Down-regulation of the Smad signaling by circZBTB46 via the Smad2-PDLIM5 axis to inhibit type I collagen expression.

BACKGROUND: Abnormal type I collagen (COL1) expression is associated with the development of many cardiovascular diseases. The TGF-beta/Smad signaling pathway and circRNAs have been shown to regulate COL1 gene expression, but the underlying molecular mechanisms are still not fully understood. METHODS: Gain- and loss-of-function experiments were prformed to study the effect of circZBTB46 on the expression of alpha 2 chain of type I collagen (COL1A2). Co-immunoprecipitation assay was performed to observe the interaction between two proteins. RNA immunoprecipitation assay and biotin pull-down assay were performed to observe the interaction of circZBTB46 with PDLIM5. RESULTS: In this study, we investigated the role of circZBTB46 in regulating COL1A2 expression in human vascular smooth muscle cells (VSMCs). We found that circZBTB46 is expressed in VSMCs and that TGF-beta inhibits circZBTB46 formation by downregulating KLF4 expression through activation of the Smad signaling pathway. CircZBTB46 inhibits the expression of COL1A2 induced by TGF-beta. Mechanistically, circZBTB46 mediates the interaction between Smad2 and PDLIM5, resulting in the inhibition of Smad signaling and the subsequent downregulation of COL1A2 expression. Furthermore, we found that the expression of TGF-beta and COL1A2 is decreased, while circZBTB46 expression is increased in human abdominal aortic aneurysm tissues, indicating that circZBTB46-mediated regulation of TGF-beta/Smad signaling and COL1A2 synthesis in VSMCs plays a crucial role in vascular homeostasis and aneurysm development. CONCLUSIONS: CircZBTB46 was identified as a novel inhibitor of COL1 synthesis in VSMCs, highlighting the importance of circZBTB46 and PDLIM5 in regulating TGF-beta/Smad signaling and COL1A2 expression.

Increased CD4+CD25+ regulatory T cells correlate with poor short-term outcomes in hepatitis B virus-related acute-on-chronic liver failure patients.

BACKGROUND: The roles of CD4(+)CD25(+) regulatory T cells (Treg) in chronicity of hepatitis B virus (HBV) infection have been confirmed. We aimed to explore alteration of Treg in patients with HBV-related acute-on-chronic liver failure (ACLF). METHODS: Thirty-two HBV-related ACLF patients, 44 chronic hepatitis B patients, and 41 healthy controls were recruited. We detected frequencies of peripheral Treg and intrahepatic forkhead winged helix transcription factor (Foxp3)(+) cells. Inhibitory activity of Treg was assessed by functional suppression assays. Serum interferon-γ and interleukin-10 were also determined. RESULTS: Peripheral Treg and intrahepatic Foxp3(+) cells were more markedly increased in ACLF than chronic hepatitis B and controls (all p < 0.001), and the Foxp3(+) cells located predominantly in the portal areas. The Treg frequency was positively correlated with HBV DNA load, international normalized ratio, model of end stage liver disease score, and serum interleukin-10 level in ACLF patients. Functional assays in vitro demonstrated that ACLF patients exhibited higher suppressive effects of Treg on proliferations of autologous CD4(+)CD25(-) T cells than controls. On logistic regression, prolonged international normalized ratio and higher peripheral Treg frequency predicted 30-day survival of ACLF. CONCLUSION: The patients with HBV-related ACLF exhibit increased amounts of Treg, of which redistribution from periphery to liver seems to modulate liver inflammation. Higher Treg amounts are associated with more severe liver disease in ACLF, and its level in combination with international normalized ratio may assist prediction of short-term outcomes of HBV-related ACLF.

Improved efficacy by individualized combination therapy with Peg IFN-a 2a and ADV in HBeAg positive chronic hepatitis B patients.

BACKGROUND/AIMS: Monotherapy with pegylated interferon alpha (Peg-IFNa) or adefovir dipivoxil (ADV) to HBeAg-positive chronic hepatitis B (CHB) patients has limited effects. This study aims to evaluate therapeutic efficacy and safety of individualized combination therapy with Peg-IFNa and ADV. METHODOLOGY: HBeAg-positive CHB patients (n=160) were enrolled in this multi-center, prospective, randomized, 'real-life' cohort study, of which received Peg IFNa-2a monotherapy or combination therapy with ADV base on the baseline features and treatment response. RESULTS: At week 24, percentages of ALT normalization, HBV DNA undetectable were both higher in individualized treatment group (ITG, 57.50%, 43.75%) than that in standard treatment group (STG, 40.00%, 27.50%; p=0.027, 0.032). The superiority of HBeAg clearance and seroconversion rates in ITG maintained from treatment termination (63.75%, 56.25%) to 48 weeks follow-up (57.50%, 53.75%). At week 96 the combined response rates were 46.25% in ITG compared with 30.00% in STG (p=0.034). Furthermore, there was no statistically significant difference in relapse rates and adverse events between the two groups. CONCLUSIONS: Individualized combination therapy can achieve higher antiviral response rates. In particular, it can accelerate undetectable HBV DNA and elevate HBeAg clearance/seroconversion rates to a greater degree than Peg-IFNa-2a monotherapy.

[Hairy roots induction of Phellodendron chinense and production of its active constituents].

OBJECTIVE: To introduce the hairy roots of Phellodendron chinense and determine the content of its active constituents. METHOD: Transformed hairy roots of P. chinense were obtained by the transformation of Agrobacterium rhizogenes A4, R1600, ATCC15834 and R1000. RESULT AND CONCLUSION: It was clearly demonstrated that T-DNA of A. rhizogenes Ri plasmid was integrated into the cells of hairy roots by PCR. The content of berberine hydrochlodride, which was determined by HPLC, was higher in hairy roots than that in the axenic plantet and callus.

Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi) gene and characterization of its protein

Chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. We used touchdown PCR to clone the chitinase (Schi) gene from Bacillus thuringiensis serovar sotto (Bt sotto) chromosomal DNA. Our DNA sequencing analysis revealed that the Bt sotto Schi gene consists of an open reading frame (ORF) of 2067 nucleotides with codes for the chitinase precursor. We also found that the putative promoter consensus sequences (the -35 and -10 regions) of the Bt soto Schi gene are identical to those of the chiA71 gene from Bt Pakistani, the chiA74 gene from Bt kenyae and the ichi gene from Bt israelensis. The Schi chitinase precursor is 688 amino acids long with an estimated molecular mass of 75.75 kDa and a theoretical isoelectric point of 5.74, and contains four domains, which are, in sequence, a signal peptide, an N-terminal catalytic domain, a fibronectin type III like domain and a C-terminal chitin-binding domain. Sequence comparison and the evolutionary relationship of the Bt sotto Schi chitinase to other chitinase and chitinase-like proteins are also discussed

[The relationship between hepatic expression, serum level of TGFbeta1 and the hepatic fibrosis in patients with viral hepatitis].

OBJECTIVE: To detect the hepatic tissue and serum level of TGFbeta1 in patients with viral hepatitis, in order to clarify their relationship of the starting, developing of hepatic fibrosis. METHODS: This study included 92 patients with viral hepatitis. Liver puncture was performed in 31 patients. Hepatic collagen staining (Masson's three colors) and TGFbeta1 immunohistochemistry staining of the liver tissue specimens were performed, morphometric quantitative measurements of hepatic histological collagen and TGFbeta1 were made. The serum level of TGFFbeta1 was detected by ELISA. RESULTS: The surface density of hepatic TGFbeta1 increased linearly with the elevation of fibrosis stage (P < 0.05), there were no significant differences between every two groups of G1, G2, G3 and G4 (P > 0.005). There was a closely positive correlation between the levels of TGFbeta1 in hepatic tissue and serum, the coefficient was 0.896 (P < 0.01). The levels of TGFbeta1 in tissue and serum both had positive correlation with hepatic collagen, coefficients were 0.863 and 0.667 (P < 0.001). The level of TGFbeta1 in tissue and serum both had positive correlation with serum levels of PCIII, HA, LN, CIV (P < 0.001). CONCLUSIONS: There was a closely relationship between the levels of TGFbeta1 in hepatic tissue and serum and liver fibrosis. The detection of TGFbeta1 in liver and serum are more sensitive than HA, LN, CIV in early period of hepatic fibrosis.

[Analysis of RAPD markers about sterility gene in cytoplasm-nucleolus interacting type rice mitochondrial DNA].

Two specific fragments OPJ18-1000 and OPJ18-1400 were obtained from mitochondrial DNAs of two rice varieties, II-32 and II you 949, by RAPD analysis. The sequenced lengths of the two differential fragments were 1068 bp and 1481 bp, respectively. The result of Southern hybridization demonstrated that the differential fragments were true. When hybridizing with II-32A, II-32B and II you 949 by OPJ18-1000 probe, we found OPJ18-1000 which had effects on rice II-32A and II you 949 cytoplasmic male sterility in transcription had not the same effects on II-32B. Though RNA dot hybridization was negative, OPJ18-1400 had a conservative domain of seven base pairs DNA sequence: 5'-TTCCCTC-3', which was homologous recombination hotspot domain in atp6 gene. It was reported that 5'-TTCCCTC-3' induced the formation of chimeric gene in mitochondrial DNA and further caused rice male sterility. As a result, the two fragments are new mitochondrial DNA fragments relevant to rice cytoplasm male sterility by homologous analysis and DNA, RNA hybridization.

[Cloning and sequencing of chitinase gene from Bacillus thuringiensis subsp israelensis].

Chitin, a linear homopolymer of N-acetyl-D-glucosamine, is a common constituent of fungal cell walls, exoskeletons of insects, and shells of invertebrates. Thus, chitinase, a chitin hydrolytic enzyme, has become of interest for potential use as biopesticides for controlling insect pests. Using a pair of specific primers, chitinase gene (ichi) was amplified by touchdown PCR from Bacillus thuringiensis subsp israelensis chromosomal DNA, and then subcloned into pGEM-T easy vector. ichi sequence (GenBank Accession Number: AF526379) with a length of 2570 bp included an open reading frame (ORF) of 2067 bp, which contained 688 amino acids with Mr = 75.79 kDa and pI = 5.90. The amino acid sequence of ichi gene shows 97.24%, 97.18%, 97.63% and 63.07% identity to that of Bacillus cereus strain 28-9 chitinase CW, Bacillus cereus CH chitinase B, Bt subsp Mexican chitinase, and Bt subsp pakistani chitinase A71, respectively. It was demonstrated that Ichi contains a signal peptide (46 amino acid residues) and three functional domains: an N-terminal catalytic domain (105 amino acid residues), a fibronectin type III like domain (74 amino acid residues), and a C-terminal chitin-binding domain (40 amino acid residues).

[Miniscale preparation for large plasmid DNA from Bacillus thuringiensis].

Miniprep of low copy number and large plasmid was done from three subspecies of Bacillus thuringiensis. The plasmid was purified using PEG 6000 instead of phenol and chloroform. Experiments demonstrate that consequences from this protocol were stable, the quality and yield of plasmid DNA produced from it have met the standard for many molecular biology experiments.

[Cloning and sequencing of p35 gene from Bombyx mori nuclear polyhedrosis virus strain Shandong].

According to the 5' and 3' untranslated region sequences of p35 gene from Bombyx mori nuclear polyhedrosis virus isolate T3, a pair of primers was designed employing computer analysis. With the primers,polymerase chain reaction (PCR) was performed and a 1080 bp fragment was obtained from BmNPV strain Shandong. Sequencing result showed that it was p35 gene (GenBank accession number: AY157746), and it included an open reading frame of 900 bp, encoding 299 amino acids with Mr=34.91 kDa and pI=6.40. BLAST analysis indicated that there were seven bases and three amino acids difference between p35 from BmNPV strain Shandong and that from BmNPV isolate T3.