glomerulosclerosis (FSGS) is a major cause of nephrotic syndrome and often leads to progressive kidney failure. Its varying clinical presentation suggests potential genetic diversity, requiring further molecular investigation. This study aims to elucidate some of the genetic and molecular mechanisms underlying FSGS. The study focuses on the use of bioinformatic analysis of gene expression data to identify genes associated with familial FSGS. A comprehensive in silico analysis was performed using the GSE99340 data set from Gene Expression Omnibus (GEO) comparing gene expression in glomerular and tubulointerstitial tissues from FSGS patients (n = 10) and Minimal Change Disease (MCD) patients (n = 8). These findings were validated using transcriptomics data obtained using RNA sequencing from FSGS (n = 3) and control samples (n = 3) from the UAE. Further validation was conducted using qRT-PCR on an independent FFPE cohort (FSGS, n = 6; MCD, n = 7) and saliva samples (FSGS, n = 3; Control, n = 7) from the UAE. Three genes (TUBB6, RPL27, and PFDN5) showed significant differential expression (p < 0.01) when comparing FSGS and MCD with healthy controls. These genes are associated with cell junction organization and synaptic pathways of the neuron, supporting the link between FSGS and the neural system. These genes can potentially be useful as diagnostic biomarkers for FSGS and to develop new treatment options.

Ascites microenvironment conditions the peritoneal pre-metastatic niche to promote the implantation of ovarian tumor spheroids: Involvement of fibrinogen/fibrin and αV and α5β1 integrins

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood.Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5β1 integrins are involved.In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.

Genomic progression for local invasion of cutaneous squamous cell carcinoma from the superficial to the deep portion.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. While many treatments exist, our understanding of its genomic progression, especially from the epidermis to the deep dermis, remains limited. This study aims to identify genetic mutations associated with the progression of cSCC into the deep dermis, providing insights into its aggressive behavior and high-risk features. We performed high-depth whole-exome sequencing on 12 cSCC tissues, along with paired normal tissues from six patients, using microdissection techniques. The mutational analysis focused on identifying alterations enriched during cSCC progression. Gene Ontology enrichment analysis, immunohistochemical assays, and external single-cell RNA data were utilized for validation. A total of 8863 non-synonymous somatic mutations were identified in 4092 genes across the superficial and deep portions of cSCCs. Analysis of deep portion mutations revealed a significant correlation with gene ontology biological processes, particularly cell junction organization, and cell-cell adhesion. Clonal mutations in these processes were more prevalent in the deep portions, indicating their impact on the cSCC mutation landscape. Genetic evolution analysis identified 29 causal genes associated with dermal invasion in cSCC. We highlight somatic mutations in cSCC, revealing heterogeneity between superficial and deep regions. Altered genes in cell junction organization and cell-cell adhesion emerged as pivotal in dermal invasion. We identified 29 causal genes primarily in deep tumor regions. Our findings emphasize analyzing multiple tumor regions to capture varied mutational landscapes. These insights advance our understanding of cSCC progression, emphasizing genetic and cellular changes during tumor evolution.

Abstract 1620: Multiregion spatial sequencing of early-stage head and neck squamous cell carcinoma revealed theranostic intratumor heterogeneity

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) is characterized by substantial intratumor heterogeneity (ITH) at the molecular level that may foster tumor evolution, adaptation, and therapeutic failure. Personalized-medicine strategies mostly rely on single tumor-biopsy samples which might not capture the mutational burden of heterogeneous tumors. We investigated genetic ITH portrayed from multiregion spatial sequencing in early-stage HNSCC patients. Patients and Method: Seventy-eight early-stage HNSCC patients treated with upfront curative-intent surgery in the prospective biobanking SCANDARE study (NCT03017573) were included. We developed a pathological measure of ITH, based on the relative contribution of multiple poorly and well-differentiated intratumor regions (n = 199) in resected tumor samples. To characterize genetic ITH, we performed targeted next-generation sequencing on multiple spatially separated intratumor regions by using a custom panel of 571 genes (named DRAGON), in combination with 3’-Tag RNA sequencing and pathway enrichment analysis. Results: Fifty-nine patients displaying a minimum of two sequenced intratumor regions (total, n = 180) were analyzed. We observed genetic ITH in 22/59 patients (37%), of whom 6/22 (27%) based on theranostic molecular alterations. Three of the six patients exhibited molecular alterations in PIK3CA (n = 2) and TSC2 (n = 1) that have supporting clinical evidence of actionability in some cancer types (OncoKB level 1) and three patients exhibited actionable molecular alterations in PTEN (n = 2) and CDKN2A/B (n = 1) supported by substantial research evidence (OncoKB level 4). Two patients exhibited molecular alterations in SMARCA4 that is likely oncogenic, albeit not yet annotated by OncoKB. The pathological measure of ITH accounted for a significant source of variation in gene expression. Notably, we identified 346 differentially expressed genes imposing p-values ≤ 0.05 and |log fold-change| ≥ 1 involved in several Reactome’s signaling pathways, including the cell junction organization (R-HSA-446728) and the tight junction interaction (R-HSA-420029) pathways. Conclusions: We reported clinically relevant genetic ITH in early-stage HNSCC, which can lead to underestimation of the mutational landscape portrayed from single tumor biopsy and may present major challenges to precision medicine. Our findings uncovered the biology behind ITH and may have translational implications for biomarkers discovery in HNSCC. Citation Format: Grégoire Marret, Sophie Vacher, Constance Lamy, Ladidi Ahmanache, Laura Courtois, Choumouss Kamoun, Maral Halladjian, Zakhia El Beaino, Jerzy Klijanienko, Charlotte Martinat, Emmanuelle Jeannot, Joey Martin, Guillaume Rougier, Nathalie Badois, Maria Lesnik, Antoine Dubray-Vautrin, Olivier Choussy, Wahib Ghanem, Rabah Taouachi, Julien Masliah Planchon, Nicolas Servant, Maud Kamal, Ivan Bièche, Christophe Le Tourneau. Multiregion spatial sequencing of early-stage head and neck squamous cell carcinoma revealed theranostic intratumor heterogeneity [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 1620.

Noninvasive urinary protein signatures combined clinical information associated with microvascular invasion risk in HCC patients

BackgroundMicrovascular invasion (MVI) is the main factor affecting the prognosis of patients with hepatocellular carcinoma (HCC). The aim of this study was to identify accurate diagnostic biomarkers from urinary protein signatures for preoperative prediction.MethodsWe conducted label-free quantitative proteomic studies on urine samples of 91 HCC patients and 22 healthy controls. We identified candidate biomarkers capable of predicting MVI status and combined them with patient clinical information to perform a preoperative nomogram for predicting MVI status in the training cohort. Then, the nomogram was validated in the testing cohort (n = 23). Expression levels of biomarkers were further confirmed by enzyme-linked immunosorbent assay (ELISA) in an independent validation HCC cohort (n = 57).ResultsUrinary proteomic features of healthy controls are mainly characterized by active metabolic processes. Cell adhesion and cell proliferation-related pathways were highly defined in the HCC group, such as extracellular matrix organization, cell–cell adhesion, and cell–cell junction organization, which confirms the malignant phenotype of HCC patients. Based on the expression levels of four proteins: CETP, HGFL, L1CAM, and LAIR2, combined with tumor diameter, serum AFP, and GGT concentrations to establish a preoperative MVI status prediction model for HCC patients. The nomogram achieved good concordance indexes of 0.809 and 0.783 in predicting MVI in the training and testing cohorts.ConclusionsThe four-protein-related nomogram in urine samples is a promising preoperative prediction model for the MVI status of HCC patients. Using the model, the risk for an individual patient to harbor MVI can be determined.

Identification of microRNA and protein interaction networks in human ovarian cancer

Ovarian cancer is one of the deadliest tumors in women, with a high mortality rate brought on by the lack of early detection. In this work, our main aim is to find promising biomarkers and pertinent mechanisms. GSE36668 was chosen from the Gene Expression Omnibus (GEO) to identify the differentially expressed genes (DEGs) using the GEO2R tool. To forecast gene ontology (GO) and pathway enrichment, online tools from ToppGene, FunRich and DAVID were employed. The protein-protein interaction (PPI) network is built via STRING v.11.5 and Cytoscape v.3.9.1. Following the detection of the hub genes, a Kaplan-Meire plotter was used to conduct additional validation survival analyses. A total of 1556 DEGs were identified using GEO2R, out of which 697 were upregulated and 859 were downregulated. According to GO analysis, DEGs were much more common in the online tools DAVID and ToppGene for cell adhesion, axoneme assembly and cilium assembly in the biological processs whereas cell surface is an essential component of the plasma membrane and extracellular matrix in the cellular component. In contrast, the plasma membranes are present in DAVID and FunRich. The DEGs are mostly linked to the MAPK, PI3K-Akt and RAP1 signaling pathways in KEGG and in the Reactome pathway, they are involved in cell-cell communication, cell and cell-cell junction organization The PPI network construct was used to find the gene clusters and to identify the hub genes MAPK1, CDH1, CBL and CCND1 by Cytoscape. The survival analysis of this hub gene CBL showed high expression in ovarian cancer which led to fewer survival chances. According to this study, ovarian cancer biomarkers are crucial to understand the molecular causes of the disease.

Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 virus and Staphylococcus aureus infections

BackgroundEarly studies indicated that vitamin D (VD) exerted pleiotropic extra-skeletal effects in the airway, but the definite linkage between VD deficiency and airway host responses remains unclear.Methods142 cases of clinical data from Department of Otolaryngology, the Seventh Affiliated Hospital of Sun Yat-sen University, were collected to characterize the relationship between VD deficiency and chronic rhinosinusitis (CRS). Based on the clinical observations, 2.5-D airway epithelial organoids cultured at the air–liquid interface (ALI) were used to simulate the effects of VD treatment in the development of airway epithelium and the modulation of the host responses against influenza H1N1 virus (representing viral infections) and Staphylococcus aureus (representing bacterial infections) infections in the airway. The intrinsic mechanisms of VD deficiency underlying epithelial remodeling were mapped by transcriptomic as well as proteomic analyses.ResultsIn this study we observed prevailing VD deficiency among inpatients suffering from CRS, a common disease predominantly characterized by epithelial impairment and remodeling. Relative to control organoids cultured without VD, long-term incubation with VD accelerated basal cell proliferation during nasal epithelial development. Under infectious conditions, VD treatment protected the organoids against influenza H1N1 virus and Staphylococcus aureus invasions by reinforcing the respiratory host defenses, including upregulation of LL37, suppression (or inhibition) of proinflammatory cytokines, strengthening of epithelial integrity, and mucociliary clearance. In silico analysis of transcriptomics and proteomics suggested that VD modulated the epithelial development and remodeling, involving epithelial cell proliferation/differentiation, epithelial–mesenchymal transition (EMT), and cytokine signaling in the immune system, as well as responses to microbe, cell junction organization, and extracellular matrix organization via PTEN signaling, independent of TGF-β signaling.ConclusionsOur findings emphasize the importance of managing VD deficiency in clinical settings for the sake of alleviating pathological epithelial remodeling. Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 and Staphylococcus aureus infections.

Genomic variants and inferred biological processes in multiplex families with Tourette syndrome.

Tourette syndrome is a developmental neuropsychiatric disorder. Its etiology is complex and elusive, although an important role of genetic factors has been established. The aim of the present study was to identify the genomic basis of Tourette syndrome in a group of families with affected members in 2 or 3 generations. Whole-genome sequencing was performed followed by co-segregation and bioinformatic analyses. Identified variants were used to select candidate genes, which were then subjected to gene ontology and pathway enrichment analysis. The study group included 17 families comprising 80 patients with Tourette syndrome and 44 healthy family members. Co-segregation analysis and subsequent prioritization of variants pinpointed 37 rare and possibly pathogenic variants shared among affected individuals within a single family. Three such variants, in the ALDH2, DLD and ALDH1B1 genes, could influence oxidoreductase activity in the brain. Two variants, in SLC17A8 and BSN genes, were involved in sensory processing of sound by inner hair cells of the cochlea. Enrichment analysis of genes whose rare variants were present in all patients from at least 2 families identified significant gene sets implicated in cell-cell adhesion, cell junction assembly and organization, processing of sound, synapse assembly, and synaptic signalling processes. We did not examine intergenic variants, but they still could influence clinical phenotype. Our results provide a further argument for a role of adhesion molecules and synaptic transmission in neuropsychiatric diseases. Moreover, an involvement of processes related to oxidative stress response and sound-sensing in the pathology of Tourette syndrome seems likely.

Transcriptome analysis based on machine learning reveals a role for autoinflammatory genes of chronic nonbacterial osteomyelitis (CNO)

Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disorder. The origin and development of CNO involve many complex immune processes, resulting in delayed diagnosis and a lack of effective treatment. Although bioinformatics analysis has been utilized to seek key genes and pathways in CNO, only a few bioinformatics studies that focus on CNO pathogenesis and mechanisms have been reported. This study aimed to identify key biomarkers that could serve as early diagnostic or therapeutic markers for CNO. Two RNA-seq datasets (GSE133378 and GSE187429) were obtained from the Gene Expression Omnibus (GEO). Weighted gene coexpression network analysis (WGCNA) and differentially expressed gene (DEG) analysis were conducted to identify the genes associated with CNO. Then, the autoinflammatory genes most associated with CNO were identified based on the GeneCards database and a CNO prediction model, which was created by the LASSO machine learning algorithm. The accuracy of the model and effects of the autoinflammatory genes according to receiver operating characteristic (ROC) curves were verified in external datasets (GSE7014). Finally, we performed clustering analysis with ConsensusClusterPlus. In total, eighty CNO-related genes were identified and were significantly enriched in the biological processes regulation of actin filament organization, cell–cell junction organization and gamma-catenin binding. The main enriched pathways were adherens junctions, viral carcinogenesis and systemic lupus erythematosus. Two autoinflammatory genes with high expression in CNO samples were identified by combining an optimal machine learning algorithm (LASSO) with the GeneCards database. An external validation dataset (GSE187429) was utilized for ROC analysis of the prediction model and two genes, and the results indicated good efficiency. Then, based on consensus clustering analysis, we found that the expression of UTS2 and MPO differed between clusters. Finally, the ceRNA network of lncRNAs and the small molecule compounds targeting the two autoinflammatory genes were predicted. The identification of two autoinflammatory genes, the HCG18/has-mir-147a/UTS2/MPO axis and signalling pathways in this study can help us understand the molecular mechanism of CNO formation and provides candidate targets for the diagnosis and treatment of CNO.

Abstract 5289: Proteomic profile of breast carcinomas with unbalanced levels of progesterone receptor isoforms

Abstract Preclinical studies indicate that progesterone receptors (PR) play a relevant role in breast carcinogenesis. Even more, a misbalanced expression of PR isoforms A (PRA) and B (PRB) differentially affect breast cancer progression and only tumors with higher levels of PRA than PRB (PRA-H) respond to an antiprogestin treatment. There are controversial results regarding the prognosis of PRA-H luminal tumors compared with those with the opposite ratio (PRB-H tumors) highlighting the necessity to further understand the role of PR isoforms in tumor progression and to develop novel methods to discriminate these tumors without performing western blot studies. Along this line we have previously studied the transcriptome of PRA-H or PRB-H breast cancer samples, thus our aim is to expand these results and compare the proteome profile of PRA-H and PRB-H samples obtained from postmenopausal breast cancer patients. Nuclear (Nuc) and cytosolic (Cyt) protein fractions were obtained from 18 breast cancer samples classified as PRA-H or PRB-H and they were studied by LC-MS/MS (UltiMate 3000 LC system - Q Exactive HF-X mass spectrometer - Thermo). We observed 289 differentially deregulated proteins in Cyt (164 down and 125 up) and 301 in Nuc extracts (131 down and 170 up; logFC &amp;gt; 1, pval &amp;lt; 0.05). Gene set enrichment analysis of the Nuc fractions showed biological processes related to Cell junction organization (pval = 8,87e-05), Collagen formation (pval = 0,0005), and Cell-Cell communication processes (pval = 0,0035) in PRB-H tumors; while in PRA-H tumors, pathways related to Innate Immune System (pval = 8,8e-05), Class I MHC mediated antigen processing &amp; presentation (pval = 8e-05) and Stabilization of p53 (pval = 0,0051) processes were enriched. Similar trends were observed in Cyt fractions. Individual candidates that were overrepresented in both fractions in PRB-H tumors are BAG3 and GH3, related to cell proliferation, and VWF, related to the activation of the Akt pathway. Overrepresented proteins in both fractions of PRA-H tumors are BST2, IFT27 related to invasion, and MIF associated with poor prognosis. As we were interested in looking for differential expressed proteins that could be tested in plasma samples to discriminate both tumor types, using the Vertebrate Secretome Database we focused on those that might be secreted. Noteworthy is CBP1 which was overrepresented in the PRB-H tumors and this was in agreement with the RNA-Seq data and with results obtained using preclinical models, repurposing this protein as one candidate of concern in the search for biomarkers. Other secretory proteins of interest are SFRP2 and SERPIND1, both highly expressed in PRA-H Cyt fractions. In conclusion, this study underscores the biological differences between PRA-H and PRB-H breast carcinomas and provides candidates that deserve to be tested as surrogate markers in plasma to discriminate patients that may benefit from an antiprogestin treatment. Citation Format: Andres M. Elia, Jana Sanchez, Rui Vitorino, Leo Saldain, Paula Martinez Vazquez, Javier Burruchaga, Eunice Spengler, Javier Muñoz, Paola Rojas, Luisa Helguero, Claudia Lanari. Proteomic profile of breast carcinomas with unbalanced levels of progesterone receptor isoforms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5289.

Light and electron microscopy continuum-resolution imaging of 3D cell cultures.

3D cell cultures, in particular organoids, are emerging models in the investigation of healthy or diseased tissues. Understanding the complex cellular sociology in organoids requires integration of imaging modalities across spatial and temporal scales. We present a multi-scale imaging approach that traverses millimeter-scale live-cell light microscopy to nanometer-scale volume electron microscopy by performing 3D cell cultures in a single carrier that is amenable to all imaging steps. This allows for following organoids' growth, probing their morphology with fluorescent markers, identifying areas of interest, and analyzing their 3D ultrastructure. We demonstrate this workflow on mouse and human 3D cultures and use automated image segmentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses identify local organization of diffraction-limited cell junctions in compact and polarized epithelia. The continuum-resolution imaging pipeline is thus suited to fostering basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy.

FLI1 and FRA1 transcription factors drive the transcriptional regulatory networks characterizing muscle invasive bladder cancer

Bladder cancer is mostly present in the form of urothelium carcinoma, causing over 150,000 deaths each year. Its histopathological classification as muscle invasive (MIBC) and non-muscle invasive (NMIBC) is the most prominent aspect, affecting the prognosis and progression of this disease. In this study, we defined the active regulatory landscape of MIBC and NMIBC cell lines using H3K27ac ChIP-seq and used an integrative approach to combine our findings with existing data. Our analysis revealed FRA1 and FLI1 as two critical transcription factors differentially regulating MIBC regulatory landscape. We show that FRA1 and FLI1 regulate the genes involved in epithelial cell migration and cell junction organization. Knock-down of FRA1 and FLI1 in MIBC revealed the downregulation of several EMT-related genes such as MAP4K4 and FLOT1. Further, ChIP-SICAP performed for FRA1 and FLI1 enabled us to infer chromatin binding partners of these transcription factors and link this information with their target genes. Finally, we show that knock-down of FRA1 and FLI1 result in significant reduction of invasion capacity of MIBC cells towards muscle microenvironment using IC-CHIP assays. Our results collectively highlight the role of these transcription factors in selection and design of targeted options for treatment of MIBC.

Host-Microbiota Communication in Spontaneously Hypertensive Rats Generates Unique IgA-Coated Gut Microbes.

Background Hypertension is associated with gut dysbiosis, altered intestinal immunity, and gut pathology in animal models and humans. Although these findings have implicated impaired interactions between gut and gut microbiota in hypertension, little is known about the specific functional gut microbes that interact with intestinal mucosa. Methods and Results To identify these microbes, we sorted Immunoglobin A (IgA)-coated (IgA+) and IgA-noncoated (IgA-) bacteria using a combination of magnetic-activated cell sorting and fluorescence-activated cell sorting, and subsequently performed 16 S rRNA gene sequencing (IgA-SEQ) to determine the microbial composition of IgA+ and IgA- fractions in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats. We observed a significant decrease in IgA+ bacteria in SHR compared with Wistar Kyoto and a distinct composition of IgA+ and IgA- bacteria between Wistar Kyoto and SHR, showing more IgA-bound Proteobacteria, Bacteroidetes and Actinobacteria but less of Firmicutes in SHR at the phylum level. We further identified enriched IgA-coated Romboutsia, Turicibacter, Ileibacterium, and Dubosiella in SHR that were negatively correlated with the various pathways including antigen presentation, immune response, cell junction organization, epithelium development, and defense response to virus. Conclusions We demonstrate new IgA-coated bacteria that participate in host-microbiota communication in hypertension, suggesting promising therapeutic interventions targeting these bacteria for hypertension management.

Proteomic and single-cell landscape reveals novel pathogenic mechanisms of HBV-infected intrahepatic cholangiocarcinoma

Despite the epidemiological association between intrahepatic cholangiocarcinoma (ICC) and hepatitis B virus (HBV) infection, little is known about the relevant oncogenic effects. A cohort of 32 HBV-infected ICC and 89 non-HBV-ICC patients were characterized using whole-exome sequencing, proteomic analysis, and single-cell RNA sequencing. Proteomic analysis revealed decreased cell-cell junction levels in HBV-ICC patients. The cell-cell junction level had an inverse relationship with the epithelial-mesenchymal transition (EMT) program in ICC patients. Analysis of the immune landscape found that more CD8 Tcells and Th2 cells were present in HBV-ICC patients. Single-cell analysis indicated that transforming growth factor beta signaling-related EMT program changes increased in tumor cells of HBV-ICC patients. Moreover, ICAM1+ tumor-associated macrophages are correlated with a poor prognosis and contributed to the EMT in HBV-ICC patients. Our findings provide new insights into the behavior of HBV-infected ICC driven by various pathogenic mechanisms involving decreased cell junction levels and increased progression of the EMT program.

Triploidization of rainbow trout affects proteins related to ovary development and reproductive activity

Rainbow trout are characterized by high amenability to chromosome manipulations during early development to produce androgens, gynogens, or sterile triploids, which are relevant in aquaculture. An understanding of the mechanisms affecting oogenesis and those resulting in diminished gonadal development in triploids is an important area of scientific study. Therefore, the aim of this study was to compare the ovary proteome from triploid (3 N) and diploid (2 N) rainbow trout females using tandem mass tag (TMT) peptide labelling coupled with liquid chromatography–mass spectrometry (LC–MS/MS) to identify differentially expressed proteins (DEPs) between 2 N and 3 N. The potential biological role of DEPs was also explored using comparative functional gene set enrichment to designate the potential protein markers of 3 N sterility. We identified a total of 3899 DEPs (q < 0.05), including 956 downregulated and 2943 upregulated proteins in 3 N ovaries compared to 2 N ovaries. The identified clusters for downregulated proteins in 3 N included lipid metabolic process, hormone biosynthesis, transport and exocytosis, cell junction organization and sperm-egg recognition, which can result in (i) the disruption of lipid, steroid hormone and vitellogenesis processes, (ii) impaired amino acid uptake, (iii) defects in cell–cell junctions reflecting tissue abnormalities and aberrant gonadal morphology, and (iv) impaired functions related to maintaining the proper structure of the vitelline envelope, oocyte formation and fertilization competence. With respect to upregulated proteins in 3 N ovaries, RNA processing, cell cycle, cytoskeleton organization and regulation of metabolic process were the primary clusters, which may reflect (i) the disturbance to RNA processing responsible for impairment of ovary development in triploid rainbow trout, (ii) disruption of spindle formation, incorrect chromosome alignment and segregation during meiotic division, (iii) increase in regulatory mechanism of apoptosis, which in consequence can provoke abnormal gonadal development and sterility, and (iv) enhanced ubiquitin–proteasome system governing aberrant gonadal morphology in 3 N females and oocyte maturation. The obtained results provide an accurate portrait of molecular changes in rainbow trout ovaries in response to triploidization and may contribute to a better understanding of the mechanisms responsible for impaired oogenesis in rainbow trout triploid females, which in turn leads to their sterility. Data are available via ProteomeXchange with identifier PXD034492.

Microglial state changes in response to LM11A‐31 promote recovery in a tauopathy mouse model of Alzheimer’s Disease

AbstractBackgroundTau pathologies in Alzheimer’s Disease (AD) contribute to neurodegeneration and synaptic degeneration via a variety of mechanisms. Neurotrophic signaling pathways present an avenue to counteract degenerative mechanisms via receptor modulating compounds, such as the p75NTR‐targeted small molecule LM11A‐31 (C31). Treatment with C31 in tauopathy models causes reductions in tau aggregation and microglial activation, restoration of synaptic plasticity, and prevention of synaptic spine loss. Using tauopathy model tauP301S mice (PS19), we examined the impact of long‐term dosing with C31 at the single‐cell transcriptional level to better understand the cell types and cellular mechanisms affected by treatment.MethodsPS19 and age‐matched (Wt) mice were dosed with either C31 or vehicle for 3 months starting at 6 months of age, when tau pathology was well established. Whole cortex was isolated for nuclear extraction and single‐nucleus RNA‐sequencing. AnalysisResultsClustering microglia for cell type annotation revealed treatment group‐specific microglial subclusters (Figure 1). Further, microglial gene expression across experimental groups showed a strong inverse correlation (R = ‐0.647; p &lt; 1.01e‐78) between C31 differential expression and PS19 differential expression in genes nominally significant (p &lt; 0.05) for both (Figure 2). In particular, microglial genes that were downregulated in the PS19 tauopathy model and upregulated by C31 were enriched for those falling into GO categories including “synapse organization” and “cell junction organization” (p‐adj: 8.95e‐20, 2.66e‐17). Conversely, expression that was up in PS19 tauopathy and down in C31 showed enrichment for “small GTPase mediated signal transduction”, and “cell junction disassembly” (p‐adj: 8.59e‐05, 0.0431).ConclusionThese findings demonstrate differences in microglial gene expression between Wt and PS19 mice some of which may be reversed by C31. Loss of homeostatic microglia has been previously associated with AD neurodegeneration, improper dendritic spine formation and inability to maintain synaptic structure. Our findings support the hypothesis that loss of homeostatic microglia exacerbates synaptic dysfunction, and their recovery may be one potential mechanism by which C31 can restore synaptic plasticity.

Pathway-level mutation analysis in primary high-grade serous ovarian cancer and matched brain metastases

Brain metastases (BMs) in ovarian cancer (OC) are a rare event. BMs occur most frequently in high-grade serous (HGS) OC. The molecular features of BMs in HGSOC are poorly understood. We performed a whole-exome sequencing analysis of ten matched pairs of formalin-fixed paraffin-embedded samples from primary HGSOC and corresponding BMs. Enrichment significance (p value; false discovery rate) was computed using the Reactome, the Kyoto Encyclopedia of Genes and Genomes pathway collections, and the Gene Ontology Biological Processes. Germline DNA damage repair variants were found in seven cases (70%) and involved the BRCA1, BRCA2, ATM, RAD50, ERCC4, RPA1, MLHI, and ATR genes. Somatic mutations of TP53 were found in nine cases (90%) and were the only stable mutations between the primary tumor and BMs. Disturbed pathways in BMs versus primary HGSOC constituted a complex network and included the cell cycle, the degradation of the extracellular matrix, cell junction organization, nucleotide metabolism, lipid metabolism, the immune system, G-protein-coupled receptors, intracellular vesicular transport, and reaction to chemical stimuli (Golgi vesicle transport and olfactory signaling). Pathway analysis approaches allow for a more intuitive interpretation of the data as compared to considering single-gene aberrations and provide an opportunity to identify clinically informative alterations in HGSOC BM.

Transcriptional mapping of the macaque retina and RPE-choroid reveals conserved inter-tissue transcription drivers and signaling pathways.

The macula and fovea comprise a highly sensitive visual detection tissue that is susceptible to common disease processes like age-related macular degeneration (AMD). Our understanding of the molecular determinants of high acuity vision remains unclear, as few model organisms possess a human-like fovea. We explore transcription factor networks and receptor-ligand interactions to elucidate tissue interactions in the macula and peripheral retina and concomitant changes in the underlying retinal pigment epithelium (RPE)/choroid. Poly-A selected, 100bp paired-end RNA-sequencing (RNA-seq) was performed across the macular/foveal, perimacular, and temporal peripheral regions of the neural retina and RPE/choroid tissues of four adult Rhesus macaque eyes to characterize region- and tissue-specific gene expression. RNA-seq reads were mapped to both the macaque and human genomes for maximum alignment and analyzed for differential expression and Gene Ontology (GO) enrichment. Comparison of the neural retina and RPE/choroid tissues indicated distinct, contiguously changing gene expression profiles from fovea through perimacula to periphery. Top GO enrichment of differentially expressed genes in the RPE/choroid included cell junction organization and epithelial cell development. Expression of transcriptional regulators and various disease-associated genes show distinct location-specific preference and retina-RPE/choroid tissue-tissue interactions. Regional gene expression changes in the macaque retina and RPE/choroid is greater than that found in previously published transcriptome analysis of the human retina and RPE/choroid. Further, conservation of human macula-specific transcription factor profiles and gene expression in macaque tissues suggest a conservation of programs required for retina and RPE/choroid function and disease susceptibility.

Efficacy of low-dose hCG on FET cycle in patients with recurrent implantation failure.

To study patients' new treatment methods and mechanisms of repeated implantation failure. A retrospective study. In vitro fertilization (IVF) unit in a Three-A hospital. Ninety-three patients with repeated implantation failure in IVF and embryo transfer. the luteal phase support. According to whether human chorionic gonadotropin(HCG) was added, the two groups were divided into an observation group and a control group, and the clinical outcomes of the two groups were compared. Furthermore, 20 patients were selected for whole exome sequencing to investigate the mechanism. The observation group's clinical pregnancy rate and live birth rate were significantly higher than those in the control group (P=0.004). Functional enrichment analysis showed that these genes were significantly enriched in embryo implantation or endometrial receptivity processes, such as microtubule-based movement, NABA CORE MATRISOME, superoxide anion generation, protein localization to vacuole, extracellular matrix organization, fertilization, microtubule-based transport, cell junction organization, microtubule cytoskeleton organization. Furthermore, variants detected in these pathway genes were missense mutations that affect the protein's biological activity but do not effectuate its inactivation. Adding HCG in the luteal phase might improve the clinical pregnancy and live birth rates in RIF patients. The potential pathogenesis of RIF genetic level may be caused by microtubule-based movement, extracellular matrix organization, and the Superoxide Anion generation pathway.