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.

Comparative transcriptome and metabolome profiles of the leaf and fruits of a Xianjinfeng litchi budding mutant and its mother plant.

Background: Litchi (Litchi chinensis) is an important sub-tropical fruit in the horticulture market in China. Breeding for improved fruit characteristics is needed for satisfying consumer demands. Budding is a sustainable method for its propagation. During our ongoing breeding program, we observed a litchi mutant with flat leaves and sharp fruit peel cracking in comparison to the curled leaves and blunt fruit peel cracking fruits of the mother plant. Methods: To understand the possible molecular pathways involved, we performed a combined metabolome and transcriptome analysis. Results: We identified 1,060 metabolites in litchi leaves and fruits, of which 106 and 101 were differentially accumulated between the leaves and fruits, respectively. The mutant leaves were richer in carbohydrates, nucleotides, and phenolic acids, while the mother plant was rich in most of the amino acids and derivatives, flavonoids, lipids and organic acids and derivatives, and vitamins. Contrastingly, mutant fruits had higher levels of amino acids and derivatives, carbohydrates and derivatives, and organic acids and derivatives. However, the mother plant's fruits contained higher levels of flavonoids, scopoletin, amines, some amino acids and derivatives, benzamidine, carbohydrates and derivatives, and some organic acids and derivatives. The number of differentially expressed genes was consistent with the metabolome profiles. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway-enriched gene expressions showed consistent profiles as of metabolome analysis. Conclusion: These results provide the groundwork for breeding litchi for fruit and leaf traits that are useful for its taste and yield.

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.

Modeling lung diseases using reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2).

BACKGROUND: A healthy alveolar epithelium is critical to the gas exchange function of the lungs. As the major cell type of alveolar epithelium, alveolar type 2 (AT2) cells play a critical role in maintaining pulmonary homeostasis by serving as alveolar progenitors during lung injury, inflammation, and repair. Dysregulation of AT2 cells may lead to the development of acute and chronic lung diseases and cancer. The lack of clinically relevant AT2 cell models hampers our ability to understand pulmonary diseases. Here, we sought to establish reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2) and investigate their potential in forming alveolar organoids to model pulmonary diseases. METHODS: Primary mouse pulmonary alveolar cells (mPACs) were isolated and immortalized with a retroviral expression of SV40 Large T antigen (LTA). Cell proliferation and survival was assessed by crystal violet staining and WST-1 assays. Marker gene expression was assessed by qPCR, Western blotting, and/or immunostaining. Alveolar organoids were generated by using matrigel. Ad-TGF-ß1 was used to transiently express TGF-ß1. Stable silencing ß-catenin or overexpression of mutant KRAS and TP53 was accomplished by using retroviral vectors. Subcutaneous cell implantations were carried out in athymic nude mice. The retrieved tissue masses were subjected to H & E histologic evaluation. RESULTS: We immortalized primary mPACs with SV40 LTA to yield the imPACs that were non-tumorigenic and maintained long-term proliferative activity that was reversible by FLP-mediated removal of SV40 LTA. The EpCAM+ AT2-enriched subpopulation (i.e., imPAC2) was sorted out from the imPACs, and was shown to express AT2 markers and form alveolar organoids. Functionally, silencing ß-catenin decreased the expression of AT2 markers in imPAC2 cells, while TGF-ß1 induced fibrosis-like response by regulating the expression of epithelial-mesenchymal transition markers in the imPAC2 cells. Lastly, concurrent expression of oncogenic KRAS and mutant TP53 rendered the imPAC2 cells a tumor-like phenotype and activated lung cancer-associated pathways. Collectively, our results suggest that the imPAC2 cells may faithfully represent AT2 populations that can be further explored to model pulmonary diseases.

Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance Alveolar Type II Cell Proliferation and Attenuate Lung Inflammation in a Rat Model of Bronchopulmonary Dysplasia.

Although it is known that exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs) alleviate hyperoxic lung injury of bronchopulmonary dysplasia (BPD) in animal models, the role of microvesicles (MVs) derived from hUCMSCs in BPD is poorly defined. Furthermore, antenatal inflammation has been linked to high risk of BPD in preterm infants. The purpose of this study was to explore whether MVs derived from hUCMSCs can preserve lung structure and function in an antenatal lipopolysaccharide- (LPS-) induced BPD rat model and to clarify the underlying mechanism. We demonstrate that antenatal LPS induced alveolar simplification, altered lung function, and dysregulated pulmonary vasculature, which restored by hUCMSCs and MVs treatment. Furthermore, MVs were large vesicles with a diameter of 100-900 nanometers and mostly uptaken by alveolar epithelial type II cells (AT2) and macrophages. Compared with the LPS-exposed group, MVs restored the AT2 cell number and SP-C expression in vivo and promoted the proliferation of AT2 cells in vitro. MVs also restored the level of IL-6 and IL-10 in lung homogenate. Additionally, PTEN/AKT and MAPK pathways were associated with the protection of MVs. Taken together, this study suggests MVs derived from hUCMSCs improve lung architecture and function in an antenatal LPS-induced BPD rat model by promoting AT2 cell proliferation and attenuating lung inflammation; thus, MVs provide a promising therapeutic vehicle for BPD treatment.

Integrated Methylome and Transcriptome Analysis Widen the Knowledge of Cytoplasmic Male Sterility in Cotton (Gossypium barbadense L.).

DNA methylation is defined as a conserved epigenetic modification mechanism that plays a key role in maintaining normal gene expression without altering the DNA sequence. Several studies have reported that altered methylation patterns were associated with male sterility in some plants such as rice and wheat, but global methylation profiles and their possible roles in cytoplasmic male sterility (CMS), especially in cotton near-isogenic lines, remain unclear. In this study, bisulfite sequencing technology and RNA-Seq were used to investigate CMS line 07-113A and its near-isogenic line 07-113B. Using integrated methylome and transcriptome analyses, we found that the number of hypermethylated genes in the differentially methylated regions, whether in the promoter region or in the gene region, was more in 07-113A than the number in 07-113B. The data indicated that 07-113A was more susceptible to methylation. In order to further analyze the regulatory network of male sterility, transcriptome sequencing and DNA methylation group data were used to compare the characteristics of near-isogenic lines 07-113A and 07-113B in cotton during the abortion stage. Combined methylation and transcriptome analysis showed that differentially expressed methylated genes were mainly concentrated in vital metabolic pathways including the starch and sucrose metabolism pathways and galactose metabolism. And there was a negative correlation between gene methylation and gene expression. In addition, five key genes that may be associated with CMS in cotton were identified. These data will support further understanding of the effect of DNA methylation on gene expression and their potential roles in cotton CMS.

Hypoxic preconditioning improves the survival and pro-angiogenic capacity of transplanted human umbilical cord mesenchymal stem cells via HIF-1α signaling in a rat model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a serious chronic respiratory disease that predominates in the neonatal period. Currently, efficacious and effective specific treatments are lacking. Mesenchymal stem cells (MSCs) transplantation has emerged as a promising option for treating BPD. However, the lower cell survival rate limits its therapeutic efficacy. Hypoxic preconditioning is a direct and effective strategy for promoting MSCs survival, proliferation, and paracrine secretion in the recipient after transplantation, which is greatly important to tissue engineering. We investigated whether hypoxia-pretreated MSCs (HPMSCs) confer superior benefit in an experimental BPD rat model. Neonatal Sprague-Dawley rats were exposed to 80-85% O2 for 14 days. Before tracheal transplantation, the MSCs were pretreated for 48 h with deferoxamine, a chemical hypoxia-mimicking agent. In vitro, the HPMSCs reduced the apoptosis rare, caspase-3 expression, and reactive oxygen species (ROS) generation and promoted proliferation, hypoxia inducible factor-1α (HIF-1α) expression, VEGF secretion, and human umbilical vein endothelial cell tube formation (p < 0.05). In vivo, the HPMSCs restored alveolar structure and lung function, ameliorated pulmonary hypertension, increased vessel density in the BPD rat model (p < 0.05). This work demonstrates for the first time that HPMSCs could have a markedly improved therapeutic effect in BPD, presenting a new potential strategy for the clinical implementation of stem cell biotechnology.

Mitochondrial gene expression analysis reveals aberrant transcription of cox3 in Gossypium barbadense CMS line H276A.

Cotton cytoplasmic male sterility (CMS) has been extensively studied; however, information regarding its molecular mechanisms has not yet been disclosed. Therefore, to explore the molecular mechanism of pollen abortion of cotton CMS line H276A, transcript profiles of 30 mitochondrial protein-encoding genes at tetrad stage were conducted with northern blot and a differential expression gene cox3 was identified. Quantitative reverse-transcribed PCR (qRT-PCR) analysis indicated that the expression level of cox3 in the CMS line H276A was 0.39-fold compared to its maintainer line H276B. In addition, the immunoblot analysis revealed that the amount of COX3 protein was decreased to 59.38% in CMS line H276A. The 5` and 3` terminals of the transcript of cox3 in two materials were determined simultaneously with circularized RNA reverse-transcribed PCR (CR-RT-PCR). The data indicated that seven 5` end of transcript of cox3 in H276A (-451/-464/-465/-467/-471/-472/-508 respect to ATG) were identified which were different from that of H276B (-411/-412). A total of 15 single nucleotide polymorphisms (SNPs) was detected by clone sequencing analysis of upstream of cox3. To our knowledge, we are the first to comprehensively analyze the transcripts of the mitochondrial genome in the cotton CMS line and to identify the 5` and 3` terminals of the transcript of cox3 in cotton. Our data will provide a framework for a better understanding of molecular mechanisms of CMS and mitochondrial gene expression in cotton.

Mechanism and Potential of Extracellular Vesicles Derived From Mesenchymal Stem Cells for the Treatment of Infectious Diseases.

Extracellular vesicles (EVs) are nano-sized membrane vesicles secreted by cells. EVs serve as a mediator for cell-to-cell communication by regulating the exchange of genetic materials and proteins between the donor and surrounding cells. Current studies have explored the therapeutic value of mesenchymal stem cells-derived EVs (MSC-EVs) for the treatment of infectious diseases extensively. MSC-EVs can eliminate the pathogen, regulate immunity, and repair tissue injury in contagious diseases through the secretion of antimicrobial factors, inhibiting the replication of pathogens and activating the phagocytic function of macrophages. MSC-EVs can also repair tissue damage associated with the infection by upregulating the levels of anti-inflammatory factors, downregulating the pro-inflammatory factors, and participating in the regulation of cellular biological behaviors. The purpose of this mini-review is to discuss in detail the various mechanisms of MSC-EV treatment for infectious diseases including respiratory infections, sepsis, and intestinal infections, as well as challenges for implementing MSC-EVs from bench to bedside.

Type 2 Alveolar Epithelial Cells Differentiated from Human Umbilical Cord Mesenchymal Stem Cells Alleviate Mouse Pulmonary Fibrosis Through ß-Catenin-Regulated Cell Apoptosis.

Pulmonary fibrosis (PF) is a chronic, progressive, and lethal disease with little response to available therapies. One of the major mechanisms of PF is the repeated injury and inadequate regeneration of the alveolar epithelium. In this study, we induced human umbilical cord mesenchymal stem cells (hUC-MSCs) to differentiate into type 2 alveolar epithelial cells (AEC2s), and we provided evidence that intratracheal transplantation of hUC-MSC-derived AEC2s (MSC-AEC2s) could improve mortality and alleviate fibrosis in bleomycin-induced PF mice. Transplantation of MSC-AEC2s could increase the AEC2 cell count in these mice, and the results of the cell tracing experiment exhibited that the increased AEC2s originated from the self-renewal of mouse alveolar epithelium. The AEC2 survival was controlled by the apoptosis of AEC2s via the expression of ß-catenin in PF mice. In in vitro experiments, MSC-AEC2s could alleviate the apoptosis of MLE-12 cells induced by transforming growth factor beta (TGF-ß1), which could be eliminated by using PRI-724, a ß-catenin inhibitor, suggesting ß-catenin signaling involved in the protection against apoptosis provided by MSC-AEC2s. Our study demonstrated that MSC-AEC2s could protect PF mice through regulating apoptosis mediated by ß-catenin, which provided a viable strategy for the treatment of PF.

Identification of chalcone synthase genes and their expression patterns reveal pollen abortion in cotton.

Chalcone synthase (CHS) is a key enzyme and producing flavonoid derivatives as well play a vital roles in sustaining plant growth and development. However, the systematic and comprehensive analysis of CHS genes in island cotton (G. barbadense) has not been reported yet especially response to cytoplasmic male sterility (CMS). To fill this knowledge gap, a genome-wide investigation of CHS genes were studied in island cotton. A total of 20 GbCHS genes were identified and grouped into five GbCHSs. The gene structure analysis revealed that most of GbCHS genes consisted of two exons and one intron, and 20 motifs were identified. Twenty five pairs duplicated events (12 GbCHS genes) were identified including 23 segmental duplication pairs and two tandem duplication events, representing that GbCHS gene family amplification mainly owned to segmental duplication events and evolving slowly. Gene expression analysis exhibited that the GbCHS family genes presented a diversity expression patterns in various organs of cotton. Coupled with functional predictions and gene expression, the abnormal expression of GbCHS06, 10, 16 and 19 might be associated with pollen abortion of CMS line in island cotton. Conclusively, GbCHS genes exhibited diversity and conservation in many aspects, which will help to better understand functional studies and a reference for CHS research in island cotton and other plants.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Alleviate Lung Injury in Rat Model of Bronchopulmonary Dysplasia by Affecting Cell Survival and Angiogenesis.

Bronchopulmonary dysplasia (BPD) is a serious chronic lung disease in premature newborns, with high morbidity and mortality rates. Mesenchymal stem cell (MSC) transplantation has developed into a promising approach to alleviate BPD. Small extracellular vesicles, which are an important therapeutic component of MSCs, have been reported to be effective in a mouse model of BPD. However, the affected cell types and detailed underlying mechanisms are unclear. In this study, we found that human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hucMSC-sEVs) were successfully absorbed by lung tissue after intratracheal administration, and remained in the lungs for at least 72 h. The results showed that hucMSC-sEVs restored alveolar structure and lung function, and ameliorated pulmonary hypertension in a rat model of BPD. The number of Ki-67-positive lung cells were improved, while the number of TUNEL-positive lung cells were reduced in our hucMSC-sEV-treated BPD model. Additionally, SP-C staining (a marker of type II alveolar epithelial cells, TIIAECs) and CD31 staining (a marker of pulmonary vascular endothelial cells, PVECs) were both increased in a hyperoxia-induced BPD model treated with hucMSC-sEVs. In vitro, under hyperoxic conditions, the tube-like structure formation was improved in human umbilical vein endothelial cells, and the proliferation was increased and the apoptosis was attenuated in MLE-12 cells treated with hucMSC-sEVs. Furthermore, we observed downregulated expression of PTEN and cleaved-caspase3, and upregulated expression of p-Akt and vascular endothelial growth factor-A in our hucMSC-sEV-treated BPD model. In conclusion, hucMSC-sEVs improved alveolarization and angiogenesis in a rat BPD model by protecting TIIAECs and PVECs, which were associated with the PTEN/Akt signaling pathway.

[Readmission of children with bronchopulmonary dysplasia in the first 2 years of life: a clinical analysis of 121 cases].

OBJECTIVE: To investigate the clinical features of readmitted children with bronchopulmonary dysplasia (BPD) in the first 2 years of life. METHODS: A retrospective analysis was performed for the clinical data of 242 children with BPD who were readmitted due to recurrent lower respiratory tract infection (LRTI) in the first 2 years of life. RESULTS: Among all the 242 children with BPD, 115(47.5%) had wheezing, and the children aged 1-2 years had a significantly higher incidence rate of wheezing than those aged less than 1 year (P<0.05). Chest imaging was performed for 193 children, among whom 31 (16.1%) had hyperlucent areas. Pulmonary function examination showed that the BPD children had significantly lower TV/kg, TPEF/TE, VPEF/VE, TEF50 and TEF75, and significantly higher respiratory rate than the controls without respiratory disease (P<0.05). Bronchoscopy was performed for 28 children, among whom 21 (75%) had airway dysplasia. All the 242 children used inhaled corticosteroids (ICS) and experienced no treatment-related adverse reactions. Six children were given intravenous infusion of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) and experienced no infusion-related events or adverse reactions, among whom one child successfully stopped oxygen therapy. CONCLUSIONS: The incidence rate of wheezing increases with the increase in age in children with BPD who are readmitted due to LRTI. Pulmonary function examination shows small airway obstruction, reduced expiratory flow rate in case of low lung capacity, and increased respiratory rate, and most children have airway dysplasia. ICS can be used to inhibit inflammatory response in the acute stage. Infusion of hUCB-MSCs is safe and feasible and may bring some benefits to the recovery from BPD.