Rapid genotyping of 32 insertion/deletion panel for human identification using fluorogenic probes-based multiplex real-time PCR.

BACKGROUND: Insertion and deletion (InDel) polymorphisms have considerable potential in the field of forensic genetics because of their low mutation rate and small amplicons. At present, InDel polymorphisms detection based on the technique of capillary electrophoresis is the main technique used in forensic DNA laboratory. However, this method is complicated and time-consuming, and is not suitable for rapid on-site paternity and personal identification. Next-generation sequencing analysis of InDels polymorphisms requires expensive instruments, large upfront reagent and supply costs, computational requirements and complex bioinformatics, increased the time to obtain results. Thus, there is an urgent need to establish a method to provide reliable, rapid, sensitive and economical genotyping for InDels. METHOD: A rapid InDels (32 InDels) panel was established using fluorogenic probes-based multiplex real-time PCR with microfluidic test cartridge and portable real-time PCR instrument. Then, we performed several validation studies including concordance, accuracy, sensitivity, stability, species specificity. RESULTS: It showed that the complete genotypes could be obtained from ≥100 pg of input DNA and from a series of challenging samples with high accuracy and specificity within 90 min. CONCLUSION: This method provides a rapid and cost-effective solution for InDels genotyping and personal identification in portable format.

Structural variant landscapes reveal convergent signatures of evolution in sheep and goats.

BACKGROUND: Sheep and goats have undergone domestication and improvement to produce similar phenotypes, which have been greatly impacted by structural variants (SVs). Here, we report a high-quality chromosome-level reference genome of Asiatic mouflon, and implement a comprehensive analysis of SVs in 897 genomes of worldwide wild and domestic populations of sheep and goats to reveal genetic signatures underlying convergent evolution. RESULTS: We characterize the SV landscapes in terms of genetic diversity, chromosomal distribution and their links with genes, QTLs and transposable elements, and examine their impacts on regulatory elements. We identify several novel SVs and annotate corresponding genes (e.g., BMPR1B, BMPR2, RALYL, COL21A1, and LRP1B) associated with important production traits such as fertility, meat and milk production, and wool/hair fineness. We detect signatures of selection involving the parallel evolution of orthologous SV-associated genes during domestication, local environmental adaptation, and improvement. In particular, we find that fecundity traits experienced convergent selection targeting the gene BMPR1B, with the DEL00067921 deletion explaining ~10.4% of the phenotypic variation observed in goats. CONCLUSIONS: Our results provide new insights into the convergent evolution of SVs and serve as a rich resource for the future improvement of sheep, goats, and related livestock.

Whole-genome selective scans detect genes associated with important phenotypic traits in goat (Capra hircus).

Goats with diverse economic phenotypic traits play an important role in animal husbandry. However, the genetic mechanisms underlying complex phenotypic traits are unclear in goats. Genomic studies of variations provided a lens to identify functional genes. In this study, we focused on the worldwide goat breeds with outstanding traits and used whole-genome resequencing data in 361 samples from 68 breeds to detect genomic selection sweep regions. We identified 210-531 genomic regions with six phenotypic traits, respectively. Further gene annotation analysis revealed 332, 203, 164, 300, 205, and 145 candidate genes corresponding with dairy, wool, high prolificacy, poll, big ear, and white coat color traits. Some of these genes have been reported previously (e.g., KIT, KITLG, NBEA, RELL1, AHCY, and EDNRA), while we also discovered novel genes, such as STIM1, NRXN1, LEP, that may be associated with agronomic traits like poll and big ear morphology. Our study found a set of new genetic markers for genetic improvement in goats and provided novel insights into the genetic mechanisms of complex traits.

Isolation and characterization of an antibacterial peptide fraction from the pepsin hydrolysate of half-fin anchovy (Setipinna taty).

Enzymatic proteolysis of food proteins is considered a promising method to generate antibacterial peptides. The objective of the present study was to isolate and characterize peptide fraction from the pepsin hydrolysate of half-fin anchovy (Setipinna taty) with antibacterial activity against Escherichia coli. The most active peptide fraction HAHp2-3-I was isolated by a series of chromatographic methods, including Sephadex G-25 chromatography, reverse high-performance liquid chromatography (RP-HPLC) and Source 5RPC ST. Peptides identification of HAHp2-3-I was carried out using UPLC-LTQ-Orbitrap mass spectrometer. HAHp2-3-I contained five cationic peptides (MLTTPPHAKYVLQW, SHAATKAPPKNGNY, PTAGVANALQHA, QLGTHSAQPVPF and VNVDERWRKL) and three anionic peptides (LATVSVGAVELCY, NPEFLASGDHLDNLQ and PEVVYECLHW). Prediction of peptide secondary structure indicated that these anionic peptides should have extended strand and random coil structures, whereas cationic peptides PTAGVANALQHA and VNVDERWRKL could form alpha helixes. In addition, results of scanning electron microscopy (SEM) revealed that treatment by HAHp2-3-I could cause the morphological changes of E. coli and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from fish protein hydrolysates.

Genome-Wide Detection of Copy Number Variations and Their Association With Distinct Phenotypes in the World's Sheep.

Copy number variations (CNVs) are a major source of structural variation in mammalian genomes. Here, we characterized the genome-wide CNV in 2059 sheep from 67 populations all over the world using the Ovine Infinium HD (600K) SNP BeadChip. We tested their associations with distinct phenotypic traits by conducting multiple independent genome-wide tests. In total, we detected 7547 unique CNVs and 18,152 CNV events in 1217 non-redundant CNV regions (CNVRs), covering 245 Mb (∼10%) of the whole sheep genome. We identified seven CNVRs with frequencies correlating to geographical origins and 107 CNVRs overlapping 53 known quantitative trait loci (QTLs). Gene ontology and pathway enrichment analyses of CNV-overlapping genes revealed their common involvement in energy metabolism, endocrine regulation, nervous system development, cell proliferation, immune, and reproduction. For the phenotypic traits, we detected significantly associated (adjusted P < 0.05) CNVRs harboring functional candidate genes, such as SBNO2 for polycerate; PPP1R11 and GABBR1 for tail weight; AKT1 for supernumerary nipple; CSRP1, WNT7B, HMX1, and FGFR3 for ear size; and NOS3 and FILIP1 in Wadi sheep; SNRPD3, KHDRBS2, and SDCCAG3 in Hu sheep; NOS3, BMP1, and SLC19A1 in Icelandic; CDK2 in Finnsheep; MICA in Romanov; and REEP4 in Texel sheep for litter size. These CNVs and associated genes are important markers for molecular breeding of sheep and other livestock species.

Whole-genome sequence analysis unveils different origins of European and Asiatic mouflon and domestication-related genes in sheep.

The domestication and subsequent development of sheep are crucial events in the history of human civilization and the agricultural revolution. However, the impact of interspecific introgression on the genomic regions under domestication and subsequent selection remains unclear. Here, we analyze the whole genomes of domestic sheep and their wild relative species. We found introgression from wild sheep such as the snow sheep and its American relatives (bighorn and thinhorn sheep) into urial, Asiatic and European mouflons. We observed independent events of adaptive introgression from wild sheep into the Asiatic and European mouflons, as well as shared introgressed regions from both snow sheep and argali into Asiatic mouflon before or during the domestication process. We revealed European mouflons might arise through hybridization events between a now extinct sheep in Europe and feral domesticated sheep around 6000-5000 years BP. We also unveiled later introgressions from wild sheep to their sympatric domestic sheep after domestication. Several of the introgression events contain loci with candidate domestication genes (e.g., PAPPA2, NR6A1, SH3GL3, RFX3 and CAMK4), associated with morphological, immune, reproduction or production traits (wool/meat/milk). We also detected introgression events that introduced genes related to nervous response (NEURL1), neurogenesis (PRUNE2), hearing ability (USH2A), and placental viability (PAG11 and PAG3) into domestic sheep and their ancestral wild species from other wild species.

Is CuO Suitable for Improving the Electrochemical Properties of g-C3N4?

G-C3N4 has a bright application prospect as an electrode material of supercapacitors, which makes it a concern. In order to increase the specific capacitance of g-C3N4, we consider to combine it with a metal oxide with high theoretical specific capacitance and utilize the synergistic effect. As a common metal oxide, CuO has the characteristics of high theoretical capacitance, high chemical stability, simple preparation and environmental friendliness. A composite containing CuO nanobelts and graphitic C3N4 (g-C3N4) was successfully synthesized by a chemical precipitation method. Various testing methods were used to explore its composition, microstructure and electrochemical properties to discuss whether CuO is suitable for improving the electrochemical properties of g-C3N4.

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.

Antitumor activities of D-glucosamine and its derivatives.

The growth inhibitory effects of D-glucosamine hydrochloride (GlcNH(2).HCl), D-glucosamine (GlcNH(2)) and N-acetyl glucosamine (NAG) on human hepatoma SMMC-7721 cells in vitro were investigated. The results showed that GlcNH(2).HCl and GlcNH(2) resulted in a concentration-dependent reduction in hepatoma cell growth as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This effect was accompanied by a marked increase in the proportion of S cells as analyzed by flow cytometry. In addition, human hepatoma SMMC-7721 cells treated with GlcNH(2).HCl resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis. NAG could not inhibit the proliferation of SMMC-7721 cells. GlcNH(2).HCl exhibited antitumor activity against Sarcoma 180 in Kunming mice at dosage of 125-500 mg/kg, dose of 250 mg/kg being the best. GlcNH(2).HCl at dose of 250 mg/kg could enhance significantly the thymus index, and spleen index and could promote T lymphocyte proliferation induced by ConA. The antitumor effect of GlcNH(2).HCl is probably host-mediated and cytocidal.

Phylogenetic analysis of euthyneuran gastropods from sea to land mainly based on comparative mitogenomic of four species of Onchidiidae (Mollusca: Gastropoda: Pulmonata).

We generated complete mitochondrial genome sequences data for 4 genera (Onchidium, Platevindex, Paraoncidium and Peronia) in Onchidiidae to construct a phylogenetic tree in conjunction with other 9 existing data among gastropods. The topology showed that the taxa clustered into two main groups of four species, one of which included Onchidium struma and the Platevindex mortoni, the other Paraoncidium reevesii and Peronia verruculata. The process in Pulmonata from sea to land in accordance with the evolution of respiratory organs from branchial gills to pulmonary cavity has been shown. This will also constitute a framework for phylogeny evolution analysis, systematic classfication of Onchidiidae and other euthyneurans (pulmonates and opisthobranchs).

Outcomes based on risk assessment of anastomotic leakage after rectal cancer surgery.

PURPOSE: Anastomotic leakage (AL) is associated with high morbidity and mortality, high reoperation rates, and increased hospital length of stay. Here we investigated the risk factors for AL after anterior resection for rectal cancer with a double stapling technique. PATIENTS AND METHODS: Data for 460 patients who underwent primary anterior resection with a double stapling technique for rectal carcinoma at a single institution from 2003 to 2007 were prospectively collected. All patients experienced a total mesorectal excision (TME) operation. Clinical AL was defined as the presence of leakage signs and confirmed by diagnostic work-up according to ICD-9 codes 997.4, 567.22 (abdominopelvic abscess), and 569.81 (fistula of the intestine). Univariate and logistic regression analyses of 20 variables were undertaken to determine risk factors for AL. Survival was analysed using the Cox regression method. RESULTS: AL was noted in 35 (7.6%) of 460 patients with rectal cancer. Median age of the patients was 65 (50-74) and 161 (35%) were male. The diagnosis of AL was made between the 6th and 12th postoperative day (POD; mean 8th POD). After univariate and multivariate analysis, age (p=0.004), gender (p=0.007), tumor site (p<0.001), preoperative body mass index (BMI) (p<0.001), the reduction of TSGF on 5th POD less than 10U/ml (p=0.044) and the pH value of pelvic dranage less than or equal to 6.978 on 3rd POD (p<0.001) were selected as 6 independent risk factors for AL. It was shown that significant differences in survival of the patients were AL-related (p<0.001), high ASA score related (p=0.036), high-level BMI related (p=0.007) and advanced TNM stage related (p<0.001). CONCLUSIONS: AL after anterior resection for rectal carcinoma is related to advanced age, low tumor site, male sex, high preoperative BMI, low pH value of pelvic drainage on POD 3 and a significant reduction of TSGF on POD 5. In addition to their high risk of immediate postoperative morbidity and mortality, AL, worse physical status, severe obesity and advanced TNM stage have similarly negative impact on survival.

Affinity purification of trypsin inhibitor with anti-Aspergillus flavus activity from cultivated and wild soybean.

Trypsin inhibitors (TI) from wild-type soybean (Glycine soya) (WBTI) and domesticated soybean (Glycine max) (SBTI) were purified using prepared chitosan resin-trypsin as filler on the affinity chromatography column. The SBTI/WBTI purification fold by affinity chromatography was 718- and 279-fold, with the activity recovery of 62% and 59%, respectively. It was found that SBTI and WBTI exerted a strong inhibition of Aspergillus. flavus growth, with IC(50) of 1.6 and 1.0 micromol/l. This growth inhibition was possibly the result of the inhibition on alpha-amylase activity of A. flavus by both the SBTI and WBTI. This was further supported by the fact that in the presence of SBTI and WBTI at 9.0 and 6.0 microg/g (peanut) on peanuts inhibited the germination and growth of A. flavus. Accordingly, characterization of the mode of action of SBTI and WBTI could constitute a first step leading to resistance to A. flavus invasion.