A Single-Cell Atlas of Bovine Skeletal Muscle Reveals Mechanisms Regulating Intramuscular Adipogenesis and Fibrogenesis

Background Intramuscular fat (IMF) and intramuscular connective tissue (IMC) are often seen in human myopathies and are central to beef quality. The mechanisms regulating their accumulation remain poorly understood. Here, we explored the possibility of using beef cattle as a novel model for mechanistic studies of intramuscular adipogenesis and fibrogenesis. Methods Skeletal muscle single-cell RNAseq was performed on three cattle breeds, including Wagyu (high IMF), Brahman (abundant IMC but scarce IMF), and Wagyu/Brahman cross. Sophisticated bioinformatics analyses, including clustering analysis, gene set enrichment analyses, gene regulatory network construction, RNA velocity, pseudotime analysis, and cell-cell communication analysis, were performed to elucidate heterogeneities and differentiation processes of individual cell types and differences between cattle breeds. Experiments were conducted to validate the function and specificity of identified key regulatory and marker genes. Integrated analysis with multiple published human and non-human primate datasets was performed to identify common mechanisms. Results A total of 32 708 cells and 21 clusters were identified, including fibro/adipogenic progenitor (FAP) and other resident and infiltrating cell types. We identified an endomysial adipogenic FAP subpopulation enriched for COL4A1 and CFD (log2FC = 3.19 and 1.92, respectively; P \u3c 0.0001) and a perimysial fibrogenic FAP subpopulation enriched for COL1A1 and POSTN (log2FC = 1.83 and 0.87, respectively; P \u3c 0.0001), both of which were likely derived from an unspecified subpopulation. Further analysis revealed more progressed adipogenic programming of Wagyu FAPs and more advanced fibrogenic programming of Brahman FAPs. Mechanistically, NAB2 drives CFD expression, which in turn promotes adipogenesis. CFD expression in FAPs of young cattle before the onset of intramuscular adipogenesis was predictive of IMF contents in adulthood (R2 = 0.885, P \u3c 0.01). Similar adipogenic and fibrogenic FAPs were identified in humans and monkeys. In aged humans with metabolic syndrome and progressed Duchenne muscular dystrophy (DMD) patients, increased CFD expression was observed (P \u3c 0.05 and P \u3c 0.0001, respectively), which was positively correlated with adipogenic marker expression, including ADIPOQ (R2 = 0.303, P \u3c 0.01; and R2 = 0.348, P \u3c 0.01, respectively). The specificity of Postn/POSTN as a fibrogenic FAP marker was validated using a lineage-tracing mouse line. POSTN expression was elevated in Brahman FAPs (P \u3c 0.0001) and DMD patients (P \u3c 0.01) but not in aged humans. Strong interactions between vascular cells and FAPs were also identified. Conclusions Our study demonstrates the feasibility of beef cattle as a model for studying IMF and IMC. We illustrate the FAP programming during intramuscular adipogenesis and fibrogenesis and reveal the reliability of CFD as a predictor and biomarker of IMF accumulation in cattle and humans

Loss of \u3ci\u3eActa2\u3c/i\u3e in Cardiac Fibroblasts Does Not Prevent the Myofibroblast Differentiation or Affect the Cardiac Repair After Myocardial Infarction

In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMαA) that is assembled into stress fibers. However, the requirement of Acta2/SMαA in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair remained unknown. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal post-MI survival rate. Moreover, Acta2 deletion did not affect the function or histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT and Acta2-null cardiac myofibroblasts. Acta2-null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a significant compensatory increase in the transcription level of non-Acta2 actin isoforms, especially Actg2 and Acta1. Moreover, in myofibroblasts, the transcription levels of cytoplasmic actin isoforms were significantly higher than those of muscle actin isoforms. In addition, we found that myocardin-related transcription factor-A is critical for myofibroblast differentiation but is not required for the compensatory effects of non-Acta2 isoforms. In conclusion, the Acta2 deletion does not prevent the myofibroblast differentiation of cardiac fibroblasts or affect the post-MI cardiac repair, and the increased expression and stress fiber formation of non-SMαA actin isoforms and the functional redundancy between actin isoforms are able to compensate for the loss of Acta2 in cardiac myofibroblasts

T\u3cem\u3ecf\u3c/em\u3e21 Marks Visceral Adipose Mesenchymal Progenitors and Functions as a Rate-Limiting Factor During Visceral Adipose Tissue Development

Distinct locations of different white adipose depots suggest anatomy-specific developmental regulation, a relatively understudied concept. Here, we report a population of Tcf21 lineage cells (Tcf21 LCs) present exclusively in visceral adipose tissue (VAT) that dynamically contributes to VAT development and expansion. During development, the Tcf21 lineage gives rise to adipocytes. In adult mice, Tcf21 LCs transform into a fibrotic or quiescent state. Multiomics analyses show consistent gene expression and chromatin accessibility changes in Tcf21 LC, based on which we constructed a gene-regulatory network governing Tcf21 LC activities. Furthermore, single-cell RNA sequencing (scRNA-seq) identifies the heterogeneity of Tcf21 LCs. Loss of Tcf21 promotes the adipogenesis and developmental progress of Tcf21 LCs, leading to improved metabolic health in the context of diet-induced obesity. Mechanistic studies show that the inhibitory effect of Tcf21 on adipogenesis is at least partially mediated via Dlk1 expression accentuation

Progenitor Cell Isolation From Mouse Epididymal Adipose Tissue and Sequencing Library Construction

Here, we present a protocol to isolate progenitor cells from mouse epididymal visceral adipose tissue and construct bulk RNA and assay for transposase-accessible chromatin with sequencing (ATAC-seq) libraries. We describe steps for adipose tissue collection, cell isolation, and cell staining and sorting. We then detail procedures for both ATAC-seq and RNA sequencing library construction. This protocol can also be applied to other tissues and cell types directly or with minor modifications. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023).1 *1 Liu, Q., Li, C., Deng, B., Gao, P., Wang, L., Li, Y., ... & Fu, X. (2023). Tcf21 marks visceral adipose mesenchymal progenitors and functions as a rate-limiting factor during visceral adipose tissue development. Cell reports, 42(3) 112166. https://doi.org/10.1016/j.celrep.2023.11216

Visual and Rapid Detection of Escherichia coli O157:H7 in Stool Samples by FTA Card-based Loop-mediated Isothermal Amplification

Escherichia coli O157:H7 ( E. coli O157:H7) can induce severe diseases in animals and humans that result in significant public health problems. Therefore, the development of rapid and visual detection methods to diagnose E. coli O157:H7 infections and monitor its prevalence is critical for the prevention and control purposes. A colorimetric loop-mediated isothermal amplification (LAMP) assay was utilized to detect E. coli O157:H7. A DNA extraction kit and Flinders Technology Associates (FTA) cards were used to extract nucleic acid in conjunction with colorimetric LAMP detection. The method developed effectively distinguished E. coli O157:H7 from other pathogens with a detection limit of 25 CFU/mL in spiked stool samples. In addition, the nucleic acid of these samples was easily extracted and transported with an FTA card at room temperature. The entire detection process was completed within 35 min using simple constant-temperature equipment. The colorimetric LAMP method with FTA card-based nucleic acid purification was shown to rapidly detect E. coli O157:H7 with sensitivity and specificity. This visual method is expected to be widely used to control E. coli O157:H7 infections, particularly in resource-limited settings

New insight into the microbiome, resistome, and mobilome on the dental waste water in the context of heavy metal environment

ObjectHospital sewage have been associated with incorporation of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) into microbes, which is considered as a key indicator for the spread of antimicrobial resistance (AMR). The compositions of dental waste water (DWW) contain heavy metals, the evolution of AMR and its effects on the water environment in the context of heavy metal environment have not been seriously investigated. Thus, our major aims were to elucidate the evolution of AMR in DWW.MethodsDWW samples were collected from a major dental department. The presence of microbial communities, ARGs, and MGEs in untreated and treated (by filter membrane and ozone) samples were analyzed using metagenomics and bioinformatic methods.ResultsDWW-associated resistomes included 1,208 types of ARGs, belonging to 29 antibiotic types/subtypes. The most abundant types/subtypes were ARGs of multidrug resistance and of antibiotics that were frequently used in the clinical practice. Pseudomonas putida, Pseudomonas aeruginosa, Chryseobacterium indologenes, Sphingomonas laterariae were the main bacteria which hosted these ARGs. Mobilomes in DWW consisted of 93 MGE subtypes which belonged to 8 MGE types. Transposases were the most frequently detected MGEs which formed networks of communications. For example, ISCrsp1 and tnpA.5/4/11 were the main transposases located in the central hubs of a network. These significant associations between ARGs and MGEs revealed the strong potential of ARGs transmission towards development of antimicrobial-resistant (AMR) bacteria. On the other hand, treatment of DWW using membranes and ozone was only effective in removing minor species of bacteria and types of ARGs and MGEs.ConclusionDWW contained abundant ARGs, and MGEs, which contributed to the occurrence and spread of AMR bacteria. Consequently, DWW would seriously increase environmental health concerns which may be different but have been well-documented from hospital waste waters

Birthdating of myenteric neuron subtypes in the small intestine of the mouse

There are many different types of enteric neurons. Previous studies have identified the time at which some enteric neuron subtypes are born (exit the cell cycle) in the mouse, but the birthdates of some major enteric neuron subtypes are still incompletely characterized or unknown. We combined 5‐ethynynl‐2′‐deoxyuridine (EdU) labeling with antibody markers that identify myenteric neuron subtypes to determine when neuron subtypes are born in the mouse small intestine. We found that different neurochemical classes of enteric neuron differed in their birthdates; serotonin neurons were born first with peak cell cycle exit at E11.5, followed by neurofilament‐M neurons, calcitonin gene‐related peptide neurons (peak cell cycle exit for both at embryonic day [E]12.5–E13.5), tyrosine hydroxylase neurons (E15.5), nitric oxide synthase 1 (NOS1) neurons (E15.5), and calretinin neurons (postnatal day [P]0). The vast majority of myenteric neurons had exited the cell cycle by P10. We did not observe any EdU+/NOS1+ myenteric neurons in the small intestine of adult mice following EdU injection at E10.5 or E11.5, which was unexpected, as previous studies have shown that NOS1 neurons are present in E11.5 mice. Studies using the proliferation marker Ki67 revealed that very few NOS1 neurons in the E11.5 and E12.5 gut were proliferating. However, Cre‐lox‐based genetic fate‐mapping revealed a small subpopulation of myenteric neurons that appears to express NOS1 only transiently. Together, our results confirm a relationship between enteric neuron subtype and birthdate, and suggest that some enteric neurons exhibit neurochemical phenotypes during development that are different from their mature phenotype. J. Comp. Neurol. 522:514–527, 2014. © 2013 Wiley Periodicals, Inc. To examine when different myenteric neuron subtypes in the mouse intestine are born, EDU labeling was combined with immunohistochemistry. Neuron subtypes were born at different, but overlapping times with serotonin interneurons first, then intrinsic sensory neurons, inhibitory motor neurons and excitatory motor neurons last, with peak birthdate around birth.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102151/1/cne23423.pd

Targeted next generation sequencing approach identifies eighteen new candidate genes in normosmic hypogonadotropic hypogonadism and Kallmann Syndrome

The genetic basis is unknown for ∼60% of normosmic hypogonadotropic hypogonadism (nHH)/Kallmann syndrome (KS). DNAs from (17 male and 31 female) nHH/KS patients were analyzed by targeted next generation sequencing (NGS) of 261 genes involved in hypothalamic, pituitary, and/or olfactory pathways, or suggested by chromosome rearrangements. Selected variants were subjected to Sanger DNA sequencing, the gold standard. The frequency of Sanger-confirmed variants was determined using the ExAC database. Variants were classified as likely pathogenic (frameshift, nonsense, and splice site) or predicted pathogenic (nonsynonymous missense). Two novel FGFR1 mutations were identified, as were 18 new candidate genes including: AMN1, CCKBR, CRY1, CXCR4, FGF13, GAP43, GLI3, JAG1, NOS1, MASTL, NOTCH1, NRP2, PALM2, PDE3A, PLEKHA5, RD3, and TRAPPC9, and TSPAN11. Digenic and trigenic variants were found in 8/48 (16.7%) and 1/48 (2.1%) patients, respectively. NGS with confirmation by Sanger sequencing resulted in the identification of new causative FGFR1 gene mutations and suggested 18 new candidate genes in nHH/KS