Krüppel-like factor KLF9 inhibits chicken intramuscular preadipocyte differentiation.

1. Poultry meat quality is affected by many factors, among which intramuscular fat (IMF) is predominant. IMF content affects tenderness, juiciness and flavour of meat. Krüppel-like transcriptional factors (KLFs) are important regulators of adipocyte differentiation. However, little is known about the KLF9 gene associated with poultry IMF deposition, especially intramuscular adipocyte differentiation.2. Previous work has shown that chicken KLF9 was differentially expressed during adipogenesis of intramuscular preadipocytes differentiation. In this study, the function of KLF9 in chicken intramuscular preadipocytes differentiation was investigated.3. In the chicken preadipocyte differentiation model, KLF9 expression showed a major increase with adipogenic induction. Overexpression of KLF9 down-regulated the expression of the adipogenic marker gene AP2, and impaired triglyceride accumulation. Knockdown of KLF9 in chicken intramuscular preadipocytes increased the expression of PPARG, CEBPA and AP2. In addition, it was proposed that KLF9 may regulate adipogenesis via lncRNAs NONGGAT002209.2, NONGGAT003346.2, NONGGAT000436.2 and NONGGAT006302.2 in chicken.4. The data supported a novel role of KLF9 in regulating chicken intramuscular preadipocyte differentiation. Such findings may contribute to a more thorough understanding of chicken IMF deposition and the improvement of poultry meat quality.

Genetic diversity of mtDNA D-loop sequences in four native Chinese chicken breeds.

1. To explore the genetic diversity of Chinese indigenous chicken breeds, a 585 bp fragment of the mitochondrial DNA (mtDNA) region was sequenced in 102 birds from the Xichuan black-bone chicken, Yunyang black-bone chicken and Lushi chicken. In addition, 30 mtDNA D-loop sequences of Silkie fowls were downloaded from NCBI. The mtDNA D-loop sequence polymorphism and maternal origin of 4 chicken breeds were analysed in this study. 2. The results showed that a total of 33 mutation sites and 28 haplotypes were detected in the 4 chicken breeds. The haplotype diversity and nucleotide diversity of these 4 native breeds were 0.916 ± 0.014 and 0.012 ± 0.002, respectively. Three clusters were formed in 4 Chinese native chickens and 12 reference breeds. Both the Xichuan black-bone chicken and Yunyang black-bone chicken were grouped into one cluster. Four haplogroups (A, B, C and E) emerged in the median-joining network in these breeds. 3. It was concluded that these 4 Chinese chicken breeds had high genetic diversity. The phylogenetic tree and median network profiles showed that Chinese native chickens and its neighbouring countries had at least two maternal origins, one from Yunnan, China and another from Southeast Asia or its surrounding area.

Promoter analysis and tissue expression of the chicken ASB15 gene.

1. This study was conducted to explore the promoter region of the chicken ASB15 gene by detecting the activities of the dual luciferase reporter gene and to assess expression profiles of the ASB15 gene in 10 different tissues from Gushi chickens. 2. Five dual luciferase reporter gene vectors were constructed and transfected into DF1 cells. The activities of recombined plasmids were measured and the core promoter was confirmed by bioinformatic analysis. Total RNA was extracted and the relative expression of the ASB15 gene was examined. 3. Data analysis indicated that the promoter was located from -955 to -212 bp. Results showed that the chicken ASB15 gene was expressed in heart, breast muscle and leg muscle. 4. This study has confirmed the promoter region and the expression profile of the chicken ASB15 gene, which provides a foundation for further exploring its transcriptional regulation and function.

Assessment of correlation between pre-miRNA-1757 polymorphism and chicken performance traits.

Single nucleotide polymorphism in microRNAs (miRNA) may influence their target gene selection and regulation efficiency, leading to animal phenotypic variation. The aim of this study was to evaluate the possible effect of single nucleotide polymorphisms in the miRNA-1757 gene precursor region (pre-mir-1757) on economic-related traits in chicken. Genotyping was performed using Sequenom MassArray® iPLEX GOLD System. Association analysis was performed using SPSS19.0. The data showed that the G/C polymorphism was significantly correlated with semi-evisceration weight, evisceration weight, carcass weight, body weight at 10 weeks of age, shank length at 4 weeks of age, pectoral angle at 8 weeks of age, and body slanting length and pelvis breadth at 12 weeks of age (P < 0.05), and led to the alteration of the RNA secondary structure of pre-mir-1757. Our results provide useful information for further annotation studies of miRNA function.

A novel 31-bp indel in the paired box 7 (PAX7) gene is associated with chicken performance traits.

1. A novel 31-bp indel polymorphism in intron 3 of the chicken paired box 7 (PAX7) gene was identified and genotyped in an F2 resource population of Gushi chicken crossed with Anka broiler to analyse its associations with chicken growth, carcass and meat quality traits. 2. Results showed that the 31-bp indel was significantly associated with body weight at 4, 6, 8, 10, 12 weeks of age and body size indices including shank length, shank girth, body slanting length at 8 and 12 weeks of age. Significant associations were found for carcass weight, semi-evisceration weight, evisceration weight, breast muscle fibre diameter, leg muscle fibre diameter, breast muscle fibre density, while no significant association with leg muscle fibre density was observed. 3. It was concluded that the 31-bp indel in intron 3 of the PAX7 gene was associated with chicken growth, carcass and meat quality traits where the 31-bp deletion had a negative effect on chicken growth and carcass traits and positive effect on meat quality traits.

Deposition rule of yolk cholesterol in two different breeds of laying hens.

The objective of this study was to investigate the deposition rule of yolk cholesterol in Lushi Green-shelled and Silky Fowl layers. A total of 90 layers of each breed were selected at an age of 15 to 51 weeks. Productive performance was recorded on a weekly basis, whereas yolk cholesterol was determined at 4-week intervals from 21 to 51 weeks of age. The average yolk cholesterol content of Silky Fowl layers during the laying period was higher than that of Lushi Green-shelled layers (58.16 and 49.67%, P > 0.05). Yolk cholesterol content decreased at 21 to 31 weeks of the laying period, whereas a non-significant increasing trend was observed during 31 to 51 weeks of laying period. In conclusion, yolk cholesterol content is not only dependent on the age of hen but also the breed of layers.

Identification and abundance of miRNA in chicken hypothalamus tissue determined by Solexa sequencing.

We used Solexa sequencing technology to identify and determine the abundance of miRNAs and compared the characteristics and expression patterns of miRNA of 1-day-old and 36-week-old chicken hypothalamuses. We obtained 17,825,753 and 10,928,745 high-quality reads from 36-week-old and 1-day-old chickens, respectively. Three hundred and seventy-one conserved miRNAs were expressed in both libraries. Among the conserved miRNAs, 22 miRNAs were up-regulated and 157 miRNAs were down-regulated in the 36-week-old chicken hypothalamus tissues. The abundance of sRNAs between 1-day-old and 36-week-old chickens differed considerably. KEGG pathway analysis suggested that the target genes of highly expressed miRNAs in the chicken hypothalamus are associated with metabolism and development. This information on differential expression of miRNAs in the hypothalamus of 1-day-old and 36-week-old chickens will help us understand the molecular mechanisms of metabolism and development.

Two novel SNPs of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene associated with growth and meat quality traits in the chicken.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is rate-limiting for metabolism of cholesterol; it plays an important role in endogenous cholesterol biosynthesis. We used DNA sequencing technology and created restriction site PCR-RFLP to detect HMGCR SNPs in an F(2) resource population of Gushi chicken and Anka broilers. We found a G/T mutation (Gln/His) in exon 17 and a T/C mutation (Pro/Pro) in exon 18. Based on association analysis of these HMGCR polymorphisms in 864 Gushi/Anka F(2) hybrids, these two mutations have significant effects on growth, carcass, meat quality, and lipid concentration.

Association study of polymorphisms inside the miR-1657 seed region with chicken growth and meat traits.

1. Polymorphisms occurring in the seed region of microRNAs (miRNAs) could influence their target gene and lead to phenotypic variation. The purpose of the research was to explore the genetic effects of the rs14934924 (G > A) mutation resident in the conserved seed region of miR-1657 on growth and meat traits of the Gushi-Anka F2 resource population. 2. The NdeI polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and association analysis were used to analyse the polymorphism. 3. The mutation was associated with body weight at 8 weeks of age, shank girth at 12 weeks of age, breast bone length at 12 weeks of age, pelvis breadth at 4 weeks of age and subcutaneous fat thickness (P < 0·05) and was associated with body weight at 4, 6, 10 and 12 weeks of age (P < 0·01). 4. Our results will be a useful resource for a subsequent study in miRNA function, and provide a basis for molecular techniques in chicken breeding.

Polymorphisms of the PNPLA3 gene and their associations with chicken growth and carcass traits.

1. An F(2) resource population of Gushi chickens crossed with Anka broilers was used to investigate the genetic effects of the chicken PNPLA3 gene on growth and adipose accumulation. 2. Associations between three SNPs (g.40006G > T, g.42344T > C and g.42404A > T) and broiler traits were determined using linkage disequilibrium, haplotype construction and association analysis. 3. The g.40006G > T mutation was associated with body weights at 4, 6, 8, 10 and 12 weeks of age, carcass weight, evisceration weight and semi-evisceration weight (P < 0.05). 4. Haplotypes of the g.42344T > C and g.42404A > T mutations were associated with body weight at 12 weeks, carcass weight, evisceration weight, and semi-evisceration weight (P < 0·05) and were associated with significant dominance effects. 5. The results suggest that the PNPLA3 gene may be in linkage with the causative mutation or a QTL controlling growth traits in chickens. In contrast to human studies, the polymorphisms were not associated with fat related traits.

Novel 9-bp indel in visfatin gene and its associations with chicken growth.

1. The effects of polymorphisms of the visfatin gene on growth performance, carcase traits, meat quality and serum variables were investigated in an F(2) resource population of Gushi chickens crossed with Anka broilers. 2. A 9-bp ('TAACCTGTG') insertion/deletion in intron10 of the visfatin gene was found and a total of 964 individuals were genotyped in the resource population. Genotypes (II, ID and DD) were identified based on the 9-bp insertion (allele I) or deletion (allele D). The insertion/deletion polymorphism was used for analysing associations of the gene with growth traits, carcase traits and meat quality traits in 414 F(2) chickens. 3. The DD genotype was not detected in those 66 F(1) chickens and the I allelic frequency (0·724-0·879) was obviously higher than the D allelic frequency (0·121-0·276) in the birds of three generations. 4. The 9-bp insertion/deletion was associated with the traits of 8-week shank length, 12-week shank length, 4-week pectoral angle and pancreas weight. The relationships with other traits: body weight, carcase traits, meat quality traits and serum variables, were not significant. 5. It was concluded that allele D (9-bp deletion) of the visfatin gene had a negative effect on skeletal growth.

CDR3 sequence motifs shared by oligoclonal rheumatoid arthritis synovial T cells. Evidence for an antigen-driven response.

T lymphocytes reactive with as yet undefined joint-localized foreign or autoantigens may be important in the pathogenesis of RA. Molecular studies demonstrating skewed T cell antigen receptor (TCR) variable gene usage and selective expansion of particular T cell clones within the synovial compartment support this view. Based on our recent study documenting selective expansion of V beta 17+ T cells in RA, we have pursued the identification of T cells relevant to the disease process, in an informative patient, by combining molecular analysis of freshly explanted RA synovial tissue V beta 17 TCR transcripts with in vitro expansion of V beta 17+ synovial tissue T cell clones. Peripheral blood V beta 17 cDNA transcripts proved heterogeneous. In contrast, two closely related sequences, not found in the peripheral blood, dominated synovial tissue V beta 17 transcripts, suggesting selective localization and oligoclonal expansion at the site of pathology. CD4+, V beta 17+ synovial tissue-derived T cell clones, isolated and grown in vitro, were found to express TCR beta chain transcripts homologous to the dominant V beta 17 synovial tissue sequences. One clone shares with a dominant synovial tissue sequence a conserved cluster of 4/5 amino acids (IGQ-N) in the highly diverse antigen binding CDR3 region, suggesting that the T cells from which these transcripts derive may recognize the same antigen. These findings have permitted a complete characterization of the alpha/beta TCR expressed by putatively pathogenic T cell clones in RA. Functional analysis suggests that the conserved CDR3 sequence may confer specificity for, or restriction by, the MHC class II antigen, DR4.

Allelic variants of human TCR BV17S1 defined by restriction fragment length polymorphism, single strand conformation polymorphism, and amplification refractory mutation system analyses.

Several human TCR BV gene subfamilies, including BV3, BV14, and BV17S1, are single member genes but are overutilized among activated CD4+ synovial T cells in the rheumatoid arthritis (RA). To define the role of these TCR BV genes in the pathogenesis of disease, it is critical to characterize the genomic organization and the allelic variations of these genes. In this study we describe allelic variations of BV17S1 defined by restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), and amplification refractory mutation system (ARMS) analyses. A single nucleotide replacement (C/T) results in an amino acid substitution (F/L) in the leader and distinguishes BV17S1*1 from BV17S1*2. This nucleotide substitution was found to create a BsmAI restriction enzyme recognition site in BV17S1*2. Therefore genotypic analyses can be performed either by the SSCP or RFLP method. The analyses of 75 unrelated individuals show that the frequency for allele BV17S1*1 is 52.7% and for allele BV17S1*2 is 47.3%. Both alleles are functionally expressed and are distributed within CD4+/CD8+ T cell subsets. Another point mutation in the CDR2 region of BV17S1, which results in the amino acid replacement of Gln by His, originally identified form a cDNA clone, has now been confirmed as an allele by ARMS analysis using genomic DNA preparations and designated to as BV17S1*3. Screening of this CDR2 related variant among normal populations indicates that this is a rare allele (1 of 75). Although this variant may be of functional significance, the genotypic analysis and functional studies are difficult due to the low frequency of BV17S1*3. In an attempt to define a correlation between BV17S1 allelic usage and susceptibility to RA, the germline distribution of BV17S1 alleles *1 and *2 has been examined in a small number of RA patients and no skewed usage has been identified.

Destruction of halogenated hydrocarbons with solvated electrons in the presence of water.

Model halogenated aromatic and aliphatic hydrocarbons and halogenated phenols were dehalogenated in seconds by solvated electrons generated from sodium in both anhydrous liquid ammonia and ammonia/water solutions. The minimum sodium required to completely dehalogenate these model compounds was determined by increasing the Na/substrate ratio until halogen loss was complete. Minimum sodium consumptions were determined in both anhydrous liquid ammonia and with a (5, 20, 50-fold molar excess of water per mole of halide). While more Na was consumed in the presence of water, these dehalogenations were still efficient when a 50-fold water excess was present. Dehalogenation is faster than competiting reactions with water. CCl4 and CH3CCl3 in the presence of a stoichiometric deficiency of sodium produced only CH4 and CH3CH3 and recovered CCl4 or CH3CCl3, respectively. No partially dechlorinated products were detected, indicating dechlorination was diffusion controlled. Na consumption per chlorine removed (as NaCl) was lower than that of Li, K or Ca and this advantage increased in the presence of water. Na consumption was lower using Na chunks instead of a thin Na mirror. Chloroaromatic compounds gave the parent aromatic hydrocarbon and aminated products in anhydrous ammonia but aminated products did not form when water was present.

[Protective effect of tetrandrine on pancreatic islet cells damaged by alloxan in rats].

Tetrandrine (TET, 100 mg/kg) injected intraperitoneally could prevent rats from diabetes induced by alloxan. After 48 h of injection of alloxan, the blood sugar of the preventive group decreased from the control value of 25.46 +/- 1.21 mmol/L to 7.63 +/- 0.44 mmol/L (P < 0.001) while the serum insulin level increased to 11.33 +/- 1.97 microU/ml and the plasma glucagon concentration to 66.85 +/- 5.07 pg/ml respectively from the control group's value of 7.13 +/- 0.45 microU/ml and 90.35 +/- 8.33 pg/ml. In glucose tolerance test, TET 100 mg/kg showed that the abnormal glucose tolerance induced by alloxan could be improved. The blood sugar area under the glucose tolerance curve of the preventive group decreased from the control groups level of 113.28 +/- 5.02 mmol.h/L to 29.45 +/- 1.63 mmol.h/L (P < 0.001). Immunohistochemical observation of islet beta cells confirmed that TET could markedly prevent beta cells from injuries induced by alloxan and there was no obvious change in the appearance of islet beta cells in the preventive group. The above results suggested that TET could protect pancreatic islet beta cells from injuries caused by Alloxan.

Clinical risk factors of delayed gastric emptying in patients after pancreaticoduodenectomy: a systematic review and meta-analysis.

BACKGROUND: The clinical risk factors of delayed gastric emptying (DGE) in patients after pancreaticoduodenectomy (PD) remains controversial. Herein, we conducted a systematic review to quantify the associations between clinical risk factors and DGE in patients after conventional PD or pylorus preserving pancreaticoduodenectomy (PPPD). METHODS: A systematic search of electronic databases (PubMed, EMBASE, OVID, Web of Science, The Cochrane Library) for studies published from 1970 to 2012 was performed. Cohort, case-control studies, and randomized controlled trials that examined clinical risk factors of DGE were included. RESULTS: Eighteen studies met final inclusion criteria (total n = 3579). From the pooled analyses, preoperative diabetes (OR 1.49, 95% CI, 1.03-2.17), pancreatic fistulas (OR 2.66, 95% CI, 1.65-4.28), and postoperative complications (OR 4.71, 95% CI, 2.61-8.50) were significantly associated with increased risk of DGE; while patients with preoperative biliary drainage (OR 0.68, 95% CI, 0.48-0.97) and antecolic reconstruction (OR 0.17, 95% CI, 0.07-0.41) had decreased risk of DGE development. Gender, malignant pathology, preoperative jaundice, intra-operative transfusion, PD vs. PPPD and early enteral feeding were not significantly associated with DGE development (all P > 0.05). CONCLUSIONS: Our findings demonstrate that preoperative diabetes, pancreatic fistulas, and postoperative complications were clinical risk factors predictive for DGE. Antecolic reconstruction and preoperative biliary drainage result in a reduction in DGE. Knowledge of these risk factors may assist in identification and appropriate referral of patients at risk of DGE.

Characterization of human TCR Vbeta gene promoter. Role of the dodecamer motif in promoter activity.

During T-lymphocyte development, the T-cell antigen receptor (TCR) gene expression is controlled by its promoter and enhancer elements and regulated in tissue- and development stage-specific manner. To uncover the promoter function and to define positive and negative regulatory elements in TCR gene promoters, the promoter activities from 13 human TCR Vbeta genes were determined by the transient transfection system and luciferase reporter assay. Although most of the TCR Vbeta gene promoters that we tested are inactive by themselves, some promoters were found to be constitutively strong. Among them, Vbeta6.7 is the strongest. 5'-Deletion and fragmentation experiments have narrowed the full promoter activity of Vbeta6.7 to a fragment of 147 base pairs immediately 5' to the transcription initiation site. A decanucleotide motif with the consensus sequence AGTGAYRTCA has been found to be conserved in most TCR Vbeta gene promoters. There are three such decamer motifs in the promoter region of Vbeta6.7, and the contribution of each such motif to the promoter activity has been examined. Further site-directed mutagenesis analyses showed that: 1) when two Ts in the decamer were mutated, the promoter activity was totally abolished; 2) when two additional nucleotides 3' to the end of decamer were mutated, the promoter activity was decreased to two-thirds of the full level; and 3) when the element with the sequence AGTGATGTCACT was inserted into other promoters, the original weak promoters become very strong. Taken together, our data suggest that the positive regulatory element in Vbeta6.7 should be considered a dodecamer rather than a decamer and that it confers strong basal transcriptional activity on TCR Vbeta genes.

Changes in the positive and negative symptoms of schizophrenic in-patients in China.

Positive and negative symptoms at admission and discharge of 401 unselected schizophrenic patients from four psychiatric hospitals around China were studied. On admission 58% of patients had prominent negative symptoms and the overall severity of negative symptoms was similar to that of positive symptoms; at discharge, negative symptoms were more prevalent and more severe. The severity of negative symptoms was not significantly correlated with duration of illness or with dosage of medication; 48% of first-episode, drug-naive patients had prominent negative symptoms on admission. Negative symptoms responded to standard neuroleptic treatment, but the improvement was less marked than that in positive symptoms (47% v. 80%). The proportion of patients classified as positive type, negative type, and mixed type schizophrenia altered dramatically with treatment. These findings highlight the importance of negative symptoms in the assessment and treatment of both acute and chronic schizophrenia.