Progress on CRISPR/Cas9 system in the genetic improvement of livestock and poultry.

CRISPR/Cas9 gene editing technology, as a highly efficient genome editing method, has been extensively employed in the realm of animal husbandry for genetic improvement. With its remarkable efficiency and precision, this technology has revolutionized the field of animal husbandry. Currently, CRISPR/Cas9-based gene knockout, gene knock-in and gene modification techniques are widely employed to achieve precise enhancements in crucial production traits of livestock and poultry species. In this review, we summarize the operational principle and development history of CRISPR/Cas9 technology. Additionally, we highlight the research advancements utilizing this technology in muscle growth and development, fiber growth, milk quality composition, disease resistance breeding, and animal welfare within the livestock and poultry sectors. Our aim is to provide a more comprehensive understanding of the application of CRISPR/Cas9 technology in gene editing for livestock and poultry.

Comparative analysis of the genetic diversity of the neutral microsatellite loci and second exon of the goat MHC-DQB1 gene.

This study compared and analyzed the genetic diversity and population structure of exon 2 of the DQB1 gene and 13 autosomal neutral microsatellite markers from 14 Chinese goat breeds to explore the potential evolutionary mechanism of the major histocompatibility complex (MHC). A total of 287 haplotypes were constructed from MHC-DQB1 exon 2 from 14 populations, and 82 nucleotide polymorphic sites (SNPs, 31.78%) and 172 heterozygous individuals (79.12%) were identified. The FST values of the microsatellites and MHC-DQB ranged between 0.01831-0.26907 and 0.00892-0.38871, respectively. Furthermore, 14 goat populations showed rich genetic diversity in the microsatellite loci and MHC-DQB1 exon 2. However, the population structure and phylogenetic relationship represented by the two markers were different. Positive selection and Tajima's D test results showed the occurrence of a diversified selection mechanism, which was primarily based on a positive and balancing selection in goat DQB. This study also found that the DQB sequences of bovines exhibited trans-species polymorphism (TSP) among species and families. In brief, this study indicated that positive and balancing selection played a major role in maintaining the genetic diversity of DQB, and TSP of MHC in bovines was common, which enhanced the understanding of the MHC evolution.

Identification of differentially expressed long non-coding RNAs and messenger RNAs involved with muscle development in Dazu black goats through RNA sequencing.

This study aimed to explore the genetic basis of muscle development in goats. The transcriptome dataset for differentially expressed lncRNAs (DELs) and differentially expressed genes (DEGs) of goat muscle at different developmental stages were obtained using RNA-Seq. A total of 447,806,481 and 587,559,465 clean reads in the longissimus dorsi muscle of Dazu black goats between 75d embryonic stage and 1d after birth were generated through Illumina paired-end sequencing, and their mapping rates were 89.82 and 90.99%, respectively. Moreover, 4517 DEGs and 648 DELs were identified, and 4784 lncRNA-mRNA targeting relationships were predicted. Gene function annotation results showed that 4101 DEGs were significantly enriched to 1098 GO terms, and 2014 DEGs were significantly enriched to 40 KEGG pathways, including many GO terms and pathways related to muscle development, such as cell differentiation and Wnt signaling pathway. Then, 10 DELs and 20 DEGs were randomly selected for RT-qPCR verification, and the agreement rate between the verification and RNA-Seq results was 90%, indicating the high reliability of the RNA-Seq data analysis. In conclusion, this study obtained several mRNAs and lncRNAs related to the muscle development of Dazu black goats and identified several targeted regulatory pairs of lncRNA-mRNA. This study may serve as a reference to understand the genetic basis and molecular mechanism of muscle development in goats.

Streptococcus humanilactis sp.nov., isolated from healthy nursing mother's breast milk.

Two bacterial strains were isolated from the breast milk of two healthy nursing mothers. The isolates were Gram-positive, catalase-negative, coccus-shaped, chain-forming organisms. Analysis of the 16S rRNA gene sequences of strain IMAU99125T shared 99.7 and 99.6% similarity with Streptococcus mitis ATCC 49456 T and Streptococcus pseudopneumoniae ATCC BAA-960 T, respectively. The nearly complete 16S rRNA gene sequences of IMAU99125T and IMAU99674 strains were very closely related (with only 0.06% difference between them). Sequence analysis of the gyrB and rpoB genes also indicated that IMAU99125T was closely related to S. mitis ATCC 49456 T (94.7% and 97.1%, respectively) and S. pseudopneumoniae ATCC BAA-960 T (94.4% and 97.1%, respectively). Average nucleotide identity (ANI) values between strain IMAU99125T and S. mitis ATCC 49456 T and S. pseudopneumoniae ATCC BAA-960 T, were 93.3% and 92.7%, respectively. Genome-to-genome distance (GGD) values between strain IMAU99125T and S. mitis ATCC 99125 T and S. pseudopneumoniae ATCC BAA-960 T were 53.4% (50.7-56.0) and 50.4% (47.7-53.0), respectively. The major fatty acids of the strain were C16:0 (51.4%). On the basis of the results of phenotypic and phylogenetic analyses, we propose that the two strains be classified as representing a novel species of the genus Streptococcus, namely Streptococcus humanilactis sp.nov. The type strain is IMAU99125T (= GDMCC 1.1876 T = KCTC 21157 T). The genome of Streptococcus humanilactis sp. nov. is comprised of 2,027,143 bp. The DNA G + C content of the strain is 40.0 mol%.

Bioinformatic Prediction of Depression-Related Signaling Pathways Regulated by miR-146a in Peripheral Blood of Patients with Post-Stroke Depression.

It was aimed to explore the differential expression of miR-146a-5p in peripheral blood of patients with post-stroke depression (PSD), and to analyze its mechanism using bioinformatics. Stroke patients were selected as the research objects, and were divided into PSD ones and non-post-stroke depression (N-PSD) ones with the National Institutes of Health stroke scale (NHISS) and Hamilton Depression Scale-17 terms (HAMD-17) scores. Peripheral blood of patients was collected for serum miR-146a-5p detection. Targetscan7.1, miRDB, DIANA TOOLS, and more databases were used to predict the target genes of miR-146a-5p. String11.0 was applied to construct a protein interaction network, and GO and KEGG pathway enrichment analysis of target genes was performed. Compared with that of N-PSD patients, serum miR-146a-5p levels in PSD patients were significantly increased (P<0.05). The receiver operator characteristic (ROC) curve suggested that the sensitivity and specificity of miR-146a-5p in predicting PSD were 0.703 and 0.811, respectively. The human miR-146a-5p sequence was highly conserved, with a total of 43 target genes. It involved analysis of activity, signaling pathways, and transcriptional regulation, as well as related signaling pathways such as Toll-like receptors (TLR), neurotrophic factors, and nuclear factor kappa-B (NF-κB). In conclusion, the expression level of miR-146a-5p was abnormally increased in PSD patients, and it could be taken as a candidate marker for the diagnosis of PSD. miR-146a-5p could affect PSD through signaling pathways of TLRs, neurotrophic factors, and NF-κB.

Identification of differentially expressed microRNAs in ovulatory and subordinate follicles in Dazu black goats.

How multiple ovulations happen in prolific goats is still unknown even though studies on ovarian physiology and folliculogenesis have made extensive progress. MicroRNAs (miRNAs) are a class of endogenous regulatory factors and regulate structural gene expression mainly at the post-transcriptional level. In this study, in the follicular phase, six large follicles were collected from prolific Dazu black goat and used to generate RNA libraries for RNA sequencing. Based on the litter size and average number of ovulatory follicles in Dazu black goats, the largest three follicles were sorted as ovulatory follicles, and the remaining as subordinate ones. In total, 418 known miRNAs and 110 novel miRNAs were found, and the expression of six randomly selected miRNAs was validated by quantitative PCR analysis. Nine miRNAs were differently expressed between the ovulatory and subordinate follicles (p < 0.01). Chi-miR-582-5p, novel-130, chi-miR-214-3p, and chi-miR-500-5p were upregulated in the ovulatory group, and chi-miR-383, chi-miR-130b-5p, chi-miR-92a-3p, chi-miR-125b-5p, and novel-9 were downregulated in the same group. Chi-miR-130b-5p and chi-miR-214-3p were predicted to target at LHR (XM_013967581.1), GDF9 (NM_001285708.1), BMP15 (NM_001285588.1), and CYP19A1 (XM_013967046.1). In conclusion, nine miRNAs were differently expressed between ovulatory and subordinate follicles, and chi-miR-130b-5p and chi-miR-214-3p were predicted to regulate the expression of genes involved in gonadotropin hormone signaling and oocyte-derived growth factors. Additional studies are needed to confirm these findings.

A nonsynonymous SNP within the AMH gene is associated with litter size in Dazu black goats.

AMH, KISS1R and GDF9 genes play a vital role in human and animal reproduction and might be used as the genetic markers for the reproduction traits selection. The aim of this study was to screen the single nucleotide polymorphisms (SNPs) within the AMH, KISS1R and GDF9 genes and to determine the correlations between these SNPs and the litter size in goats. Nine single SNPs within these genes were used for genotyping of the 190 Dazu black goat populations by SNaPshot technique. The polymorphisms of nine SNPs within these genes were detected in Dazu black goats. The significant correlation was observed between one SNP (g.89172108A > C) within the AMH gene and the litter size of second born in Dazu black goats (p < 0.05). The SNP was located in exon 4 (XM_018050765.1) of the AMH gene and was one nonsynonymous substitution, which resulted in a change of an amino acid from Glutamine to Proline (Gln38Pro). These results suggested that the nonsynonymous SNP g.89172108A > C of AMH gene could be used as a potential genetic marker for Marker-assisted selection (MAS) in goats breeding programs.

Interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of CD4+ T cells.

Interleukin 7 (IL-7) and T cell antigen receptor signals have been proposed to be the main drivers of homeostatic T cell proliferation. However, it is not known why CD4(+) T cells undergo less-efficient homeostatic proliferation than CD8(+) T cells do. Here we show that systemic IL-7 concentrations increased during lymphopenia because of diminished use of IL-7 but that IL-7 signaling on IL-7 receptor-alpha-positive (IL-7Ralpha(+)) dendritic cells (DCs) in lymphopenic settings paradoxically diminished the homeostatic proliferation of CD4(+) T cells. This effect was mediated at least in part by IL-7-mediated downregulation of the expression of major histocompatibility complex class II on IL-7Ralpha(+) DCs. Our results indicate that IL-7Ralpha(+) DCs are regulators of the peripheral CD4(+) T cell niche and that IL-7 signals in DCs prevent uncontrolled CD4(+) T cell population expansion in vivo.

SNP screening of the MSTN gene and correlation analysis between genetic polymorphisms and growth traits in Dazu black goat.

The aim of this study was to detect SNPs in myostatin (MSTN) gene of four goat breeds, and analyze the correlation of these markers on body measurement traits in the Dazu black goat breed. In total, twenty polymorphic sites were found in one hundred forty-eight individuals, and all SNPs were distributed in introns 1 and 2, except g. 425 C > T, which was found in the regulatory region. Three SNPs (g. 2732 C > T, g. 2752 G > A and g. 4552 A > C) were polymorphic in all four breeds. None of the tag SNPs (g. 425 C > T, g. 1583 A > G, 2732 C > T, g. 4552 A > C and g. 5167 C > T) were significantly correlated with body measurement traits (p > 0.05) in the Dazu black goat. However, individuals with genotype combination 3 (GtC 3) of tag SNPs had higher birth weight and weaning weight than individuals with the other genotype combinations (p < 0.05). Moreover, the genotype combination 4 (GtC 4) was significantly associated with body length and height at the age of 2 months (p < 0.05), and genotype combination 13 (GtC 13) was significantly correlated with body height at 6 months (p < 0.05). Briefly, the combined tag SNP markers might be useful for goat marker-associated selective breeding.

Immunization with oral KISS1 DNA vaccine inhibits testicular Leydig cell proliferation mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes in goats.

This study aimed to analyze the effect and mechanism of immunization of oral KISS1 DNA vaccine on the proliferation of goat testicular Leydig cells. Ten 8-week-old male goats were randomly divided into KISS1 DNA vaccine and control groups for immunization (five goats each group). These goats were sacrificed at 8 weeks after primary immunization, and the tissue samples of hypothalamus, pituitary, and testis and Leydig cell samples were collected for RT-PCR and CCK8 assay. Immunization with the oral KISS1 DNA vaccine effectively inhibited the proliferation of Leydig cells, the expression of hypothalamus KISS1, GPR54, and GnRH mRNA, pituitary GnRHR and LH mRNA, testicular LHR mRNA, and apoptosis-inhibitory gene Bcl-2 mRNA in Leydig cells. By contrast, the immunization enhanced the mRNA expression of apoptosis-promoting gene Bax and Clusterin in Leydig cells. These findings indicate that immunization with the oral KISS1 DNA vaccine can inhibit the proliferation of goat testicular Leydig cells mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes.

Comparative and selection sweep analysis of CNV was associated to litter size in Dazu black goats.

This study aims to identify the relative Copy number variation (CNV) associated with the litter size of Dazu black goats based on the unpublished CNV analytical results of our previously published sequencing data, in which the litter-size groups were classified into extreme low- and high-yield groups. Firstly, to compare the existence of valuable CNV in Dazu black goats with different fertility levels with mixed pools. We obtained 4992 and 4888 CNVs from the HY and LY, which overlapping 1461 genes, and classified on the original CNV type. Three genes [LOC108633278, PPP1R12A, and YIPF4] were observed in the intersection between the HY deletion and the LY duplication groups. Secondly, on individuals level, we identified a novel candidate CNV (Chr1_50215501, FST = 0.148, VST = 0.347) from 214 autosomal credible CNVs to be significant with litter size in the Dazu black goat, which located in the CBLB gene. This finding indicates the CBLB gene may affect the litter size of the Dazu black goats through structural variations, and Chr1_50215501 can be an effective genetic marker for marker-assisted selection breeding, and this study was also helps understand the molecular mechanism related to the goat litter size.

Three SNPs within exons of INHA and ACVR2B genes are significantly associated with litter size in Dazu black goats.

The aim of this study was to analyse the association between single-nucleotide polymorphisms within INHA and ACVR2B and litter size in Dazu black goats. In total, twenty-two SNPs were genotyped in 190 individuals by SNaPshot and resequencing. The results showed that three SNPs (SNP_1, SNP_12 and SNP_13 in this study) were detected to have significant additive genetic effect on the recorded goat litter size (p < .05). The SNP_1 (NC_030809.1), a non-synonymous substitution of G for T at chr2-g. 28314990 in the exon 2 of INHA gene (NM_001285606.1), resulted in homozygote 2 (HOM2) contributed 0.25 and heterozygote (HET) contributed 0.12 larger litter than homozygote 1 (HOM1). Meanwhile, SNP_12 (Chr22-g. 11721225 A > T) and SNP_13 (Chr22-g. 11721227 A > C) (NC_030829.1) simultaneously mutated at the first and third position of a triplet AAA (lysine, K) in the exon 4 of ACVR2B gene (XM_018066623.1) had estimated genetic effects of HOM1 (0.00) and HOM2 (0.03) larger than HET (-0.12). In conclusion, one SNPs (chr2-g. 28314990 T > G) within the exon 2 of INHA and two SNPs (Chr22-g. 11721225 A > T and Chr22-g. 11721227 A > C) in the exon 4 of ACVR2B gene were highly recommended as candidate markers of litter size in Dazu black goats. A large-scale association study to assess the impact of these variants on litter size is still necessary.

Relevance research between the expression of p16INK4a , Notch1, and hTERC genes: The development of HPV16-positive cervical cancer.

BACKGROUND: GLOBOCAN 2018 latest data show cervical cancer ranks fourth in morbidity and mortality among women. Many genes in cervical lesions differ in sensitivity and specificity. However, the diagnostic molecules for early cervical cancer are not very clear. This paper screens biomarkers for early molecular diagnosis of Mongolian patients with cervical cancer. METHODS: Immunohistochemical SP method was used to detect the expression of p16INK4a and Notch1 protein in paraffin sections of 226 Mongolian patients with HPV16-positive cervical lesions after pathological examination, and 100 of them were randomly selected by fluorescence in situ hybridization to detect hTERC gene. The HPV16-binding human cervical cancer SiHa cell line was used to silence the expression of HPV16 E6/E7 gene by RNA interference, and the expression of p16INK4a , Notch1, and hTERC genes and protein expression levels were detected by RT-PCR and Western blot. RESULTS: The positive expression rates of p16INK4a , Notch1, and hTERC genes in HPV16-positive cervical cancer, CIN-III, CIN-II, CIN-I, uterine leiomyoma, and chronic cervicitis were significantly different (P < .05); the positive expression rates of the three genes were also significantly different in the same type of cervical lesions (P < .05); RNA interference can effectively inhibit HPV16 E6/E7, p16INK4a and Notch1 gene expression, but has no effect on hTERC gene expression. CONCLUSION: The p16INK4a gene can be used as a biomarker for early screening of cervical cancer, and the hTERC gene can be used to confirm the clinical diagnosis of cervical cancer.

Metabolic profile of Rhizoma coptidis in human plasma determined using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

RATIONALE: Rhizoma coptidis extract and its alkaloids show various pharmacological activities, but its metabolic profile in human plasma has not been thoroughly investigated. In the present research, the metabolism of Rhizoma coptidis at a clinical dose (5 g/60 kg/day) was systematically analyzed to determine its biotransformation processes in human plasma. METHODS: In this research, the metabolites of Rhizoma coptidis in human plasma after oral administration of Rhizoma coptidis extract at a clinical dose were investigated using ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution LTQ-Orbitrap mass spectrometry. The structural elucidation of the constituents was confirmed by comparing their retention times (tR ) and MSn fragments with those of standards and literature reports. RESULTS: In total, two prototypes and twelve metabolites were detected in human plasma. The two prototypes were confidently identified using reference standards. Of the compounds detected, M7 (berberrubinen-9-O-glucuronide) was the most abundant based on its peak area, which indicates that this compound might be a pharmacokinetic marker for Rhizoma coptidis alkaloids in humans. Based on the metabolites detected in human plasma, a possible metabolic pathway for Rhizoma coptidis in vivo was proposed. CONCLUSIONS: The results indicated that the alkaloids in Rhizoma coptidis were extensively biotransformed in vivo mainly via conjugation with glucuronic acid (GluA) or sulfuric acid (SulA) to form phase II metabolites, and the GluA metabolites are likely the dominant form in human plasma. To the best of our knowledge, this is the first in vivo evaluation of the metabolic profile of the whole Rhizoma coptidis extract in human plasma, which is essential for determining the chemicals responsible for the pharmacological activities of Rhizoma coptidis in vivo. Moreover, it would be beneficial for us to further systematically study the pharmacokinetic behavior of Rhizoma coptidis in humans.

Himawari-8 Satellite Based Dynamic Monitoring of Grassland Fire in China-Mongolia Border Regions.

In this study, we used bands 7, 4, and 3 of the Advance Himawari Imager (AHI) data, combined with a Threshold Algorithm and a visual interpretation method to monitor the entire process of grassland fires that occurred on the China-Mongolia border regions, between 05:40 (UTC) on April 19th to 13:50 (UTC) on April 21st 2016. The results of the AHI data monitoring are evaluated by the fire point product data, the wind field data, and the environmental information data of the area in which the fire took place. The monitoring result shows that, the grassland fire burned for two days and eight hours with a total burned area of about 2708.29 km². It mainly spread from the northwest to the southeast, with a maximum burning speed of 20.9 m/s, a minimum speed of 2.52 m/s, and an average speed of about 12.07 m/s. Thus, using AHI data can not only quickly and accurately track the dynamic development of a grassland fire, but also estimate the spread speed and direction. The evaluation of fire monitoring results reveals that AHI data with high precision and timeliness can be highly consistent with the actual situation.

Development of mammary luminal progenitor cells is controlled by the transcription factor STAT5A.

Mammary alveologenesis is abrogated in the absence of the transcription factors STAT5A/5B, which mediate cytokine signaling. To reveal the underlying causes for this developmental block, we studied mammary stem and progenitor cells. While loss of STAT5A/5B did not affect the stem cell population and its ability to form mammary ducts, luminal progenitors were greatly reduced and unable to form alveoli during pregnancy. Temporally controlled expression of transgenic STAT5A in mammary epithelium lacking STAT5A/5B restored the luminal progenitor population and rescued alveologenesis in a reversible fashion in vivo. Thus, STAT5A is necessary and sufficient for the establishment of luminal progenitor cells.

Massively Parallel CRISPR-Cas9 Knockout Screening in Sheep Granulosa Cells for FSH Response Genes.

Follicle-stimulating hormone (FSH) regulates ovarian follicle development through specific gene expression programs. Granulosa cells (GCs) are somatic cells surrounding the oocytes, secreting gonadotropins to regulate ovulation and promote follicular development. By analyzing the effects of different doses of FSH on the proliferation of GCs, we found that adding 10 ng/mL of FSH, as the optimal concentration, could promote the growth of GCs. Furthermore, we have successfully constructed the first CRISPR-Cas9 knockout library targeting the genes on chromosomes 2 and 3 and the X chromosomes of the sheep massively parallel coding gene, as well as an ovarian GCs knockout cell library. For the first time, we have exposed the knockout cell library to a concentration of 10 ng/mL FSH to explore the underlying mechanisms. Through this screening, we have identified 836 positive-negative screening genes that are responsive to FSH, thereby revealing the regulatory mechanisms and screening the functionality of candidate genes. Next, RNA-Seq of control (0 ng/mL), low (10 ng/mL), and high (100 ng/mL) doses of FSH revealed 1708 differentially expressed genes, and combined with 836 genes, we obtained 129 FSH dose-dependent genes with extremely significant differences. This enables us to delve deeper into investigating and identifying the mechanisms by which FSH regulates GCs. More generally, we have discovered new regulatory factors and identified reproductivity-associated major effectors. These findings provide novel research directions for further studies on sheep reproduction.

A novel "five-in-one" comprehensive medical care framework for rehabilitation and nursing.

With the evolution of society, the world has entered a moderate stage of aging. Not surprisingly, the aging problem in the world is getting more intense, resulting in the increasing demand for higher-quality and well-organized medical and elderly care services. To cope with that, many researchers have dedicated themselves to advancing the medical care system based on data or platforms. However, they have ignored the life cycle, health service and management and the inevitable shift of living scenarios for the elderly. Therefore, the study aims to improve health conditions and enhance senior citizens' life quality and happiness index. In this paper, we build a unified body for people in their old age, bridging the disconnection between medical care and elderly care and constructing the "five-in-one" comprehensive medical care framework. It should be mentioned that the system takes the human life cycle as its axis, relies on the supply side and supply chain management, integrates medicine, industry, literature and science as methods, and takes health service management as a requirement. Furthermore, a case study on upper limb rehabilitation is elaborated along the "five-in-one" comprehensive medical care framework to confirm the effectiveness of the novel system.

Recent advancements in digital health management using multi-modal signal monitoring.

Healthcare is the method of keeping or enhancing physical and mental well-being with its aid of illness and injury prevention, diagnosis, and treatment. The majority of conventional healthcare practices involve manual management and upkeep of client demographic information, case histories, diagnoses, medications, invoicing, and drug stock upkeep, which can result in human errors that have an impact on clients. By linking all the essential parameter monitoring equipment through a network with a decision-support system, digital health management based on Internet of Things (IoT) eliminates human errors and aids the doctor in making more accurate and timely diagnoses. The term "Internet of Medical Things" (IoMT) refers to medical devices that have the ability to communicate data over a network without requiring human-to-human or human-to-computer interaction. Meanwhile, more effective monitoring gadgets have been made due to the technology advancements, and these devices can typically record a few physiological signals simultaneously, including the electrocardiogram (ECG) signal, the electroglottography (EGG) signal, the electroencephalogram (EEG) signal, and the electrooculogram (EOG) signal. Yet, there has not been much research on the connection between digital health management and multi-modal signal monitoring. To bridge the gap, this article reviews the latest advancements in digital health management using multi-modal signal monitoring. Specifically, three digital health processes, namely, lower-limb data collection, statistical analysis of lower-limb data, and lower-limb rehabilitation via digital health management, are covered in this article, with the aim to fully review the current application of digital health technology in lower-limb symptom recovery.