Biomedical subjects

Bao Zhang

Publications and source records attributed to Bao Zhang.

At least 19 recordsLinked to original sources

PubMed2026

NS2A V89F mutation in a DENV1 clinical isolate enhances neurotropism and neuroinvasion.

INTRODUCTION: Dengue virus (DENV) neurological complications are increasingly reported, yet the viral genetic determinants of neurotropism remain poorly characterized. METHODS: We screened 25 DENV1 clinical isolates from the 2014 outbreak in Guangdong, China, for neurotropism in suckling mice, and integrated comparative genomics, pre-expression functional assays, population-scale sequence analysis, and OpenFold3 structural modeling to identify mutations associated with enhanced neuroinvasion. RESULTS: We found that only strain P1253 induced neurological symptoms and mortality via subcutaneous inoculation, producing cortical-selective lesions distinct from the diffuse encephalitic damage observed after intracranial inoculation, and P1253 replicated preferentially in human brain microvascular endothelial cells (HBMEC) compared to contemporaneous strains. Comparative genomics identified three unique mutations in P1253 (NS1 175Y→H, NS2A 89V→F, NS4A 2V→I), and pre-expression assays demonstrated that only NS2A 89V→F significantly enhanced viral replication and cytopathic effect in HBMEC. Analysis of 1,990 complete DENV1 genomes revealed five natural mutant types in the NS2A 89 -96 residue region, with P1253 representing the FIPI quadruple-mutant type, and OpenFold3 structural prediction showed that 89V→F introduced on the VIPI background induced the most significant distal domain reorientation (RMSD 1.605 Å), increasing the centroid-to-centroid distance between residues 89 -96 and 185 -218 from 18.221 Å to 27.462 Å. DISCUSSION: These findings identify NS2A 89V→F as a candidate adaptive mutation associated with enhanced neurotropism in DENV1 and provide a framework for monitoring neurovirulent variants.

Dengue Virus↗

PubMed2025

The MTORC1 signaling pathway related gene POLR3G serves as a potential prognostic biomarker in Hepatocellular Carcinoma.

This study aims to investigate the prognostic significance and potential biological functions of the MTORC1 signaling pathway-associated gene POLR3G in Hepatocellular carcinoma (HCC). A prognostic risk model for HCC was developed by integrating HCC-related datasets and associated clinical data obtained from The Cancer Genome Atlas (TCGA) database. The GSVA website was employed to analyze the model genes across pan-cancer datasets, focusing on copy number variations (CNV), single nucleotide variations (SNV), methylation differences, drug sensitivity and immune cell infiltration profiles. Subsequently, we examined the expression levels and prognostic significance of POLR3G in HCC. Utilizing Spearman correlation analysis, we identified genes associated with POLR3G. Furthermore, Gene Set Enrichment Analysis (GSEA) was employed to elucidate the potential signaling pathways in which POLR3G may be involved. The relationship between POLR3G expression and immune cell abundance in HCC samples was assessed using the ssGSEA algorithm. Finally, the impact of POLR3G on HCC cell proliferation was validated through CCK-8 and EDU cell proliferation assays. Through univariate Cox regression analysis and LASSO regression analysis, we established a prognostic risk model for HCC comprising 13 genes. The analysis revealed that individuals categorized in the low-risk group had a markedly improved overall survival probability relative to those in the high-risk group. POLR3G exhibited a markedly elevated expression in HCC tissues when compared to adjacent normal tissues. The expression of POLR3G was correlated with tumor grade, and elevated POLR3G expression was associated with poor prognosis in HCC patients. Furthermore, the expression level of POLR3G was found to be correlated with the level of immune cell infiltration. Knockdown of POLR3G significantly inhibited the proliferative capacity of hepatocellular carcinoma cells. The findings suggest that POLR3G may serve as a potential biomarker influencing the prognosis of hepatocellular carcinoma patients by modulating the tumor immune microenvironment.

Humans↗

PubMed2007

mtDNA diversity and genetic lineages of eighteen cattle breeds from Bos taurus and Bos indicus in China.

In order to clarify the origin and genetic diversity of indigenous cattle breeds in China, we carried out phylogenetic analysis of representatives of those breeds by employing mitochondrial gene polymorphism. Complete cyt b gene sequences, 1140 bp in length, were determined for a total of 136 individuals from 18 different breeds and these sequences were clustered into two distinct genetic lineages: taurine (Bos taurus) and zebu (Bos indicus). In analysis of the cyt b gene diversity, Chinese cattle showed higher nucleotide (0.00923) and haplotype diversity (0.848) than the reports from other studies, and the animals from the taurine lineage indicated higher nucleotide diversity (0.00330) and haplotype diversity (0.746) than the ones from the zebu lineage (0.00136; 0.661). The zebu mtDNA dominated in the southern breeds (63.3-100%), while the taurine dominated in the northern breeds (81.8-100%). Six cattle breeds from the central area of China exhibited intermediate frequencies of zebu mtDNA (25-71.4%). This polymorphism revealed a declining south-to-north gradient of female zebu introgression and a geographical hybrid zone of Bos taurus and Bos indicus in China.

Animals↗

PubMed2006

Neuroprotective effect of A20 on TNF-induced postischemic apoptosis.

Focal cerebral ischemia causes apoptosis in neural cells during the postischemia period. TNF is critically involved in such neuronal apoptosis mediated by caspase pathways. A20 can inhibit TNF-induced apoptosis in many cell types. However, little work has been carried out in central nervous system. In the present study, gene transfer of A20 resulted in reduction of infarct volume and improvement of neurological deficit in ischemia rats. Results of flow cytometry, TUNEL and DNA fragmentation assay all indicated A20 could inhibit TNF-induced apoptosis both in primary rat hippocampal neurons and SH-SY5Y cells. Moreover, we found A20 targeted the TNF apoptotic pathway by inhibiting proteolytic cleavage of caspase 8 and 3 in SH-SY5Y cells. These data demonstrated A20 could effectively protect neurons from postischemic apoptosis and may function partly on death receptor caspase pathway. Gene transfer of A20 may be a promising approach to gene therapy for cerebral ischemia in the future.

Animals↗

PubMed2006

[Preparation of small interfering RNA expression cassette based on PCR technique].

OBJECTIVE: To modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference. METHODS: The U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference. RESULTS: The sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection. CONCLUSION: The siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.

Gene Silencing↗

PubMed2006

[Encapsulating hepatocytes with chitosan in physiological conditions].

Prepared from 15.3% N-acetylated chitosan (FNC), half N-acetylated chitosan (HNC) possesses a good solubility in a weak basic solution, guaranteeing the formation of microcapsules by the coacervating reaction between HNC and methacrylic acid (MAA)-hydroxyethyl methacrylate (HEMA)-methyl methacrylate (MMA) (MAA-HEMA-MMA) terpolymer under physiological conditions. When hepatocytes were encapsulated in such 3-dimensional microenvironment, as compared to monolayer culture, cell functions, including P450 activity, urea production and albumin release, were well supported. The prepared microcapsules have good mechanical stability and permeability.

Animals↗

PubMed2005

Application of restriction display PCR technique in the preparation of cDNA microarray probes.

AIM: To develop a simplified and efficient method for the preparation of hepatitis C virus (HCV) cDNA microarray probes. METHODS: With the technique of restriction display PCR (RD-PCR), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and re-amplified by RD-PCR. We separated the differential genes by polyacrylamide gel electrophoresis and silver staining. Single bands cut out from the polyacrylamide gel were isolated. The third-round PCR was performed using the single bands as PCR template. The RD-PCR fragments were purified and cloned into the pMD18-T vector. The recombinant plasmids were extracted from positive clones, and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting RD-PCR products to the surface of amino-modified glass slides using a robot. We validated the detection of microarray by hybridization and sequence analysis. RESULTS: A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced, which were the specific gene fragments of HCV. These fragments could be further used as probes in microarray preparation. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The results of hybridization and sequence analysis showed that the specificity, sensitivity, accuracy, reproducibility, and linearity in detecting HCV RNA were satisfactory. CONCLUSION: The RD-PCR technique is of great value in obtaining a large number of size-comparable gene probes, which provides a speedy protocol in generating probes for the preparation of microarrays. Microarray prepared as such could be further optimized and applied in the clinical diagnosis of HCV.

DNA Probes↗

PubMed2005

Improving alachlor biodegradability by ferrate oxidation.

Alachlor can be recalcitrant when present at high concentrations in wastewater. Ferrate oxidation was used as a pretreatment to improve its biodegradability and was evaluated by monitoring alachlor elimination and removal of COD(Cr) (chemical oxygen demand determined by potassium dichromate) during the oxidation process up to a value compatible with biological treatment. Ferrate oxidation resulted in elimination of alachlor followed by degradation of its intermediates. High pH suppressed alachlor removal and COD(Cr) removal due to the low redox potential of ferrate ions. Although alachlor can be totally eliminated within 10 min under optimized conditions (alachlor, 40 mg l(-1); ferrate:alachlor molar ratio, 2; and pH 7.0), its complete mineralization cannot be achieved by ferrate oxidation alone. Alachlor solution treated by ferrate for 10 min inhibited an up-flow biotreatment with activated sludge. The biodegradability of ferrate-pretreated solution improved when the treatment was increased to 20 min, at the point of which BOD(5)/COD(Cr) ratio of the treated solution was increased to 0.87 from 0.35 after 10 min treatment. Under optimized conditions, ferrate oxidation for 20 min resulted in total elimination of alachlor, partial removal of COD(Cr) and the ferrate-treated solution could be effectively treated by the up-flow activated sludge process.

Acetamides↗

PubMed2005

Characterization of amphoteric multilayered thin films by means of zeta potential measurements.

Multilayer films of amphoteric methylated collagen were assembled on SOURCE 15S or SOURCE 15Q beads by sequential electrostatic deposition with negatively charged methylacrylic acid-hydroxyethyl methacrylate-methyl methacrylate (MAA-HEMA-MMA) terpolymer. Methylated collagen and terpolymer were deposited under conditions where they were oppositely charged to one another, thereby facilitating growth of the films through electrostatic interactions. Measurements revealed alternating positive and negative zeta-potential with the deposition of each methylated collagen and terpolymer layer, respectively. Assembly pH had a remarkable influence on zeta-potential of the assembled multilayers and the deposition of methylated collagen will be frustrated when the assembly pH is up to 9.0. In addition, ionic strength (NaCl concentration) showed an intricate effect on zeta-potential of the films of amphoteric methylated collagen.

Collagen↗

PubMed2005

Growth inhibition of K562 cells by cyclin E gene-specific small interfering RNA.

OBJECTIVE: To study the inhibitory effect of small interference RNA(siRNA) of cyclin E gene on the growth of K562 cells. METHODS: siRNA targeting the 940 bp site of the cyclin E mRNA were designed and generated by PCR amplification. The PCR products containing U6 promoter and the siRNA were then transfected into K562 cells via Lipofectamine2000. The cells transfected with non-functional siRNA served as the negative control group and those only treated with serum-free RPMI1640 as the blank control group. Cell counting, reverse transcriptase (RT)-PCR and flow cytometry were employed to evaluate the effect of RNA interference. RESULTS: Compared with the negative and blank control groups, the viable cell count in the interference group was decreased by approximately 80%, the ratio of G(1)-phase cells increased by nearly 30%, and growth arrest was observed. Cyclin E mRNA expression in the cells of the interference group was significantly lowered by about 70%; as compared with that of the negative and blank control groups, whereas the latter two groups had similar expression levels. CONCLUSION: RNA interference induces obvious inhibition of cyclin E gene expression, which consequently affects the proliferation of K562 cells.

Cell Proliferation↗

PubMed2005

[Construction and preliminary identification of subtracted cDNA library of leukemia cell line K562].

OBJECTIVE: Subtractive hybridization technology is a common method to screen and clone differentially expressed genes. This study was to construct subtracted cDNA library of leukemia cell line K562, and screen for differentially expressed genes. METHODS: cDNA fragments of K562 cells (tester), prepared by restriction display (RD), were subtracted with the Sau3A I-digested cDNA fragments of normal lymphocytes (driver). The subtracted cDNA fragments were re-amplified, and cloned into pMD18-T vectors. Positive clones were selected by blue-white screening. The inserts in plasmid were amplified by polymerase chain reaction (PCR), and some of which were sequenced. RESULTS: The subtracted library contained 360 positive clones with cDNA fragments distributed mainly from 200 to 800 bp. The 50 randomly sequenced clones were derived from 42 known genes. CONCLUSION: Specific subtracted cDNA library of K562 cells was successfully constructed with reliable quality, and may be used to further screen and clone differentially expressed genes of K562 cells.

Cloning, Molecular↗

PubMed2005

[c-myc gene silencing in K562 cells with RNA interference].

OBJECTIVE: To study the inhibitory effect of small interfering RNA (siRNA) targeting c-myc gene in K562 cells. METHODS: siRNAs targeting the site 1357 of c-myc mRNA was designed and synthesized. In vitro cultured K562 cells were transfected with lipofectamine 2000 and the inhibitory effect was detected by reverse transcriptase (RT)-PCR, cell count, MTT assay and fluorescence-activated cell sorting. RESULTS: Compared with the negative and blank control group, the transfection group showed marked decrease in the c-myc expression and the K562 cells exhibited increased apoptosis rate. CONCLUSION: RNA interference can effectively inhibit c-myc expression and induce apoptosis in K562 cells.

Apoptosis↗

PubMed2005

[Research of restriction display technique in cDNA microarrays preparation for detecting of HCV].

The cDNA microarrays for HCV detection was prepared. With the restriction display technique (RD), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and amplified by RD-PCR. We separated the differential genes through polyacrylamide gel electrophoresis and sliver staining. Single bands were isolated which were cut out from the polyacrylamide gel. The third-round PCR could be performed by using the single bands as PCR template. The RD-PCR fragments were purified and cloned into the pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting PCR products to the surface of amido modified glass slides by the robotics. We validated the detection of microarray by the hybridization and the results of sequence analysis. A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced, which were the specific gene fragments of HCV. These fragments could be further used as probes in the microarray preparations. From the results of hybridization and sequence date analysis, the specificity, sensitivity, accuracy, reproducibility and linearity in detecting HCV RNA were satisfactory. RD technique is of great value in obtaining a large number of size-comparable gene probes, which provide a swift protocol in generating probes for the preparation of microarrays, and the optimized microarray is sensitive and effective in clinical diagnosis of HCV.

DNA Probes↗

PubMed2005

[Application of oligonucleotide microarray primer extension to detection of p53 single nucleotide polymorphisms].

BACKGROUND & OBJECTIVE: Increasing evidences imply that single nucleotide polymorphisms (SNPs) are involved in etiopathology, individual therapy, and prognosis of many diseases. Rapid and accurate SNPs detection in disease genes is vitally important for genomic research. To detect p53 SNPs, the method of an arrayed primer extension based on oligonucleotide microarray was developed. METHODS: Twelve extension primers were designed in exon 3 of p53 gene. These primers were synthesized and printed onto a microarray with 7x8 spots. The fragments of p53 DNA were extracted from K562 cells by nested polymerase chain reaction (PCR), and hybridized with the microarray. After hybridization, the primer extension reactions were carried out with DNA polymerase Klenow, labeled with Cy3-dUTP or Cy5-dCTP; the product was washed and scaned, and the fragments of p53 was sequenced by ABI3730 DNA Analyzer. RESULTS: The scanning result showed that the primer extension reactions were successful, and single base labeled with Cy3 or Cy5 was extended correctly at the end of 3'primers. The results of microarray SNPs detection were consistent with the results of sequencing verification, while much less complicated. CONCLUSION: These results indicate that the arrayed primer extension techniques are useful in parallel detecting SNPs of genes of interest, which is not only sensitive and accurate but also miniaturized the assays when analyzing multiple DNA targets with minimal reagents.

DNA Primers↗

PubMed2005

[Comparison of two amine-modified chemical platforms for DNA microarray preparation].

OBJECTIVE: To study two amine modification procedures for DNA microarray preparation based on polymeric coatings. METHODS: One of the proposed approaches utilized poly-amine coating of silanized slides activated by 1,4-phenylene diisothiocyanate, and the other employed acrylic acid-co-acrylamide copolymer and 1-(3-dimethylamino propyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide as the coating agents and activator, respectively. The modified slides were used for preparing lambda phage DNA microarrays, whose properties were analyzed by hybridization. RESULTS: Formation of dendrimeric structure and polymer was observed on the surface of the slides. The signal spots in uniform, steady and regular shape, in comparison with the commercial CMT-GAPS slides, indicated successful manufacture of the microarrays. CONCLUSION: The two platforms are suitable for microarray preparation, and the method of acrylic acid-co-acrylamide copolymer modification is more preferred.

Acrylamides↗

PubMed2005

A novel sample labeling method with restriction display PCR for 60-mer oligonucleotide microarray.

OBJECTIVE: To investigate the value of restriction display PCR (RD-PCR) as a novel and expedient sample labeling method for high-density 60-mer oligonucleotide microarray. METHODS: Peripheral blood samples from three volunteers were collected and the total RNA was extracted from the peripheral blood mononuclear cells and labeled with RD-PCR protocol, followed by hybridization with Agilent Human 1B oligonucleotide microarrays in a two-color comparison format. The RNA from the same subject was divided into two aliquot and labeled with Cy3 and Cy5 respectively. The spots with significant difference between the foreground and local background intensities and those without significant difference between Cy5 and Cy3 signal intensities were selected for analysis. SPSS software was used to perform the statistical tests and plot generation. VSN packages were used under R language to remove the systematic array and dye biases. RESULTS: Totally 8744 common spots of the 3 microarrays were evaluated. The results demonstrated that RD-PCR could be a promising novel method for efficient labeling of microarray samples. Further analysis indicated the presence of adjustable biases derived from the array and incorporated dye in the labeling processes. The RD-PCR labeling showed better performance than the conventional approaches in regards to reproducibility of the quantitative signals for gene intensity and capability to label RNAs of lowly expressed genes. CONCLUSION: Given the evidence of the feasibility of using RD-PCR labeling in the field of high-density long oligonucleotide microarray, further optimization of the protocol may unleash the full potential of this novel labeling method.

Humans↗

PubMed2005

[Expression of SARS spike gene in Shizomycete pombe].

The viral spike protein is the main surface antigen of the coronavirus, and it could be useful in the research of clinical diagnosis, SARS vaccine and the structure biology.According to the analysis of the main antigen of the SARS spike protein, 5 fragments of the whole spike gene were cloned, and ligated to the vector pNMT1. Through electroporation transformantion to TCP1, the recombinant S. pombe strains capable of expressing the 5 fragments were constructed. SDS-PAGE or Western blot analysis of the induced expression products demonstrated that the 5 recombinant proteins were expressed in the fission yeast respectively.

Cloning, Molecular↗

PubMed2004

A modified hybridization protocol for low-density diagnostic DNA microarrays.

To promote the application of DNA microarrays for clinical diagnosis, the problems of cross-hybridization and low signal intensity in the hybridization processes has been addressed. We tested a new hybridization protocol for low-density diagnostic DNA microarrays, by skipping the purification step during sample labeling, while elevating the hybridization temperature from 42 degrees C to 52 degrees C, adding a step of distilled water rinsing immediately after hybridization and before the low stringency washing steps. It was found that the modified hybridization protocol works well in our study, which increased detection sensitivity and eliminated nonspecific signals.

DNA, Bacterial↗