Genome-wide single nucleotide polymorphism array and whole-genome sequencing reveal the inbreeding progression of Banna minipig inbred line.

Inbred pigs are promising animal models for biomedical research and xenotransplantation. Established in 1980, the Banna minipig inbred (BMI) line originated from a sow and its own male offspring. It was selected from a small backcountry minority Lahu village, where records show that no other pig breed has ever been introduced. During the inbreeding process, we perfomed extreme inbreeding over 23 consecutive generations using full-sibling or parent-offspring mating. In order to investigate the inbreeding effects in BMI pigs across generations over the past 40 years, in this study we conducted a genome-wide SNP genotyping of the last 10 generations, representing generations 14-23. In total, we genotyped 57,746 SNPs, corresponding to an average decrease in heterozygosity rate of 0.0078 per generation. Furthermore, we were only able to identify 18,216 polymorphic loci with a MAF larger than 0.05, which is substantially lower than the values in previous reports on other pig breeds. In addition, we sequenced the genome of the first pig in the twenty-third generation (inbreeding coefficient 99.28%) to an average coverage of 12.4× to evaluate at the genome level the impact of advanced inbreeding. ROH analysis indicates that BMI pigs have longer ROHs than Wuzhishan and Duroc pigs. Those long ROH regions in BMI pigs are enriched for distinct functions compared with the highly polymorphic regions. Our study reveals a genome-wide allele diversity loss during the progress of inbreeding in BMI pigs and characterizes ROH and polymorphic regions as a result of inbreeding. Overall, our results indicate the successful establishment of the BMI line, which paves the way for further in-depth studies.

Incidence and mortality of nasopharyngeal carcinoma: interim analysis of a cluster randomized controlled screening trial (PRO-NPC-001) in southern China.

BACKGROUND: Previous mass screening studies have shown that IgA antibodies against Epstein-Barr Virus (EBV) can facilitate early detection of nasopharyngeal carcinoma (NPC), but the impact of EBV-antibody screening for NPC-specific mortality remains unknown. PATIENTS AND METHODS: A prospective, cluster randomized, controlled trial for NPC screening (PRO-NPC-001) was conducted in 3 selected towns of Zhongshan City and 13 selected towns of Sihui City in southern China beginning in 2008. Serum samples of the screening group were tested for two previously selected anti-EBV antibodies. Subjects with serological medium risk were subsequently retested annually for 3 years, and those with serological high risk were referred to otorhinolaryngologists for diagnostic check-up. An interim analysis was carried out to evaluate the primary end points of the NPC-specific mortality and the early diagnostic rate, and the secondary end point of the NPC incidence, through linkage with the database of Zhongshan City. RESULTS: Among 70 296 total subjects, 29 413 screened participants (41.8% of the total subjects) in the screening group and 50 636 in the control group, 153 (43.3 per 100 000 person-year), 62 (55.3 per 100 000 person-year) and 99 (33.1 per 100 000 person-year) NPC cases were identified. The early diagnostic rates of NPC were significantly higher in the participants (79.0%, P < 0.0001) and the screening group (45.9%, P < 0.0001) compared with the control group (20.6%). Although no differences were found between NPC-specific mortality of the screening group and the control group [relative risk (RR)= 0.82, 95% confidence interval (CI) 0.37-1.79], lower NPC-specific mortality was noticed among participants from the screening group versus the control group (RR = 0.22, 95% CI 0.09-0.49). CONCLUSION: IgA antibodies against EBV can identify high-risk population and was effective in screening for early asymptomatic NPC. Although the mortality reduction was not significant in the primary end point, we noted encouraging evidence of a mortality reduction in screening participants in this interim analysis. CLINICAL TRIAL NUMBER: NCT00941538.

Complementary DNA cloning, sequence analysis, and tissue transcription profile of a novel U2AF2 gene from the Chinese Banna mini-pig inbred line.

U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an important gene for pre-messenger RNA splicing in higher eukaryotes. In this study, the Banna mini-pig inbred line (BMI) U2AF2 coding sequence (CDS) was cloned, sequenced, and characterized. The U2AF2 complete CDS was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) technique based on the conserved sequence information of cattle and known highly homologous swine expressed sequence tags. This novel gene was deposited into the National Center for Biotechnology Information database (Accession No. JQ839267). Sequence analysis revealed that the BMI U2AF2 coding sequence consisted of 1416 bp and encoded 471 amino acids with a molecular weight of 53.12 kDa. The protein sequence has high sequence homology with U2AF65 of 6 species - Homo sapiens (100%), Equus caballus (100%), Canis lupus (100%), Macaca mulatta (99.8%), Bos taurus (74.4%), and Mus musculus (74.4%). The phylogenetic tree analysis revealed that BMI U2AF65 has a closer genetic relationship with B. taurus U2AF65 than with U2AF65 of E. caballus, C. lupus, M. mulatta, H. sapiens, and M. musculus. RT-PCR analysis showed that BMI U2AF2 was most highly expressed in the brain; moderately expressed in the spleen, lung, muscle, and skin; and weakly expressed in the liver, kidney, and ovary. Its expression was nearly silent in the spinal cord, nerve fiber, heart, stomach, pancreas, and intestine. Three microRNA target sites were predicted in the CDS of BMI U2AF2 messenger RNA. Our results establish a foundation for further insight into this swine gene.

Effects of different activation regimens on pronuclear formation and developmental competence of in vitro-matured porcine oocytes after intracytoplasmic sperm injection.

To improve the activation protocol for in vitro-maturated porcine oocytes after intracytoplasmic sperm injection (ICSI), we examined the combined effect of U0126, a specific inhibitor of mitogen-activated protein kinase kinases, and an electrical pulse on pronuclear formation and developmental competence. Two approaches were tested: (i) 6-h treatment of ICSI oocytes with U0126 applied at different intervals (0, 2, 3 or 4 h) after the electrical pulse and (ii) treatment of ICSI oocytes with U0126 applied 4 h after the electrical pulse over an additional 4, 6 or 8 h. Another protein kinase inhibitor, 6-dimethylaminopurine, was used as a chemical activator in control experiments. The highest rates of diploid embryo formation, normal fertilization and blastocyst formation were observed after 6 h of exposure to U0126 starting 4 h after the electrical pulse. Therefore, U0126 can be used as an activating agent for porcine oocytes fertilized by ICSI.

Screening and identification of various components in Thalictrum fortunei using a combination of liquid chromatography/time-of-flight tandem mass spectrometry.

An approach for screening and identification of various components in a traditional Chinese medicine (TCM), using a combination of LC/TOF-MS technique was described in this paper. The chemical profile of Thalictrum fortunei, well-known in TCM, was studied using the established method. The possibilities of screening and identifying non-target components inside TCM with modern data acquisition methods of acceleration time of flight mass spectrometers, such as data-dependent MS to MS/MS switching were investigated. As a result, 27 components were identified. This study was aimed to screen and identify the main components of T. fortunei using LC/TOF-MS, expecting to provide a rapid, sensitive, economical and systematical method for the identification and further quality evaluation of TCM preparation.

Fertility in single-ovulating and superovulated dairy heifers after insemination with low dose sex-sorted sperm.

The present study was performed to test fertility in single-ovulating and superovulated dairy heifers after insemination with low dose sex-sorted sperm under field conditions. Some parameters, including the dosage, deposition site and timing, were assessed with the pregnancy rates after artificial insemination (AI). Moreover, the use of oestrus synchronization in combination with sorted sperm was evaluated. Besides that, we also improved the embryo production efficiency in superovulated dairy heifers by optimizing the timing of inseminations and repartitioning the sexed sperm dosage among multiple inseminations. The conception rate (52.8%) in heifers after low dose (2 × 10(6) ) insemination with sorted sperm deep into the uterine horn did not differ (p > 0.05) from that (59.6%) of conventional AI (1 × 10(7) non-sorted sperm) and that of deep insemination with low dose non-sorted sperm (57.7%). There was also no difference (p > 0.05) between conception rates after single (51.7%) and double (53.8%) deep insemination with sorted semen. Heifers inseminated with sorted sperm at synchronous oestrus had a lower pregnancy rate (48.1%) than heifers at spontaneous oestrus (53.6%), but this did not reach statistical difference (p > 0.05). The average number of transferable embryos collected in vivo from heifers inseminated with sorted sperm (4.81 ± 2.04) did not differ (p > 0.05) from that obtained from heifers after insemination with non-sorted sperm (5.36 ± 2.74). Thus, we concluded that the pregnancy rate after deep intra-uterine insemination with low dose sorted sperm was similar to that of non-sorted sperm, which was either also deposited at a low dose deep intra-uterine or into the uterine body. Synchronization of oestrus can be beneficial in combination with sorted sperm to optimize the organization and management of dairy herds. The results from superovulated heifers demonstrated that our insemination regime can be used to obtain a comparable embryo production efficiency with sorted sperm than with non-sorted sperm.

[Chemical constituents of Rosa laevigata Michx].

Six compounds were isolated from Rosa laevigata. Five of them were obtained from the ethanolic extract and identified as 2 alpha, 3 beta, 19 alpha, 23-tetrahydroxyurs-12-en-28-oic acid, 2 alpha, 3 alpha, 19 alpha, 23-tetrahydroxyurs-12-en-28-oic acid, euscaphic acid, beta-sitosterol and daucosterol. The other one was obtained from the acetate of emulsive layer of the petroleum ether and elucidared as 2 alpha, 3 beta-dihydrolup-28-methyl ester diacetate.

Effect of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. leaf and MAPK signal transduction pathway on inducible nitric oxide synthase expression in alveolar macrophage of chronic bronchitis rats.

The goal of this study was to investigate the possible therapy mechanism of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. Leaf (TAL) in alveolar macrophage (AM) of chronic bronchitis (CB) rats. CB model was established by injection of bacillus calmette guein (BCG) plus lipopolisacharide (LPS) in rats. TAL significantly inhibited the increased NO concentration, iNOS expression and phosphorylation of p38 MAPK in alveolar macrophages (AMs) of CB rats. Using in vivo test, we found that SB203580, a p38 MAPK inhibitor, (10 muM) significantly inhibited inducible nitric oxide synthase (iNOS) mRNA expression in AM. This data indicate that TAL highly decreases excessive iNOS expression and NO induction, and p38 MAPK signal transduction participates in iNOS expression and NO induction in AM of CB rats. The effect of TAL on iNOS expression in AM may be related to its inhibition of p38 MAPK signal transduction.

Fasting regulates the expression of adiponectin receptors in young growing pigs.

Adiponectin is an adipocyte-derived hormone that can improve insulin sensitivity. Its functions in regulating glucose utilization and fatty acid metabolism in mammals are mediated by 2 subtypes of adiponectin receptors (AdipoR1 and AdipoR2). This study was conducted to determine the effect of fasting on the expression of adiponectin and its receptors. The expression of adiponectin was not affected in s.c. adipose tissue, but adiponectin expression increased in visceral adipose tissue after fasting. In contrast, expression of both AdipoR mRNA was increased in the liver and s.c. adipose tissue of 24-h-fasted pigs compared with fed pigs, but the mRNA in muscle and visceral adipose tissue was not affected by fasting. A third putative adiponectin receptor, T-cadherin, was cloned and the mRNA expression was determined. T-Cadherin has been recognized to act as a vascular adiponectin receptor in vascular endothelial and smooth muscle cells. Our data showed that the expression of T-cadherin was decreased in the muscle of fasted pigs, suggesting that the expression of T-cadherin can be regulated by feeding status. In summary, in young pigs, adiponectin mRNA was up-regulated by fasting in visceral, but not s.c., adipose tissue, whereas AdipoR1 and AdipoR2 mRNA were increased in s.c., but not visceral, adipose tissue. The adiponectin receptor, T-cadherin, was expressed in s.c. and visceral adipose tissue and in muscle, but only muscle mRNA expression was decreased by fasting.

Three major urinary metabolites of sinomenine in rats.

Urinary metabolites of sinomenine were investigated in rats after intragastric administration. Three major metabolites were obtained and characterised as 4-hydroxy-3,7,7-trimethoxy-17-methyl-(9alpha,13alpha,14alpha)-morphinan-6-one (1), 7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methyl-N-oxide-(9alpha,13alpha,14alpha)-morphinan-6-one (2), and 7,8-didehydro-4-hydroxy-3,7-dimethoxy-(9alpha,13alpha,14alpha)-morphinan-6-one (3). Their structures have been elucidated on the base of spectral analysis, among which 1 and 2 were new compounds.

Effect of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. leaf on inflammatory cytokine and mediator induction from alveolar macrophages of chronic bronchitic rats.

OBJECTIVE: The study was to evaluate the effect of triterpene acids of Eriobotrya japonica (thunb.) lindl. leaf (TAL) on inflammatory cytokine and mediator expression in alveolar macrophages (AM) of chronic bronchitic (CB) rats. METHODS: CB was induced by endotracheal instillation of lipopolysaccharide (LPS) followed by Bacillus Calmette Guerin (BCG) injection via the caudal vein one week later. Treatment groups received TAL at there different doses (50, 150, or 450 mg/kg daily i. g.), Ketotifen fumarate (5 mg/kg daily i. g.) or dexamethasone (1.2 mg/kg daily i. g.) for two weeks, 7 days after LPS injection. AM were then isolated and incubated for 24 h. IL-1, TNF-alpha and PGE2 levels in cultured supernatants were measured by thymocyte co-stimulating assay and radioimmunoassay. Immunocytochemistry staining and western-blot were used for intracellular location and activation of p65 subunit of NF-kB. LTB(4) level was analyzed by reverse-phase high performance liquid chromatography (RP-HPLC). RESULTS: The levels of TNF-alpha, IL-1, NF-kB, PGE2 and LTB(4) expression in AM of TAL groups were significantly decreased compared to the CB group (P < 0.05 or P < 0.01), in a dose dependent manner. CONCLUSION: TAL inhibited NF-kB activation in AM from CB rats and led to down regulation of TNF-alpha, IL-1, PGE(2) and LTB(4) expression, which might be a mechanism for its anti-inflammatory effects in CB rats.

Sustained elevation of Epstein-Barr virus antibody levels preceding clinical onset of nasopharyngeal carcinoma.

We have monitored Epstein-Barr virus (EBV) IgA antibody levels of 39 nasopharyngeal carcinoma (NPC) cases for up to 15 years before clinical onset of NPC, and assessed preclinical serologic status of another 68 cases. Our results identify a serologic window preceding diagnosis when antibody levels are raised and sustained. This window can persist for as long as 10 years, with a mean duration estimated to as 37+/-28 months. Ninety-seven of these 107 NPC cases exhibited such a window. Cases that did not may reflect individual antibody response to EBV. Serologic screening at enrollment identified those cases who had already entered the window and became clinically manifested earlier (median=28 months) than those who entered the window after enrollment (median=90 months). The former account for 19 of 21 cases diagnosed within 2 years of screening. Nasopharyngeal carcinoma risk levels among seropositive subjects were also highest during this period. Both prediction rates and risk levels declined thereafter; cases detected at later times were composed of increasing proportions of individuals who entered the serological window after screening. Our findings establish EBV antibody as an early marker of NPC and suggest that repeated screening to monitor cases as they enter this window has considerable predictive value, with practical consequences for cancer treatment.