Whole-Genome Resequencing of Worldwide Wild and Domestic Sheep Elucidates Genetic Diversity, Introgression, and Agronomically Important Loci.

Domestic sheep and their wild relatives harbor substantial genetic variants that can form the backbone of molecular breeding, but their genome landscapes remain understudied. Here, we present a comprehensive genome resource for wild ovine species, landraces and improved breeds of domestic sheep, comprising high-coverage (∼16.10×) whole genomes of 810 samples from 7 wild species and 158 diverse domestic populations. We detected, in total, ∼121.2 million single nucleotide polymorphisms, ∼61 million of which are novel. Some display significant (P < 0.001) differences in frequency between wild and domestic species, or are private to continent-wide or individual sheep populations. Retained or introgressed wild gene variants in domestic populations have contributed to local adaptation, such as the variation in the HBB associated with plateau adaptation. We identified novel and previously reported targets of selection on morphological and agronomic traits such as stature, horn, tail configuration, and wool fineness. We explored the genetic basis of wool fineness and unveiled a novel mutation (chr25: T7,068,586C) in the 3'-UTR of IRF2BP2 as plausible causal variant for fleece fiber diameter. We reconstructed prehistorical migrations from the Near Eastern domestication center to South-and-Southeast Asia and found two main waves of migrations across the Eurasian Steppe and the Iranian Plateau in the Early and Late Bronze Ages. Our findings refine our understanding of genome variation as shaped by continental migrations, introgression, adaptation, and selection of sheep.

Historical Introgression from Wild Relatives Enhanced Climatic Adaptation and Resistance to Pneumonia in Sheep.

How animals, particularly livestock, adapt to various climates and environments over short evolutionary time is of fundamental biological interest. Further, understanding the genetic mechanisms of adaptation in indigenous livestock populations is important for designing appropriate breeding programs to cope with the impacts of changing climate. Here, we conducted a comprehensive genomic analysis of diversity, interspecies introgression, and climate-mediated selective signatures in a global sample of sheep and their wild relatives. By examining 600K and 50K genome-wide single nucleotide polymorphism data from 3,447 samples representing 111 domestic sheep populations and 403 samples from all their seven wild relatives (argali, Asiatic mouflon, European mouflon, urial, snow sheep, bighorn, and thinhorn sheep), coupled with 88 whole-genome sequences, we detected clear signals of common introgression from wild relatives into sympatric domestic populations, thereby increasing their genomic diversities. The introgressions provided beneficial genetic variants in native populations, which were significantly associated with local climatic adaptation. We observed common introgression signals of alleles in olfactory-related genes (e.g., ADCY3 and TRPV1) and the PADI gene family including in particular PADI2, which is associated with antibacterial innate immunity. Further analyses of whole-genome sequences showed that the introgressed alleles in a specific region of PADI2 (chr2: 248,302,667-248,306,614) correlate with resistance to pneumonia. We conclude that wild introgression enhanced climatic adaptation and resistance to pneumonia in sheep. This has enabled them to adapt to varying climatic and environmental conditions after domestication.

Paternal Origins and Migratory Episodes of Domestic Sheep.

The domestication and subsequent global dispersal of livestock are crucial events in human history, but the migratory episodes during the history of livestock remain poorly documented [1-3]. Here, we first developed a set of 493 novel ovine SNPs of the male-specific region of Y chromosome (MSY) by genome mapping. We then conducted a comprehensive genomic analysis of Y chromosome, mitochondrial DNA, and whole-genome sequence variations in a large number of 595 rams representing 118 domestic populations across the world. We detected four different paternal lineages of domestic sheep and resolved, at the global level, their paternal origins and differentiation. In Northern European breeds, several of which have retained primitive traits (e.g., a small body size and short or thin tails), and fat-tailed sheep, we found an overrepresentation of MSY lineages y-HC and y-HB, respectively. Using an approximate Bayesian computation approach, we reconstruct the demographic expansions associated with the segregation of primitive and fat-tailed phenotypes. These results together with archaeological evidence and historical data suggested the first expansion of early domestic hair sheep and the later expansion of fat-tailed sheep occurred ∼11,800-9,000 years BP and ∼5,300-1,700 years BP, respectively. These findings provide important insights into the history of migration and pastoralism of sheep across the Old World, which was associated with different breeding goals during the Neolithic agricultural revolution.

Whole-genome resequencing of wild and domestic sheep identifies genes associated with morphological and agronomic traits.

Understanding the genetic changes underlying phenotypic variation in sheep (Ovis aries) may facilitate our efforts towards further improvement. Here, we report the deep resequencing of 248 sheep including the wild ancestor (O. orientalis), landraces, and improved breeds. We explored the sheep variome and selection signatures. We detected genomic regions harboring genes associated with distinct morphological and agronomic traits, which may be past and potential future targets of domestication, breeding, and selection. Furthermore, we found non-synonymous mutations in a set of plausible candidate genes and significant differences in their allele frequency distributions across breeds. We identified PDGFD as a likely causal gene for fat deposition in the tails of sheep through transcriptome, RT-PCR, qPCR, and Western blot analyses. Our results provide insights into the demographic history of sheep and a valuable genomic resource for future genetic studies and improved genome-assisted breeding of sheep and other domestic animals.

Effects of berberine on the growth performance, antioxidative capacity and immune response to lipopolysaccharide challenge in broilers.

This study evaluated the effects of berberine on growth performance, immunity, haematological parameters, antioxidant capacity, and the expression of immune response-related genes in lipopolysaccharide (LPS)-challenged broilers. We assigned 120 one-day-old male broilers (Ross 308) to two treatment groups; each group included two subgroups, each of which included six replicates of five birds per replicate. The experiment used a 2 × 2 factorial arrangement with berberine treatment (0 or 60 mg/kg dietary) and challenge status [injection of saline (9 g/L w/v) or LPS (1.5 mg/kg body weight)] as the main factors. On days 14, 16, 18 and 20, broilers were intraperitoneally injected with LPS or physiological saline. Blood and liver samples were collected on day 21. Dietary berberine supplementation significantly alleviated the compromised average daily gain and average daily feed intake (p < 0.05) caused by LPS. The LPS challenge led to increased lymphocyte and white blood cell (WBC) counts, malondialdehyde (serum and liver) content, and immunoglobulin G and M, tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) expression (p < 0.05) and significantly reduced serum total superoxide dismutase (T-SOD) activity (p < 0.05). Dietary berberine significantly mitigated the LPS-induced decreases in the mRNA expression of nuclear factor-kappa B (NF-κB), TNF-α, IL-1ß, inducible nitrite synthase and cyclooxygenase-2 (p < 0.05) in the liver. In conclusion, berberine supplementation has a positive effect on LPS challenge, which may be related to the increase in antioxidant enzyme activity and inhibition of both NF-κB signalling and the expression of inflammatory mediators.

Expression profile analysis of microRNAs during hair follicle development in the sheep foetus.

MicroRNAs (miRNAs) regulate the development and growth cycle of hair follicles (HFs). The molecular mechanism by which miRNAs determine the development of HFs in the sheep foetus remains elusive. In this study, the expression profiles of miRNAs at 11 development periods (45, 55, 65, 75, 85, 95, 105, 115, 125, 135 and 145 d) in sheep foetus skin were analysed by high-throughput sequencing and bioinformatics analysis. A total of 72 conserved miRNAs, 44 novel miRNAs and 32 known miRNAs were significantly differentially expressed. qRT-PCR results for 18 miRNAs were consistent with the sequencing data. 85 d of foetal development was the starting point for secondary hair follicle (SF) development according to tissue morphology and cluster analysis. In SF development, the prolactin signalling pathway and platelet activation played important roles, and 10 miRNAs were potential candidate miRNAs in SF initiation.

[Predictive value of radiological parameter for re-displacement of conservative treatment of distal radius fracture].

OBJECTIVE: To analyze the radiological parameters of the conservatively in treating distal radius fracture and investigate whether the final re-displacement can be predicted after primary reduction. METHODS: From January 2013 to June 2014,212 patients with distal radial fracture conservatively were treated in our hospital, 107 patients of them were excluded because of their incomplete radiological parameters;the remaining 105 patients were available for radiological were assessed after injury, there were 56 male patients and 49 female patients in this study, the average age of the patients was 51 years old (ranged from 22 to 80 years). According to AO classification, there were 47 cases of type A2 and C1, and 58 cases of type A3, C2, C3. All patients were treated by closed reduction and below-elbow cast immobilization for 4 to 6 weeks. All patients were followed up for 3 to 6 months (means 4.5 months) by X-ray, all fractures were healed. Standard AP and lateral radiographic examination was conducted before reduction and after reduction and bony consolidation,the dorsal angulation and the radial angle were measured at each time point. The linear regression was used for the analysis to find out whether the final re-displacement can be predicted after primary reduction. RESULTS: Among 105 patients,the significant correlations were found for the dorsal angulation between the reduction time and the end time (r = 0.82) and for the radial angulation between the reduction time and end time (r = 0.85). CONCLUSION: The dorsal angulation and the radial angulation after complete healing can be predicted from linear the regression functions. Due to the possibility of predicting the end result, whether the fracture should receive further conservative treatment or surgical treatment can be decided immediately.

[Molecular cloning and tissue expression of the CCNG1 gene in sheep].

The CCNG1 gene encodes cyclin G1, which is an important cell cycle regulator and has been reported to be involved in reproductive biological processes, such as oocyte maturation and granule cell proliferation in mammals. But the study of CCNG1 in sheep has been rarely reported. To examine the effects of CCNG1 on estrous control and seasonal breeding in sheep, we first cloned and characterized the expression level of the sheep CCNG1 gene. Then by Real-time PCR, we detected and analyzed the expressions of CCNG1 gene at mRNA levels in the hypothalamus-pituitary-ovary (HPO) axis in different stages of an estrous cycle in Duo Lang sheep (non-seasonal breeding) and Merino sheep (seasonal breeding). The results showed that the open reading frame of the sheep CCNG1 gene is 885 bp in length and encodes 294 amino acids. Bioinformatic analysis indicated that the secondary structure of the sheep CCNG1 protein contained multiple phosphorylation sites and some Protein Kinase C phosphorylation sites. CCNG1 mRNA was identified in all tissues tested, with the levels in ovary and kidney higher than others. The expression profiles of CCNG1 in the HPO axis in different stages of an estrous cycle were similar in different sheep breeds: the expression levels of CCNG1 in the ovary, uterus, pineal gland and pituitary gland all peaked in the estrus phase. But there were significant differences for expression change extent of CCNG1 in ovaries in the oestrus and metestrus phase between different sheep breeds. The results suggested that CCNG1 probably participated in the regulation of estrous behavior and seasonal reproduction through controling the growth and development of follicles in sheep.

NADPH oxidase-2 is a key regulator of human dermal fibroblasts: a potential therapeutic strategy for the treatment of skin fibrosis.

The proliferation of human skin dermal fibroblasts (HDFs) is a critical step in skin fibrosis, and transforming growth factor-beta1 (TGF-ß1) exerts pro-oxidant and fibrogenic effects on HDFs. In addition, the oxidative stress system has been implicated in the pathogenesis of skin disease. However, the role of NADPH oxidase as a mediator of TGF-ß1-induced effects in HDFs remains unknown. Thus, our aim was to investigate the role of NADPH in human skin dermal fibroblasts. Primary fibroblasts were cultured and pretreated with various stimulants. Real-time Q-PCR and Western blotting analyses were used for mRNA and protein detection. In addition, siRNA technology was applied for gene knock-down analysis. Hydrogen peroxide production and 2',7'-dichlorofluorescein diacetate (DCFDA) measurement assay were performed. Here, our findings demonstrated that HDFs express key components of non-phagocytic NADPH oxidase mRNA. TGF-ß1 induced NOX2 and reactive oxygen species formation via NADPH oxidase activity. In contrast, NOX3 was barely detectable, and other NOXs did not display significant changes. In addition, TGF-ß1 phosphorylated MAPKs and increased activator protein-1 (AP-1) in a redox-sensitive manner, and NOX2 suppression inhibited baseline and TGF-ß1-mediated stimulation of Smad2 phosphorylation. Moreover, TGF-ß1 stimulated cell proliferation, migration, collagen I and fibronectin expression, and bFGF and PAI-1 secretion: these effects were attenuated by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and NOX2 siRNA. Importantly, NOX2 siRNA suppresses collagen production in primary keloid dermal fibroblasts. These findings provide the proof of concept for NADPH oxidase as a potential target for the treatment of skin fibrosis.

[Correlation analysis between polymorphism of the 59383635th locus on X chromosome and fat-tail trait in sheep].

Fat tail or fat rump is one of essential traits for surviving in harsh environments, and the mechanism of fat deposition and its inheritable characters in sheep are still unclear. Therefore, the 59383635th locus on X chromosome in our unpublished chip data was chosen as candidate SNP, PCR-SSCP method was used to detect genotypes in five sheep breeds which have extreme differences in tail types (Altay, Small Tail Han Sheep, Hu, Chinese Merino and Suffolk), and the mathematical model was employed to analyze the correlation between the polymorphism and the trait of fat tail or fat rump. The results in this study showed that the high frequency of allele T exists in Altay flock, and the frequency of allele C appears to be particularly high in the thin tail sheep breeds. The result of mathematical model showed that the ratio of T/C increased exponentially with the increase of phenotype score. These results suggest that there is a big difference in the SNP distribution between fat tail (rump) and thin tail sheep populations, and the SNP can be used as an ideal molecular marker in high-fat or low-fat sheep breeding. However, the biological function of the SNP remains to be further studied.

[Detection and analysis of polymorphisms of 59571364 and 59912586 loci on X chromosome in fat-tail and thin-tail sheep flocks].

In order to analyze the correlation of tail fat deposition and two SNP loci on Ovis arise chromosome X and provide a theoretical basis for using molecular assisted selection technology in further low-fat sheep breeding, the breeds with extreme differences in tail types (Altay, Small Tail Han Sheep, Hu, Chinese Merino and Suffolk) were used to detect the polymorphisms of two SNP loci on X chromosome and the haplotypes with PCR-RFLP method. The results showed that the TT genotype at 59571364 locus and GG genotype at 59912586 locus were preponderant genotypes in thin-tailed Chinese Merino and Suffolk sheep flocks, while the percentage of the two genotypes in fat-tailed (fat-rmup) Altay and Hu flocks is less than 2%. Haplotype analysis showed that CA haplotype is the main haplotype, the percentage of CA is up to 55%, and the percentage of CA and TA haplotypes together was 88.33% in Altay sheep flock. These results suggest that there are great differences in the SNP distribution of the 59571364 and 59912586 loci among different tail-typed sheep flocks, which can be used as molecular markers in high or low fat sheep breeding.

[Cloning and expression in follicle anagen of ILK gene in sheep].

Integrin linked kinase (ILK) is a scaffold protein, which plays important roles in hair follicle development. The cDNA sequence of novel ILK gene in sheep was cloned by PCR method and analyzed by bioinformatics. Tissue expression profiling in eight tissues and temporal profiling at different wool follicle anagen stages in skin was analyzed. The results showed that the whole open reading frame (ORF) of ILK gene was 1 359 bp in length, which encoded 452 amino acids. Bioinformatic analysis indicated that the secondary structure of ILK gene was mainly made up of three ankyrin repeats and a kinase domain, and there were multiple phosphorylation and Protein Kinase C sites in this gene. The RT-PCR result confirmed that ILK mRNA was expressed in heart, liver, spleen, lung, skeletal muscle, skin, and small intestine, and the expression level was much higher in skin, spleen, and liver than others. The q-PCR analysis demonstrated that the ex-pression level of ILK was significantly increased from March to May (early follicle anagen initiation) in both sheep breeds, Chinese Merino and Kazakh sheep, and there were certain differences from June to October between the two breeds. The above results indicated that ILK gene may play key roles in regulating secondary follicle growth.

[Effect of ginsenoside Rg1 on the spatial learning-memory ability in dementia rats after transplanted with bone marrow mesenchymal stem cells].

OBJECTIVE: To study the effect and mechanism of ginsenoside Rg1 on the spatial learning-memory ability in rats with Alzheimer's disease after transplanted with bone marrow mesenchymal stem cells (BMSCs). METHODS: Using digital randomization table method, seventy-five male SD rats were divided into the bilateral FF transection model group (as the model group: ambi-hippocampal fimbria-fomix transected), the sham-operative control group (the SOC group: receiving the same modeling process as the model group, but without ambi-hippocampal fimbria-fomix transected), the ginsenoside Rg1 treatment group (as the treatment group: Two weeks after modeling ginsenoside Rg1 was peritoneally injected at the dose of 5 mg/kg, once daily for four weeks in total), the BMSCs transplanted treatment group [as the control group: Two weeks after modeling every rat received transplantation of BMSCs (10 microL, 1 x 10(6) cells)], and the ginsenoside Rg1 + BMSCs treatment group (as the combination group: They received both transplantation of BMSCs and peritoneal injection of ginsenoside Rgl). The spatial learning-memory ability of rats was detected by Morris water maze and the escape latency (s) was recorded, mRNA expression of nerve growth factor (NGF) was detected using Real-time PCR. RESULTS: Six weeks after the hippocampal fimbria-fomix (FF) transection, the escape latency o feach medication group was obviously shorter than that of the model group, and the spatial learning-memory ability of dementia rats was somewhat improved. The spatial learning-memory ability of rats in the combination group was (29.95 +/- 2.03) and the mRNA expression level of NGF was (1.13 +/- 0.15), better than those in the BMSCs group (44.36 +/- 1.43, 0.78 +/- 0.09, P<0.05). CONCLUSIONS: Ginsenoside Rg1 could strengthen the spatial learning-memory ability in dementia rats after transplanted with BMSCs. Its mechanism might be possibly correlated with up-regulating mRNA expression of NGF in basal forebrain after BMSCs transplantation.

One-pot fabrication and antimicrobial properties of novel PET nonwoven fabrics.

Recently, with the ever-growing demand for healthy living, more and more research is focused on materials capable of killing harmful microorganisms around the world. It is believed that designing such protective materials for hygienic and biomedical applications can benefit people in professional areas and daily life. Thus, in this paper, one novel kind of antibacterial poly(ethylene terephthalate) (PET) nonwoven fabrics was conveniently one-pot prepared, with the combined immobilization of two biological antimicrobial agents, i.e. ε-polylysine and natamycin, by using the soft methacrylate nonwoven fabrics adhesives. Then, the antimicrobial activities of the functional fabrics were investigated by using the standard shaking-flask method, showing excellent antibacterial efficiency (AE) against both Escherichia coli (8099) and Staphylococcus aureus (ATCC 6538) (AE > 99.99%) compared with untreated PET nonwoven fabrics. The anti-bioaerosol tests also showed similar trends. Meantime, scanning electron microscopy analysis indicated that the bacteria on the antibacterial PET appeared to be partly bacteriolyzed and showed much less viability than those on the pristine ones. Moreover, the long residual biocidal action of such modified PET fabrics was also evaluated, and the antibacterial activity of antibacterial fibers was unaffected by the 3 month artificially accelerated aging.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist inhibits transforming growth factor-beta1 and matrix production in human dermal fibroblasts.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists are increasingly used in patients with diabetes, and some studies have suggested a beneficial effect on organ fibrosis, but their effects on dermal cell growth and extracellular matrix (ECM) turnover are unknown. To investigate the effect of the PPAR-gamma agonist troglitazone on cell growth and matrix production in human dermal fibroblasts (HDF), HDF were cultured and grown in a different concentration of troglitazone. PPAR-gamma expression and matrix production were measured in HDF in the presence of troglitazone. The mRNA expressions of TGF-beta1, collagen I (Col I) and fibronectin (FN) were determined by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). The protein of transforming growth factor-beta1 (TGF-beta1) was determined by enzyme-linked immunosorbent assay (ELISA) and proteins of Col I and FN were determined by Western blotting. The mRNA expression of TGF-beta1, Col I and FN were significantly decreased in HDF in 15-30 micromol l(-1) troglitazone compared to the control group with Dulbecco's modified Eagle's medium (P<0.01). An obvious decrease of TGF-beta1 protein was found in troglitazone-treated groups as compared to the control group (P<0.05). Exposure of HDF to troglitazone reduced col I secretion (P<0.05), and fibronectin secretion (P<0.05). This study suggests that PPAR-gamma agonist will provide a novel approach with therapeutic potential in dermal fibrosis, such as hypertrophic scar, keloid and so on.

Activation of peroxisome proliferator-activated receptor-gamma inhibits transforming growth factor-beta1 induction of connective tissue growth factor and extracellular matrix in hypertrophic scar fibroblasts in vitro.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands have been recently reported to have beneficial effects on organ fibrosis. However, their effects on extracellular matrix (ECM) turnover in hypertrophic scar fibroblasts (HSFs), and the related molecular mechanisms are unknown. HSFs were cultured and exposed to different concentration PPAR-gamma ligands in the presence of transforming growth factor-beta1 (TGF-beta1). In growth-arrested HSFs, a PPAR-gamma natural ligand (15-deoxy-D12,14-prostaglandin J2, 15d-PGJ2) and a synthetic ligand (GW7845) dose-dependently attenuated TGFbeta1-induced expression of Connective tissue growth factor (CTGF), collagens and fibronectin. Furthermore, the suppression of CTGF mRNA and protein expression are relieved by pretreatment with an antagonist of PPAR-gamma (GW9662). Moreover, GW7845 and 15d-PGJ2 partially inhibited the expression and phosphorylation of the TGF-beta1/Smad pathway. These results suggest that in TGFbeta1-stimulated HSFs, PPAR-gamma ligands caused an antiproliferative effect and reduced ECM production through mechanisms that included reducing CTGF expression, and a crosstalk between PPAR-gamma and Smad may be involved in the inhibitory effects of PPAR-gamma ligands.

[Free deep inferior epigastric artery perforator flap techniques for the immediate post modified radial mastectomy reconstruction].

OBJECTIVE: To discuss the feasibility and method of immediate breast reconstruction right after modified radical mastectomy in early breast carcinoma patients. METHODS: Deep inferior epigastric artery perforation flaps were immediately applied on patients to reconstruct the breast after the skin-sparing mastectomy. The breasts were shaped after the deep inferior epigastric artery and vein were anastomosed to the thoracodorsal artery and vein. RESULTS: In 10 cases of breast reconstruction by DIEP flaps since 2005, there were completely survival in 8 flaps, distal skin necrosis in 1 flap, adiponecrosis in 1 flap. With the follow-up of 9-28 months, the reconstructed breasts were well-shaped and there were no abdominal complication in dnor sites. Ninety percent patients were satisfied with the results from the good to the best level. CONCLUSION: Most patients were satisfied with the results of mastectomy reconstruction.

[Microenvironment of positive pressure powered air purifying medical protective equipment].

OBJECTIVE: To study the filtration efficiency of a positive pressure powered air purifying medical protective equipment and the effect of the flow rate on the microenvironment of the equipment. METHODS: The filtration efficiency of high efficiency particulate air (HEPA) filter was measured with the biologic aerosol of simulating virus (Escherichia coli bacteriophage f(2)). The simulation work was done at the walk rate of 4 km/h in summer. The effect of the flow rate on the oxygen content, the carbon dioxide content, the temperature and the humidity of the microenvironment of the equipment was investigated. The clinical experiments were conducted in three appointed hospital for fighting against SARS. RESULTS: The HEPA filter could filtrate 99.99% simulating viruses in the air. When the flow rate ranged from 75 to 125 L/min, the microenvironment parameters of the equipment were: the oxygen content was between 19.6% and 20.1% (the physiological safety limit is more than 14.6%); the carbon dioxide content ranged from 0.43% to 0.57% (the physiological safety limit is less than 1.0%); the temperature was between 32.0 degrees C to 32.2 degrees C; the humidity ranged from 49.7% to 59.4% (the physiological safety limit is the temperature 31 degrees C and the humidity 85% or temperature 38 degrees C and humidity 50%). Each microenvironment parameter met the demand of a healthy person under the normal workload. In the clinical experiments, the doctors wearing the equipment who performed the tracheotomy for a SARS patient in a deep coma were not infected. CONCLUSION: The medical protective equipment can protect the doctor and nurse in SARS contaminated areas effectively and improve their work conditions.