Intra-amniotic delivery of tropomodulin 3 rescues cell apoptosis induced by miR-200b-3p upregulation via non-canonical nuclear factor kappa B pathways in ethylene thiourea induced anorectal malformations fetal rat.

Ethylene thiourea (ETU), a metabolite of the fungicide ethylene bisdithiocarbamate (EBDC), has received great concern because of its harmful effects. ETU-induced anorectal malformations (ARMs) in rat models have been reported and widely used in the study of ARMs embryogenesis. Dysplasia of the lumbosacral spinal cord (LSSC), pelvic floor muscles (PFMs), and hindgut (HG) during intrauterine life affects postoperative defecation in patients with ARMs. However, the underlying toxic effects of ETU and pathological mechanisms in the three defecation-related tissues of fetuses with ARMs have not been reported. Thus, this study aimed to elucidate the molecular mechanisms involved in ARMs, with a focus on the dysregulation of miR-200b-3p and its downstream target tropomodulin 3 (TMOD3). The mRNA and protein levels of miR-200b-3p and TMOD3 in LSSC, PFMs, and HG of fetal rats with ARMs were evaluated by reverse transcription quantitative polymerase chain reaction and Western blotting (WB) on embryonic day 17 (E17). Further, a dual-luciferase reporter assay confirmed their targeting relationship. Gene silencing and overexpression of miR-200b-3p and TMOD3 were performed to verify their functions in HEK-293 T cells. Fetal rats with ARMs also received intra-amniotic microinjection of Ad-TMOD3 on E15, and key molecules in nuclear factor kappa (NF-κB) signaling and apoptosis were evaluated by WB on E21. Abnormally high levels of miR-200b-3p inhibited TMOD3 expression by binding with its 3'-untranslated region, leading to the activation of the non-canonical NF-κB signaling pathway, which is critical in the maldevelopment of LSSC, PFMs, and HG in ARMs rats. Furthermore, miR-200b-3p triggered apoptosis by directly targeting TMOD3. Notably, intra-amniotic Ad-TMOD3 microinjection revealed that the upregulation of TMOD3 expression mitigates the effects of miR-200b-3p on the activation of non-canonical NF-κB signaling and apoptosis in fetal rat model of ARMs. A novel miR-200b-3p/TMOD3/non-canonical NF-κB signaling axis triggered the massive apoptosis in LSSC, PFMs, and HG of ARMs, which was restored by the intra-amniotic injection of Ad-TMOD3 during embryogenesis. Our results indicate the potential of TMOD3 as a treatment target to restore defecation.

Inflammatory microenvironment of moderate pulpitis enhances the osteo-/odontogenic potential of dental pulp stem cells by autophagy.

AIM: This study investigated the effects of the inflammatory microenvironment of moderate pulpitis on biological properties of human dental pulp stem cells (DPSCs) and further explored the mechanism involved in osteo-/odontogenic induction of the inflammatory microenvironment. METHODOLOGY: Healthy DPSCs (hDPSCs) and inflammatory DPSCs (iDPSCs) were isolated from human-impacted third molars free of caries and clinically diagnosed with moderate pulpitis, respectively. Healthy DPSCs were treated with lipopolysaccharides (LPS) to mimic iDPSCs in vitro. The surface markers expressed on hDPSCs and iDPSCs were detected by flow cytometry. A CCK-8 assay was performed to determine cell proliferation. Flow cytometric analysis was used to evaluate cell apoptosis. The osteo-/odontogenic differentiation of DPSCs was evaluated by western blot, alkaline phosphatase staining, and Alizarin Red S staining. The functions of the genes of differentially expressed mRNAs of hDPSCs and iDPSCs were analysed using gene set enrichment analysis. Transmission electron microscopy and western blot were used to evaluate the autophagy changes of LPS-treated DPSCs. RESULTS: Compared with hDPSCs, iDPSCs showed no significant difference in proliferative capacity but had stronger osteo-/odontogenic potential. In addition, the mRNAs differentially expressed between iDPSCs and hDPSCs were considerably enriched in autophagosome formation and assembly-related molecules. In vitro mechanism studies further found that low concentrations of LPS could upregulate DPSC autophagy-related protein expression and autophagosome formation and promote its odontogenic/osteogenic differentiation, whereas the inhibition of DPSC autophagy led to the weakening of the odontogenic/osteogenic differentiation induced by LPS. CONCLUSIONS: This explorative study showed that DPSCs isolated from teeth with moderate pulpitis possessed higher osteo-/odontogenic differentiation capacity, and the mechanism involved was related to the inflammatory microenvironment-mediated autophagy of DPSCs. This helps to better understand the repair potential of inflamed dental pulp and provides the biological basis for pulp preservation and hard tissue formation in minimally invasive endodontics.

Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Anti-miR-141-3p maintains homeostasis between autophagy and apoptosis by targeting Yy1 in the fetal lumbosacral defecation center of rats.

Anorectal malformations (ARMs) are congenital diseases that lead to postoperative fecal incontinence, constipation, and soiling, despite improvements in surgery; however, their pathological mechanisms remain unclear. Here, we report the role of microRNA-141-3p in maintaining homeostasis between apoptosis and autophagy in the lumbosacral defecation center of fetal rats with ARMs. Elevated microRNA-141-3p expression inhibited YIN-YANG-1 expression by binding its 3' UTR, and repressed autophagy and triggered apoptosis simultaneously. Then, adenylate cyclase 3 was screened to be the downstream target gene of YIN-YANG-1 by chromatin immunoprecipitation sequencing experiments, and Yin Yang 1 could positively activate the transcription of adenylate cyclase 3 by directly interacting with the motif GAGATGG and ATGG in its promoter. Intraamniotic microinjection of adeno-rno-microRNA-141-3p-sponge-GFP in fetal rats with ARMs on embryonic day 15 restored apoptosis-autophagy homeostasis. These findings reveal that microRNA-141-3p upregulation impaired homeostasis between apoptosis and autophagy by inhibiting the YIN-YANG-1/adenylate cyclase 3 axis, and that intraamniotic injection of anti-microRNA-141-3p helped maintain homeostasis in the lumbosacral defecation center of ARMs during embryogenesis.

Regulatory role of m6A epitranscriptomic modifications in normal development and congenital malformations during embryogenesis.

The discovery of N6-methyladenosine (m6A) methylation and its role in translation has led to the emergence of a new field of research. Despite accumulating evidence suggesting that m6A methylation is essential for the pathogenesis of cancers and aging diseases by influencing RNA stability, localization, transformation, and translation efficiency, its role in normal and abnormal embryonic development remains unclear. An increasing number of studies are addressing the development of the nervous and gonadal systems during embryonic development, but only few are assessing that of the immune, hematopoietic, urinary, and respiratory systems. Additionally, these studies are limited by the requirement for reliable embryonic animal models and the difficulty in collecting tissue samples of fetuses during development. Multiple studies on the function of m6A methylation have used suitable cell lines to mimic the complex biological processes of fetal development or the early postnatal phase; hence, the research is still in the primary stage. Herein, we discuss current advances in the extensive biological functions of m6A methylation in the development and maldevelopment of embryos/fetuses and conclude that m6A modification occurs extensively during fetal development. Aberrant expression of m6A regulators is probably correlated with single or multiple defects in organogenesis during the intrauterine life. This comprehensive review will enhance our understanding of the pivotal role of m6A modifications involved in fetal development and examine future research directions in embryogenesis.

A novel genotype-phenotype between persistent-cloaca-related VACTERL and mutations of 8p23 and 12q23.1.

The mechanism underlying anorectal malformations (ARMs)-related VACTERL (vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, and renal and limb abnormalities) remains unclear. Copy number variation (CNV) contributed to VACTERL pathogenicity. Here, we report a novel CNV in 8p23 and 12q23.1 identified in a case of ARMs-related VACTERL association. This 12-year-old girl presented a cloaca (urethra, vagina, and rectum opening together and sharing a single tube length), an isolated kidney, and a perpetuation of the left superior vena cava at birth. Her intelligence, growth, and development were slightly lower than those of normal children of the same age. Array comparative genomic hybridization revealed a 9.6-Mb deletion in 8p23.1-23.3 and a 0.52-Mb duplication in 12q23.1 in her genome. Furthermore, we reviewed the cases involving CNVs in patients with VACTERL, 8p23 deletion, and 12q23.1 duplication, and our case was the first displaying ARMs-related VACTERL association with CNV in 8p23 and 12q23.1. These findings enriched our understanding between VACTERL association and the mutations of 8p23 deletion and 12q23.1 duplication. IMPACT: This is a novel case of a Chinese girl with anorectal malformations (ARMs)-related VACTERL with an 8p23.1-23.3 deletion and 12q23.1 duplication. Cloaca malformation is presented with novel copy number variation in 8p23.1-23.3 deletion and 12q23.1 duplication.

Small but strong: the emerging role of small nucleolar RNA in cardiovascular diseases.

Cardiovascular diseases (CVDs) are the leading cause of mortality and disability worldwide. Numerous studies have demonstrated that non-coding RNAs (ncRNAs) play a primary role in CVD development. Therefore, studies on the mechanisms of ncRNAs are essential for further efforts to prevent and treat CVDs. Small nucleolar RNAs (snoRNAs) are a novel species of non-conventional ncRNAs that guide post-transcriptional modifications and the subsequent maturation of small nuclear RNA and ribosomal RNA. Evidently, snoRNAs are extensively expressed in human tissues and may regulate different illnesses. Particularly, as the next-generation sequencing techniques have progressed, snoRNAs have been shown to be differentially expressed in CVDs, suggesting that they may play a role in the occurrence and progression of cardiac illnesses. However, the molecular processes and signaling pathways underlying the function of snoRNAs remain unidentified. Therefore, it is of great value to comprehensively investigate the association between snoRNAs and CVDs. The aim of this review was to collate existing literature on the biogenesis, characteristics, and potential regulatory mechanisms of snoRNAs. In particular, we present a scientific update on these snoRNAs and their relevance to CVDs in an effort to cast new light on the functions of snoRNAs in the clinical diagnosis of CVDs.

A corticoamygdalar pathway controls reward devaluation and depression using dynamic inhibition code.

Reward devaluation adaptively controls reward intake. It remains unclear how cortical circuits causally encode reward devaluation in healthy and depressed states. Here, we show that the neural pathway from the anterior cingulate cortex (ACC) to the basolateral amygdala (BLA) employs a dynamic inhibition code to control reward devaluation and depression. Fiber photometry and imaging of ACC pyramidal neurons reveal reward-induced inhibition, which weakens during satiation and becomes further attenuated in depression mouse models. Ablating or inhibiting these neurons desensitizes reward devaluation, causes reward intake increase and ultimate obesity, and ameliorates depression, whereas activating the cells sensitizes reward devaluation, suppresses reward consumption, and produces depression-like behaviors. Among various ACC neuron subpopulations, the BLA-projecting subset bidirectionally regulates reward devaluation and depression-like behaviors. Our study thus uncovers a corticoamygdalar circuit that encodes reward devaluation via blunted inhibition and suggests that enhancing inhibition within this circuit may offer a therapeutic approach for treating depression.