WT1 Inhibits Human Renal Carcinoma Cell Proliferation and Induces G2/M Arrest by Upregulating IL-24 Expression.

The transcription factor Wilms' tumor 1 (WT1) is involved in development, tissue homeostasis, and disease. However, the exact roles and the mechanisms of WT1 in renal carcinoma are not well understood. Therefore, in this study, we evaluated the ability of WT1 to block proliferation in renal carcinoma cells in vitro. Experimental analysis showed that WT1 overexpression inhibited the proliferation of renal carcinoma A498 cells and promoted arrest at the G2/M checkpoint. RNA-Seq identified differentially expressed genes, including IL-24, related to both the cell proliferation and the cell cycle. WT1 overexpression upregulated IL-24 expression, and IL-24 overexpression induced G2/M arrest. ChIP-Seq identified JUN as a direct target of WT1 in A498 cells, in which positive regulation was shown by RT-qPCR. It has been shown that the transcription factor JUN can regulate IL-24 expression, and therefore, we hypothesize that WT1 might regulate the IL-24 through JUN. Furthermore, analysis based on TCGA datasets showed that the expression of WT1-regulated genes, including TXNIP and GADD45A, was significantly correlated with the stage and histological grade of tumors, with high levels linked to favorable prognoses. Our results demonstrated that the overexpression of WT1 upregulates IL-24, leading to G2/M checkpoint arrest to reduce proliferation. These results indicate that regulation of IL-24 by WT1 inhibits proliferation and may represent a potential target for treating renal carcinoma.

A novel single-nucleotide polymorphism of the visfatin gene and its associations with performance traits in the chicken.

Visfatin is a peptide that is predominantly expressed in visceral adipose tissue and is hypothesized to be related to obesity and insulin resistance. In this study, a novel silent single-nucleotide polymorphism (SNP) was found in exon 7 of the chicken visfatin gene (also known as PBEF1) by single-stranded conformation polymorphism (SSCP) and DNA sequencing. In total, 836 chickens forming an F2 resource population of Gushi chicken crossed with Anka broiler were genotyped by XbaI forced RFLP, and the associations of this polymorphism with chicken growth, carcass characteristics, and meat quality were analyzed. Significant associations were found between the polymorphism and 4-week body weight (BW4), 6-week body weight (BW6), 4-week body slanting length (BSL4), fat bandwidth (FBW), breast muscle water loss rate (BWLR) and breast muscle fiber density (BFD) (P < 0.05), as well as 4-week breastbone length (BBL4) (P < 0.01). These observations suggested that the polymorphism in exon7 of the visfatin gene had significant effects on the early growth traits of chicken.

Two novel SNPs in the coding region of the bovine PRDM16 gene and its associations with growth traits.

As a zinc-finger protein, PR domain containing 16 (PRDM16) controls brown fat determination by stimulating brown fat-selective genes expression while suppressing the expression of genes selective for white fat cells, whose mutations were associated with myelodysplastic syndrome (MDS) and leukemogenesis in human and murine model of leukemia. To date, no polymorphisms of PRDM16 gene in bovine had been reported. Herein, PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation within PRDM16 gene in 1031 Chinese indigenous bovine. The results revealed two novel silent mutations: XM_001788152: m.1641T>C (547aa), 1881G>A (627aa). Hence, we described the PvuII and HaeIII forced PCR-RFLP methods for detecting these mutations, respectively. In the forced PCR-RFLP analysis with PvuII, the frequencies of bovine PRDM16-C allele varied from 0.044 to 0.506 in four Chinese native breeds. In the forced PCR-RFLP analysis with HaeIII, the frequencies of bovine PRDM16-G allele were 0.474, 0.494, 0.576 and 0.906 for Jiaxian (JX), Nanyang (NY), Qinchuan (QC) and Chinese Holstein (CH) population. Significant statistical differences between genotypic frequencies implied that both of the polymorphic loci were significantly associated with cattle breeds by the chi square test (chi2 = 190.058, P < 0.001 and chi2 = 118.239, P < 0.001 for PvuII; chi2 = 209.842, P < 0.001 and chi2 = 108.711, P < 0.001 for HaeIII). The associations of the PvuII and HaeIII forced PCR-RFLPs of bovine PRDM16 loci with growth traits were analyzed in Nanyang breed. The two SNPs were associated with body weight and average daily gain in Nanyang aged 12 months, individuals with genotype TT and AA showed significantly better body weight (P < 0.05) and average daily gain (P < 0.01) at 12 months, respectively.

A coding SNP of LHX4 gene is associated with body weight and body length in bovine.

Heterozygous mutations in LHX4 are associated with combined pituitary hormone deficiency. In this study, the polymorphism of LHX4-HaeIII locus was revealed in 822 individuals from four Chinese cattle breeds. The PCR-RFLP analysis showed that there were three genotypes: GG, GA, AA. The frequencies of genotype GG ranged from 0.6620 to 0.9789 in analyzed populations. The genotypic frequencies of LHX4 locus in the four populations all agreed with Hardy-Weinberg equilibrium (P > 0.05). Distributions of genotypic frequencies of different breeds (QC, NY, JX, CH) at this locus were found to be significantly different based on a chi(2) test (P < 0.001). The genetic diversity analysis revealed the JX cattle possessed intermediate genetic diversity, and the other three Chinese cattle breeds belonged to poor genetic diversity. Correlation analysis with growth traits in the NY breed indicated that: the animals with genotype GA had greater body weight than those with genotype GG (P < 0.05); the animals with GA genotype owned significantly longer body length than the ones with GG genotype (P < 0.05) at 18 and 24 months.

Analysis of caprine pituitary specific transcription factor-1 gene polymorphism in indigenous Chinese goats.

Since mutations on POU1F1 gene possibly resulted in deficiency of GH, PRL, TSH and POU1F1, this study revealed the polymorphism of goat POU1F1-AluI locus and analyzed the distribution of alleles on 13 indigenous Chinese goat breeds. The PCR-RFLP analysis showed the predominance of TT genotype and the frequencies of allele T varied from 0.757 to 0.976 in the analyzed populations (SBWC, Bo, XH and HM). Further study, distributions of genotypic and allelic frequencies at this locus were found to be significantly different among populations based on a chi(2)-test (P < 0.001), suggesting that the breed factor significantly affected the molecular genetic character of POU1F1 gene. The genetic diversity analysis revealed that Chinese indigenous populations had a wide spectrum of genetic diversity in goat POU1F1-AluI locus. However, the ANOVA analysis revealed no significant differences for gene homozygosty, gene heterozygosty, effective allele numbers and PIC (polymorphism information content) among meat, dairy and cashmere utility types (P > 0.05), suggesting that goat utility types had no significant effect on the spectrum of genetic diversity.

Purification and characterization of a thermal stable antimicrobial protein from housefly larvae, Musca domestica, induced by ultrasonic wave.

This work describes the induction, purification and partial biochemical characterizations of an antimicrobial protein from the housefly larvae induced by ultrasonic wave. It has been purified to apparent homogeneity by ammonium sulfate precipitation followed by Sephadex G-75, Bio-gel P6 gel filtration, and CM-Sepharose Fast Flow cation exchange chromatography. The protein is a cationic protein with an apparent molecular weight of 16315 Da determined by no-denaturing electrophoresis and SDS-PAGE, respectively. Biochemical profile assays show that this protein has good thermal stability, and repeatedly frozen and defrosted durability. The optimum pH for antimicrobial activity is around pH5. The antimicrobial range of the protein includes Gram-positive, Gram-negative bacteria and some fungi. Results of the membrane permeability assays suggest that the probable mode of action of this protein is membrane-disrupting mechanism.

Three novel single-nucleotide polymorphisms of the bovine LHX3 gene.

The LHX3 gene encodes LIM homeodomain class transcription factors that have important roles to play in pituitary and nervous system development.On the one hand,mutations of LHX3 are associated with deficiencies of growth hormone (GH),prolactin (PRL),luteotrophic hormone (LH),follicle-stimulating hormone (FSH)and thyroid- stimulating hormone (TSH);on the other hand,mutations of LHX3 are also associated with combined pituitary hormone deficiency (CPHD) diseases in human and animal models.To date,few polymorphisms of the bovine LHX3 gene have been reported.In this study,polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)and DNA sequencing methods were employed to screen the genetic variations within the bovine LHX3 gene in 802 Chinese indigenous cattle.The results revealed three novel single-nucleotide polymorphisms (SNPs): AY923832: g.7553G >A, 7631C>T and 7668C>G.Among them,a synonymous mutation of exon II was identified: GAG ((Glu)> GAA (Glu) at position 72 aa (AY923832:g.7553G>A) of LHX3 ((403aa) in the four Chinese bovine breeds.Significant statistical differences in genotypic frequencies for exon II and its flanking region of the LHX3 gene implied that the polymorphic locus was significantly associated with cattle breeds by the X2-test (X2=68.975,df=6, P <0.001).Hence,the three novel SNPs not only extend the spectrum of genetic variations of the bovine LHX3 gene, but could also possibly contribute to conducting association analysis and evaluating these as genetic markers in bovine breeding and genetics,and CPHD detection.

Studies on the antibacterial activities and mechanisms of chitosan obtained from cuticles of housefly larvae.

Chitosan was obtained from cuticles of the housefly (Musca domestica) larvae. Antibacterial activities of different Mw chitosans were examined against six bacteria. Antibacterial mechanisms of chitosan were investigated by measuring permeability of bacterial cell membranes and observing integrity of bacterial cells. Results show that the antibacterial activity of chitosan decreased with increase in Mw. Chitosan showed higher antibacterial activity at low pH. Ca2+ and Mg2+ could markedly reduce the antibacterial activity of chitosan. The minimum inhibitory concentrations of chitosans ranged from 0.03% - 0.25% and varied with the type of bacteria and Mw of chitosan. Chitosan could cause leakage of cell contents of the bacteria and disrupt the cell wall.