Linlin Hao

Identification of Four SNPs in LHB Gene and Their Associations with Sperm Qualities of Chinese Buffaloes

ABSTRACT Luteinizing hormone beta polypeptide (LHB) gene has been considered important for sexual behavior and has associations with sperm quality. In this study, four SNPs (g.276 T>C, g.377A>C, g.401T>C, and g.412A>G) were detected in the LHB gene of 165 water buffaloes by direct sequencing and identification of overlap peaks, each of which was associated with at least one sperm quality trait of ejaculate volume, sperm concentration, post-thaw sperm motilities, and sperm abnormalities by chi-square analysis. Among them, g.276 T>C was associated with ejaculate volume (F = 2.857, p < 0.05), sperm concentration (F = 2.052, p < 0.05), and post-thaw sperm motilities (F = 3.480, p < 0.05); g.377A>C was related to ejaculate volume (F = 4.178, p < 0.05), g.401T>C had a marker effect on sperm abnormalities (F = 3.332, p < 0.05), g.412A>G was associated with sperm concentration (F = 3.579, p < 0.05), and sperm abnormalities (F = 3.408, p < 0.05). Furthermore, four haplotypes (H1: ACG, H2: CCG, H3: CTA, H4: CCA) were generated by linkage disequilibrium analysis, which composed seven genotypes. Among them, the buffaloes with combined genotype H2H2 had the higher ejaculate volume and the individuals with the combined haplotypes H1H4 had higher sperm concentration. In summary, our study showed that there was a significant association between SNPs of LHB gene and Chinese water buffalo sperm quality traits. To the best of our knowledge, this is the first report addressing the associations between the SNPs in the LHB gene and the sperm qualities of Chinese buffaloes.

Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter

The RNA-guided endonuclease clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) derived from CRISPR systems is a simple and efficient genome-editing technology applied to various cell types and organisms. So far, the extensive approach to detect the cleavage activity of customized Cas9/guide RNA (gRNA) is T7 endonuclease I (T7EI) assay, which is time and labor consuming. In this study, we developed a visualized fluorescent reporter system to detect the specificity and cleavage activity of gRNA. Two gRNAs were designed to target porcine immunoglobulin M and nephrosis 1 genes. The cleavage activity was measured by using the traditional homology-directed repair (HDR)-based fluorescent reporter and the single-strand annealing (SSA)-based fluorescent reporter we established in this study. Compared with the HDR assay, the SSA-based fluorescent reporter approach was a more efficient and dependable strategy for testing the cleavage activity of Cas9/gRNA, thereby providing a universal and efficient approach for the application of CRISPR/Cas9 in generating gene-modified cells and organisms.

Reduced Somatostatin in Hypothalamus of Young Male Mouse Increases Local but not Circulatory GH

The release of growth hormone (GH) from the pituitary gland is primarily inhibited by somatostatin (SRIF) from the hypothalamus via interactions with five types of SRIF receptors (SSTRs). However, the inhibition mechanism of SRIF on GH has not been fully examined. In this study, we repressed the hypothalamic SRIF in young male mice by stereotaxic injection of the lentiviral-shRNA against SRIF to investigate the role of hypothalamic SRIF on hormone secretion in the GH/IGF-1 axis. We found that the reduction of SRIF in hypothalamus was associated with an increase in the protein, but not the mRNA level, of the GH in the pituitary where SSTR 2 and SSTR 5 act importantly. Interestingly, the level of blood circulatory SRIF, GH, IGF-1 and the body weight were not significantly influenced by the downregulation of hypothalamic SRIF. Our findings provide insights into the mechanisms underlying the inhibition of SRIF on GH secretion.

Growth hormone and receptor gene mutations in Chinese Banna miniature pig

Abstract The Banna miniature pig (BNMP) is a representative miniature pig breed in China. Even though BNMP dwarfism is obvious, its underlying causative mutations remain unknown. In this study, the BNMP and Large White pig (LWP) serum growth hormone (GH) and insulin-like growth factor (IGF-1) levels were detected by ELISA and compared. BNMP serum IGF-1 levels were significantly lower than LWP levels (P<0.05). The miniature condition may arise from mutations in the GH and GH receptor (GHR) genes. Therefore, GH and GHR cDNA from the BNMP were cloned into a pMD18-T vector by RT-PCR using the total RNA obtained from the BNMPs pituitary and liver tissues. Sequencing results indicated that the open reading frame of the BNMP GH gene is composed of a 26-residue signal peptide and a 191-residue mature peptide. The coding sequence of the BNMP GHR gene contained 639 amino acids, including a signal peptide that is 18 amino acids long. Two amino acid substitutions, A09V and R22Q, were found in the signal peptide of the GH gene. Additionally, the S104P mutation was found in the BNMPs mature GH protein. Four mutations in the cytoplasmic domain of GHR may influence the downstream signal transduction of GHR, which needs further experimental evidence.

Novel short antimicrobial peptide isolated from Xenopus laevis skin: Novel Short Antimicrobial Peptide

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright

The effect of heat stress on bull sperm quality and related HSPs expression

Heat stress dramatically decreases bull sperm quality and has recently received more attention due to the warmer global climate and more intensive production. However, no data exist regarding sperm quality or the related molecular mechanisms under heat stress. Recent studies showed that inducible heat shock proteins (HSPs) play an important role in the dairy heat stress regulation. In this article, to investigate the impacts of heat stress on sperm quality and the associated molecular mechanisms, sperm quality and enzyme activities concerning acrosome reaction were assessed in Simmental, Limousin and Yanbian bulls under heat stress. Subsequently, changes in heat shock protein expression profiles of Simmental bulls were observed, because we observed that sperm quality of these bulls was most sensitive to heat stress. Finally, the relationship between sperm quality and heat shock protein expression under heat stress was analyzed. The results show that summer heat stress decreased the sperm quality of the three bull breeds significantly. Moreover, different levels of heat stimulation induced various enzyme activity changes, among which the activity change in acrosomal enzyme was the most remarkable. Furthermore, the expression of heat shock proteins in the sperm was influenced by the imposed heat stress, among which the expression levels of HSP60 and HSP70 were increased while HSP90 decreased. In summary, our data show that heat stress seriously affects sperm quality and that HSP90 was most sensitive, although it should be noted that seasonal effects may confound these results. This change in heat shock protein expression may be the major factor that affected the sperm quality of the bulls. The findings may provide a new hypothesis for how heat stress impacts reproduction mechanistically.

Enhancing the mechanical performance of poly(ether ether ketone)/zinc oxide nanocomposites to provide promising biomaterials for trauma and orthopedic implants

Poly(ether ether ketone)/zinc oxide (PEEK/ZnO) composites were manufactured by using the injection molding technique. Before blending with the PEEK resin matrix, some ZnO nanoparticles were modified by γ-aminopropyltriethoxylsilane (APTES). The effect of surface modification of ZnO nanoparticles by amino groups and Si–O bonds was investigated. PEEK/ZnO composites were characterized by scanning electron microscopy (SEM), thermogravimetric analysis, and X-ray diffraction. The scanning electron micrographs showed that ZnO nanoparticles were encapsulated in the PEEK phase; within this phase, the nanoparticles were homogeneously dispersed. Mechanical and tribological properties were measured by tensile strength, flexural strength, coefficient of friction, and wear rate. It was shown that the interfacial compatibility between ZnO nanoparticles and PEEK matrix was significantly enhanced due to the amino and Si–O bonds decorated on the ZnO nanoparticles. More importantly, the thermal stability of PEEK improved upon the incorporation of ZnO nanoparticles into this matrix. Cell viability studies with mouse osteoblasts demonstrated that cell growth on PEEK and PEEK/ZnO was significantly enhanced. On the basis of the obtained results, PEEK/ZnO composites are recommended as promising candidates for orthopaedic materials and trauma implants.

Linkage disequilibrium and functional analysis of PRE1 insertion together with SNPs in the promoter region of IGFBP7 gene in different pig breeds

Polymorphisms in regions upstream of transcription initiation site may modify the transcriptional activity of target genes by changing promoter activity. This study aims to determine whether or not polymorphisms at porcine IGFBP7 promoter region affect gene expression. In this study, eight SNPs and one PRE1 insertion in this region were first confirmed. The PRE1 insertion was widespread in 20 Chinese indigenous breeds, but was not observed in three commercial breeds. A perfect linkage disequilibrium, consisting of six of those SNPs and a PRE1, was observed with two haplotypes (h1 and h2) in five pig breeds. The h1 haplotype had an overwhelming superiority distribution in Large White, Landrace, and Bama mini-pig; in turn, the h2 only existed in the PRE1 presence breeds. As the haplotypes and PRE1 were located at gene promoter regions, we further investigated the transfection of plasmids with three different fragments of IGFBP-7 promoter region (H1, H2, RF). The CMV promoter of the pEGFP-N1 was substituted by these three different fragments, respectively. Different transcriptional and translational activities of EGFP in PK-15 cells were observed in these three recombinant plasmids by quantitative real-time PCR and flow cytometric analysis. The results indicated that H1 had the higher transcriptional and translational activities of EGFP as compared to the H2 (P < 0.05, P < 0.05). As compared to the RF group, EGFP mRNA expression level was significantly higher in H1 groups (P < 0.05). The IGFBP-7 promoter polymorphisms detected in this study may be important functional variants and potential genetic markers for pig population genetic study.

Porcine IGF1 synonymous mutation alter gene expression and protein binding affinity with IGF1R

CONTEXT Insulin like growth factor 1 (IGF1) is privotal in the regulation of animal growth and is a single-chain globular protein composed of B, C, A, and D regions, of which the C region is involved in maintaining high affinity binding to the IGF1 receptor (IGF1R). PURPOSE In this study, significant expression differences between large pigs and miniature pigs were detected and only one synonymous SNP (c.258G>A) in the C region of the coding sequence of IGF1 gene was screened. The aim of this manuscript was to clear the function of the SNP and clarify the mechanism of its influnce. METHODS The expression vectors contained A allele and G allele were constructed, and the expression assays of the two groups were determined by qRT-PCR and western blotting, then the stability assays of the mRNA and protein were carried out under the inhibitation of actinomycin D and cycloheximide, respectively. At last, the binding affinity of IGF1-G and IGF1-A with IGF1R were indicated by co-immunoprecipitation and double immunofluorescence labeling methods, the conformation difference was detected by differential immunodetection. RESULTS The IGF1-G expressed higher than IGF1-A in both transcription and translation levels, and the mRNA and protein stabilities of IGF1-G were lower than IGF1-A (P < 0.05). Furthermore, the relative binding affinity of GG-genotype IGF1 with IGF1R was significantly higher than that of the AA-genotype IGF1 (P < 0.05), and there was a difference in the conformation of the IGF1 with two genotypes. CONCLUSION Our findings indicated the synonymous mutation altered the IGF1 gene expression and confirmed the synonymous mutation affected the IGF1 folding and the interactions with the IGF1R preliminarily.

Systematic review on the efficacy and safety of immune checkpoint inhibition in renal cell carcinoma

To perform a systematic review of the relevant literature about clinical trials on efficacy and safety of immune checkpoint inhibition, whether it is used alone, in combination or with other targeted therapies in patients with advanced and metastatic renal cell carcinoma (RCC), two team members reviewed the abstracts and selected pertinent articles from the relevant databases. A narrative review of randomized controlled trials was performed and seven randomized controlled trials were identified in this systematic review. In treatment of RCC, nivolumab has superior efficacy and safety compared with second-line everolimus. Combination strategies, especially those combined with anti-VEGF agents presents better efficacy but worse outcomes in term of safety than monotherapy and conventional treatment and might guide treatment choice for patients with RCC.