Development and characterization of microsatellite markers of the eastern keelback mullet (Liza affinis).

Twenty-four polymorphic microsatellite loci were isolated and characterized for Liza affinis using a (GT)13-enriched genomic library. The number of alleles per locus ranged from 3 to 9, with a mean number of 6.250. The observed and expected heterozygosities ranged from 0.417 to 1.000 and from 0.550 to 0.861, with an average of 0.859 and 0.779, respectively. Deviation from Hardy-Weinberg proportions was detected at three loci. Evidence of null alleles was found at two loci. These markers will be useful in further studies investigating the genetic variation and population structure of this species, and may provide insights into the maintenance and efficient management of eastern keelback mullet resources.

Toll-like receptor (TLR)-2/4 expression in retinal ganglion cells in a high-glucose environment and its implications.

Diabetic retinopathy (DR), a major complication of diabetes mellitus, is the leading cause of adult blindness. The Toll-like receptor (TLR) family is believed to be involved in the pathogenesis and progression of DR. Here, we investigated the expression profiles of TLR-2 and TLR-4 in retinal ganglion cells (RGCs), in an attempt to elucidate the role of these molecules in the etiology of DR. In vitro cultured RGCs were divided into control and high-glucose groups. The mRNA and protein levels of TLR-2, TLR-4, and nuclear factor (NF)-κB were detected by real-time PCR and western blotting. RGCs were further transfected with specific siRNA targeting TLR2/TLR4; the proliferation of transfected RGCs and their tumor necrosis factor (TNF)-α and interleukin (IL)-8 secretory capacity were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and enzyme-linked immunosorbent assays (ELISA), respectively. In a high-glucose environment, TLR-2/4 expression was significantly upregulated in RGCs (while their viability decreased); additionally, NF-κB expression and secretion of TNF-α and IL-8 were significantly increased. Co-silencing of the TLR-2 and TLR-4 genes inhibited NF- κB expression and TNF-α/IL-8 secretion, while increasing the survival rate of RGCs. Therefore, a high-glucose environment can potentiate the expression of TLR-2 and TLR-4 in RGCs, activate the downstream signaling pathway, and increase the secretion of pro-inflammatory factors, thereby aggravating DR.

Effects of choice of month of treatment and parity order on bovine superovulation traits.

In this study, the performance of 300 Changbaishan Black cattle treated for superovulation from June to September was evaluated to determine the optimal conditions and herds for bovine embryo production. Data analysis revealed that cattle treated in July and August had higher numbers of available embryos (NAE), M1 embryos (NM1), and total embryos (NTE), as well as a higher percentage of M1 embryos (PM1). The temperature and precipitation observed during July and August were greater than those seen in the other two months; strong correlations were observed between these traits and the choice of month of treatment. In addition, multiparous cattle showed a better performance, higher NTE, NAE, NM1, and PM1 values, higher percentages of available embryos, and a lower percentage of degenerated embryos. The co-efficient correlation analysis showed that the month chosen for the treatment did not affect the superovulation traits of nulliparous cattle; however, the choice of the month affected multiparous cattle. Multiparous and nulliparous cattle exhibited many significant differences when treated in July and in August. In addition, the superovulatory traits of multiparous cattle, and not the nulliparous cattle, were strongly correlated to the choice of month of treatment. The results suggested that superovulation is more effective during a period with appropriate environmental temperature and humidity, and that multiparous cattle are more suitable for morula production.

Association of a hypoxia-inducible factor-3α gene polymorphism with superovulation traits in Changbaishan black cattle.

This study was designed to examine a single nucleotide polymorphism (SNP) in the HIF-3α gene in three hundred Changbaishan black cattle using PCR-restriction fragment length polymorphism to determine whether there is an association between this SNP and superovulation. The cloning and sequencing results indicate that the polymorphism is due to a point mutation at the 278-bp position in the HIF-3α gene, resulting in 3 genotypes (AA, AB, and BB). Association analysis indicated that the polymorphism has a significant effect on the number of unfertilized embryos (NUE) (P < 0.05) in the cattle. Cattle with genotype BB had a higher NUE than those with genotype AA, but the difference in NUE between AB and AA or BB was not significant. The polymorphism also has a highly significant effect on the number of degenerative embryos (NDE) and the number of total embryos (NTE) (P < 0.01). Genotype BB was associated with a higher NDE than AA, but the difference in NDE between AB and AA or BB was not significant. Genotype BB showed a higher NTE than AA or AB, but the difference in NTE between AA and AB was not significant. No significant conclusions could be drawn with respect to susceptibility to other traits. HIF-3α could serve as a useful biomarker for donor selection, superovulation improvement, and assisted fertility.

Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products.

In this study, we optimized a restriction-ligation-free (RLF) method to save time and cost of constructing multiple plasmids with the same gene insert, and examined the efficacy of RLF on high-throughput multi-plasmid cloning. This method utilizes the precise DNA repair and recombination systems within Escherichia coli, which allows to bypass the in vitro restriction and ligation enzyme reactions commonly included in routine cloning procedures. A homologous arm is linked to the 5'-end of the forward primer used to amplify both the target gene and vector. A different homologous arm is linked to the 5'-end of the reverse primer. Therefore, genes can be cloned into the vectors by homologous recombination after co-transformation of the amplified target gene and the linearized vector, which bear the same homologous arm on either end. More than twenty-four different plasmids were generated by this method, which uses two simple polymerase chain reaction steps. This method is highly efficient in cloning any gene of interest into any vector at any site without sequence constraints, as no restriction and ligation reactions are required.

Isolation and characterization of novel polymorphic microsatellite markers for Lutjanus erythropterus.

We isolated and characterized 22 polymorphic microsatellite loci in the crimson snapper (Lutjanus erythropterus) using a (GT)13-enriched genomic library. We found three to 15 alleles per locus, with a mean of 6.68. The observed and expected heterozygosities ranged from 0.087 to 0.978 and from 0.125 to 0.904, respectively, with averages of 0.576 and 0.650, respectively. Only three loci showed significant deviation from the Hardy-Weinberg equilibrium after Bonferroni correction. Four loci showed evidence for null alleles. These markers will be useful for analyzing the population genetic structure and gene flow of L. erythropterus.

Proton pump inhibitor use and risk of spontaneous bacterial peritonitis in cirrhotic patients: a systematic review and meta-analysis.

We used a meta-analysis approach to investigate the association between proton pump inhibitor (PPI) use and risk of spontaneous bacterial peritonitis (SBP) in cirrhotic patients. We searched Ovid Medline, Embase, and the Cochrane Library to identify eligible studies. We included studies that compared cirrhotic patients who did or did not use PPIs. The primary outcome was SBP, and the secondary outcome was overall bacterial infection. Results were pooled using random-effect models. This process led to identification of 12 journal articles and 5 conference abstracts. The pooled data showed that PPI use in patients with cirrhosis and ascites was significantly associated with an increased risk of SBP [odds ratio (OR) = 2.17; 95% confidence interval (CI) = 1.46-3.23; P < 0.05; I2 = 85.6%] and overall risk of bacterial infection (OR = 1.98; 95%CI = 1.36-2.87; P < 0.05; I2 = 0). Subgroup analysis revealed that journal articles and studies reporting adjusted effect estimates demonstrated that PPI users had a significantly increased risk of SBP (OR = 2.13; 95%CI = 1.61-2.82; P < 0.05; I2 = 29.4%; and OR = 1.98; 95%CI = 1.42-2.77; P < 0.05; I2 = 67%, respectively). In conclusion, PPI use increased the risk of SBP and overall bacterial infection in patients with cirrhosis and ascites. PPIs should be administered after careful assessment of the indications in cirrhotic patients. Future well-designed prospective studies are warranted to clarify the dose relationships and to compare infection risks associated with different classes of PPIs.

Isolation and characterization of microsatellite loci in the purpleback flying squid (Sthenoteuthis oualaniensi).

The purpleback flying squid (Sthenoteuthis oualaniensis) is a pelagic squid with tremendous potential for commercial exploitation. We isolated and characterized 21 polymorphic microsatellite loci for S. oualaniensis using a (GT)13-enriched genomic library. The number of alleles per locus varied from 6 to 32. The observed and expected heterozygosities ranged from 0.188 to 0.890, and 0.537 to 0.968, respectively. No significant linkage disequilibrium was detected at these loci. Five loci significantly deviated from the Hardy-Weinberg equilibrium, and four loci may have exhibited null alleles. These microsatellite markers will facilitate further studies in population genetics and the sustainable utilization of S. oualaniensis.

Expression pattern of JMJD1C in oocytes and its impact on early embryonic development.

Cell reprogramming mediated by histone methylation and demethylation is crucial for the activation of the embryonic genome in early embryonic development. In this study, we employed quantitative real-time polymerase chain reaction (qRT-PCR) to detect mRNA levels and expression patterns of all known histone demethylases in early germinal vesicle stage and in vitro-matured metaphase II (MII) oocytes (which are commonly used as donor cells for nuclear transfer). On screening, the Jumonji domain containing 1C (JMJD1C) gene had the highest level of expression and hence was used for subsequent experiments. We also found that JMJD1C was primarily expressed in the nucleus and showed relatively high levels of expression at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages of embryos developed from MII oocytes fertilized in vitro. Further, we knocked down the JMJD1C gene in MII oocytes using siRNA and monitored the cleavage of zygotes and development of early embryos after in vitro fertilization. The results showed that the zygote cleavage and blastocyst rates of the transfection group were reduced by 57.1 ± 0.07 and 50 ± 0.01% respectively, which were significantly lower than those of the negative control group (P < 0.05). These data suggest that JMJD1C plays a key role in the normal development of early bovine embryos. Our results also provide a theoretical basis for the investigation of the role and molecular mechanism of histone demethylation in the early development of bovine embryos.

Study on the ERCC1 gene polymorphism response to chemotherapy and prognosis of gastric cancer.

We conducted a cohort study to investigate the role of 2 single-nucleotide polymorphisms of the excision repair cross-complimentary group 1 (ERCC1) gene polymorphism in response to chemotherapy and clinical outcomes of gastric cancer. A total of 231 patients with newly diagnosed and histopathologically confirmed primary gastric cancer participated in the study. ERCC1 rs11615 and rs3212986 were genotyped. Individuals with the ERCC1 rs11615 TT genotype and the T allele showed a significant poorer response to chemotherapy compared to the wild-type genotype. Patients carrying the rs11615 TT genotype (22.8 months) and the T allele (24.2 months) showed a significantly shorter median survival time when compared with the GG genotype (33.7 months). Cox proportional hazard regression analysis showed that adjusted hazard ratios of overall survival in those carrying the rs11615 TT genotype and the T allele were 2.79 (1.15-7.26) and 1.84 (1.19-2.87) when using the wild-type genotype as a reference variable. In conclusion, this study reports that the ERCC1 rs11615 TT polymorphism can be used as a prognostic marker to determine the clinical outcome of gastric cancer patients treated with 5-fluorouracil-based chemotherapy.

Surgical management of biliary cystadenoma and cystadenocarcinoma of the liver.

Biliary cystadenoma (BCA) and biliary cystadenocarcinoma (BCAC) are rare biliary duct neoplasms. This study investigated reasonable management strategies of cystic neoplasms in the liver. Charts of 39 BCA/BCAC patients (9 males, 30 female; median age 53.74 ± 14.50 years) who underwent surgery from January 1999 to December 2009 were reviewed retrospectively. Cyst fluid samples of 32 BCA/BCAC patients and 40 simple hepatic cyst patients were examined for the tumor markers carbohydrate associated antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA). The most frequent symptoms were abdominal pain (N = 10), abdominal mass (N = 7), abdominal distension (N = 4), jaundice (N = 2), and fever (N = 3); the remaining patients showed no clinical symptoms. Liver resection (N = 17) or enucleation (N = 22) was performed in the 39 patients. Ultimately, 35 patients were diagnosed with intrahepatic BCA and four patients were diagnosed with BCAC. The median CA19-9 level was significantly higher in BCA/BCAC patients than in simple hepatic cyst patients. The median CEA levels in BCA/BCAC patients and controls were 6.83 ± 2.43 and 4.21 ± 2.91 mg/L, respectively. All symptoms were resolved after surgery, and only one BCAC patient showed recurrence. The incidence of intrahepatic cystic lesions was 1.7%. Increased CA19-9 levels in the cyst fluid is a helpful marker for distinguishing BCA/BCAC from common simple cysts. The presence of coarse calcifications is suggestive of BCAC. Complete surgical removal of these lesions yielded satisfying long-term outcomes with a very low recurrence rate.

Polymorphic microsatellite loci for the crimson snapper (Lutjanus erythropterus).

We isolated and characterized 22 polymorphic microsatellite loci in Lutjanus erythropterus using a (GT)13-enriched genomic library. We found between 2 and 8 alleles per locus, with a mean of 4.85. The observed and expected heterozygosities ranged from 0.065 to 0.867 and from 0.085 to 0.832, respectively, with means of 0.461 and 0.529, respectively. Allele frequencies in three loci were found to deviate from Hardy-Weinberg equilibrium. Evidence for null alleles was found for three loci. These markers will be useful for distinguishing released captive-bred L. erythropterus individuals from wild individuals.

Polymorphic microsatellite loci isolated from the yellowbelly threadfin bream, Nemipterus bathybius.

Twenty-two polymorphic microsatellite loci were isolated and characterized from a (GT)13-enriched Nemipterus bathybius genomic library. The number of alleles per locus ranged from 4 to 13, with an average of 7.86. The observed and expected heterozygosity was 0.167-0.889 and 0.278-0.904, respectively, with averages of 0.590 and 0.690. Three loci deviated from Hardy-Weinberg proportions, and 2 loci showed evidence of null alleles. No significant linkage disequilibrium was detected in the pairwise comparisons among the 22 loci. These markers are expected to be useful for the population genetic analysis of N. bathybius.

Identification and assessment of differentially expressed genes involved in growth regulation in Apostichopus japonicus.

Rapid and efficient growth is a major consideration and challenge for global mariculture. The differential growth rate of the sea cucumber, Apostichopus japonicus, has significantly hampered the total production of the industry. In the present study, forward and reverse suppression subtractive hybridization libraries were constructed and sequenced from a fast-growth group and a slow-growth group of the sea cucumber. A total of 142 differentially expressed sequence tags (ESTs) with insertions longer than 150 bp were identified and further analyzed. Fifty-seven of these ESTs (approximately 40%) were functionally annotated for cell structure, energy metabolism, immunity response, and growth factor categories. Six candidate genes, arginine kinase, cytochrome c oxidase subunit I, HSP70, ß-actin, ferritin, and the ADP-ribosylation factor, were further validated by quantitative PCR. Significant differences were found between the fast- and slow-growth groups (P < 0.05) for the expression levels of arginine kinase, cytochrome c oxidase, HSP70, the ADP-ribosylation factor, and ß-actin. However, no significant difference was observed for ferritin. Our results provide promising candidate gene markers for practical size screening, and also further promote marker-assisted selective breeding of this species.

Alteration of Phascolosoma esculenta heat shock protein 90 expression under heavy metal exposure and thermal stress.

The full-length complementary DNA (cDNA) of heat shock protein 90 was cloned from Phascolosoma esculenta (PeHSP90) using expressed sequence tag and rapid amplification of cDNA end approaches. The cDNA of PeHSP90 was 2521 bp including a 5'-untranslated region of 110 bp, a 3'-untranslated region of 230 bp, and an open reading frame of 2181 bp. All of the characteristic motifs of the HSP90 family were completely conserved in the deduced amino acid of PeHSP90. The expression of PeHSP90 was induced by 3 heavy metals or elevated temperature, under which Zn²âº displayed effects were more toxic than those of Cd²âº and Cu²âº. The polyclonal antibodies generated from the recombinant product of PeHSP90 were specifically identified not only in the recombinant product but also in the native protein from hemocytes. These results strongly suggested that PeHSP90 was involved in heavy metal challenge and thermal stress regulation in P. esculenta.