Establishment and Characterization of a Chicken Myoblast Cell Line.

Skeletal muscle, which is predominantly constituted by multinucleated muscle fibers, plays a pivotal role in sustaining bodily movements and energy metabolism. Myoblasts, which serve as precursor cells for differentiation and fusion into muscle fibers, are of critical importance in the exploration of the functional genes associated with embryonic muscle development. However, the in vitro proliferation of primary myoblasts is inherently constrained. In this study, we achieved a significant breakthrough by successfully establishing a chicken myoblast cell line through the introduction of the exogenous chicken telomerase reverse transcriptase (chTERT) gene, followed by rigorous G418-mediated pressure screening. This newly developed cell line, which was designated as chTERT-myoblasts, closely resembled primary myoblasts in terms of morphology and exhibited remarkable stability in culture for at least 20 generations of population doublings without undergoing malignant transformation. In addition, we conducted an exhaustive analysis that encompassed cellular proliferation, differentiation, and transfection characteristics. Our findings revealed that the chTERT-myoblasts had the ability to proliferate, differentiate, and transfect after multiple rounds of population doublings. This achievement not only furnished a valuable source of homogeneous avian cell material for investigating embryonic muscle development, but also provided valuable insights and methodologies for establishing primary cell lines.

The typical developmental trajectory and energy requirements of Shitou goose during the embryonic stage.

Low hatchability has been a persistent challenge in the goose industry. Establishing standard atlases and comprehending embryonic development patterns are essential to improving the hatching rates of goose eggs. However, comprehensive descriptions of normal atlases, embryonic development, and energy requirements in geese are lacking. In this study, a total of 120 fertile eggs from well-known large Shitou goose were incubated using 12 nesting purebred female geese. During hatching, both the temperature of the eggshells and the weight of eggs were recorded, and daily photographs of the embryos were captured to monitor their development closely. After hatching, counted the number of pores per unit area of eggshells by choosing eggs from without sperm, dead embryos, and normally hatched. Furthermore, 150 Shitou goose eggs were hatched by automatic incubator, with adjustments made based on observed normal developmental stages that incubated by female geese. The eggs were carefully opened to meticulously document embryonic morphology and create a detailed development map. Measurements were taken of the eye diameter, length of the lower beak, tarsometatarsus bone, and embryo length. Subsequently, an analysis was conducted to assess the calcium, phosphorus, crude protein, and crude fat content to study the energy requirements for embryo development. characteristics on the 7th, 15th, 23rd and 28th days of Shitou goose hatching corresponded to the 5th, 10th, 17th and 19th days of chicken egg incubation, respectively. These days were distinguished individually by "visible embryo's eye", "closure", "sealing the door", and "flashing hair". Besides, the hatch rate of the incubator reached 86.67%, and the cumulative water loss rate increased with embryo age. Notably, normally developing embryos displayed a significantly higher number of pores on the eggshell surface compared to dead embryos (P < 0.05). Additionally, embryonic body length, eyeball diameter, and lower beak length exhibited continuous growth until day 19 of incubation, while tarsometatarsus length increased steadily from days 12 to 31. Liver size measurement began on the 10th day of incubation, while both leg and chest muscles showed continuous growth from the 12th day. For energy demand, the embryo primarily relied on protein sourced from the egg yolk within the first 10 days of development. Afterward, the egg yolk provided both protein and fat for embryonic growth. In summary, this study has generated a comprehensive developmental map for Shitou goose embryos, offering valuable insights into their growth and morphological changes throughout the incubation period. This map can serve as a reference for optimizing machine incubation techniques to enhance goose egg hatching rates and provide fresh perspectives on the development of geese.

Novel Circular RNA CircSLC2A13 Regulates Chicken Muscle Development by Sponging MiR-34a-3p.

The skeletal muscle is the major muscle tissue in animals, and its production is subject to a complex and strict regulation. The proliferation and differentiation of myoblasts are important factors determining chicken muscle development. Circular RNAs (circRNAs) are endogenous RNAs that are widely present in various tissues of organisms. Recent studies have shown that circRNA plays key roles in the development of skeletal muscles. The solute carrier (SLC) family functions in the transport of metabolites such as amino acids, glucose, nucleotides, and essential nutrients and is widely involved in various basic physiological metabolic processes within the body. In this study, we have cloned a novel chicken circular RNA circSLC2A13 generated from the solute carrier family 2 member 13 gene (SLC2A13). Also, circSLC2A1 was confirmed by sequencing verification, RNase R treatment, and reverse transcription analysis. Currently, our results show that circSLC2A13 promoted the proliferation and differentiation of chicken myoblasts. The double luciferase reporter system revealed that circSLC2A13 regulated the proliferation and differentiation of myoblasts by competitive binding with miR-34a-3p. In addition, results indicated that circSLC2A13 acts as a miR-34a-3p sponge to relieve its inhibitory effect on the target SMAD3 gene. In summary, this study found that chicken circSLC2A13 can bind to miR-34a-3p and weaken its inhibitory effect on the SMAD family member 3 gene (SMAD3), thereby promoting the proliferation and differentiation of myoblasts. This study laid foundations for broiler industry and muscle development research.

Chicken muscle antibody array reveals the regulations of LDHA on myoblast differentiation through energy metabolism.

Myoblast proliferation and differentiation are highly dynamic and regulated processes in skeletal muscle development. Given that proteins serve as the executors for the majority of biological processes, exploring key regulatory factors and mechanisms at the protein level offers substantial opportunities for understanding the skeletal muscle development. In this study, a total of 607 differentially expressed proteins between proliferation and differentiation in myoblasts were screened out using our chicken muscle antibody array. Biological function analysis revealed the importance of energy production processes and compound metabolic processes in myogenesis. Our antibody array specifically identified an upregulation of LDHA during differentiation, which was associated with the energy metabolism. Subsequent investigation demonstrated that LDHA promoted the glycolysis and TCA cycle, thereby enhancing myoblasts differentiation. Mechanistically, LDHA promotes the glycolysis and TCA cycle but inhibits the ETC oxidative phosphorylation through enhancing the NADH cycle, providing the intermediate metabolites that improve the myoblasts differentiation. Additionally, increased glycolytic ATP by LDHA induces Akt phosphorylation and activate the PI3K-Akt pathway, which might also contribute to the promotion of myoblasts differentiation. Our studies not only present a powerful tool for exploring myogenic regulatory factors in chicken muscle, but also identify a novel role for LDHA in modulating myoblast differentiation through its regulation of cellular NAD+ levels and subsequent downstream effects on mitochondrial function.

Molecular mechanisms underlying the impact of muscle fiber types on meat quality in livestock and poultry.

In the past, the primary emphasis of livestock and poultry breeding was mainly on improving the growth rate, meat production efficiency and disease resistance. However, the improvement of meat quality has become a major industrial focus due to the ongoing advancements in livestock and poultry breeding. Skeletal muscles consist of multinucleated myofibers formed through the processes of myoblast proliferation, differentiation and fusion. Muscle fibers can be broadly classified into two main types: slow-twitch (Type I) and fast-twitch (Type II). Fast-twitch fibers can be further categorized into Type IIa, Type IIx, and Type IIb. The proportion of Type I and Type IIa muscle fibers is positively associated with meat quality, while the presence of Type IIb muscle fibers in skeletal muscle tissue is inversely related to meat quality. Consequently, muscle fiber composition directly influences meat quality. The distribution of these fiber types within skeletal muscle is governed by a complex network, which encompasses numerous pivotal regulators and intricate signaling pathways. This article aims to succinctly outline the parameters utilized for assessing meat quality, elucidate the relationship between muscle fiber composition and meat quality as well as elaborate on the relevant genetic factors and their molecular mechanisms that regulate muscle fiber types in livestock and poultry. This summary will enrich our comprehension of how to improve meat quality in livestock and poultry, providing valuable insights for future improvements.

Circular RNA circIGF2BP3 Promotes the Proliferation and Differentiation of Chicken Primary Myoblasts.

The quality and quantity of animal meat are closely related to the development of skeletal muscle, which, in turn, is determined by myogenic cells, including myoblasts and skeletal muscle satellite cells (SMSCs). Circular RNA, an endogenous RNA derivative formed through specific reverse splicing in mRNA precursors, has the potential to influence muscle development by binding to miRNAs or regulating gene expression involved in muscular growth at the transcriptional level. Previous high-throughput sequencing of circRNA in chicken liver tissue revealed a circular transcript, circIGF2BP3, derived from the gene encoding insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3). In this study, we confirmed the presence of the natural circular molecule of circIGF2BP3 through an RNase R enzyme tolerance assay. RT-qPCR results showed high circIGF2BP3 expression in the pectoral and thigh muscles of Yuexi frizzled feather chickens at embryonic ages 14 and 18, as well as at 7 weeks post-hatch. Notably, its expression increased during embryonic development, followed by a rapid decrease after birth. As well as using RT-qPCR, Edu, CCK-8, immunofluorescence, and Western blot techniques, we demonstrated that overexpressing circIGF2BP3 could promote the proliferation and differentiation of chicken primary myoblasts through upregulating genes such as proliferating cell nuclear antigen (PCNA), cyclin D1 (CCND1), cyclin E1 (CCNE1), cyclin dependent kinase 2 (CDK2), myosin heavy chain (MyHC), myoblast-determining 1 (MyoD1), myogenin (MyoG), and Myomaker. In conclusion, circIGF2BP3 promotes the proliferation and differentiation of myoblasts in chickens. This study establishes a foundation for further investigation into the biological functions and mechanisms of circIGF2BP3 in myoblasts proliferation and differentiation.

Combined Inhibition of UBE2C and PLK1 Reduce Cell Proliferation and Arrest Cell Cycle by Affecting ACLY in Pan-Cancer.

Various studies have shown that the cell-cycle-related regulatory proteins UBE2C, PLK1, and BIRC5 promote cell proliferation and migration in different types of cancer. However, there is a lack of in-depth and systematic research on the mechanism of these three as therapeutic targets. In this study, we found a positive correlation between the expression of UBE2C and PLK1/BIRC5 in the Cancer Genome Atlas (TCGA) database, revealing a potential combination therapy candidate for pan-cancer. Quantitative real-time PCR (qRT-PCR), Western blotting (WB), cell phenotype detection, and RNA-seq techniques were used to evidence the effectiveness of the combination candidate. We found that combined interference of UBE2C with PLK1 and UBE2C with BIRC5 affected metabolic pathways by significantly downregulating the mRNA expression of IDH1 and ACLY, which was related to the synthesis of acetyl-CoA. By combining the PLK1 inhibitor volasertib and the ACLY inhibitor bempedoic acid, it showed a higher synergistic inhibition of cell viability and higher synergy scores in seven cell lines, compared with those of other combination treatments. Our study reveals the potential mechanisms through which cell-cycle-related genes regulate metabolism and proposes a potential combined targeted therapy for patients with higher PLK1 and ACLY expression in pan-cancer.

The Mouse CircGHR Regulates Proliferation, Differentiation and Apoptosis of Hepatocytes and Myoblasts.

The anterior pituitary gland of animals secretes growth hormone (GH) to bind to the growth hormone receptor (GHR) on the liver cell membrane through the blood circulation, thereby promoting the downstream gene insulin-like growth factor-1 (IGF1) expression, which is the canonical GH-GHR-IGF1 signaling pathway. Therefore, the amount of GHR and the integrity of its structure will affect animal growth and development. In the previous study, we found that the mouse GHR gene can transcribe a circular transcript named circGHR. Our group cloned the full-length of the mouse circGHR and analyzed its spatiotemporal expression profile. In this study, we further predicted the open reading frame of circGHR with bioinformatics, subsequently constructed a Flag-tagged protein vector and preliminarily verified its coding potential with western blot. Additionally, we found that circGHR could inhibit the proliferation of NCTC469 cells and has a tendency to inhibit cell apoptosis, while for C2C12 cells, it showed a tendency to inhibit cell proliferation and promote its differentiation. Overall, these results suggested that the mouse circGHR had the potential to encode proteins and affect cell proliferation, differentiation and apoptosis.

Investigating the heat tolerance and production performance in local chicken breed having normal and dwarf size.

Heat stress significantly impairs the growth performance of broilers, which causes serious losses to the poultry industry every year. Thus, understanding the performance of indigenous chicken breeds under such environment is crucial to address heat stress problem. The purpose of this study was to investigate the effects of heat stress (HS) on production performance, tissue histology, heat shock response (HSP70, HSP90), and muscle growth-related genes (GHR, IGF-1, and IGF-1R) of Normal yellow chicken (NYC) and Dwarf yellow chicken (DYC). Seventy-two female birds from each strain were raised under normal environmental conditions up to 84 days, with birds from each strain being divided into two groups (HS and control). In the HS group, birds were subjected to high temperature at 35 ± 1 °C for 8 h daily and lasted for a week, while in the control group, birds were raised at 28 ± 1 °C. At 91 days old, bird's liver, hypothalamus, and breast muscle tissues were collected to evaluate the gene expression, histological changes, and the production performance. The Feed intake, weight gain ratio, total protein intake and protein efficiency ratio showed a significant reduction in the treatments (P < 0.01) and treatment × strain interaction (P < 0.05) with breast muscle rate significantly reducing among the treatments (P < 0.01) after 7 days of HS. Correspondingly, total abdominal fat showed significant change among treatment and strain (P < 0.01, P < 0.05), respectively. Besides, HS markedly upregulated the mRNA expression of HSP70 and HSP90 in the pectoralis major of both chicken strains, but no significant increase (P < 0.05) was found in mRNA expression of HSP90 in liver and hypothalamus tissues of both chicken strains. Moreover, HS significantly upregulated (P < 0.05) the expression of lipogenic genes (FASN, ACC) in liver tissues of NYC, while mRNA expression of these genes showed no variation in DYC. Similarly, HS downregulated the mRNA expression of muscle growth-related genes (GHR, IGF-1, and IGF-1R). Consequently, the histopathological analysis showed that histological changes were accompanied by inflammatory cell infiltration in liver tissues of both chicken strains; however, histopathological changes were more severe in NYC than dwarf chicken strain. Conclusively, this study depicted that the production performance and growth rate varied significantly between treatment and control group of NYC. However, heat treatment in DYC has not shown significant damaging consequences as compared to the control group that signifies the vital role of the dwarf trait in thermal tolerance.

Mining of chicken muscle growth genes and the function of important candidate gene RPL3L in muscle development.

The birth weight of chickens does not significantly affect the weight at slaughter, while the different growth rate after birth was one of the important reasons for the difference in slaughter weight. Also, the increase in chickens' postnatal skeletal muscle weight is the main cause of the slaughter weight gain, but which genes are involved in this biological process is still unclear. In this study, by integrating four transcriptome datasets containing chicken muscles at different developmental times or different chicken tissues in public databases, a total of nine candidate genes that may be related to postnatal muscle development in chickens were obtained, including RPL3L, FBP2, ASB4, ASB15, CKMT2, PGAM1, YIPF7, PFKM, and LDHA. One of these candidate genes is RPL3L, whose 42 bp insertion/deletion (indel) mutation significantly correlated with multiple carcass traits in the F2 resource population from Xinghua chickens crossing with White Recessive Rock (WRR) chickens, including live weight, carcass weight, half eviscerated weight, eviscerated weight, breast meat weight, wing weight, leg muscle shear force, and breast muscle shear force. Also, there was a very significant difference between different genotypes of the RPL3L 42 bp indel mutation in these trains. Further experiments showed that RPL3L was highly expressed in chicken skeletal muscle, and its overexpression could promote the proliferation and inhibit the differentiation of chicken myoblasts by regulating ASB4 and ASB15 expression. Our findings demonstrated that the RPL3L 42 bp indel may be one of the molecular markers of chicken weight-related traits.

KAT2A/E2F1 Promotes Cell Proliferation and Migration via Upregulating the Expression of UBE2C in Pan-Cancer.

Various studies have shown that lysine acetyltransferase 2A (KAT2A), E2F transcription factor 1 (E2F1), and ubiquitin conjugating enzyme E2 C (UBE2C) genes regulated the proliferation and migration of tumor cells through regulating the cell cycle. However, there is a lack of in-depth and systematic research on their mechanisms of action. This study analyzed The Cancer Genome Atlas (TCGA) to screen potential candidate genes and the regulation network of KAT2A and E2F1 complex in pan-cancer. Quantitative real-time PCR (qRT-PCR) and Western blotting (WB), cell phenotype detection, immunofluorescence co-localization, chromatin immunoprecipitation assay (ChIP), and RNA-Seq techniques were used to explore the functional of a candidate gene, UBE2C. We found that the expression of these three genes was significantly higher in more than 10 tumor types compared to normal tissue. Moreover, UBE2C was mainly expressed in tumor cells, which highlighted the impacts of UBE2C as a specific therapeutic strategy. Moreover, KAT2A and E2F1 could promote cell proliferation and the migration of cancer cells by enhancing the expression of UBE2C. Mechanically, KAT2A was found to cooperate with E2F1 and be recruited by E2F1 to the UBE2C promoter for elevating the expression of UBE2C by increasing the acetylation level of H3K9.

Sox6 Differentially Regulates Inherited Myogenic Abilities and Muscle Fiber Types of Satellite Cells Derived from Fast- and Slow-Type Muscles.

Adult skeletal muscle is primarily divided into fast and slow-type muscles, which have distinct capacities for regeneration, metabolism and contractibility. Satellite cells plays an important role in adult skeletal muscle. However, the underlying mechanisms of satellite cell myogenesis are poorly understood. We previously found that Sox6 was highly expressed in adult fast-type muscle. Therefore, we aimed to validate the satellite cell myogenesis from different muscle fiber types and investigate the regulation of Sox6 on satellite cell myogenesis. First, we isolated satellite cells from fast- and slow-type muscles individually. We found that satellite cells derived from different muscle fiber types generated myotubes similar to their origin types. Further, we observed that cells derived from fast muscles had a higher efficiency to proliferate but lower potential to self-renew compared to the cells derived from slow muscles. Then we demonstrated that Sox6 facilitated the development of satellite cells-derived myotubes toward their inherent muscle fiber types. We revealed that higher expression of Nfix during the differentiation of fast-type muscle-derived myogenic cells inhibited the transcription of slow-type isoforms (MyH7B, Tnnc1) by binding to Sox6. On the other hand, Sox6 activated Mef2C to promote the slow fiber formation in slow-type muscle-derived myogenic cells with Nfix low expression, showing a different effect of Sox6 on the regulation of satellite cell development. Our findings demonstrated that satellite cells, the myogenic progenitor cells, tend to develop towards the fiber type similar to where they originated. The expression of Sox6 and Nfix partially explain the developmental differences of myogenic cells derived from fast- and slow-type muscles.

Establishment methods and research progress of livestock and poultry immortalized cell lines: A review.

An infinite cell line is one of the most favored experimental tools and plays an irreplaceable role in cell-based biological research. Primary cells from normal animal tissues undergo a limited number of divisions and subcultures in vitro before they enter senescence and die. On the contrary, an infinite cell line is a population of non-senescent cells that could proliferate indefinitely in vitro under the stimulation of external factors such as physicochemical stimulation, virus infection, or transfer of immortality genes. Cell immortalization is the basis for establishing an infinite cell line, and previous studies have found that methods to obtain immortalized cells mainly included physical and chemical stimulations, heterologous expression of viral oncogenes, increased telomerase activity, and spontaneous formation. However, some immortalized cells do not necessarily proliferate permanently even though they can extend their lifespan compared with primary cells. An infinite cell line not only avoids the complicated process of collecting primary cell, it also provides a convenient and reliable tool for studying scientific problems in biology. At present, how to establish a stable infinite cell line to maximize the proliferation of cells while maintaining the normal function of cells is a hot issue in the biological community. This review briefly introduces the methods of cell immortalization, discusses the related progress of establishing immortalized cell lines in livestock and poultry, and compares the characteristics of several methods, hoping to provide some ideas for generating new immortalized cell lines.

PRR11 promotes cell proliferation by regulating PTTG1 through interacting with E2F1 transcription factor in pan-cancer.

The upregulated proline rich 11 (PRR11) plays a critical role in cancer progression. The relevant biological functions of PRR11 in pan-cancer development are not well understood. In the current study, we found that PRR11 was upregulated in 19 cancer types compared with that of normal tissues and high-expressed PRR11 was a predictor of poor prognosis in 10 cancer types by bioinformatics. Then we showed that interfering PRR11 on three cancer cell lines could greatly inhibit cell proliferation and migration and arrest cells to S phase in vivo. Based on RNA-seq, downregulation of PRR11 expression could extremely suppress the expression of PTTG1 and the cell cycle pathway identified by a differentially expressed gene analysis and an enrichment analysis. The expression of PRR11 and PTTG1 was positively correlated in TCGA and independent GEO data sets. Importantly, we revealed that the PRR11 could express itself in the nucleus and interact with E2F1 on the PTTG1 promoter region to increase the expression of PTTG1. Further results indicated that the expression of PTTG1 was also associated with poor prognosis in 10 cancer types, while downregulation of PTTG1 expression could inhibit cancer cell proliferation and migration. Therefore, we found that PRR11 served as an oncogene in pan-cancer and could influence the cell cycle progression through regulating the expression of PTTG1 by interacting with the transcription factor E2F1.

Identification and Validation of Serum CST1 as a Diagnostic Marker for Differentiating Early-Stage Non-Small Cell Lung Cancer from Pulmonary Benign Nodules.

BACKGROUND: Effective means for early diagnosis are imperative to reduce death rate of non-small cell lung cancer (NSCLC) patients. We aimed to find out high-performance serologic markers to distinguish early-stage NSCLC patients from benign pulmonary nodule patients and healthy controls (HC). Cystatin-SN (CST1) is an active cysteine protease inhibitor of the CST superfamily, involving in the processes of inflammation and tumorigenesis. This is the first exploration of the diagnostic and prognostic values of serum CST1 in NSCLC. METHODS: We analyzed the transcriptome data from The Cancer Genome Atlas and the Gene Expression Omnibus database, screened biomarkers for NSCLC, and verified the candidate markers via the ONCOMINE database. Then, we performed ELISA, western blotting, and immunohistochemistry analysis to detect the expression levels of CST1 in NSCLC cell lines, tumor tissues, and serum samples of clinical cohorts. RESULTS: We identified 3 up-regulated secreted protein-encoding genes, validated the expression levels of CST1 in NSCLC tumor tissues and cell lines, and found that serum CST1 levels of NSCLC (4289 ± 2405 pg/mL) were significantly higher than those of PBN patients (1558 ± 441 pg/mL, P < .0001) and healthy controls (1529 ± 416 pg/mL, P < .0001). The AUC of the combination of CST1, Cytokeratin 19 fragment (Cyfra21-1), and Carcinoembryonic antigen (CEA) for distinguishing early-stage NSCLC from PBN/HC was as high as .914/0.925. Furthermore, our results suggested that the NSCLC patient with low serum CST1 level had a better survival rate. CONCLUSIONS: Serum CST1 may serve as a novel diagnostic marker for differentiating early-stage NSCLC from PBN and HC, and could be used as a prognosis predictor in NSCLC patients.

Multi-Omics Perspective Reveals the Different Patterns of Tumor Immune Microenvironment Based on Programmed Death Ligand 1 (PD-L1) Expression and Predictor of Responses to Immune Checkpoint Blockade across Pan-Cancer.

Immune checkpoint inhibitor (ICI) therapies have shown great promise in cancer treatment. However, the intra-heterogeneity is a major barrier to reasonably classifying the potential benefited patients. Comprehensive heterogeneity analysis is needed to solve these clinical issues. In this study, the samples from pan-cancer and independent breast cancer datasets were divided into four tumor immune microenvironment (TIME) subtypes based on tumor programmed death ligand 1 (PD-L1) expression level and tumor-infiltrating lymphocyte (TIL) state. As the combination of the TIL Z score and PD-L1 expression showed superior prediction of response to ICI in multiple data sets compared to other methods, we used the TIL Z score and PD-L1 to classify samples. Therefore, samples were divided by combined TIL Z score and PD-L1 to identify four TIME subtypes, including type I (3.24%), type II (43.24%), type III (6.76%), and type IV (46.76%). Type I was associated with favorable prognosis with more T and DC cells, while type III had the poorest condition and composed a higher level of activated mast cells. Furthermore, TIME subtypes exhibited a distinct genetic and transcriptional feature: type III was observed to have the highest mutation rate (77.92%), while co-mutations patterns were characteristic in type I, and the PD-L1 positive subgroup showed higher carbohydrates, lipids, and xenobiotics metabolism compared to others. Overall, we developed a robust method to classify TIME and analyze the divergence of prognosis, immune cell composition, genomics, and transcriptomics patterns among TIME subtypes, which potentially provides insight for classification of TIME and a referrable theoretical basis for the screening benefited groups in the ICI immunotherapy.

AutoVEM: An automated tool to real-time monitor epidemic trends and key mutations in SARS-CoV-2 evolution.

With the global epidemic of SARS-CoV-2, it is important to effectively monitor the variation, haplotype subgroup epidemic trends and key mutations of SARS-CoV-2 over time. This is of great significance to the development of new vaccines, the update of therapeutic drugs, and the improvement of detection methods. The AutoVEM tool developed in the present study could complete all mutations detections, haplotypes classification, haplotype subgroup epidemic trends and candidate key mutations analysis for 131,576 SARS-CoV-2 genome sequences in 18 h on a 1 core CPU and 2 GB RAM computer. Through haplotype subgroup epidemic trends analysis of 131,576 genome sequences, the great significance of the previous 4 specific sites (C241T, C3037T, C14408T and A23403G) was further revealed, and 6 new mutation sites of highly linked (T445C, C6286T, C22227T, G25563T, C26801G and G29645T) were discovered for the first time that might be related to the infectivity, pathogenicity or host adaptability of SARS-CoV-2. In brief, we proposed an integrative method and developed an efficient automated tool to monitor haplotype subgroup epidemic trends and screen for the candidate key mutations in the evolution of SARS-CoV-2 over time for the first time, and all data could be updated quickly to track the prevalence of previous key mutations and new candidate key mutations because of high efficiency of the tool. In addition, the idea of combinatorial analysis in the present study can also provide a reference for the mutation monitoring of other viruses.

Effects of Circular RNA of Chicken Growth Hormone Receptor Gene on Cell Proliferation.

Animal growth and development are regulated by neural and endocrine growth axes, in which cell proliferation plays key roles. Recently, many research showed that circular RNAs were involved in hepatocyte and myoblast proliferation. Previously, we identified a circular RNA derived from the chicken GHR gene, named circGHR. However, the function of circGHR is unclear. The objective of this study was to investigate circGHR expression pattern and its roles in cell proliferation. Results indicated that circGHR was a closed-loop structure molecule, and it was richer in the nucleus of hepatocytes and myoblast. Real-time PCR showed that circGHR was increased from E13 to the 7th week in the liver but decreased in the thigh and breast muscle. The CCK-8 assay displayed that circGHR promoted cell proliferation. Simultaneously, the biomarker genes PCNA, CCND1, and CDK2 and the linear transcripts GHR and GHBP were upregulated when circGHR was overexpressed. Altogether, these data exhibited that circGHR could promote cell proliferation possibly by regulating GHR mRNA and GHBP expression.

Polymorphisms of AMY1A gene and their association with growth, carcass traits and feed intake efficiency in chickens.

Investigations on the association between chicken traits and genetic variations can provide basic information to improve production performance in chickens. In our previous work, we genotyped 450 male chickens with a 600 K SNP array [1] and found that several SNPs in the genomic regions of the amylase alpha 1A (AMY1A) gene were significantly associated with feed intake efficiency and carcass traits. Given the lower accuracy of the SNP array, we performed direct sequencing with male and female chickens to further test chicken AMY1A polymorphisms and investigate their association with 17 traits in chickens. The results showed that 7 SNPs in the 5' flanking region, exon, intron and 3' UTR (3' untranslated region) of AMY1A, were significantly associated with daily gain (DG), average daily feed intake (ADFI), leg muscle weight (LMW) and abdominal fat (AF) (p < 0.05). Additionally, the haplotypes based on three SNPs, rs15910189, rs314354067 and rs316026696, showed significant associations with DG (p < 0.01), ADFI and AF (p < 0.05). To better understand the transcriptional regulation of AMY1A, we cloned its 5' flanking region and found that the SNPs rs316436216 and rs314213090 which might change the transcriptional regulator binding sites, were in the suppressor and enhancer regions, respectively. In addition, luciferase assays revealed that the SNP rs314613110 in the 3' UTR influenced the binding of the miRNA gga-miR-1764-3p. To validate whether there is any copy number variation in AMY1A in our population, we performed a genome-wide assessment of CNVs through whole-genome resequencing data. However, no CNV was found in AMY1A in our population, which is different from the increased copy number of AMY1A found in humans who consume a high-starch diet. Therefore, the present study provides substantial evidence for the association of AMY1A polymorphisms with growth traits and feed intake efficiency, which might contribute to chicken breeding programs.

Comprehensive evolution and molecular characteristics of a large number of SARS-CoV-2 genomes reveal its epidemic trends.

OBJECTIVES: To further reveal the phylogenetic evolution and molecular characteristics of the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on a large number of genomes and provide a basis for the prevention and treatment of SARS-CoV-2. METHODS: Various evolution analysis methods were employed. RESULTS: The estimated ratio of the rates of non-synonymous to synonymous changes (Ka/Ks) of SARS-CoV-2 was 1.008 or 1.094 based on 622 or 3624 SARS-CoV-2 genomes and nine key specific sites of high linkage, and four major haplotypes were found: H1, H2, H3 and H4. The results of Ka/Ks, detected population size and development trends of each major haplotype showed that H3 and H4 subgroups were going through a purify evolution and almost disappeared after detection, indicating that they might have existed for a long time. The H1 and H2 subgroups were going through a near neutral or neutral evolution and globally increased with time, and the frequency of H1 was generally high in Europe and correlated with the death rate (r >0.37), suggesting that these two haplotypes might relate to the infectivity or pathogenicity of SARS-CoV-2. CONCLUSIONS: Several key specific sites and haplotypes related to the infectivity or pathogenicity of SARS-CoV-2, and the possible earlier origin time and place of SARS-CoV-2 were indicated based on the evolution and epidemiology of 16,373 SARS-CoV-2 genomes.