Role of HBV DNA polymerase in mediating the immune escape of tumor cells in HBV-related hepatocellular carcinoma / 临床肝胆病杂志

ObjectiveTo determine whether HBV DNA polymerase is associated with T-cell failure and thus mediates the immune escape of HBV-related hepatocellular carcinoma （HCC） tumor cells， and to investigate the specific molecular mechanisms. MethodsLiver cancer cell lines Huh7 and HepG2 stably transfected with HBV DNA polymerase expression plasmid with Flag （Flag-HBV-P） and intercellular adhesion molecule-1 （ICAM1） were co-cultured with Jurkat cells， and MTT assay， qRT-PCR， and ELISA were used to measure Jurkat cell proliferation， activation （CD69 expression）， and secretion of the cytokine IFN-γ. RNA-seq was used to screen for differentially expressed immune-associated molecules between stably transfected cell lines and control cells， and mRNA half-life and protein half-life assays were used to determine the specific levels of the immune-associated molecules that were affected by HBV DNA polymerase. Related websites were used to predict the transcription factors that may bind to the promoter region of this immune-associated molecule， Western blot was used to verify the effect of transcription factors on the immune-associated molecule， and rescue experiment was used to determine whether HBV DNA polymerase affects the expression level of the immune-associated molecule through this transcription factor. The independent-samples t test was used for comparison between two groups. ResultsThe experimental group had significant reductions in Jurkat cell proliferation， activation， and cytokine secretion compared with the control group （all P<0.01）. Compared with the control group， the experimental group （Huh7 and HepG2 cell lines） had significant reductions in the mRNA and protein expression levels of ICAM1 （all P<0.01）. Website prediction identified the ICAM1 promoter and preliminarily highlighted NFKB1， RELA， and STAT3. Compared with the control group， the experimental group （Huh7 and HepG2 cell lines） had a significant reduction in the protein expression level of p65 （all P<0.01）. After p65 overexpression， there was a significant increase in the protein expression level of ICAM1， and after the expression of p65 was reduced， there was a significant reduction in the protein expression level of ICAM1 （all P<0.01）. In the rescue experiment， there was no significant difference in the protein expression level of ICAM1 between the control group and the experimental group after p65 overexpression （all P>0.05）. After the overexpression of ICAM1， there were no significant differences in the proliferation， activation， and cytokine secretion of Jurkat cells between the control group and the experimental group （Huh7 and HepG2 cell lines） （all P>0.05）. ConclusionHBV DNA polymerase downregulates the level of ICAM1 to mediate HCC immune escape by inhibiting the expression of p65 in NF-κB.

Characterization of a family B DNA polymerase from Thermococcus eurythermalis A501 and its application in PCR / 生物工程学报

DNA polymerases are widely used in PCR and play important roles in life science research and related fields. Development of high-performance DNA polymerases is of great commercial interest as the current commercial DNA polymerases could not fully satisfy the requirements of scientific research. In this study, we cloned and expressed a family B DNA polymerase (NCBI accession number TEU_RS04875) from Thermococcus eurythermalis A501, characterized its enzymatic property and evaluated its application in PCR. The recombinant Teu-PolB was expressed in E. coli and purified with affinity chromatography and ion-exchange chromatography. The enzymatic properties of Teu-PolB were characterized using fluorescence-labeled oligonucleotides as substrates. The application potential of Teu-PolB in PCR was evaluated using the phage λ genomic DNA as a template. Teu-PolB has DNA polymerase and 3'→5' exonuclease activities, and is highly thermostable with a half-life of 2 h at 98 ℃. The most suitable PCR buffer is consisted of 50 mmol/L Tris-HCl pH 8.0, 2.5 mmol/L MgCl2, 60 mmol/L KCl, 10 mmol/L (NH4)2SO4, 0.015% Triton X-100 and 0.01% BSA, and the optimal extension temperature is 68 ℃. Under the optimized conditions, a 4 kb target fragment was successfully amplified with an extension rate of 2 kb/min. The yield of the Teu-PolB amplified-DNA was lower than that of Taq DNA polymerase, but its extension rate and fidelity was higher than that of Taq and Pfu DNA polymerases. The biochemical properties of Teu-PolB demonstrate that this enzyme can be used in PCR amplification with high thermostability, good salt tolerance, high extension rate and high fidelity.

Recent advances of continuous in vivo evolution / 生物工程学报

Laboratory evolution is an important approach to improve the performance of microorganisms. In the past decades, the methods for laboratory evolution have developed rapidly and applied widely. However, the commonly used evolution strategies for strains or specific proteins cannot achieve continuous mutation, and require multiple rounds of operation, therefore they are considered as a labor intensive process. The development of mutation and screening technologies have facilitated the development of continuous evolution in vivo and greatly improved the efficiency of laboratory evolution. The continuous in vivo evolution achieves in vivo mutation, perfectly combining mutation with screening to evolve a specific phenotype with minimal human intervention. This review summarizes the recent advances of in vivo continuous evolution technologies for either genome-scale mutation or evolution of specific proteins. The principles of these technologies and their applications are introduced. On this basis, the advantages and limitations of these technologies are discussed. We also give a perspective of future development of continuous in vivo evolution.

Preliminary study on the changes of Pole2 expression in the lymphocytes with aging as well as in the age-related diseases / 中华检验医学杂志

Objective:To explore the age-related changes of Pole2 expression levels in peripheral blood lymphocytes of healthy people, and analyze the differences of Pole2 expression levels in peripheral blood lymphocytes of four common senile disease patients between normal peers.Methods:Healthy people and patients in the physical examination center of Xuanwu Hospital of Capital Medical University from December 2018 to December 2019 were selected as the research objects, the mRNA and protein level of Pole2 in heathy individual as 20-29,30-39,40-49,50-59,60-69,70-79 y were checked by RT-PCR and Western blot,and the age-related curve was drew. The mRNA and protein expression levels of Pole2 were also detected in the peripheral blood lymphocytes of patients of DM,CHD,AD,CVD. And the deviation with healthy people of the same age was analyzed from one way ANOVA and repeated measurement were used to compare the mean of multiple groups.Results:The mRNA and protein levels of Pole2 increased in the lymphocytes of healthy people at 20-29 y and 50-59 y grouy, and decreased after 60-69 y and 70-79 y ( P<0.001). The Pole2 levels of mRNA and protein in the lymphocytes of patients of CHD,AD,DM and CVD cerebral atherosclerosis were significantly higher than that of healthy people of the same age. Moreover, the Pole2 level of lymphocytes of AD patients was significantly higher than that of other patient groups ( P<0.001). Conclusion:The expression level of Pole2 in peripheral blood lymphocytes increases with age before the age of 60 years old and decreases with age after 60 years old ;and the expression in four common senile diseases was higher than that in normal people.

Application and clinical significance of TCGA molecular classification in endometrial cancer / 中华妇产科杂志

Objective:To explore the application and clinical significance of the cancer genome atlas (TCGA) molecular classification in endometrial cancer (EC).Methods:Sixty-six EC patients collected from December 2018 to March 2021 from Peking University People′s Hospital were categorized into four subgroups based on TCGA molecular classification tested by next generation sequencing. The correlation among four molecular subgroups and the clinical-pathological features including prognosis were analyzed.Results:(1) Clinical and pathological features: median age at diagnosis was 56 years (range: 24-78 years). The cases were distributed as follows: 3 (5%) cases DNA polymerase epsilon (POLE) ultra-mutated, 11 (17%) cases high microsatellite instability (MSI-H) including 2 Lynch syndrome, 42 (64%) cases low copy-number (CN-L) and 10 (15%) cases high copy-number (CN-H). There were significant differences among four subtypes in the combination of other tumors, tumor family history, surgical method, International Federation of Gynecology and Obstetrics (FIGO, 2009) stage, depth of muscle invasion and lymph vascular space invasion (all P<0.05). The proportions of patients in CN-H subgroup with advanced FIGO stage (stage Ⅲ-Ⅳ), deep muscle invasion and positive lymph-vascular space invasion were significantly increased. There were no significant differences in age, menopausal status, body mass index, metabolic syndrome-related complications, preoperative serum CA 125 and human epididymis protein 4 levels, tumor size, pathological grade (only endometrioid cancer), and lymph node metastasis among the 4 TCGA molecular types (all P>0.05). (2) Immuno-related molecular analysis: among 66 EC patients, 27 patients underwent immunohistochemical analysis of programmed cell death 1 ligand 1 (PD-L1) protein, and 28 patients underwent tumor mutation burden (TMB) detection. POLE and MSI-H subgroups contained TMB than those in CN-L and CN-H ( P<0.05).(3) Prognosis: the median follow-up time was 10 months (range: 0-28 months). The progression-free survival rate of TCGA molecular types were 100% (POLE ultra-mutated), 100% (MSI-H), 98% (CN-L), and 80% (CN-H) respectively and had significant differences ( P=0.034). The overall survival were 100% (POLE ultra-mutated), 100% (MSI-H), 98% (CN-L), and 90% (CN-H) respectively, but there were not statistically significant difference ( P=0.361). POLE ultra-mutated and MSI-H subgroups had the best survival, while CN-H had the worst. Conclusion:TCGA molecular classification has feasibility and clinical value in clinical application of EC, which is helpful to identify the prognosis of patients.

Pcr Testing On Diagnosis Of Covid–19

There are multiple issues with setting up new diagnostic testing capacity outside ofregular diagnostic laboratories. This is not an overnight solution. Research laboratories havedifferent approaches, equipment and staff. There are multiple steps to ensure that themethods used and results are a correctly validated, as there is nothing more dangerous at themoment than reporting incorrect negative or positive results to staff or patients. That beingsaid we can adapt and validate assays and make them work using our equipment that can thenbe rolled out to support the NHS. PCR is a method widely used to rapidly make millions tobillions of copies of a specific DNA sample, allowing scientists to take a very small sample ofDNA and amplify it to a large enough amount to study in detail. It is fundamental too much ofgenetic testing including analysis of ancient samples of DNA and identification of infectiousagents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplifiedin a series of cycles of temperature changes. PCR is now a common and often indispensabletechnique used in medical laboratory and clinical laboratory research for a broad variety ofapplications including biomedical research and criminal forensics.

Production and evaluation of egg derived hot start antibodies

BACKGROUND: Hot start can greatly improve specificity, sensitivity and yield of PCR. Non-specific amplification can occur in PCR when reaction mixture is prepared at room temperature, because Taq DNA polymerase is active and the primers can hybridize non-specifically. Hot start Taq DNA polymerases remain inactive at room temperature and are activated after heating at 95°C preventing non-specific amplification. Monoclonal antibodies against Taq DNA polymerase is the first line of reagents used for turn on regular Taq DNA polymerase into Hot start one. The goal of this research was to produce and evaluate Hot Start antibodies derived from chicken eggs. RESULTS: We performed affinity purification of yolk immunoglobulin (IgY) and obtained polyclonal Hot Start antibodies. The yield of specific antibodies was 0.36 mg per egg or 0.2% of total yolk antibodies. The protocol for real time measurement and Hot start IgY activity assessment was developed. We found that Hot start IgY can reversibly block Taq DNA polymerase activity at 50°C and have no negative impact neither on the Taq DNA polymerase activity after denaturation nor on the reverse transcriptase. We estimated that 1.0 µg of Hot start IgY effectively blocks 5 U activity of Taq DNA polymerase. CONCLUSIONS: Egg derived Hot Start polyclonal antibodies are the cheapest source of Hot start antibodies, from one immune egg we can isolate 0.36 mg IgY, this quantity is enough for producing 1800 U activity of Hot start Taq DNA Polymerase.

Desarrollo y validación del método de amplificación isotérmica mediada en lazo para la detección del virus Zika / Development and validation of loop-mediated isothermal amplification for the detection of the Zika virus

RESUMEN Se desarrolló un método de amplificación isotérmica mediada en lazo de transcriptasa inversa (RT-LAMP) para detectar Zika. Los primers se diseñaron basándose en la región NS5 de 64 genomas completos. Se usó reactivo LAMP liofilizado. Inicialmente, se probaron siete arbovirus diferentes y solo las muestras de Zika resultaron positivas. Además, las diluciones seriadas de una de los ARN de Zika se compararon mediante RT-LAMP y qRT-PCR, demostrando que RTLAMP es 1000 veces más sensible. También se evaluó 300 muestras de suero usando RT-LAMP y los resultados se compararon con los métodos de qRT-PCR estándar y obtuvimos un 99,3% (IC95%: 97,7 - 100,0) de sensibilidad, 100% (IC95%: 99,7 - 100,0) de especificidad, 100% (IC95%: 99,7 -100,0) de valor predictivo positivo y 99,3% (IC95%: 97,7 - 100,0) de valor predictivo negativo. En conclusión, este método brinda una alternativa de bajo costo, alto rendimiento, viabilidad y confiabilidad para el diagnóstico rápido de Zika en instalaciones de atención primaria de salud.

ABSTRACT A Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method was developed to detect Zika. The primers were designed based on the NS5 region of 64 complete genomes. Lyophilized LAMP reagent was used. Initially, seven different arboviruses were tested and only Zika samples tested positive. Additionally, serial dilutions of one of Zika's RNA were compared using RT-LAMP and qRT-PCR, demonstrating that RT-LAMP is 1,000 times more sensitive. We also evaluated 300 serum samples with RT-LAMP comparing the results with standard qRT-PCR methods, and we obtained a 99.3% sensitivity, 100% specificity, 100% positive predictive value, and 99.3% negative predictive value. In conclusion, this method provides a low-cost, high-performance, viable, and reliable alternative for the rapid diagnosis of Zika in primary health-care facilities.

Effect of Capsaicin in Inhibiting Migration and Invasion of Breast Cancer MCF-7 Cells by Down-regulating SIRT1 / 中国实验方剂学杂志

Objective: To investigate the effect of capsaicin on the migration and invasion of human breast cancer MCF-7 cells and the underlying molecular mechanism. Method: Three capsaicin intervention groups of different concentrations (25, 50, 75 μmol·L-1) and a blank group were set up. After MCF-7 cells were treated with different concentrations of capsaicin (25, 50, 75 μmol·L-1) for 24 h, the cell migration and invasion abilities were assessed by Transwell migration and invasion assay, respectively. Meanwhile, the mRNA level of silent information regulator 2 homolog 1 (SIRT1) and DNA polymerase δ catalytic subunit p125 encoding gene POLD1 (POLD1) were detected by Real-time polymerase chain reaction (Real-time PCR). The protein levels of SIRT1 and DNA polymerase δ catalytic subunit p125 (p125) were detected by Western blot. Result: Compared with the blank group, the number of transmembrane cells was significantly reduced, and the mobility was significantly decreased (P-1) in MCF-7 cells for 24 h. Capsaicin (25, 50, 75 μmol·L-1) significantly down-regulated the mRNA and protein expressions of SIRT1 (P-1) in MCF-7 cells for 24 h. Furthermore, capsaicin (25, 50, 75 μmol·L-1) also significantly down-regulated the mRNA expression of POLD1 and the protein expression of p125 (P-1) in MCF-7 cells for 24 h. Conclusion: Capsaicin remarkably inhibits the cell migration and invasion of breast cancer MCF-7 cells, and the possible mechanism may be related to the down-regulation of SIRT1 and POLD1 mRNA expression levels and SIRT1 and p125 protein expression levels.

Genetic Mutation Screening of DNA Polymerase in Human Lung Cancer / 中国肺癌杂志

BACKGROUND@#DNA polymerase β is one of the key enzymes for DNA repair and it was reported that about 30 percent of different types of cancers carried mutations in its coding gene Polb. However, it is still controversial whether it is true or false because of the small sample size in these studies. In current study, we performed genetic screening of promoter and coding regions of Polb gene in 69 Chinese lung cancer patients using Sanger sequencing method, so as to elucidate real mutation frequency of Polb mutations in Chinese Han population.@*METHODS@#Salting out extraction method was used to get the genome DNAs from tumor and normal matched tissues of 69 lung cancer patients. The promoter and 14 coding regions of Polb gene were then amplified using these DNAs as the template. After purification, amplicons were sequenced and aligned to the wild type Polb gene in NCBI database, in order to find out the mutated sites of Polb gene in Chinese lung cancer patients.@*RESULTS@#In this study, we totally found only 5 mutated sites in Polb gene. In detail, 3 mutations (-196G>T, -188_-187insCGCCC, -168C>A) were located in the promoter region; 2 mutations (587C>G, 612A>T) were found in coding regions. Specially, mutations of -188_-187insCGCCC and 587C>G (resulting to the amino acid substitution of Thr to Ser at position 196) had never been reported by other groups before. However, all these 5 mutated sites could be detected in both tumor and matched normal tissues, which inferred that they are not lung tumor specific mutations.@*CONCLUSIONS@#No lung tumor specific mutations of Polb gene could be found in Chinese lung cancer patients and Polb gene mutation might not be a molecular marker for Chinese lung cancer patients.

Variabilidad genética en regiones codificantes del antígeno de superficie y el dominio de la transcriptasa inversa de la polimerasa del virus de la hepatitis B, Colombia, 2002-2014 / Genetic variability in coding regions of the surface antigen and reverse transcriptase domain of hepatitis B virus polymerase, Colombia, 2002-2014

Introducción. Se estima que 240 millones de personas en el mundo tienen infección crónica con el virus de la hepatitis B (HBV). En Colombia, la endemia es variable y circulan diferentes genotipos virales. Las mutaciones a lo largo del genoma se han asociado con resistencia antiviral, el escape ante la reacción de anticuerpos neutralizadores tras la vacunación o a la infección natural, la infección oculta y la progresión a carcinoma hepatocelular. Objetivo. Identificar los genotipos y las mutaciones presentes en la región codificante del antígeno de superficie (S) y del dominio de la transcriptasa inversa (reverse transcriptase, RT) de la polimerasa del HBV en muestras de suero remitidas al Instituto Nacional de Salud de Colombia para el diagnóstico de hepatitis B, entre el 2002 y el 2014. Materiales y métodos. En 495 muestras de suero positivas para el antígeno de superficie de la hepatitis B (HBsAg) se buscó el ADN viral, se amplificó y secuenció un fragmento de 1.591 nucleótidos y, posteriormente, se hizo el análisis filogenético correspondiente. Resultados. En 66 de las muestras se logró detectar el genoma viral y 28 de ellas se secuenciaron exitosamente. El análisis filogenético permitió identificar los genotipos y subgenotipos F3 y A2. Una muestra presentó simultáneamente las sustituciones de resistencia L180M y M204V, otra presentó la sustitución I169L y en una se identificó la mutación P120Q, previamente asociada con variantes de escape. Dos muestras presentaron una deleción de 105 nucleótidos en la región preS1-preS2. Conclusiones. Se corroboró la circulación en Colombia de los genotipos y subgenotipos F3 y A2, así como la presencia de mutaciones de resistencia y escape. El presente estudio constituye un aporte a la epidemiologia molecular del HBV en Colombia.

Introduction: Despite the availability of an effective vaccine and treatment to reduce the viral load and progressive hepatocellular injury, approximately 240 million people worldwide are chronically infected with the hepatitis B virus (HBV). In Colombia, the circulation of different viral genotypes has been confirmed. Mutations in the genome have been associated to antiviral therapy resistance, viral escape to neutralizing antibodies, occult infection and progression to hepatocellular carcinoma. Objective: To identify the genotypes and the presence of mutations in the coding region of the surface (S) antigen and the reverse transcriptase (RT) domain of the polymerase of HBV obtained from serum samples for hepatitis B diagnosis received by the Instituto Nacional de Salud during the period 2002-2014. Materials and methods: A total of 495 serum samples with previous HBsAg reactive result were used for molecular detection. A fragment of 1,591 nucleotides was sequenced, and the corresponding phylogenetic analysis was performed. Results: We detected the viral genome of HBV in 66 samples and 28 were successfully sequenced. The phylogenetic analysis allowed the identification of subgenotypes F3 and A2. The L180M and M204V resistance mutations were simultaneously identified in one sample, while the I169L resistance mutation was identified in another one. A single escape mutation, P120Q, was identified in one more. Two samples showed a deletion of 105 nucleotides in the preS1-preS2 region. Conclusions: The circulation of genotypes/subgenotypes F3 and A2 of HBV in Colombia was corroborated, as well as the presence of some resistance and escape mutations. The present study constitutes a contribution to the molecular epidemiology of HBV in Colombia.

Research progress on translesion synthesis DNA polymerase ζ-Rev7 in tumor development / 基础医学与临床

Translesion DNA synthesis is a kind of post-replication repair process, which mainly involves DNA polymerase kappa, eta and zeta.Translesion synthesis is a tolerance mechanism of DNA damage inside the cell, in which DNA polymerase zeta plays an important role.DNA polymerase zeta is mainly composed of subunits Rev7 and Rev3, and play a main role of participate in the damage repair in cell metabolism.Rev7(also known as MAD2L2)is a multifunctional protein that can be combined with a variety of proteins to participate in DNA dam-age repair, cell cycle regulation, gene expression and carcinogenesis, which is also involved in the prognosis of various adverse tumors.

Five Chinese patients with mitochondrial diseases caused by POLG gene mutations / 中华神经科杂志

Objective To report the clinical features, myopathological changes, and gene mutations in five Chinese patients with mitochondrial diseases caused by POLG gene mutations. Methods Clinical materials of five unrelated patients who were referred to Department of Neurology, Peking University First Hospital from April 2012 to January 2018, carrying POLG gene mutations, were retrospectively analyzed. Muscle/nerve biopsies and targeted second-generation gene sequencing were performed on the patients. Results Among the five patients, three were male and two were female. Two cases were dominant inheritance and three were sporadic or recessive inheritance. The ages of onset were from 15 to 40 years with disease course of one to 26 years. One of them showed atypical SANDO (sensory ataxic neuropathy, dysarthria, and ophthalmoparesis) syndrome accompanied by cardiac preexcitation syndrome. There were two cases with autosomal dominant and one case with recessive progressive external ophthalmoplegia plus syndrome. One case presented with cognitive delay and sensory neuropathy. The pathological changes of mitochondrial myopathy were observed in all four patients with muscle involvement. Sural nerve biopsy in the patient with cognitive delay and sensory ataxia revealed chronic axonal pathological changes. POLG gene mutations were found in all five patients by targeted next generation sequencing, including single heterozygous mutations in two dominant inherited patients (c. 914 G＞A and c. 2864A＞G, respectively), and compound heterozygous POLG gene mutations in the other three sporadic/recessive inherited patients (c. 2591 A＞G/c. 1790 G＞A, c. 924G＞T/c. 3002delG and c. 1613A＞T/c. 1612 G＞T, respectively). There were six novel mutations not reported before, i.e., c.914G＞A(p.S305N), c.924G＞T(p.Q308H), c.1613A＞T(p.E538V), c.1612G＞T(p.E538*), c.1790 G＞A(p.R597Q) and c.3002delG. Conclusions POLG gene mutations can lead to different clinical spectrums. Progressive external ophthalmoplegia, limb weakness and axonal sensory neuropathy are common presentations in this group of patients with POLG gene related mitochondrial neuromuscular diseases. Novel mutations found in this study expand the mutational spectrum of POLG gene.

Interactions of aminoglycosides with RNAs and proteins via carbohydrate microarray / 生物工程学报

Aminoglycosides are broad-spectrum antibacterials to treat bacterial infections, especially gram-negative bacteria infections. However, aminoglycosides are losing efficacy because of the increase in antibiotic resistance and their inherent toxicity, attracting more interests in developing new aminoglycosides. Several clinically used aminoglycosides are mainly exerted by inhibition of protein synthesis through binding to bacterial rRNA. The bacterial ribosome RNA is the most currently exploited RNA drug target. Identification of new compounds that target RNAs is indispensable to fight with the growing threat that bacteria pose to human safety. In this work, we used carbohydrate microarrays to probe interactions of low molecular weight ligands with RNAs and proteins. Carbohydrate microarrays, comprising hundreds to thousands of different glycan structures on surfaces in a spatially discrete pattern, are sensitive and versatile tools to study the interactions between biological macromolecules. Herein, aminoglycosides have been immobilized onto the modified glass microscope slides and their interactions with RNAs and proteins are then measured through the labeled fluorescence. The results displayed that microarray can be used to detect the binding of aminoglycosides with three types of target molecules, including the small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), the large group I ribozyme RNA (approximately 400 nucleotide) and certain proteins (toxicity-causing enzymes, such as DNA polymerase and phospholipase C). For rRNA A-site mimics, the fluorescence intensities of 16S rRNA is stronger than that of 18S rRNA, illustrating that as a screen technique, the microarray method can not only determine the binding affinity to RNA but also detect the specific binding to bacterial rRNA mimic. The ability to screen group I ribozyme RNA can be helpful to the discovery of new RNA therapeutic targets. Binding of immobilized aminoglycosides to toxicity-causing proteins (DNA polymerase and phospholipase C) is a new method to study of aminoglycoside toxicity. These studies lay the foundation for rapid identification of new RNA-binding ligands with strong and specific binding affinity for their desired targets.

A simple and efficient method for extraction of Taq DNA polymerase

Background Thermostable DNA polymerase (Taq Pol ?) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. The method used ammonium sulfate to precipitate the enzyme, and it saved effort and money but not time. Moreover, we found that 30-40% activity of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small amount of DNA. Results We provided a novel, simplified and low-cost method to purify the Taq Pol ? after overproduction of the enzyme in Escherichia coli, which used ethanol instead of ammonium sulfate to precipitate the enzyme. The precipitate can be directly dissolved in the storage buffer without dialysis. In addition, DNA and RNA contamination was removed with DNase I and RNase A before precipitation, and the extraction procedure was optimized. Our improvements increase recovery rate and specific activity of the enzyme, and save labor, time, and cost. Conclusions Our method uses ethanol, DNase I, and RNase A to purify the Taq Pol ?, and simplifies the operation, and increases the enzyme recovery rate and quality.

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.

Construction of human telomerase reverse transcriptase-immortalized rat bone marrow mesenchemal stem cell strains / 中国组织工程研究

BACKGROUND:Because of convenient source, multi-lineage differentiation and low immunogenicity, bone marrow mesenchymal stem cels are the ideal cel type to serve as vectors of transgenic cels in pain management. However, the replicative senescence and smal amount of cels obtained from the bone marrow restrict the application of bone marrow mesenchymal stem cels in pain research. OBJECTIVE:To construct human telomerase reverse transcriptase (hTERT)-immortalized rat bone marrow mesenchymal stem cels as transgenic celular vectors for pain therapy. METHODS:Bone marrow mesenchymal stem cels were obtained from whole rat bone marrow, and then transfected with a lentivirus containing the hTERT (pLV-Puro-EF1α-hTERT) folowed by puromycin selection. hTERT expression and telomerase activity in these transfected cels were determined by RT-PCR and TRAP. Morphological changes, capacity of cel growth and multi-lineage differentiation, chromosome karyotype and tumorigenicity were observed in vitro. Moreover, the expression of cel surface molecule, Nestin, MHC-I and MHC-II in transfected cels were also detected by flow cytometry and immunocytochemistry. RESULTS AND CONCLUSION: The bone marrow mesenchymal stem cels geneticaly modified by hTERT could be cultured and passaged through 30 generations in vitro. Compared to the primary and negative transfected cels, the hTERT-modified bone marrow mesenchymal stem cels showed higher expression of hTERT mRNA, telomerase activity and cel proliferation. Most of transfected cels stayed at G2/M and S stages. The proliferation index of the transfected cels were increased dramaticaly. The positive rates of CD29, CD44 and CD90 were over 70%, but the positive rates of CD34 and CD45 were less than 5%. Transfected cels were positive for Nestin in the cytoplasm, but negative for MHC-1 and MHC-11. In addition, this cel line continued to exhibit the characteristics of fibroblastic bone marrow mesenchymal stem cels, including phenotype, differentiation into osteoblasts, adipocytes and neuron-like cels. No chromosome abnormality and tumor formation were observed in this experiment. Taken together, these data suggests that the rat bone marrow mesenchymal stem cels immortalized by hTERT gene are constructed successfuly and stil maintain major stem cels characteristics, which provide safe and stable cel vectors as research base for pain therapy.

Establishment of a new strategy for studying the phenotype resistance of hepatitis B virus isolates / 中华传染病杂志

Objective To establish a novel and convenient method to study the phenotype of drug resistant hepatitis B virus (HBV) isolates,and to analyze the drug susceptibility by replacing the reverse transcriptase (RT) domain of wild-type HBV with that of the drug resistant HBV isolates.Methods Full length of HBV isolates was amplified and cloned from the sera of patients prior to nucleoside/nucleotide analogues (NA) treatment.Wild-type full-length HBV genomes was used to construct the recombinant expression plasmids PHY536207 (genotype B) and PHY97 (genotype C).The restriction enzyme sites were introduced in the upstream and downstream region of reverse transeription (RT) domain to construct plasmid,which were named as mPHY536207 and mPHY97,respectively.Lamivudine (LAM) resistant mutant and adefovir (ADV) resistant mutant were isolated and cloned to construct recombinant expression plasmids PHY634 and PHY6923,respectively.Subsequently,the RT domain of mPHY536207 was replaced by that of drug resistant mutant to construct the plasmids RT634 (LAM-resistant) and RT6923 (ADVresistant).The HBV constructs were transfected into Huh7 cells.The HBsAg levels in supernatant were determined by enzyme-linked immunosobent assay (ELISA),and the amount of intracellular HBV DNA was assayed by real-time polymerase chain reaction and Southern blot analysis.Results The plasmids PHY536207 and PHY97 containing genotype B and genotype C wild-type fulllength HBV genomes were constructed successfully,both of which could replicate in Huh7 cells.Intracellular HBV DNA extracted from cells in each of six-well culture plates was more than 1 × 107 copy/ mL,and the introduction of Pst Ⅰ restriction enzyme site did not affect the viral replication and HBsAg secretion.PHY634 and RT634,in which mutant RT domain was replaced into a wild type HBV expressing vector,exhibited the same HBV DNA replication under the treatment with different doses of LAM,the value of 50％ inhibitory concentration (IC50) was ＞100 μmol/L,while the IC50 of mPHY536207 was 0.18μmol/L.Moreover,wild-type isolate was sensitive to ADV (IC50 =1.2 μmol/L),while PHY6923 and RT6923 were resistant to ADV treatment (IC50 ＞100 μmol/L).Conclusion The phenotypic assay is successfully developed in this study based on replacing RT domain of wild-type HBV strains with that of clinical isolated drug resistant strain.

Comparison of sxx commercially-available dna polymerases for direct ccr / Comparacao de seis polimerases de DNA disponiveis comercialmente para o PCR direto

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens. .

RESUMO O propósito deste estudo foi avaliar 6 polimerases de DNA disponíveis comercialmente que são resistentes aos inibidores do PCR para uma amplificação potencial de DNA de amostras de sangue total. O DNA genômico do parasita humano da malária, Plasmodium falciparum, foi analisado sob condições que incluíram os componentes inibidores do sangue extraído de sangue ressacado em papel de filtro. Nossos resultados sugerem que a polimerase KOD FX DNA é superior a outras polimerases. .

DNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism.

To analyze the active sites of various prokaryotic and eukaryotic DNA polymerases and propose a plausible mechanism of action for the polymerases with the Escherichia coli DNA polymerase I as a model system. Study Design: Bioinformatics, Biochemical and X-ray crystallographic data were analyzed. Place and Duration of Study: Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India. From 2007 to 2012. Methodology: The advanced version of T-COFFEE was used to analyze both prokaryotic and eukaryotic DNA polymerase sequences. Along with this bioinformatics data, X-ray crystallographic and biochemical data were used to confirm the possible amino acids in the active sites of different types of polymerases from various sources. Results: Multiple sequence analyses of various polymerases from different sources show only a few highly conserved motifs among these enzymes except eukaryotic epsilon polymerases where a large number of highly conserved sequences are found. Possible catalytic/active site regions in all these polymerases show a highly conserved catalytic amino acid K/R and the YG/A pair. A distance conservation is also observed between the active sites. Furthermore, two highly conserved Ds and DXD motifs are also observed. Conclusion: The highly conserved amino acid K/R acts as the proton abstractor in catalysis and the YG/A pair acts as a “steric gate” in selection of only dNTPS for polymerization reactions. The two highly conserved Ds act as the “charge shielder” of dNTPs and orient the alpha phosphate of incoming dNTPs to the 3’-OH end of the growing primer.