Yunyun Cheng

Lipase catalysis in organic solvents

The conditions for esterification and transesterification catalyzed by porcine pancreatic lipase in organic media were studied. It was found that the enzyme reaction was dependent on the following factors: the pH at which the enzyme powder was prepared from its solution, the polarity of organic media, the reaction temperature, the water content in reaction system, and the substrate structures. Effects of the above factors on enzyme activity were discussed.

Esterification and transesterification with immobilized lipase in organic solvent

In this paper, we report the reactions of esterification and transesterification catalyzed by the following lipase adducts in organic solvents: (1) glass-adsorbed; (2) acetone precipitated on porous glass, kieselghur-adsorbed; (3) Al2O3-adsorbed; and (4) agar bead-adsorbed. The optimal water content varied for different forms of the enzymes. Under the most favorable conditions, kieselguhr-adsorbed and agar bead-adsorbed lipases, which have higher catalytic activities in organic solvents, are the best of all forms of lipases.

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.

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.

Genome-wide characterization of PRE-1 reveals a hidden evolutionary relationship between suidae and primates

We identified and characterized a free PRE-1 element inserted into the promoter region of the porcine IGFBP7 gene whose integration mechanisms into the genome, including copy number, distribution preferences, capacity to exonize and phyloclustering pattern are similar to that of the primate Alu element. 98% of these PRE-1 elements also contain two conserved internal AluI restriction enzyme recognition sites, and the RNA structure of PRE1 can be folded into a two arms model like the Alu RNA structure. It is more surprising that the length of the PRE-1 fragments is nearly the same in 20 chromosomes and positively correlated to its fracture site frequency. All of these fracture sites are close to the mutation hot spots of PRE-1 families, and most of these hot spots are located in the non-complementary fragile regions of the PRE-1 RNA structure. Sequence homology analysis showed that the PRE-1 element seemed to share a common ancestor 7SL RNA with primates but was generated by different evolutionary model, which suggests that the suidae may be the closest relatives to primates in laurasiatheria.

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.

The Mechanism of Dietary Protein Modulation of Bone Metabolism via Alterations in Members of the GH/IGF Axis

Dietary protein intake as a critical regulatory factor of bone metabolism is a vital element to regulate nutritional status of mammals. Under the action of protease, dietary protein is digested into peptides and free amino acids (FAAs). Then, the metabolites are absorbed by enterocytes and metabolized in various organs of mammals. The dietary protein intake regulates bone metabolism generally via two aspects, dietary itself and signaling transduction. At the dietary level, different kinds of amino acids (AAs) of dietary protein may affect various protein metabolism of bone by regulating proteasome depending on proteolysis and protein synthesis. In addition, dietary protein from multiple sources such as animal, vegetal and healthcare products, presents distinct influences on bone metabolism via regulating calcium balance; At the cellular level, these products can regulate several biological functions via regulating signaling transduction. For example, the significant member of growth hormone/insulin-like growth factor (GH/IGF) axis can be regulated by dietary protein, which has an influence on bone metabolism through different approaches. This review mainly discusses the relationship between dietary protein and GH/IGF axis and illustrates the regulation of bone metabolism in mammals by dietary protein and its signaling transduction.