Identification of 19 loci for reproductive traits in a local Chinese chicken by genome-wide study.

Reproductive traits have long been studied and have an important influence on chicken breeding. To identify quantitative trait loci affecting reproductive traits, a genome-wide analysis of a Chinese chicken breed was performed to analyze age at first egg body weight at first egg, first egg weight, egg weight at the age of 300 days, egg weight at the age of 462 days, egg number at the age of 300 days, egg number between the ages of 300 and 462 days and egg number at the age of 462 days. Nineteen SNPs related to reproductive traits were presented (P < 1.80E-6). Nine of the 19 SNPs had a significant effect on BWF, six SNPs were significantly associated with egg weight, and four SNPs were significantly associated with egg number. These SNPs were located near to or in 17 genes including FAM184B, HTT, KCNH7, CDC42BPA, KCNIP4, GJA5, CBFB, and GPC6. The present results may be beneficial for reproductive research and may be used in marker-assisted selection in future studies. These results could potentially benefit further breeding programs, especially in Jinghai Yellow Chicken.

Effect of miR-29c and miR-129-5p on epithelial-mesenchymal transition in experimental biliary atresia mouse models.

Biliary atresia (BA) is a destructive bile duct disease occurring in newborn children within a few weeks after birth. In this study, the effect of miR-29c and miR-129-5p on epithelial-mesenchymal transition (EMT) in experimental BA was explored by constructing BA mouse models via Rhesus rotavirus vaccine infection. miR-29c and miR-129-5p expression was analyzed by real-time quantitative polymerase chain reaction. EMT was established by induction with transforming growth factor (TGF)-ß1. miR-29c and miR-129-5p were overexpressed and inhibited, respectively, by Lipofectamine transfection. EMT-related protein (formin-like 2, FMNL2; E-cadherin; vimentin; and cytokeratin-19, CK-19) expression was analyzed by western blot and immunofluorescent assay. The results indicated that miR-29c and miR-129-5p were downregulated and upregulated in BA mice. TGF-ß1 induction caused a time-dependent decrease and increase in miR-29c and miR-129-5p, respectively. Additionally, TGF-ß1 induced an increase in FMNL2 and vimentin expression and a decrease in E-cadherin and CK-19 expression (P < 0.05). Overexpression or suppression of miRNA-29c or miR-129-5p, respectively, induced the inhibition of FMNL2 and vimentin, and promotion of E-cadherin and CK-19 expression, in the test groups compared to the non-intervention group (P < 0.05). However, the FMNL2, vimentin, E-cadherin, and CK- 19 expression did not differ between the control and non-intervention groups (P > 0.05). Thus, miR-29c upregulation or miR-129-5p downregulation effectively prevented EMT in BA by regulating the expression of EMT pathway-related proteins. Therefore, miR-29c and miR-129-5p could be utilized as therapeutic targets for BA in the future.

Myf5 and MyoG gene SNPs associated with Bian chicken growth trait.

The growth trait is important in poultry production. We analyzed the association between single nucleotide polymorphisms (SNPs) in the Myf5 and MyoG gene and Bian chicken growth traits. SNPs in candidate genes of the Bian chickens were detected by the polymerase chain reaction-single strand conformation polymorphism method. Two mutation loci and six genotypes were identified in each candidate gene. In terms of growth traits, least square analysis showed that the FF genotype of the MyoG was the advantage genotype and the IJ genotype of the Myf5 was the disadvantage genotype for growth trait in Bian chicken. Correlation analysis suggested that the different combination genotypes between Myf5 and MyoG genes had a significant effect on growth traits in Bian chickens. The result suggested that MyoG and Myf5 genes can be used in marker-assisted selection for improving the growth trait in Bian chicken.

Genome-wide association study of growth traits in the Jinghai Yellow chicken.

Growth is one of the most economically important traits in the poultry industry. In this study, we identified single-nucleotide polymorphisms (SNPs) and candidate genes associated with growth traits of the Jinghai Yellow chicken. Genome-wide association studies were conducted using the Illumina 60 K SNP Chicken array to genotype 400 Jinghai Yellow chickens. For each bird, the body weights at hatching and at 2, 4, 6, 8, 12, 14, and 16 weeks were recorded. The SNPs that were significantly associated with the growth traits were identified using the general linear regression model. The results revealed a total of 18 SNPs that reached Bonferroni genome-wide significance (P < 1.80E-6). Three proximal genes (BTRC, NLK, and NF1) were found to participate in the Wnt-signaling pathway and mitogen-activated protein kinase signaling pathway. Haplotype analysis identified 19 significant haplotypes and identified a region 152.4-156.3M on GGA1 affecting 3 growth traits (BW4, BW14, and BW16). These results may help identify the exact locations of body weight quantitative trait loci on a genome level and indicate variants that can be used for subsequent investigations for Jinghai Yellow chicken.

Single nucleotide polymorphisms associated with growth traits in Jinghai yellow chickens.

Body weight is one of the most important economic traits in the poultry industry. In the present study, a custom SNP Beadchip was used to analyze the association between those 15 SNPs and 12 growth traits of Jinghai yellow chickens, and other important genetic parameters were also calculated and analyzed. The results indicated that nine of the 15 SNPs were associated with growth traits in Jinghai yellow chickens (P < 0.05), and the identified SNPs were also in linkage disequilibrium. Five of the nine identified SNPs were mainly associated with all of the growth traits, which indicated that those five SNPs might have significant influence on Jinghai yellow chicken growth traits. Polymorphism information content (PIC) analyses indicated that five of the nine SNPs exhibited moderate polymorphism (0.25 < PIC < 0.5), which reflected intermediate genetic diversity. Six candidate genes surrounding the significant SNPs were obtained and subjected to Gene Ontology annotation analyses and pathway analyses. The functions of six important candidate genes (SETDB2, ATP7B, INTS6, KPNA3, DLEU7, and FOXO1A) were discussed. The present study provided basic data for marker-assisted selection in Jinghai yellow chickens.

Association between a functional single nucleotide polymorphism in the brain-derived neurotrophic factor gene and risk of child asthma.

Brain-derived neurotrophic factor (BDNF) promotes synaptic remodeling and modulates the function of other neurotransmitters. Allergic inflammation triggers neuronal dysfunction and structural changes in the airways. Genetic polymorphisms in functional regions of the BDNF gene have a plausible role in modulating the risk of child asthma (CA). This study examined the potential association between CA and three single nucleotide polymorphisms (SNPs) in BDNF (rs2030323, rs6265, and rs16917204 in the promoter, exon 4, and 3'-untranslated regions, respectively). The study was conducted in 350 children with asthma and 356 healthy controls. The genotype and allele frequencies and difference between groups were analyzed using HaploView 4.0 and SPSS 20.0 software platforms. The analysis revealed a strong association between the rs6265 genotype distribution and CA. The frequency of the G allele was significantly higher in CA patients than in healthy controls (P = 0.0007, odds ratio = 1.323, 95% confidence interval = 1.073-1.632). Strong linkage disequilibrium was observed between rs16917204 and rs6265. A significantly higher number of G-G haplotypes were observed in CA patients than in controls (P = 0.024 after Bonferroni correction), while the G-A haplotypes were more significant in controls (P = 0.013 after Bonferroni correction). This suggested that BDNF gene polymorphisms confer susceptibility to CA, and also support the notion that BDNF dysfunction is involved in the pathophysiological process of CA.

Association between the -607 C > A polymorphism in interleukin-18 gene promoter with gastrointestinal cancer risk: a meta-analysis.

The interleukin-18 (IL-18) gene -607 C/A polymorphism has been reported to be associated with gastrointestinal cancer, but there are conflicting results from previous studies on said topic. Therefore, the aim of this meta-analysis is to derive a more precise estimation of the association between the -607 C/A polymorphism in the IL-18 gene and gastrointestinal cancer risk. Literature searches of PubMed, Google Scholar, and Web of Science databases were carried out in 2015. Five studies were assessed with a total of 1618 cases and 1155 healthy controls. When results from all eligible studies were pooled into the meta-analysis, we found significant association between the IL-18 gene -607 C/A polymorphism and gastrointestinal cancer risk (CC vs AA: OR = 0.93, 95%CI = 0.72- 1.20; CC vs CA: OR = 0.76, 95%CI = 0.62-0.92; dominant model: OR = 1.25, 95%CI = 1.03-1.50; recessive model: OR = 1.09, 95%CI = 0.87-1.37). In the subgroup analysis, significant associations between the -607 C/A polymorphism and gastrointestinal cancer risk were found in esophageal cancer. However, this polymorphism did not appear to have any influence on gastric cancer and colorectal cancer susceptibility. In conclusion, this meta-analysis suggests that the -607 C/A polymorphism in the IL-18 gene may be associated with susceptibility to esophageal cancer. Further studies with large sample sizes are needed to confirm these conclusions.

A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

Genome-wide association study of meat quality traits in chicken.

Meat quality traits are very important in the poultry industry. To identify single nucleotide polymorphisms (SNPs) and candidate genes affecting meat quality traits, a genome-wide association study was performed using the Illumina chicken 60K SNP beadchip in Jinghai yellow chicken. Four meat quality traits were measured. Two SNPs reached 5% Bonferroni genome-wide significance (P < 1.8E-6) and 7 SNPs reached "suggestive" genome-wide significance (P < 3.59E-6) with meat quality. These SNPs were located nearby or in 7 candidate genes, including CBLN2, HPGDS, SETD2, and ANKRD46, among others. A total of 5650 haplotpyes were established and only 1 was found to be associated with fat content in leg muscle. These results indicate that the 9 SNPs and 7 genes are important candidate markers and may influence meat quality traits in chicken.

Adjuvant chemotherapy may improve survival of patients with luminal A breast cancer and positive lymph nodes.

The study examined the clinicopathological characteristics and treatment options in patients with luminal A breast cancer. This retrospective cohort included 1580 patients with luminal A breast cancer treated between January 2005 and June 2007. Patients were divided into four subgroups according to lymph node status. Prognostic factors and 5-year overall survival (OS) and disease-free survival (DFS) of patients were analyzed. The median duration of follow-up was 67 months. Multivariate Cox-regression analysis revealed that patients in the LN2 and LN3 subgroups had a higher risk of recurrence and death than patients in the LN0 subgroup (LN2: HR = 2.2 for DFS and HR = 2.1 for OS; LN3: HR = 4.7 for DFS and HR = 4.7 for OS). In the LN2 subgroup, there was a trend towards reduced risk of recurrence and death for patients receiving adjuvant chemotherapy plus endocrine therapy, although this difference did not reach statistical significance. In the LN0 and LN1 subgroups, there was a trend towards an increased risk of death in patients receiving chemotherapy. Although lymph node status remains one of the most important independent prognostic predictors for luminal A breast cancer, in patients with 0-3 positive lymph nodes endocrine therapy can be considered sufficient. However, patients with ≥4 positive lymph nodes, and especially in those with ≥ 10, should receive chemotherapy.

Expression and antibody generation of the cancer-testis antigen, BIOT2-S.

Biot2-S is a mouse cancer-testis antigen gene that was identified using the cross-reactive serological analysis of recombinant cDNA expression libraries (SEREX) technique in the State Key Laboratory of Biotherapy, West China Hospital, Sichuan University. To express BIOT2-S and generate its antibody for further investigation, the Biot2-S prokaryotic recombinant expression vector Biot2-S/pGEX6P-1 was constructed with Escherichia coli DH5α as a cloning vector, and BIOT2-S was expressed in E. coli Rosetta (DE3). The recombinant BIOT2-S was expressed in the form of an inclusion body and the targeted recombinant BIOT2-S was produced at the level of approximately 25% total bacterial proteins after being induced with optimum conditions (0.2 mM isopropyl-ß-D-thiogalactopyranoside for 6 h at 37°C). The target protein was purified by glutathione S-transferase (GST)-trap FF affinity chromatography and detected by western blot. The purified recombinant protein was further confirmed by electrospray ionization quadrupole time-of-flight mass spectrometry after removal of the GST-tags. Then the purified BIOT2-S was used to immunize adult rabbits to generate its antibody. The antibody was purified and its specificity determined. The titer of the antibody was shown to reach 10(4) and the antibody was demonstrated to be able recognize the corresponding protein in the testes of mouse and chicken; the tumor cell lines CT-26 and S180 also reacted with the antibody. This study provides a valuable foundation for further research on the cancer-testis antigen BIOT2-S.

Molecular cloning and characterization, and prokaryotic expression of the GnRH1 gene obtained from Jinghai yellow chicken.

The gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Recent studies have reported the occurrence of GnRH molecular variants in numerous species. In this study, the GnRH1 gene from Jinghai yellow chicken was cloned by reverse transcriptase-polymerase chain reaction and transformed into BL21 (DE3) competent cells. The GnRH1 gene and amino acid sequences were subjected to bioinformatic analyses. The GnRH1 gene nucleotide sequence was discovered to be 352 bp long, containing a coding, promoter, and section of the 3'-regions. The GnRH1 gene shared 93, 81, 54, 58, 61, 76, 76, 59, 76, and 66% sequence identity with Meleagris gallopavo, Columba livia, Homo sapiens, Bos taurus, swines, Capra hircus, Ovis aries, Pantholops hodgsonii, Equus caballus, and Rattus norvegicus, respectively. The GnRH1 gene showed conserved domains. The GnRH1 protein was a secreted protein comprising 92 amino acids, with a molecular weight of 10205.6 Da and a theoretical pI of 5.67. Most of the amino acid residues were observed to be hydrophilic, indicating water solubility. The predicted secondary structures of proteins included α-helices (h; 23.08%), ß-extensions (e; 10.92%), and random coils (c; 66.0%). The successful construction of prokaryotic expression vector pET32a-GnRH1 was confirmed by restriction and sequence analysis. SDS-PAGE analysis showed the successful expression of recombinant plasmid in Escherichia coli BL21 (molecular weight = 25-28 kDa). Larger quantities of protein were expressed in supernatant, indicating greater expression in soluble form. Western blot analysis confirmed the expression of the target protein.

Molecular genetic diversity and maternal origin of Chinese black-bone chicken breeds.

Chinese black-bone chickens are valued for the medicinal properties of their meat in traditional Chinese medicine. We investigated the genetic diversity and systematic evolution of Chinese black-bone chicken breeds. We sequenced the DNA of 520 bp of the mitochondrial cyt b gene of nine Chinese black-bone chicken breeds, including Silky chicken, Jinhu black-bone chicken, Jiangshan black-bone chicken, Yugan black-bone chicken, Wumeng black-bone chicken, Muchuan black-bone chicken, Xingwen black-bone chicken, Dehua black-bone chicken, and Yanjin black-bone chicken. We found 13 haplotypes. Haplotype and nucleotide diversity of the nine black-bone chicken breeds ranged from 0 to 0.78571 and 0.00081 to 0.00399, respectively. Genetic diversity was the richest in Jinhu black-bone chickens and the lowest in Yanjin black-bone chickens. Analysis of phylogenetic trees for all birds constructed based on hyplotypes indicated that the maternal origin of black-bone chickens is predominantly from three subspecies of red jungle fowl. These results provide basic data useful for protection of black-bone chickens and help determine the origin of domestic chickens.

Induction of apoptosis in human cervical carcinoma Hela cells with active components of Menispermum dauricum.

Menispermum dauricum DC possesses a wide range of pharmacological effects. In this study, the mechanism of apoptosis induced by active components of M. dauricum was investigated in the human cervical carcinoma HeLa cell line. HeLa cells were treated with different M. dauricum concentrations over different time periods. The proliferation-inhibitory rate and cytotoxic effect of HeLa cells were measured by using the methyl thiazolyl tetrazolium (MTT) assay, and the apoptotic rate was detected by flow cytometry. Expressions of caspase-9, caspase-8, caspase-3, Bcl-2, and Fas proteins, in the apoptotic pathway, and the expression of nuclear factor-kappa B (NF-κB) were detected by SP immunocytochemistry. The MTT assay showed that active components of M. dauricum could significantly inhibit the growth of HeLa cells in a dose- and time-dependent manner (P<0.01). The Sub-Gl peak was found by flow cytometry, and the maximal apoptosis rate was 24.93%. Immunocytochemistry showed that after treatment with M. dauricum, the expressions of caspase-8, caspase-9, caspase-3, Fas protein, and NF-κB all increased, and the expression of the Bcl-2 protein decreased, with significant differences relative to the control group (P<0.01). Apoptosis in HeLa cells could be induced by active components of M. dauricum through the NF-κB signal transduction pathway and the caspase pathway, which was related to the downregulation of Bcl-2 expression and the upregulation of Fas expression.

Complete sequence analysis of mitochondrial DNA of aplastic anemia patients.

This study was primarily undertaken to test the hypothesis that mitochondrial DNA (mtDNA) mutations may be associated with aplastic anemia (AA). We analyzed mtDNA sequences from 15 patients with AA. The samples were obtained from bone marrow, and patients' oral epithelial cells were collected for normal tissue comparison. Total DNA was amplified by PCR after extraction, and these segments were then sent for sequencing. The results were compared with those of oral epithelial tissues as well as mtDNA sequences in the revised Cambridge Reference Sequence (rCRS) database. We detected 61 heteroplasmic mutations in 11 genes, including those encoding NADH dehydrogenase (ND)1-2 and 4-6, tRNA glutamic acid (TRNE), ribosomal RNA (RNR) 1 and 2, cytochrome c oxidase (COX1), cytochrome b (CYTB), and tRNA glycine (TRNG); mutation rates were particularly high in ND2 (34.4%) and ND4 (21.3%) in the patients' mtDNA genomes. The products of these genes are involved in oxidation in the respiratory chain, and a large number of homoplasmic mutations were found. Interestingly, these 162 polymorphisms were mostly in the D-loop DNA structure (54.3%), in which numerous mutations associated with leukemia and myelodysplastic syndromes are found. We conclude that functional impairment of the mitochondrial respiratory chain induced by mutation may be an important reason for hematopoietic failure in AA patients.

Prospective analysis of KRAS wild-type patients with metastatic colorectal cancer using cetuximab plus FOLFIRI or FOLFOX4 treatment regimens.

Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor, has proven to be efficient in the treatment of metastatic colorectal cancer. We made a prospective study of the efficacy and toxicities of cetuximab-combination first-line (FOLFOX4) versus second/third-line (FOLFIRI) chemotherapy in 98 KRAS wild-type patients who had metastatic colorectal cancer. Wild-type KRAS had been identified by direct sequencing. Associations between clinical response/progression-free survival/overall survival/toxicities and cetuximab-combination chemotherapy timing were evaluated. The overall response rate was significantly higher for first-line treatment than for second/third-line treatment (relative risk = 1.707, 95% confidence interval = 1.121-2.598). Both progression-free survival and overall survival indicated significantly longer survival of first-line treatment than second/third-line treatment patients. This study is a validation of a molecular analysis of KRAS wild-type status for the prediction of response to cetuximab-combination chemotherapy for metastatic colorectal cancer patients; its predictive role was less prominent in the second/third-line than in the first-line treatment patients.

Leishmania mexicana: the circular DNA 1 (CD1) element contains genes encoding nucleotide-binding protein.

OBJECTIVE: To determine the nucleotide sequence of cloned CD1 fragments from Leishmania mexicana and find ORFs predicted to have protein coding function. METHODS: CD1 element was separated by CHEF and recovered by agarase, and the digested CD1 fragments were cloned into pZero vector. Nucleotide sequences were determined by the dideoxy chain termination method with the automatic sequencing system ALF using the M13 universal primers. Sequences were analyzed using GCG-PCGENE computer programs. RESULTS: The sequence with 4,385 nucleotides was determined and two ORFs were considered to have protein coding function (encoding nucleotide-binding protein). CONCLUSION: Genes encoding nucleotide-binding protein were identified from the amplified CD1 element of Leishmania mexicana.

Population-based study of the incidence of Shigella diarrhea and causative serotypes in Santiago, Chile.

BACKGROUND: Shigella is an important cause of diarrheal disease in children in developing countries. The increasing prevalence of antibiotic-resistant strains has stimulated interest in the use of multivalent Shigella vaccines. Because Shigella vaccines under development are based on eliciting immunity to O antigens, monitoring the distribution of serotypes in defined target populations is critical. We initiated health center-based surveillance in a poor semirural community in Colina, Santiago (7489 children <60 months of age) to determine the age-specific incidence of Shigella disease and the responsible serotypes. FINDINGS: Surveillance was maintained at the 2 health centers during warm seasons (November 1 through April 30) for 4 successive years (1994 to 1998). Shigella was recovered from 54 of 243 cases of dysentery (22%) and from 215 of 3966 cases of nondysenteric diarrhea (5.4%) (P < 0.001). The peak mean annual incidence of shigellosis occurred among children 12 to 47 months of age (9.0 to 12.6 cases/10(3) children), although the incidence in infants (5.2/10(3)) and children 48 to 59 months of age (6.2/10(3)) was also substantial. During the 1995 through 1996 season, an age-matched healthy control was cultured for every child <60 months of age with diarrhea. Shigella isolation from cases (34 of 576, 5.9%) was >8-fold higher than controls (4 of 576, 0.7%) (P < 0.01). Four serotypes, Shigella sonnei (45%), Shigella flexneri 2b (19%), S. flexneri 2a (14%) and S. flexneri 6 (11%), accounted for 89% of all cases. INTERPRETATION: Shigella remains an important pediatric pathogen in Santiago. The serotype distribution from Colina, which closely resembles data from a population-based surveillance study in Santiago in the mid-1980s, demonstrates a remarkable degree of serotype stability in Santiago during a 15-year period.