Identification of signaling pathways that specify a subset of migrating enteric neural crest cells at the wavefront in mouse embryos.

During enteric nervous system (ENS) development, pioneering wavefront enteric neural crest cells (ENCCs) initiate gut colonization. However, the molecular mechanisms guiding their specification and niche interaction are not fully understood. We used single-cell RNA sequencing and spatial transcriptomics to map the spatiotemporal dynamics and molecular landscape of wavefront ENCCs in mouse embryos. Our analysis shows a progressive decline in wavefront ENCC potency during migration and identifies transcription factors governing their specification and differentiation. We further delineate key signaling pathways (ephrin-Eph, Wnt-Frizzled, and Sema3a-Nrp1) utilized by wavefront ENCCs to interact with their surrounding cells. Disruptions in these pathways are observed in human Hirschsprung's disease gut tissue, linking them to ENS malformations. Additionally, we observed region-specific and cell-type-specific transcriptional changes in surrounding gut tissues upon wavefront ENCC arrival, suggesting their role in shaping the gut microenvironment. This work offers a roadmap of ENS development, with implications for understanding ENS disorders.

Cinchophen induces RPA1 related DNA damage and apoptosis to impair ENS development of zebrafish.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have become contaminants widely distributed in the environment due to improper disposal and discharge. Previous study has found several components might involve in impairing enteric nervous system (ENS) development of zebrafish, including NSAIDs cinchophen. Deficient ENS development in fetal could lead to Hirschsprung disease (HSCR), a congenital neurocristopathy characterized by absence of enteric neurons in hindgut. However, the intrinsic mechanism of neurotoxicity of cinchophen is unclear. We confirmed that cinchophen could impair ENS development of zebrafish and transcriptome sequencing revealed that disfunction of Replication protein A1 (RPA1), which is involved in DNA replication and repairment, might be relevant to the neurotoxicity effects induced by cinchophen. Based on previous data of single cell RNA sequencing (scRNA-seq) of zebrafish gut cells, we observed that rpa1 mainly expressed in proliferating, differentiating ENS cells and neural crest progenitors. Interestingly, cinchophen induced apoptosis and impaired proliferation. Furthermore, cinchophen caused DNA damage and abnormal activation of ataxia telangiectasia mutated/ Rad3 related (ATM/ATR) and checkpoint kinase 2 (CHK2). Finally, molecular docking indicated cinchophen could bind and antagonize RPA1 more effectively. Our study might provide a better understanding and draw more attention to the role of environmental factors in the pathogenesis of HSCR. And the mechanism of cinchophen neurotoxicity would give theoretical guidance for clinical pharmacy.

Abnormally elevated expression of ACTA2 of circular smooth muscle leads to hyperactive contraction in aganglionic segments of HSCR.

BACKGROUND: Actin Alpha 2 (ACTA2) is expressed in intestinal smooth muscle cells (iSMCs) and is associated with contractility. Hirschsprung disease (HSCR), one of the most common digested tract malformations, shows peristaltic dysfunction and spasm smooth muscles. The arrangement of the circular and longitudinal smooth muscle (SM) of the aganglionic segments is disorganized. Does ACTA2, as a marker of iSMCs, exhibit abnormal expression in aganglionic segments? Does the ACTA2 expression level affect the contraction function of iSMCs? What are the spatiotemporal expression trends of ACTA2 during different developmental stages of the colon? METHODS: Immunohistochemical staining was used to detect the expression of ACTA2 in iSMCs of children with HSCR and Ednrb-/- mice, and the small interfering RNAs (siRNAs) knockdown technique was employed to investigate how Acta2 affected the systolic function of iSMCs. Additionally, Ednrb-/- mice were used to explore the changes in the expression level of iSMCs ACTA2 at different developmental stages. RESULTS: The expression of ACTA2 is higher in circular SM in the aganglionic segments of HSCR patients and Ednrb-/- mice than in normal control children and mice. Down regulation of Acta2 weakens the contraction ability of intestinal smooth muscle cells. Abnormally elevated expression of ACTA2 of circular smooth muscle occurs since embryonic day 15.5 (E15.5d) in aganglionic segments of Ednrb-/- mice. CONCLUSIONS: Abnormally elevated expression of ACTA2 in the circular SM leads to hyperactive contraction, which may cause the spasm of aganglionic segments in HSCR.

Comprehensive characterization of the genetic landscape of familial Hirschsprung's disease.

BACKGROUND: Hirschsprung's disease (HSCR) is one of the most common congenital digestive tract malformations and can cause stubborn constipation or gastrointestinal obstruction after birth, causing great physical and mental pain to patients and their families. Studies have shown that more than 20 genes are involved in HSCR, and most cases of HSCR are sporadic. However, the overall rate of familial recurrence in 4331 cases of HSCR is about 7.6%. Furthermore, familial HSCR patients show incomplete dominance. We still do not know the penetrance and genetic characteristics of these known risk genes due to the rarity of HSCR families. METHODS: To find published references, we used the title/abstract terms "Hirschsprung" and "familial" in the PubMed database and the MeSH terms "Hirschsprung" and "familial" in Web of Science. Finally, we summarized 129 HSCR families over the last 40 years. RESULTS: The male-to-female ratio and the percentage of short segment-HSCR in familial HSCR are much lower than in sporadic HSCR. The primary gene factors in the syndromic families are ret proto-oncogene (RET) and endothelin B receptor gene (EDNRB). Most families show incomplete dominance and are relevant to RET, and the RET mutation has 56% penetrance in familial HSCR. When one of the parents is a RET mutation carrier in an HSCR family, the offspring's recurrence risk is 28%, and the incidence of the offspring does not depend on whether the parent suffers from HSCR. CONCLUSION: Our findings will help HSCR patients obtain better genetic counseling, calculate the risk of recurrence, and provide new insights for future pedigree studies.

Down-Regulation of Double C2 Domain Alpha Promotes the Formation of Hyperplastic Nerve Fibers in Aganglionic Segments of Hirschsprung's Disease.

Hirschsprung's disease (HSCR) is a common developmental anomaly of the gastrointestinal tract in children. The most significant characteristics of aganglionic segments in HSCR are hyperplastic extrinsic nerve fibers and the absence of endogenous ganglion plexus. Double C2 domain alpha (DOC2A) is mainly located in the nucleus and is involved in Ca2+-dependent neurotransmitter release. The loss function of DOC2A influences postsynaptic protein synthesis, dendrite morphology, postsynaptic receptor density and synaptic plasticity. It is still unknown why hyperplastic extrinsic nerve fibers grow into aganglionic segments in HSCR. We detected the expression of DOC2A in HSCR aganglionic segment colons and established three DOC2A-knockdown models in the Neuro-2a cell line, neural spheres and zebrafish separately. First, we detected the protein and mRNA expression of DOC2A and found that DOC2A was negatively correlated with AChE+ grades. Second, in the Neuro-2a cell lines, we found that the amount of neurite outgrowth and mean area per cell were significantly increased, which suggested that the inhibition of DOC2A promotes nerve fiber formation and the neuron's polarity. In the neural spheres, we found that the DOC2A knockdown was manifested by a more obvious connection of nerve fibers in neural spheres. Then, we knocked down Doc2a in zebrafish and found that the down-regulation of Doc2a accelerates the formation of hyperplastic nerve fibers in aganglionic segments in zebrafish. Finally, we detected the expression of MUNC13-2 (UNC13B), which was obviously up-regulated in Grade3/4 (lower DOC2A expression) compared with Grade1/2 (higher DOC2A expression) in the circular muscle layer and longitudinal muscle layer. The expression of UNC13B was up-regulated with the knocking down of DOC2A, and there were protein interactions between DOC2A and UNC13B. The down-regulation of DOC2A may be an important factor leading to hyperplastic nerve fibers in aganglionic segments of HSCR. UNC13B seems to be a downstream molecule to DOC2A, which may participate in the spasm of aganglionic segments of HSCR patient colons.

A novel nine-microRNA-based model to improve prognosis prediction of renal cell carcinoma.

BACKGROUND: With the improved knowledge of disease biology and the introduction of immune checkpoints, there has been significant progress in treating renal cell carcinoma (RCC) patients. Individual treatment will differ according to risk stratification. As the clinical course varies in RCC, it has developed different predictive models for assessing patient's individual risk. However, among other prognostic scores, no transparent preference model was given. MicroRNA as a putative marker shown to have prognostic relevance in RCC, molecular analysis may provide an innovative benefit in the prophetic prediction and individual risk assessment. Therefore, this study aimed to establish a prognostic-related microRNA risk score model of RCC and further explore the relationship between the model and the immune microenvironment, immune infiltration, and immune checkpoints. This practical model has the potential to guide individualized surveillance protocols, patient counseling, and individualized treatment decision for RCC patients and facilitate to find more immunotherapy targets. METHODS: Downloaded data of RCC from the TCGA database for difference analysis and divided it into a training set and validation set. Then the prognostic genes were screened out by Cox and Lasso regression analysis. Multivariate Cox regression analysis was used to establish a predictive model that divided patients into high-risk and low-risk groups. The ENCORI online website and the results of the RCC difference analysis were used to search for hub genes of miRNA. Estimate package and TIMER database were used to evaluate the relationship between risk score and tumor immune microenvironment (TME) and immune infiltration. Based on Kaplan-Meier survival analysis, search for immune checkpoints related to the prognosis of RCC. RESULTS: There were nine miRNAs in the established model, with a concordance index of 0.702 and an area under the ROC curve of 0.701. Nine miRNAs were strongly correlated with the prognosis (P < 0.01), and those with high expression levels had a poor prognosis. We found a common target gene PDGFRA of hsa-miR-6718, hsa-miR-1269b and hsa-miR-374c, and five genes related to ICGs (KIR2DL3, TNFRSF4, LAG3, CD70 and TNFRSF9). The immune/stromal score, immune infiltration, and immune checkpoint genes of RCC were closely related to its prognosis and were positively associated with a risk score. CONCLUSIONS: The established nine-miRNAs prognostic model has the potential to facilitate prognostic prediction. Moreover, this model was closely related to the immune microenvironment, immune infiltration, and immune checkpoint genes of RCC.

APOA1 Level is Negatively Correlated with the Severity of COVID-19.

OBJECTIVE: We used public data to analyze the proteomics, metabolomics and transcriptomics characteristics of COVID-19 patients to identify potential therapeutic targets. More importantly, we also collected clinical data for verification to make the analysis results more reliable. METHODS: Download the serum proteomics and metabolomics data of COVID-19 patients and describe their changes in serum proteins and metabolites, and use bioinformatics analysis methods to identify potential biomarkers and therapeutic targets. Finally, clinical data and experimental data of cell infection were combined for verification. RESULTS: It was found that the serum apolipoprotein A1 (APOA1) protein level in COVID-19 patients was down-regulated (log2FC = -0.39, false discovery rate (FDR) < 0.001), and the degree of reduction in the severe group was more significant (kruskal-test p = 2.5e-05). What is more, APOA1 was not only expressed lower in male patients (Wilcox-test p = 0.012), but also negatively correlated with C-reactive protein (CRP, r = -0.37, p = 0.019). The experiment data from SARS-CoV-2 infected cells further showed that the protein and transcript level of APOA1 gradually decreased as the infection time increased, and the transcription level (log2FC = -8.3, FDR = 0.0015) was more down-regulated than protein level (log2FC = -0.95, FDR = 0.0014). More importantly, the collected clinical data also confirmed that APOA1 was down-regulated in COVID-19 patients (kruskal-test p = 0.001), and APOA1 levels are negatively correlated with IL6 (r = -0.396, p = 2.22e-07), D-dimers (DD, r = -0.262, p = 8.19e-04), prothrombin time (PT, r = -0.464, p = 6.68e-10) and thrombin time (TT, r = -0.279, p = 3.46e-04). CONCLUSION: The degree of down-regulation of APOA1 is positively correlated with the severity of COVID-19, and the expression level of APOA1 is negatively correlated with CRP, IL6, DD, PT, TT, and positively correlated with HD and LDL. This indicates that APOA1 may be a key molecule in tandem acute inflammatory response, coagulation abnormalities and cholesterol metabolism disorder in COVID-19, and could be a potential therapeutic target.

The incidence of influenza in children was decreased in the first flu season after COVID-19 pandemic in Wuhan.

Wuhan, China was the first city to discover COVID-19. With the government's macro-control and the active cooperation of the public, the spread of COVID-19 has been effectively controlled. In order to understand the additional impact of these measures on the prevalence of common influenza, we have collected flu test data from the Pediatric Clinic of Zhongnan Hospital of Wuhan University from September to December 2020, and compared them with the same period in 2018 and 2019. It is found that compared with the same period in 2018 and 2019, the rate of children's influenza activity in 2020 has significantly decreased, which indicates that the protective measures against COVID-19 have effectively reduced the level of influenza activity.

A novel immune-related prognostic index for predicting breast cancer overall survival.

PURPOSE: To find immune-related genes with prognostic value in breast cancer, and construct a prognostic risk assessment model to make a more accurate assessment. Moreover, looking for potential immune markers for breast cancer immunotherapy. METHODS: The breast cancer (BC) data were retrieved from The Cancer Genome Atlas (TCGA) database as a training set. Through the Weighted gene co-expression network analysis (WGCNA), Kaplan-Meier (KM) analysis, lasso regression analysis and stepwise backward Cox regression analysis, screening for prognosis-related immune genes, a prognostic index was built, and external validation with two data sets of Gene Expression Omnibus (GEO) database was performed. Transcription factor (TF) regulatory network was constructed to identify key transcription factors that regulate prognostic immune genes. Gene set enrichment analysis (GSEA) was used to explore the signal pathways differences between high and low-risk groups, estimate package and TIMER database were used to evaluate the relationship between risk score and tumor immune microenvironment. RESULTS: We obtained 10 prognosis-related immune genes, and the index showed accurate prognostic value. We also identified 7 prognostic transcription factors. Multiple signaling pathways that inhibit tumor progression were enriched in the low-risk group, and risk score was significantly negatively related to the degree of immune infiltration and the expression level of immune checkpoint genes. CONCLUSION: We successfully constructed an independent prognostic index, which not only has a stronger predictive ability than the tumor pathological stage, but also can reflect the immune infiltration of breast cancer patients.

Linc-ROR promotes the progression of breast cancer and decreases the sensitivity to rapamycin through miR-194-3p targeting MECP2.

linc-ROR is reported to be a potential biomarker of breast cancer, but the detailed mechanism of linc-ROR-mediated breast cancer regulation has not been fully studied. We aimed to explore how linc-ROR affects proliferation, metastasis, and drug sensitivity in breast cancer. Cell lines in which linc-ROR was overexpressed or knocked down were constructed, and the cell proliferation, colony formation, cell migration, and invasion abilities of these lines were explored. A CCK-8 assay was performed to determine the sensitivity of the breast cancer cells to rapamycin. Next-generation sequencing was conducted to explore the detailed regulatory mechanism of linc-ROR; differentially expressed RNAs in the linc-ROR-overexpressing cell line compared with the negative control were screened out, and their target genes were chosen to perform Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, protein-protein interaction network analysis, and competing endogenous RNA (ceRNA) network analysis. The ceRNA mechanism of linc-ROR for miR-194-3p, which targets MECP2, was determined through dual-luciferase reporter assay, RT-qPCR, western blot, and rescue experiments. Finally, we found that linc-ROR was upregulated in breast tumor tissues. linc-ROR promoted the cell proliferation, colony formation, cell migration, and invasion of breast cancer and decreased the sensitivity of breast cancer cells to rapamycin. The overexpression of linc-ROR triggered changes in the whole transcriptome of breast cancer cells, and a total of 85 lncRNAs, 414 microRNAs, 490 mRNAs, and 92 circRNAs were differentially expressed in the linc-ROR-overexpressing cell line compared with the negative control. Through a series of bioinformatic analyses, the 'linc-ROR/miR-194-3p/MECP2' ceRNA regulatory axis was confirmed to be involved in the linc-ROR-mediated progression and drug sensitivity of breast cancer. In conclusion, linc-ROR serves as an onco-lncRNA in breast cancer and promotes the survival of breast cancer cells during rapamycin treatment by functioning as a ceRNA sponge for miR-194-3p, which targets MECP2.