Comparative analyses of dynamic transcriptome profiles highlight key response genes and dominant isoforms for muscle development and growth in chicken.

BACKGROUND: Modern breeding strategies have resulted in significant differences in muscle mass between indigenous chicken and specialized broiler. However, the molecular regulatory mechanisms that underlie these differences remain elusive. The aim of this study was to identify key genes and regulatory mechanisms underlying differences in breast muscle development between indigenous chicken and specialized broiler. RESULTS: Two time-series RNA-sequencing profiles of breast muscles were generated from commercial Arbor Acres (AA) broiler (fast-growing) and Chinese indigenous Lushi blue-shelled-egg (LS) chicken (slow-growing) at embryonic days 10, 14, and 18, and post-hatching day 1 and weeks 1, 3, and 5. Principal component analysis of the transcriptome profiles showed that the top four principal components accounted for more than 80% of the total variance in each breed. The developmental axes between the AA and LS chicken overlapped at the embryonic stages but gradually separated at the adult stages. Integrative investigation of differentially-expressed transcripts contained in the top four principal components identified 44 genes that formed a molecular network associated with differences in breast muscle mass between the two breeds. In addition, alternative splicing analysis revealed that genes with multiple isoforms always had one dominant transcript that exhibited a significantly higher expression level than the others. Among the 44 genes, the TNFRSF6B gene, a mediator of signal transduction pathways and cell proliferation, harbored two alternative splicing isoforms, TNFRSF6B-X1 and TNFRSF6B-X2. TNFRSF6B-X1 was the dominant isoform in both breeds before the age of one week. A switching event of the dominant isoform occurred at one week of age, resulting in TNFRSF6B-X2 being the dominant isoform in AA broiler, whereas TNFRSF6B-X1 remained the dominant isoform in LS chicken. Gain-of-function assays demonstrated that both isoforms promoted the proliferation of chicken primary myoblasts, but only TNFRSF6B-X2 augmented the differentiation and intracellular protein content of chicken primary myoblasts. CONCLUSIONS: For the first time, we identified several key genes and dominant isoforms that may be responsible for differences in muscle mass between slow-growing indigenous chicken and fast-growing commercial broiler. These findings provide new insights into the regulatory mechanisms underlying breast muscle development in chicken.

Selection of candidate genes affecting meat quality and preliminary exploration of related molecular mechanisms in the Mashen pig.

OBJECTIVE: The aim of this study was to select the candidate genes affecting meat quality and preliminarily explore the related molecular mechanisms in the Mashen pig. METHODS: The present study explored genetic factors affecting meat quality in the Mashen pig using RNA sequencing (RNA-Seq). We sequenced the transcriptomes of 180-day-old Mashen and Large White pigs using longissimus dorsi to select differentially expressed genes (DEGs). RESULTS: The results indicated that a total of 425 genes were differentially expressed between Mashen and Large White pigs. A gene ontology enrichment analysis revealed that DEGs were mainly enriched for biological processes associated with metabolism and muscle development, while a Kyoto encyclopedia of genes and genomes analysis showed that DEGs mainly participated in signaling pathways associated with amino acid metabolism, fatty acid metabolism, and skeletal muscle differentiation. A MCODE analysis of the protein-protein interaction network indicated that the four identified subsets of genes were mainly associated with translational initiation, skeletal muscle differentiation, amino acid metabolism, and oxidative phosphorylation pathways. CONCLUSION: Based on the analysis results, we selected glutamic-oxaloacetic transaminase 1, malate dehydrogenase 1, pyruvate dehydrogenase 1, pyruvate dehydrogenase kinase 4, and activator protein-1 as candidate genes affecting meat quality in pigs. A discussion of the related molecular mechanisms is provided to offer a theoretical basis for future studies on the improvement of meat quality in pigs.

Novel splice isoforms of pig myoneurin and their diverse mRNA expression patterns.

OBJECTIVE: The aim of this study was to clone alternative splicing isoforms of pig myoneurin (MYNN), predict the structure and function of coding protein, and study temporal and spatial expression characteristics of each transcript. METHODS: Alternative splice isoforms of MYNN were identified using RNA sequencing (RNA-seq) and cloning techniques. Quantitative real-time polymerase chain reaction (qPCR) was employed to detect expression patterns in 11 tissues of Large White (LW) and Mashen (MS) pigs, and to study developmental expression patterns in cerebellum (CE), stomach (ST), and longissimus dorsi (LD). RESULTS: The results showed that MYNN had two alternatively spliced isoforms, MYNN-1 (GenBank accession number: KY470829) and MYNN-2 (GenBank accession number: KY670835). MYNN-1 coding sequence (CDS) is composed of 1,830 bp encoding 609 AA, whereas MYNN-2 CDS is composed of 1,746 bp encoding 581 AA. MYNN-2 was 84 bp less than MYNN-1 and lacked the sixth exon. MYNN-2 was found to have one C2H2 type zinc finger protein domain less than MYNN-1. Two variants were ubiquitously expressed in all pig tissues, and there were significant differences in expression of different tissues (p<0.05; p<0.01). The expression of MYNN-1 was significantly higher than that of MYNN-2 in almost tissues (p<0.05; p<0.01), which testified that MYNN-1 is the main variant. The expression of two isoforms decreased gradually with increase of age in ST and CE of MS pig, whereas increased gradually in LW pig. In LD, the expression of two isoforms increased first and then decreased with increase of age in MS pig, and decreased gradually in LW pig. CONCLUSION: Two transcripts of pig MYNN were successfully cloned and MYNN-1 was main variant. MYNN was highly expressed in ST, CE, and LD, and their expression was regular. We speculated that MYNN plays important roles in digestion/absorption and skeletal muscle growth, whereas the specific mechanisms require further elucidation.

Coxsackievirus A10 impairs nail regeneration and induces onychomadesis by mimicking DKK1 to attenuate Wnt signaling.

Coxsackievirus A10 (CV-A10) infection, a prominent cause of childhood hand-foot-and-mouth disease (HFMD), frequently manifests with the intriguing phenomenon of onychomadesis, characterized by nail shedding. However, the underlying mechanism is elusive. Here, we found that CV-A10 infection in mice could suppress Wnt/ß-catenin signaling by restraining LDL receptor-related protein 6 (LRP6) phosphorylation and ß-catenin accumulation and lead to onychomadesis. Mechanistically, CV-A10 mimics Dickkopf-related protein 1 (DKK1) to interact with Kringle-containing transmembrane protein 1 (KRM1), the CV-A10 cellular receptor. We further found that Wnt agonist (GSK3ß inhibitor) CHIR99021 can restore nail stem cell differentiation and protect against nail shedding. These findings provide novel insights into the pathogenesis of CV-A10 and related viruses in onychomadesis and guide prognosis assessment and clinical treatment of the disease.

Deciphering a reliable synergistic bispecific strategy of rescuing antibodies for SARS-CoV-2 escape variants, including BA.2.86, EG.5.1, and JN.1.

The game between therapeutic monoclonal antibodies (mAbs) and continuously emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has favored the virus, as most therapeutic mAbs have been evaded. Addressing this challenge, we systematically explored a reproducible bispecific antibody (bsAb)-dependent synergistic effect in this study. It could effectively restore the neutralizing activity of the bsAb when any of its single mAbs is escaped by variants. This synergy is primarily attributed to the binding angle of receptor-binding domain (RBD)-5, facilitating inter-spike cross-linking and promoting cryptic epitope exposure that classical antibody cocktails cannot achieve. Furthermore, RBD-5 with RBD-2, RBD-6, and RBD-7, alongside RBD-8, also exhibit significantly enhanced effects. This study not only shifts the paradigm in understanding antibody interactions but paves the way for developing more effective therapeutic antibodies against rapidly mutating SARS-CoV-2, with Dia-19 already showing promise against emerging variants like BA.2.86, EG.5.1, and JN.1.

Integrated LC/MS-based lipidomics and transcriptomics analyses revealed lipid composition heterogeneity between pectoralis intramuscular fat and abdominal fat and its regulatory mechanism in chicken.

Intramuscular fat (IMF) content is conducive to multiple meat quality properties, while abdominal fat (AF) is treated as waste product in chicken industry. However, the heterogeneity and distinct regulatory mechanisms of lipid composition between the IMF and AF are still unclear. In this study, we carried out non-targeted lipidomics analyses of pectoralis IMF and AF, and detected a total of 423 differential lipid molecules (DLMs) between chicken IMF and AF, including 307 up-regulated and 116 down-regulated DLMs in pectoral IMF. These DLMs exhibited the definite alteration of lipid composition. The up-reglated DLMs in IMF were mainly glycerophospholipids (GPs), including the bulk of phosphatidylcholines (PC, PC (P) and PC (O)), phosphatidylethanolamines (PE, PE (P) and PE (O)), phosphatidylglycerols (PG) and phosphatidylinositol (PI), while the up-reglated DLMs in AF were mainly glycerolipids (GLs), including most of triacylglycerols (TG) and diacylglycerols (DG). We further identified 28 main DLMs contributing to the heterogeneous deposition of IMF and AF, including 11 TGs common to IMF and AF, 12 PCs/PC (P)s specific to IMF and 5 DGs specific to AF. Further integration of transcriptome with the main DLMs by weighted gene co-expression network analysis (WGCNA), we found five key gene sets that included 386 unique genes promoting IMF deposition in pectoralis, 213 unique genes promoting AF deposition, 6 unique genes detrimental to AF deposition, 7 common genes that promote IMF deposition in pectoralis while adversely affect AF deposition, and 28 genes that only promoted IMF deposition in pectoralis but had no effect on AF deposition. In addition, we also observed the expression characteristics of key genes in vivo and in vitro, and found that transmembrane protein family gene TMEM164 might be mainly involved in the positive regulation of intramuscular fat deposition in pectoralis and zinc finger protein family gene ZNF488 had a potential unique positive regulatory function on abdominal fat deposition. These findings provide new perspectives for understanding IMF and AF heterodeposition and will serve as a valuable information resource for improving meat quality via breeding selection in chicken.

Glutamic-oxaloacetic transaminase 1 regulates adipocyte differentiation by altering nicotinamide adenine dinucleotide phosphate content.

OBJECTIVE: This study was performed to examine whether the porcine glutamic-oxaloacetic transaminase 1 (GOT1) gene has important functions in regulating adipocyte differentiation. METHODS: Porcine GOT1 knockout and overexpression vectors were constructed and transfected into the mouse adipogenic 3T3-L1 cells. Lipid droplets levels were measured after 8 days of differentiation. The mechanisms through which GOT1 participated in lipid deposition were examined by measuring the expression of malate dehydrogenase 1 (MDH1) and malic enzyme (ME1) and the cellular nicotinamide adenine dinucleotide phosphate (NADPH) content. RESULTS: GOT1 knockout significantly decreased lipid deposition in the 3T3-L1 cells (p<0.01), whereas GOT1 overexpression significantly increased lipid accumulation (p<0.01). At the same time, GOT1 knockout significantly decreased the NADPH content and the expression of MDH1 and ME1 in the 3T3-L1 cells. Overexpression of GOT1 significantly increased the NADPH content and the expression of MDH1 and ME1, suggesting that GOT1 regulated adipocyte differentiation by altering the NADPH content. CONCLUSION: The results preliminarily revealed the effector mechanisms of GOT1 in regulating adipose differentiation. Thus, a theoretical basis is provided for improving the quality of pork and studies on diseases associated with lipid metabolism.

Biological activity reduction and mitochondrial and lysosomal dysfunction of mesenchymal stem cells aging in vitro.

BACKGROUND: Mesenchymal stem cells (MSCs) have been extensively used for the treatment of various diseases in preclinical and clinical trials. In vitro propagation is needed to attain enough cells for clinical use. However, cell aging and viability reduction caused by long-time culture have not been thoroughly investigated, especially for the function of mitochondria and lysosomes. Therefore, this study was designed to detect mitochondrial and lysosomal activity, morphological and functional changes in human umbilical cord MSCs (UMSCs) after long-time culture. METHODS: First, we examined cell activities, including proliferation and immigration ability, differentiation potential, and immunosuppressive capacity of UMSCs at an early and late passages as P4 (named UMSC-P4) and P9 (named UMSC-P9), respectively. Then, we compared the mitochondrial morphology of UMSC-P4 and UMSC-P9 using the electronic microscope and MitoTracker Red dyes. Furthermore, we investigated mitochondrial function, including mitochondrial membrane potential, antioxidative ability, apoptosis, and ferroptosis detected by respective probe. Cell energy metabolism was tested by mass spectrometry. In addition, we compared the lysosomal morphology of UMSC-P4 and UMSC-P9 by electronic microscope and lysoTracker Red dyes. Finally, the transcriptome sequence was performed to analyze the total gene expression of these cells. RESULTS: It was found that UMSC-P9 exhibited a reduced biological activity and showed an impaired mitochondrial morphology with disordered structure,  reduced mitochondrial crista, and mitochondrial fragments. They also displayed decreased mitochondrial membrane potential, antioxidative ability, tricarboxylic acid cycle activity and energy production. At the same time, apoptosis and ferroptosis were increased. In addition, UMSC-P9, relative to UMSC-P4, showed undegraded materials in their lysosomes, the enhancement in lysosomal membrane permeability, the reduction in autophagy and phagocytosis. Moreover, transcriptome sequence analysis also revealed a reduction of cell function, metabolism, mitochondrial biogenesis, DNA replication and repair, and an increase of gene expression related to cell senescence, cancer, diseases, and infection in UMSC-P9. CONCLUSION: This study indicates that in vitro long-time culturing of MSCs can cause mitochondrial and lysosomal dysfunction, probably contributing to the decline of cell activity and cell aging. Therefore, the morphology and function of mitochondria and lysosomes can be regarded as two important parameters to monitor cell viability, and they can also serve as two important indicators for optimizing in vitro culture conditions.

Characteristics of Brucella abortus vaccine strain A19 reveals its potential mechanism of attenuated virulence.

Brucella vaccination is one of the most important strategies for controlling brucellosis in livestock. The A19 strain was the effective vaccine used to control brucellosis in China. However, the characteristics of physiological and attenuated virulence of the A19 strain are not investigated in detail. In this study, we compared the phenotypic characteristics of the A19 to the wild-type strain S2308. Virulence test showed that the A19 was significantly attenuated at chronic infection stage in infected mouse model. In growth analysis, the A19 exhibited a quick growth at exponential phase and premature at stationary phase. The inflammatory response of macrophages infected by the A19 was detected using TaqMan qPCR assay, indicating that the inflammatory level of the A19-infected macrophages was higher than that of the S2308 infection. Cell death analysis showed that the A19 was not cytotoxic for macrophages. Cell infection showed that the A19 reduced its ability to invade, survive and traffic within host cells, and the intracellular A19 hardly excludes lysosome-associated marker LAMP-1, suggesting that the A19 can't escape the lysosome degradation within host cells. In further study, the sensitivity test exhibited that the A19 is more sensitive to stress and bactericidal factors than the S2308 strain, Western blot and silver staining analysis exhibited that the A19 has a different expression pattern of OMPs and reduces LPS O-antigen expression relative to the S2308 strain. Those data give us a more detailed understanding about the A19 vaccine strain, which will be beneficial for improvement of current Brucella vaccine and overcoming its defects.

Pathogenicity and Immune Responses of Aspergillus fumigatus Infection in Chickens.

Aspergillus fumigatus is a ubiquitous pathogen in poultry farms, causing aspergillosis in chickens. To study the pathogenicity of A. fumigatus, 14-days-old chickens were infected with fungal conidia (2 × 107 CFU/mL) via thoracic intra-air sacs inoculation. The clinical symptoms, gross and histopathological lesions, and fungal load in the lungs were examined. Additionally, the mRNAs of Toll like receptors (TLR) and pro-inflammatory cytokines were evaluated by quantitative PCR to explore the immune responses induced by A. fumigatus. The results showed that overt depression, ruffled feathers, and dyspnea were observed in the infected chickens as early as 3 days post infection (dpi). Eleven out of 25 infected chickens died from 5 to 9 dpi, and A. fumigatus could also be reisolated from the infected lung. Histopathological examination revealed obvious airsacculitis and pneumonia, characterized by inflammatory cell infiltration (heterophils and macrophages), and granulomatous lesions in the lung. The mRNA expressions of TLR1 and TLR2 were upregulated in the lung and spleen, and most pro-inflammatory cytokines including IL-1ß, Cxcl-8, TNF-α, IL-12, and IFN-γ were increased in both the lung and spleen during the tested period, suggesting that the innate immune responses were triggered by A. fumigatus infection, and these cytokines participated in the inflammatory responses against A. fumigatus. These results indicate that A. fumigatus infection by thoracic intra-air sacs inoculation can cause severe respiratory damage in chickens, activate TLR1 and TLR2 mediated immune responses, and elicit large expression of pro-inflammatory cytokines such as IL-1ß, Cxcl-8, and IFN-γ. These data will help further understanding of the pathogenesis and immune responses of A. fumigatus infection in the chicken.