Ephrin Receptor Signaling Research Articles

Proteomics analysis of carotid body tumor revealed potential mechanisms and molecular differences among Shamblin classifications.

Carotid body tumors (CBTs) are a rare type of paraganglioma, and surgical resection is the only effective treatment. Because of the proximity of CBTs to the carotid artery, jugular vein, and cranial nerve, surgery is extremely difficult, with high risks of hemorrhage and neurovascular injury. The Shamblin classification is used for CBT clinical evaluation; however, molecular mechanisms underlying classification differences remain unclear. This study aimed to investigate pathogenic mechanisms and molecular differences between CBT types. In Shamblin I, II, and III tumors, differentially expressed proteins (DEPs) were identified using direct data-independent acquisition (DIA). DEPs were validated using immunohistochemistry. Proteomics profiling of three Shamblin subtypes differed significantly. Bioinformatics analysis showed that adrenomedullin signaling, protein kinase A signaling, vascular endothelial growth factor (VEGF) signaling, ephrin receptor signaling, gap junction signaling, interleukin (IL)-1 signaling, actin cytoskeleton signaling, endothelin-1 signaling, angiopoietin signaling, peroxisome proliferator-activated receptor (PPAR) signaling, bone morphogenetic protein (BMP) signaling, hypoxia-inducible factor 1-alpha (HIF-1α) signaling, and IL-6 signaling pathways were significantly enriched. Furthermore, 60 DEPs changed significantly with tumor progression. Immunohistochemistry validated several important DEPs, including aldehyde oxidase 1 (AOX1), mediator complex subunit 22 (MED22), carnitine palmitoyltransferase 1A (CPT1A), and heat shock transcription factor 1 (HSF1). To our knowledge, this is the first application of proteomics quantification in CBT. Our results will deepen the understanding of CBT-related pathogenesis and aid in identifying therapeutic targets for CBT treatment.

Distinct H3K27me3 and H3K27ac Modifications in Neural Tube Defects Induced by Benzo[a]pyrene.

The pathological mechanisms of neural tube defects (NTDs) are not yet fully understood. Although the dysregulation of histone modification in NTDs is recognized, it remains to be fully elucidated on a genome-wide level. We profiled genome-wide H3K27me3 and H3K27ac occupancy by CUT&Tag in neural tissues from ICR mouse embryos with benzo[a]pyrene (BaP)-induced NTDs (250 mg kg-1) at E9.5. Furthermore, we performed RNA sequencing (RNA-seq) to investigate the regulation of histone modifications on gene expressions. Gene ontology and KEGG analysis were conducted to predict pathways involved in the development of NTDs. Our analysis of histone 3 lysine 27 modification in BaP-NTD neural tissues compared to BaP-nonNTD revealed 6045 differentially trimethylated regions and 3104 acetylated regions throughout the genome, respectively. The functional analysis identified a number of pathways uniquely enriched for BaP-NTD embryos, including known neurodevelopment related pathways such as anterior/posterior pattern specification, ephrin receptor signaling pathway, neuron migration and neuron differentiation. RNA-seq identified 423 differentially expressed genes (DEGs) between BaP-NTD and BaP-nonNTD group. The combination analysis of CUT&Tag and RNA-seq found that 55 DEGs were modified by H3K27me3 and 25 by H3K27ac in BaP-NTD, respectively. In the transcriptional regulatory network, transcriptional factors including Srsf1, Ume6, Zbtb7b, and Cad were predicated to be involved in gene expression regulation. In conclusion, our results provide an overview of histone modifications during neural tube closure and demonstrate a key role of genome-wide alterations in H3K27me3 and H3K27ac in NTDs corresponding with changes in transcription profiles.

Use of methylation and fragmentation signals in the detection of early-stage colorectal cancer.

201 Background: Colorectal cancer (CRC) remains a leading cause of cancer related mortality worldwide. We utilized cell-free DNA (cfDNA) methylation and fragmentation characteristics of selected cancer-related biomarker regions and applied tumor-derived signal deduction and a machine learning algorithm to refine a blood test for the early detection of CRC. Methods: This was a prospective, international (Spain, Ukraine, Germany and USA [part of NCT04792684 study] population), observational cohort study. Plasma samples were collected either prior to a scheduled screening colonoscopy or prior to colonic surgery for primary CRC. 95 cfDNA samples from 48 early stage (I-II), 47 late-stage (III-IV) CRC patients (mean age 65 [48-83], female 45%, distal cancers 51%) and 204 age, gender and country of origin matched colonoscopy-checked controls were analyzed. 79 of the control patients had a negative colonoscopy finding (cNEG), 96 had benign findings of diverticulosis, hemorrhoids and/or hyperplastic polyps (BEN), 29 had non-advanced adenomas (NAA). Samples were analyzed utilizing previously described hybrid-capture based sequencing methodology. Panel of targeted biomarkers was previously identified through tissue- and plasma-based discovery and further narrowed down through cancer-related biological pathways analysis workflow. Individual cfDNA fragments belonging to each biomarker region were scored for cancer-specific signal. Finally, calculated scores were used in prediction model building and testing for establishing panel accuracy for CRC detection. Results: Prediction model utilizing a panel of methylation and fragmentation scores originating from cfDNA biomarkers belonging to relevant cancer development and progression related pathways, such as axonal guidance, ephrin receptor signaling, epithelial-mesenchymal transition and FGF signaling, correctly classified 92% (87/95) of CRC patients. Sensitivity per cancer stage ranged from 91% (21/23) for stage I, 92% (23/25) for stage II, 91% (30/33) for stage III and 93% (13/14) for stage IV. Fragmentation signals contributed most to early-stage cancers (I-II), while methylation signals were more significant for late stage (III-IV) detection. Specificity of the model was 94% (199/204), with 97% (28/29) NAA, 91% (87/96) BEN and 96% (76/79) cNEG patients correctly identified. Lesion location, gender, age and country of origin were not significantly correlated to prediction outcome. Conclusions: Use of methylation and fragmentation characteristics of cancer-related cfDNA regions, combined with a machine-learning algorithm is highly accurate for early-stage (I-II) CRCs (92% sensitivity at 94% specificity). The study is being further expanded on larger cohort for validation of a highly accurate and minimally invasive blood-based CRC screening test.

Abstract P028: Methylation biomarkers for colorectal cancer early detection and survival prognostics impact gene expression and link to cancer-related biological pathways

Abstract Background: DNA methylation has been previously shown to have diagnostic and predictive potential for colorectal cancer (CRC). Aim of this study was to evaluate putative methylation markers in the context of early cancer development and diagnostics as well as further investigate the biological significance of these regions. Methods: Biomarker discovery was done by whole genome bisulfite sequencing (WGBS) of 88 CRC, 48 advanced adenoma (AA) and corresponding adjacent normal tissue (NAT) samples. Short-list of significantly hypermethylated regions (DMRs) was correlated to transcriptomics data from 512 CRC patients in The Cancer Genome Atlas (TCGA) cohort. Pathway enrichment for biological pathway analysis of the DMRs was done by using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Survival analysis was performed using Kaplan–Meier method on sub-groups of patients divided by the methylation status of individual markers. Finally, individual marker significance of selected regions was evaluated by analyzing 26 plasma samples from early stage (stage I-IIA) CRC samples and 42 colonoscopy verified controls (CNT) with targeted methylation sequencing assay. Results: 4167 putative marker regions were identified from biomarker discovery with WGBS. Differential signal could be observed both between AA and NAT and CRC and NAT, while several of these regions were differentially methylated also between AA and CRC samples, indicating biological signal change with adenoma progression to cancer. 84 hypermethylated DMRs from several verification studies were further evaluated against transcriptome data from TCGA, where overlap for 69 genes was found. 19 of these genes showed a significant down- regulation (p< 0.05), indicating a link between hypermethylation and gene expression. 2 genes showed significant up-regulation (p< 0.05), which could indicate other epigenic processes to be in place. KEGG pathway analysis revealed that the top pathways involved were axonal guidance, ephrin receptor signaling, epithelial-mesenchymal transition and FGF signaling, which all play significant role in the context of cancer development and progression. Kaplan-Meier analysis showed significant correlation to patients 5- year survival prediction linked to 3 genes: FGF14 (p=0.025, HR = 1.75) DPY19L2P1 (p=0.012, HR = 1.86), PTPRO (p=0.046, HR = 1.63). Targeted sequencing analysis on plasma samples of patients with early stage (I-IIA) colorectal cancer and age and gender matching colonoscopy-verified controls, showed high individual marker accuracy with AUC= 0.78 for FGF14, AUC= 0.81 for DPY19L2P1 and AUC= 0.73 for PTPRO. Conclusions: Methylation markers have distinct signals in early development of CRC, with high individual accuracy for separating early-stage cancers from matching controls. These regions have impact on gene expression and can be linked to relevant biological pathways. Extending early detection potential of the markers to further prognostics and stratification, could lead to better outcomes and improved survival of the patients. Citation Format: Pol Canal Noguer, Alejandro Requena Bermejo, Francesco Mattia Mancuso, Juan Carlos Higareda, Marina Manrique López, Marko Chersicola, Pablo Pérez Martínez, Pablo Antonio Camino, Primoz Knap, Vivian Erklavec Zajec, Kristi Kruusmaa. Methylation biomarkers for colorectal cancer early detection and survival prognostics impact gene expression and link to cancer-related biological pathways. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P028.

Chronic Inhalation Exposure to Antimony Trioxide Exacerbates the MAPK Signaling in Alveolar Bronchiolar Carcinomas in B6C3F1/N Mice.

Antimony trioxide (AT) is used as a flame retardant in fabrics and plastics. Occupational exposure in miners and smelters is mainly through inhalation and dermal contact. Chronic inhalation exposure to AT particulates in B6C3F1/N mice and Wistar Han rats resulted in increased incidences and tumor multiplicities of alveolar/bronchiolar carcinomas (ABCs). In this study, we demonstrated Kras (43%) and Egfr (46%) hotspot mutations in mouse lung tumors (n = 80) and only Egfr (50%) mutations in rat lung tumors (n = 26). Interestingly, there were no differences in the incidences of these mutations in ABCs from rats and mice at exposure concentrations that did and did not exceed the pulmonary overload threshold. There was increased expression of p44/42 mitogen-activated protein kinase (MAPK) (Erk1/2) protein in ABCs harboring mutations in Kras and/or Egfr, confirming the activation of MAPK signaling. Transcriptomic analysis indicated significant alterations in MAPK signaling such as ephrin receptor signaling and signaling by Rho-family GTPases in AT-exposed ABCs. In addition, there was significant overlap between transcriptomic data from mouse ABCs due to AT exposure and human pulmonary adenocarcinoma data. Collectively, these data suggest chronic AT exposure exacerbates MAPK signaling in ABCs and, thus, may be translationally relevant to human lung cancers.

Gene Expression Sets and Renal Profiling from the Rain (Renal AL Amyloid Involvement and NEOD00) Trial

INTRODUCTION: Despite the high prevalence of kidney involvement in patients with light chain (AL) amyloidosis, a significant knowledge gap exists in understanding how immunoglobulin light chains drive kidney injury. The chief reason is the lack of molecular data from high-quality kidney tissue samples from this patient population. The RAIN trial (NCT03168906) was a phase 2B randomized placebo-controlled, open-label clinical trial that evaluated the efficacy of NEOD001, a monoclonal antibody targeting amyloid fibrils, versus placebo in patients with AL nephropathy. OBJECTIVES: Identifying amyloid-specific molecular signatures in kidney biopsy tissue obtained from AL patients with nephropathy.MATERIALS& METHODS: Renal biopsies were collected on all RAIN participants before randomization to NEOD001 or placebo. Trial clinical data was incorporated to link tissue, molecular, and kidney function data. The histologic assessment included a composite scarring injury score (CSIC) as determined by the degree of glomerular and tubulointerstitial scarring, and a novel amyloid score (AS), as quantified by the extent and distribution of amyloid deposition. Glomerular (G) and tubulointerstitial compartments were micro-dissected and sequenced separately. To identify enriched pathways in AL nephropathy, the expression data was compared to the glomerular and tubular expression profiles of healthy living donor (LD) from the Nephrotic Syndrome Study Network (NEPTUNE) study. Differentially expressed genes (DEGs) were determined with the R limma package based on the voom transformed log2 count data with an FDR of <=0.05. The AL and NEPTUNE datasets were merged and batch-corrected using ComBat. RESULTS: Ten patients were enrolled and randomized in the RAIN trial (5 NEOD001 and five placeboes) before trial closure in April 2018. Kidney tissue was available in nine of the ten patients. Cluster analysis revealed two distinct patient clusters (G1 and G2) within the glomerular and tubulointerstitial compartments. Key data of kidney function and biopsy morphology by compartment and cluster type are shown in Table 1. Despite similar eGFR, there was a higher AS and a non-significant increase in 24-hour urine protein in the glomeruli of individuals in the G2 patient cluster. Subcomponent analysis of AS revealed that the increase in amyloid score in G2 was driven almost entirely by an increase in mesangial and capillary wall amyloid deposition. Pathway enrichment in Amyloid kidney disease versus LD: IPA analysis identified unique activation signatures in the glomeruli and tubules. In the glomeruli of biopsies from the G2 cluster, there was evidence of increased TNF, IFNG, IL33 and VEGFA cytokine activity and a predicted increase in canonical PI3K/Akt signaling and innate immune responses. Tubules showed increased activation of transcriptional regulators TP53, TP73, increased cytokine signaling of IFNG, TGFb1 and VEGF. There was also a predicted activation of major canonical pathways including B cell receptor signaling, PI3K/AKT and Ephrin receptor signaling (Fig 1). Pathway Enrichment in G2 versus G1: Glomeruli showed activation of fibrotic pathways accompanied by a reduction in metabolic processes including gluconeogenesis, lipid transport and xenobiotics. There was an increase in canonical signaling of LPS/IL-1 mediated inhibition of RXR function that drives increased IL1 and TNF release from macrophages. Transcriptomic evidence of TNF activation was also noted in the TI. CONCLUSIONS: The RAIN trial closed prematurely, and thus no conclusions can be reached about the efficacy of NEOD001 in patients with AL nephropathy. However, these limited data revealed the presence of two distinct molecular subtypes of AL nephropathy. Whether these distinct molecular subtypes reflect different stages of the disease along a continuous spectrum or are of unique prognostic entities in themselves remains unclear. Detailed studies, of a larger cohort of patients including investigating diseased tissue at a single cell resolution can help identify the response of individual kidney cell types. Understanding the cellular responses of key cell types to injury from amyloid deposits could provide novel therapeutic targets to mitigate renal injury and improve patient outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Network Analyses Implicate Death-Associated Protein Kinase 3 (DAPK3) as a Key Factor in Colitis-Associated Dysplasia Progression.

Ulcerative colitis (UC) is a progressive disorder that elevates the risk of colon cancer development through a colitis-dysplasia-carcinoma sequence. Gene expression profiling of colitis-associated lesions obtained from patients with varied extents of UC can be mined to define molecular panels associated with colon cancer development. Differential gene expression profiles of 3 UC clinical subtypes and healthy controls were developed for the GSE47908 microarray data set of healthy controls, left-sided colitis, pancolitis, and colitis-associated dysplasia (CAD) using limma R. A gene ontology enrichment analysis of differentially expressed genes (DEGs) revealed a shift in the transcriptome landscape as UC progressed from left-sided colitis to pancolitis to CAD, from being immune-centric to being cytoskeleton-dependent. Hippo signaling (via Yes-associated protein [YAP]) and Ephrin receptor signaling were the top canonical pathways progressively altered in concert with the pathogenic progression of UC. A molecular interaction network analysis of DEGs in left-sided colitis, pancolitis, and CAD revealed 1 pairwise line, or edge, that was topologically important to the network structure. This edge was found to be highly enriched in actin-based processes, and death-associated protein kinase 3 (DAPK3) was a critical member and sole protein kinase member of this network. Death-associated protein kinase 3 is a regulator of actin-cytoskeleton reorganization that controls proliferation and apoptosis. Differential correlation analyses revealed a negative correlation for DAPK3-YAP in healthy controls that flipped to positive in left-sided colitis. With UC progression to CAD, the DAPK3-YAP correlation grew progressively more positive. In summary, DAPK3 was identified as a candidate gene involved in UC progression to dysplasia.

Fetal Zone Steroids Show Discrete Effects on Hyperoxia-Induced Attenuation of Migration in Cultured Oligodendrocyte Progenitor Cells.

Cerebral oxygenation disturbances contribute to the pathogenesis of brain lesions in preterm infants with white matter damage. These children are at risk of developing long-term neurodevelopmental disabilities. Preterm birth is associated with sudden hormonal changes along with an untimely increase in oxygen tissue tension. There is a persistent high postnatal production of fetal zone steroids (FZS), which serve in the fetoplacental unit as precursors for placental estrogen synthesis during pregnancy. The role of FZS in events associated with oxygenation differences and their impact on the developing white matter is not well understood. Therefore, we investigated the effect of hyperoxia (80% O2) and subsequent administration of FZS on the protein composition and migration capabilities of immature oligodendrocytes using the OLN93 (rat-derived OPC) cell line as an experimental model. We tested the effect of the FZS, dehydroepiandrosterone (DHEA), 16α-OH-DHEA, and adiol (5-androstene-3β, 17β-diol). After 24-hour exposure to hyperoxia, we monitored the changes in the proteome profile following treatment and observed significant alterations in pathways regulating cytoskeletal remodelling, cell migration, and cell survival. Additionally, hyperoxia leads to impaired migration of the OLN93 cells in culture. Administration of the FZS showed positive effects on the migration process under normoxic conditions in general. However, under hyperoxic conditions, the trend was less prominent. The observed effects could be related to changes in levels of cofilin/LIMK pathway-associated proteins. Adiol had a negative effect when administered together with estradiol, and the proteomic data reveal the activation of ephrin receptor signalling that might be responsible for the attenuation of migration. The results suggest that FZS can differentially regulate pathways involved in the migration of OLN93 cells. A deeper insight into the precise role of endogenous FZS would be an essential prerequisite for developing new treatment strategies including supplementation of estradiol and other steroids in preterm infants.

Persistent SARS‐CoV‐2 Effects Induce Neuropathy Signature in Dorsal Root Ganglia Underlying Hypersensitivity in a Hamster Model

Post‐acute sequelae of COVID‐19, commonly known as long‐COVID, is defined as a persistent symptom(s) that is unexplainable by alternative diagnosis and lasts beyond three months after the onset of COVID‐19. Several studies have now identified long‐COVID in &gt;50% of COVID‐19 patients, emphasizing the need for elucidating mechanisms underlying these symptoms. Pain is a prevalent symptom in long‐COVID patients and presents as headache, persistent muscle pain, joint pain, stomach pain, chest pain, respiratory discomfort, and dysesthesia or paresthesia. Understanding the molecular underpinnings of pain maintenance in these patients could provide information on novel therapeutic strategies. Our lab has previously identified novel treatments for pain due to peripheral inflammation from dysesthesia‐inducing mechanisms associated with acute SARS‐CoV‐2 (SCV2) respiratory infection in hamsters (1‐4 days post‐infection, dpi). We have also previously demonstrated the validity of long‐term infection of hamsters (30‐60 dpi) as a pre‐clinical model of long‐COVID. By utilizing the Von Frey assay, we found that SCV2, but not Influenza A, causes mechanical hypersensitivity at 28 dpi in both male and female hamsters. Bulk RNA sequencing of 31 dpi thoracic dorsal root ganglia (DRGs) revealed a unique transcriptional perturbation signature (168 DEGs, 47 up &amp; 121 down, p‐adj.&lt;0.1), despite viral clearance at approximately 7 dpi. Ingenuity Pathway Analysis of this sequencing (853 DEGs, p‐nom.&lt;0.05) identified several injury‐related canonical pathways, including Production of NO &amp; ROS in Macrophages, Signaling by Rho Family GTPases, mTOR Signaling, Estrogen Receptor Signaling, and Ephrin Receptor Signaling. The Enrichr DisGeNET browser associated these transcriptional changes with clinical neurodegeneration phenotypes, including Amyotrophic Lateral Sclerosis, Alzheimer’s Disease, Neurodegenerative Disorders, and Parkinson Disease. Of note, TUNEL staining did not demonstrate any sign of SCV2‐induced sensory neuron apoptosis at 31 dpi. Further investigation of the sequencing dataset revealed a broad downregulation of TubbmRNA isoforms and Mbp, suggesting microtubule and myelin dysregulation. We also observed a drastic increase in Scn8areads, which could point to Nav1.6‐induced DRG neuron hyperexcitability. In conclusion, our findings suggest that SCV2 leaves a lasting hypersensitivity‐associated transcriptomic signature in DRGs despite early viral clearance that appears to be associated with neurodegeneration mechanisms. As we continue investigating the mechanisms underlying SCV2‐induced actions in DRGs and peripheral nerves, we will also use an upstream regulator analysis of our RNA sequencing data to identify promising therapeutic targets.

Cerebrospinal Fluid MicroRNA Changes in Cognitively Normal Veterans With a History of Deployment-Associated Mild Traumatic Brain Injury.

A history of traumatic brain injury (TBI) increases the odds of developing Alzheimer’s disease (AD). The long latent period between injury and dementia makes it difficult to study molecular changes initiated by TBI that may increase the risk of developing AD. MicroRNA (miRNA) levels are altered in TBI at acute times post-injury (<4 weeks), and in AD. We hypothesized that miRNA levels in cerebrospinal fluid (CSF) following TBI in veterans may be indicative of increased risk for developing AD. Our population of interest is cognitively normal veterans with a history of one or more mild TBI (mTBI) at a chronic time following TBI. We measured miRNA levels in CSF from three groups of participants: (1) community controls with no lifetime history of TBI (ComC); (2) deployed Iraq/Afghanistan veterans with no lifetime history of TBI (DepC), and (3) deployed Iraq/Afghanistan veterans with a history of repetitive blast mTBI (DepTBI). CSF samples were collected at the baseline visit in a longitudinal, multimodal assessment of Gulf War veterans, and represent a heterogenous group of male veterans and community controls. The average time since the last blast mTBI experienced was 4.7 ± 2.2 years [1.5 – 11.5]. Statistical analysis of TaqManTM miRNA array data revealed 18 miRNAs with significant differential expression in the group comparisons: 10 between DepTBI and ComC, 7 between DepC and ComC, and 8 between DepTBI and DepC. We also identified 8 miRNAs with significant differential detection in the group comparisons: 5 in DepTBI vs. ComC, 3 in DepC vs. ComC, and 2 in DepTBI vs. DepC. When we applied our previously developed multivariable dependence analysis, we found 13 miRNAs (6 of which are altered in levels or detection) that show dependencies with participant phenotypes, e.g., ApoE. Target prediction and pathway analysis with miRNAs differentially expressed in DepTBI vs. either DepC or ComC identified canonical pathways highly relevant to TBI including senescence and ephrin receptor signaling, respectively. This study shows that both TBI and deployment result in persistent changes in CSF miRNA levels that are relevant to known miRNA-mediated AD pathology, and which may reflect early events in AD.

Regulatory Network Analysis in Estradiol-Treated Human Endothelial Cells.

Background/Aims: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a comprehensive regulatory network (miRNA–transcription factor–downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. Methods: miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17β-estradiol (E2) (1 nmol/L, 24 h). miRNA–mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. Results: miRNA–target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship according to miRNA target prediction databases. The analysis identified 588 miRNA–target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion molecule binding pathways. Conclusion: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.

Genomic Effects of Arginine on Cell Behavior of Conceptus Trophectoderm in Ungulates

Abstract Hatched ungulate (e.g., pigs, sheep and other ruminants) blastocysts undergo dramatic morphological transitions from spherical to tubular to filamentous forms to conceptuses (embryo/fetus and associated extraembryonic membranes) before implantation. L-Arginine (Arg), a conditionally essential amino acid, is required for this process to activate the mTOR cell signaling pathway to induce proliferation of both porcine and ovine conceptus trophectoderm cells. However, the genomic effects of arginine on trophectoderm cells is unknown. RNA-seq was used for a comparative transcriptome analysis of porcine and ovine trophectoderm cells to further understand effects of Arg on regulation of metabolism in trophectoderm cells. An established porcine trophectoderm (pTr) cell line isolated from D12 porcine conceptuses, as well as an established ovine trophectoderm (oTr) cell line isolated from D15 ovine conceptuses were used to determine response to Arg at the physiological concentration of 0.2 mM in a 48-h culture. In pTr cells, a total of 2,723 differentially expressed genes (DEG; 1,482 up and 1,241 down) were identified in response to Arg. In oTr cells, a total of 5,369 DEG (2,819 up and 2,550 down) were detected. Comparison analyses showed that the Arg-treated pTr and oTr transcriptomes share 873 common DEG (273 up and 342 down). Canonical pathway analyses identified the top enriched pathways in both pTr and oTr cells, including activation of actin cytoskeleton signaling, adrenomedullin signaling, and IGF-1 signaling; and inhibition of cell cycle G2/M checkpoint regulation, and p53 signaling. In response to Arg, pathways associated with cholesterol biosynthesis, and estrogen-mediated S-phase entry were exclusively activated in the pTr cells; whereas interferon signaling, ephrin receptor signaling, and integrin signaling were specifically activated in the oTr cells. Results from this study advance understanding of mechanisms responsible for elongation of ovine and porcine conceptuses and enable the rational design of future experiments.

Detection of pro angiogenic and inflammatory biomarkers in patients with CKD

Cardiovascular disease (CVD) is the most common cause of death in patients with native and post-transplant chronic kidney disease (CKD). To identify new biomarkers of vascular injury and inflammation, we analyzed the proteome of plasma and circulating extracellular vesicles (EVs) in native and post-transplant CKD patients utilizing an aptamer-based assay. Proteins of angiogenesis were significantly higher in native and post-transplant CKD patients versus healthy controls. Ingenuity pathway analysis (IPA) indicated Ephrin receptor signaling, serine biosynthesis, and transforming growth factor-β as the top pathways activated in both CKD groups. Pro-inflammatory proteins were significantly higher only in the EVs of native CKD patients. IPA indicated acute phase response signaling, insulin-like growth factor-1, tumor necrosis factor-α, and interleukin-6 pathway activation. These data indicate that pathways of angiogenesis and inflammation are activated in CKD patients’ plasma and EVs, respectively. The pathways common in both native and post-transplant CKD may signal similar mechanisms of CVD.

Transcriptome of rhesus macaque (Macaca mulatta) exposed to total-body irradiation

The field of biodosimetry has seen a paradigm shift towards an increased use of molecular phenotyping technologies including omics and miRNA, in addition to conventional cytogenetic techniques. Here, we have used a nonhuman primate (NHP) model to study the impact of gamma-irradiation on alterations in blood-based gene expression. With a goal to delineate radiation induced changes in gene expression, we followed eight NHPs for 60 days after exposure to 6.5 Gy gamma-radiation for survival outcomes. Analysis of differential gene expression in response to radiation exposure yielded 26,944 dysregulated genes that were not significantly impacted by sex. Further analysis showed an increased association of several pathways including IL-3 signaling, ephrin receptor signaling, ErbB signaling, nitric oxide signaling in the cardiovascular system, Wnt/β-catenin signaling, and inflammasome pathway, which were associated with positive survival outcomes in NHPs after acute exposure to radiation. This study provides novel insights into major pathways and networks involved in radiation-induced injuries that may identify biomarkers for radiation injury.

Coculture of porcine luteal cells during in vitro porcine oocyte maturation affects blastocyst gene expression and developmental potential

Oocyte maturation in culture is still the weakest part of in vitro fertilization (IVF) and coculture with somatic cells may be an alternative to improve suboptimal culture conditions, especially in the pig in which maturation takes more than 44 h. In the present study, we investigated the effect of a coculture system of porcine luteal cells (PLC) during in vitro maturation (IVM) on embryo development and gene expression. Cumulus-oocyte complexes were matured in vitro in TCM-199 with human menopausal gonadotrophin (control) and in coculture with PLC. IVF was performed with frozen-thawed boar semen in Tris-buffered medium. Presumptive zygotes were cultured in PZM for 7 days. The coculture with PLC significantly increased blastocysts rates. Gene expression changes were measured with a porcine embryo-specific microarray and confirmed by RT-qPCR. The global transcription pattern of embryos developing after PLC coculture exhibited overall downregulation of gene expression. Following global gene expression pattern analysis, genes associated with lipid metabolism, mitochondrial function, endoplasmic reticulum stress, and apoptosis were found downregulated, and genes associated with cell cycle and proliferation were found upregulated in the PLC coculture. Canonical pathway analysis by Ingenuity Pathway revealed that differential expression transcripts were associated with the sirtuin signaling pathway, oxidative phosphorylation pathway, cytokines and ephrin receptor signaling. To conclude, the coculture system of PLC during IVM has a lasting effect on the embryo until the blastocyst stage, modifying gene expression, with a positive effect on embryo development. Our model could be an alternative to replace the conventional maturation medium with gonadotrophins with higher rates of embryo development, a key issue in porcine in vitro embryo production.

Identification of CDK1 as a candidate marker in cutaneous squamous cell carcinoma by integrated bioinformatics analysis.

BackgroundCutaneous squamous cell carcinoma (cSCC) is a relatively common cancer that accounts for nearly 50% of non-melanoma skin cancer cases. However, the genotypes that are linked with poor prognosis and/or high relapse rates and pathogenic mechanisms of cSCC are not fully understood. To address these points, three gene expression datasets were analyzed to identify candidate biomarker genes in cSCC.MethodsThe GSE117247, GSE32979, and GSE98767 datasets comprising a total of 32 cSCC samples and 31 normal skin tissue samples were obtained from the National Center for Biotechnology Information Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified and underwent pathway enrichment analyses with the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG). A putative DEG protein–protein interaction (PPI) network was also established that included hub genes. The expression of CDK1, MAD2L1, BUB1 ans CDC20 were examined in the study.ResultsA total of 335 genes were identified, encompassing 219 found to be upregulated and 116 genes that were downregulated in cSCC, compared to normal tissue. Enriched functions of these DEGs were associated with Ephrin receptor signaling and cell division; cytosol, membrane, and extracellular exosomes; ATP-, poly(A) RNA-, and identical protein binding. We also established a PPI network comprising 332 nodes and identified KIF2C, CDC42, AURKA, MAD2L1, MYC, CDK1, FEN1, H2AFZ, BUB1, BUB1B, CKS2, CDC20, CCT2, ACTR2, ACTB, MAPK14, and HDAC1 as candidate hub genes. The expression of CDK1 are significantly higher in the cSCC tissues than that in normal skin.ConclusionsThe DEGs identified in this study are potential therapeutic targets and biomarkers for cSCC. CDK1 is a gene closely related to the occurrence and development of cSCC, which may play an important role. Bioinformatics analysis shows that it is involved in the important pathway of the pathogenesis of cSCC, and may be recognized and applied as a new biomarker in the future diagnosis and treatment of cSCC.

Single Cell Transcriptomics Implicate Novel Monocyte and T Cell Immune Dysregulation in Sarcoidosis.

Sarcoidosis is a systemic inflammatory disease characterized by infiltration of immune cells into granulomas. Previous gene expression studies using heterogeneous cell mixtures lack insight into cell-type-specific immune dysregulation. We performed the first single-cell RNA-sequencing study of sarcoidosis in peripheral immune cells in 48 patients and controls. Following unbiased clustering, differentially expressed genes were identified for 18 cell types and bioinformatically assessed for function and pathway enrichment. Our results reveal persistent activation of circulating classical monocytes with subsequent upregulation of trafficking molecules. Specifically, classical monocytes upregulated distinct markers of activation including adhesion molecules, pattern recognition receptors, and chemokine receptors, as well as enrichment of immunoregulatory pathways HMGB1, mTOR, and ephrin receptor signaling. Predictive modeling implicated TGFβ and mTOR signaling as drivers of persistent monocyte activation. Additionally, sarcoidosis T cell subsets displayed patterns of dysregulation. CD4 naïve T cells were enriched for markers of apoptosis and Th17/Treg differentiation, while effector T cells showed enrichment of anergy-related pathways. Differentially expressed genes in regulatory T cells suggested dysfunctional p53, cell death, and TNFR2 signaling. Using more sensitive technology and more precise units of measure, we identify cell-type specific, novel inflammatory and regulatory pathways. Based on our findings, we suggest a novel model involving four convergent arms of dysregulation: persistent hyperactivation of innate and adaptive immunity via classical monocytes and CD4 naïve T cells, regulatory T cell dysfunction, and effector T cell anergy. We further our understanding of the immunopathology of sarcoidosis and point to novel therapeutic targets.

Single-cell transcriptomic analysis reveals key immune cell phenotypes in the lungs of patients with asthma exacerbation

Asthma exacerbations are associated with heightened asthma symptoms, which can result in hospitalization in severe cases. However, the molecular immunologic processes that determine the course of an exacerbation remain poorly understood, impeding the progression of development of effective therapies. Our aim was to identify candidate genes that are strongly associated with asthma exacerbation at a cellular level. Subjects with asthma exacerbation and healthy control subjects were recruited, and bronchoalveolar lavage fluid was isolated from these subjects via bronchoscopy. Cells were isolated through fluorescence-activated cell sorting, and single-cell RNA sequencing was performed on enriched cell populations. We showed that the levels of monocytes, CD8+ T cells, and macrophages are significantly elevated in the bronchoalveolar lavage fluid of patients. Aset of cytokines and intracellular transduction regulators are associated with asthma exacerbations and are shared across multiple cell clusters, forming a complicated molecular framework. An additional group of core exacerbation-associated modules is activated, including eukaryotic initiation factor 2 signaling, ephrin receptor signaling, and C-X-C chemokine receptor type 4 signaling in the subpopulations of CD8+ T cells (C1-a) and monocyte clusters (C7 clusters), which are associated with infection. Our study identified a significant number of severe asthma-associated genes that are differentially expressed by multiple cell clusters.

Ephrin and Eph receptor signaling in female reproductive physiology and pathology†.

Ephrins are ligands of Eph receptors (Ephs); both of which are sorted into two classes, A and B. There are five types of ephrin-As (ephrin-A1-5) and three types of ephrin-Bs (ephrin-B1-3). Also, there are 10 types of EphAs (EphA1-10) and six types of EphBs (EphB1-6). Binding of ephrins to the Eph receptors activates signaling cascades that regulate several biological processes such as cellular proliferation, differentiation, migration, angiogenesis, and vascular remodeling. Clarification of their roles in the female reproductive system is crucial to understanding the physiology and pathology of this system. Such knowledge will also create awareness regarding the importance of these molecules in diagnostic, prognostic, and therapeutic medicine. Hence, we have discussed the involvement of these molecules in the physiological and pathological events that occur within the female reproductive system. The evidence so far suggests that the ephrins and the Eph receptors modulate folliculogenesis, ovulation, embryo transport, implantation, and placentation. Abnormal expression of some of these molecules is associated with polycystic ovarian syndrome, ovarian cancer, tubal pregnancy, endometrial cancer, uterine leiomyoma (fibroids), cervical cancer, and preeclampsia, suggesting the need to utilize these molecules in the clinical setting. To enhance a quick development of this gradually emerging field in female reproductive medicine, we have highlighted some "gaps in knowledge" that need prospective investigation.

Pten and Dicer1 loss in the mouse uterus causes poorly differentiated endometrial adenocarcinoma.

Endometrial cancer remains the most common gynecological malignancy in the United States. While the loss of the tumor suppressor, PTEN (phosphatase and tensin homolog), is well studied in endometrial cancer, recent studies suggest that DICER1, the endoribonuclease responsible for miRNA genesis, also plays a significant role in endometrial adenocarcinoma. Conditional uterine deletion of Dicer1 and Pten in mice resulted in poorly differentiated endometrial adenocarcinomas, which expressed Napsin A and HNF1B (hepatocyte nuclear factor 1 homeobox B), markers of clear-cell adenocarcinoma. Adenocarcinomas were hormone-independent. Treatment with progesterone did not mitigate poorly differentiated adenocarcinoma, nor did it affect adnexal metastasis. Transcriptomic analyses of DICER1 deleted uteri or Ishikawa cells revealed unique transcriptomic profiles and global miRNA downregulation. Computational integration of miRNA with mRNA targets revealed deregulated let-7 and miR-16 target genes, similar to published human DICER1-mutant endometrial cancers from TCGA (The Cancer Genome Atlas). Similar to human endometrial cancers, tumors exhibited dysregulation of ephrin-receptor signaling and transforming growth factor-beta signaling pathways. LIM kinase 2 (LIMK2), an essential molecule in p21 signal transduction, was significantly upregulated and represents a novel mechanism for hormone-independent pathogenesis of endometrial adenocarcinoma. This preclinical mouse model represents the first genetically engineered mouse model of poorly differentiated endometrial adenocarcinoma.