Protective effect of liraglutide on the myocardium of type 2 diabetic rats by inhibiting polyadenosine diphosphate-ribose polymerase-1

BACKGROUND In recent years,studies have found that the occurrence and development of diabetic cardiomyopathy(DCM)is closely related to an increase in polyadenosine diphosphate-ribose polymerase-1(PARP-1)activity.PARP-1 activation could be involved in the pathophysiological process of DCM by promoting oxidative stress,the inflammatory response,apoptosis and myocardial fibrosis.AIM To investigate the mechanism of liraglutide in improving myocardial injury in type 2 diabetic rats,further clarified the protective effect of liraglutide on the heart,and provided a new option for the treatment of DCM.METHODS Forty healthy male SD rats aged 6 wk were randomly divided into two groups,a normal control group(n=10)and a model group(n=30),which were fed an ordinary diet and a high-sugar and high-fat diet,respectively.After successful modeling,the rats in the model group were fed a high-glucose and high-fat diet for 4 wk and randomly divided into a model group and an intervention group(further divided into a high-dose group and a low-dose group).The rats were fed a high-glucose and high-fat diet for 8 wk and then started drug intervention.Blood samples were collected from the abdominal aorta to detect fasting blood glucose and lipid profiles.Intact heart tissue was dissected,and its weight was used to calculate the heart weight index.Haematoxylin and eosin staining was used to observe the pathological changes in the myocardium and the expression of PARP-1 in the heart by immunohistochemistry.RESULTS The body weight and heart weight index of rats in the model group were significantly increased compared with those in the normal control group,and those in the intervention group were decreased compared with those in the model group,with a more obvious decrease observed in the high-dose group(P<0.05).In the model group,myocardial fibers were disordered,and inflammatory cells and interstitial fibrosis were observed.The cardiomyopathy of rats in the intervention group was improved to different degrees,the myocardial fibers were arranged neatly,and the myocardial cells were clearly striated;the improvement was more obvious in the high-dose group.Compared with the normal control group,the expression of PARP-1 in myocardial tissue of the model group was increased,and the difference was statistically significant(P<0.05).After liraglutide intervention,compared with the model group,the expression of PARP-1 in myocardial tissue was decreased,and the reduction was more obvious in the high-dose group(P<0.05)but still higher than that in the normal control group.CONCLUSION Liraglutide may improve myocardial injury in type 2 diabetic rats by inhibiting the expression of myocardial PARP-1 in a dose-dependent manner.

Predictions in Clinical Efficiency of SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp) Inhibitors by Molecular Docking

This study utilizes the enzyme-substrate complex theory to predict the clinical efficacy of COVID-19 treatments at the biological systems level, using molecular docking stability indicators. Experimental data from the Protein Data Bank and molecular structures generated by AlphaFold 3 were used to create macromolecular complex templates. Six templates were developed, including the holo nsp7-nsp8-nsp12 (RNA-dependent RNA polymerase) complex with dsRNA primers (holo-RdRp-RNA). The study evaluated several ligands—Favipiravir-RTP, Remdesivir, Abacavir, Ribavirin, and Oseltamivir—as potential viral RNA polymerase inhibitors. Notably, the first four of these ligands have been clinically employed in the treatment of COVID-19, allowing for comparative analysis. Molecular docking simulations were performed using AutoDock 4, and statistical differences were assessed through t-tests and Mann-Whitney U tests. A review of the literature on COVID-19 treatment outcomes and inhibitors targeting RNA polymerase enzymes was conducted, and the inhibitors were ranked according to their clinical efficacy: Remdesivir > Favipiravir-RTP > Oseltamivir. Docking results obtained from the second and third templates aligned with clinical observations. Furthermore, Abacavir demonstrated a predicted efficacy comparable to Favipiravir-RTP, while Ribavirin exhibited a predicted efficacy similar to that of Remdesivir. This research, focused on inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase, establishes a framework for screening AI-generated drug templates based on clinical outcomes. Additionally, it develops a drug screening platform based on molecular docking binding energy, enabling the evaluation of novel or repurposed drugs and potentially accelerating the drug development process.

Systematic analysis of DNA polymerases as therapeutic targets in pan-cancers

Introduction:DNA polymerases are crucial for maintaining genome stability and influencing tumorigenesis.However,the clinical implications of DNA polymerases in tumorigenesis and their potential as anti-cancer therapy targets are not well understood.Methods:We conducted a systematic analysis using TCGA Pan-Cancer Atlas data and Gene Set Cancer Analysis results to examine the expression profiles of 15 DNA polymerases(POLYs)and their clinical correlations.We also evaluated the prognostic value of POLYs by analyzing their expression levels in relation to overall survival time(OS)using Kaplan-Meier survival curves.Additionally,we investigated the correlations between POLY expression and immune cells,DNA damage repair(DDR)pathways,and ubiquitination.Drug sensitivity analysis was performed to assess the relationship between POLY expression and drug response.Results:Our analysis revealed that 14 out of 15 POLYs exhibited significantly distinct expression patterns between tumor and normal samples across most cancer types,except for DNA nucleotidylexotransferase(DNTT).Specifically,POLD1 and POLE showed elevated expression in almost all cancers,while POLQ exhibited high expression levels in all cancer types.Some POLYs showed heightened expression in specific cancer subtypes,while others exhibited low expression.Kaplan-Meier survival curves demonstrated significant prognostic value of POLYs in multiple cancers,including PAAD,KIRC,and ACC.Cox analysis further validated these findings.Alteration patterns of POLYs varied significantly among different cancer types and were associated with poorer survival outcomes.Significant correlations were observed between the expression of POLY members and immune cells,DDR pathways,and ubiquitination.Drug sensitivity analysis indicated an inverse relationship between POLY expression and drug response.Conclusion:Our comprehensive study highlights the significant role of POLYs in cancer development and identifies them as promising prognostic and immunological biomarkers for various cancer types.Additionally,targeting POLYs therapeutically holds promise for tumor immunotherapy.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus,hepatitis B virus,and human immunodeficiency virus 1

BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Low testing rates and high BRCA prevalence: Poly (ADP-ribose) polymerase inhibitor use in Middle East BRCA/homologous recombination deficiency-positive cancer patients

BACKGROUND Poly(ADP-ribose)polymerase inhibitors(PARPis)are approved as first-line therapies for breast cancer gene(BRCA)-positive,human epidermal growth factor receptor 2-negative locally advanced or metastatic breast cancer.They are also effective for new and recurrent ovarian cancers that are BRCA-or homologous recombination deficiency(HRD)-positive.However,data on these mutations and PARPi use in the Middle East are limited.AIM To assess BRCA/HRD prevalence and PARPi use in patients in the Middle East with breast/ovarian cancer.METHODS This was a single-center retrospective study of 57 of 472 breast cancer patients tested for BRCA mutations,and 25 of 65 ovarian cancer patients tested for HRD.These adult patients participated in at least four visits to the oncology service at our center between August 2021 and May 2023.Data were summarized using descriptive statistics and compared using counts and percentages.Response to treatment was assessed using Response Evaluation Criteria in Solid Tumors criteria.RESULTS Among the 472 breast cancer patients,12.1%underwent BRCA testing,and 38.5%of 65 ovarian cancer patients received HRD testing.Pathogenic mutations were found in 25.6%of the tested patients:26.3%breast cancers had germline BRCA(gBRCA)mutations and 24.0%ovarian cancers showed HRD.Notably,40.0%of gBRCA-positive breast cancers and 66.0%of HRD-positive ovarian cancers were Middle Eastern and Asian patients,respectively.PARPi treatment was used in 5(33.3%)gBRCA-positive breast cancer patients as first-line therapy(n=1;7-months progression-free),for maintenance(n=2;>15-months progression-free),or at later stages due to compliance issues(n=2).Four patients(66.6%)with HRD-positive ovarian cancer received PARPi and all remained progression-free.CONCLUSION Lower testing rates but higher BRCA mutations in breast cancer were found.Ethnicity reflected United Arab Emirates demographics,with breast cancer in Middle Eastern and ovarian cancer in Asian patients.

Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Deoxyribonucleic Acid-Polymerase Chain Reaction Status of HIV Exposed Infants in a Sub Regional Prevention of Mother-to-Child Transmission of HIV Programme during the Period 2009-2020

Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV infection status in HIVexposed infants who had their first DNA polymerase chain reaction test in our molecular Laboratory. Subjects, Materials and Methods: Dried Blood Spots for HIV DNA results from 5 states between 2009 and 2020 were analyzed in the PCR laboratory of the Federal Teaching Hospital, Gombe. Results: Nine thousand eight hundred and twenty-three Human Immunodeficiency Virus Deoxyribonucleic acid polymerase Chain Reaction results were analysed;4937 (50.2%) were males. During the study period, there was an overall declining trend in the mother-to-child transmission rate from 3.8% in 2009 to 1.0% in 2020. 6120 (62.3%) of HIV + mothers received Highly active antiretroviral therapy HAART before pregnancy. 7845 (76.2%) of the infants received Nevirapine prophylaxis. Dried blood spot samples were collected from 4077 (41.5%) at 6 - 8 weeks. 8438 (85.9%) received cotrimoxazole. 9469 (96.4%) were ever breastfed. Of the 9823 HIV DNA PCR results, 255 (2.6%) were positive while 69/4077 (1.7%) and 109/2662 (4.1%) were positive for HIV DNA at 6 - 8 weeks and > 12 weeks respectively. (p = 0.001). 86/747 (11.5%) of infants whose HIV-positive mothers received no ARVS were HIV DNA positive. (p = 0.001). 106/884 (12.0%) of infants who had no Antiretroviral prophylaxis had positive HIV DNA results;7/413 (1.7%) with Zidovudine/Nevirapine prophylaxis had positive results. (p = 0.001). 246/9469 (2.6%) of infants that were ever breastfed were positive for HIV DNA;11/354 (3.0%) that never breastfed had positive HIV DNA. Conclusion: Lack of maternal/infant ARVs and prolonged breastfeeding increased the risk of infant HIV infection.

Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections,rt269L and rt269I

BACKGROUND The presence of two distinct hepatitis B virus(HBV)Pol RT polymorphisms,rt269L and rt269I,could contribute to the unique clinical or virological phenotype of HBV genotype C2.Therefore,a simple and sensitive method capable of identifying both types in chronic hepatitis B(CHB)patients infected with genotype C2 should be developed.AIM To develop a novel simple and sensitive locked nucleic acid(LNA)-real timepolymerase chain reaction(RT-PCR)method capable of identifying two rt269 types in CHB genotype C2 patients.METHODS We designed proper primer and probe sets for LNA-RT-PCR for the separation of rt269 types.Using synthesized DNAs of the wild type and variant forms,melting temperature analysis,detection sensitivity,and endpoint genotyping for LNA-RT-PCR were performed.The developed LNA-RT-PCR method was applied to a total of 94 CHB patients of genotype C2 for the identification of two rt269 polymorphisms,and these results were compared with those obtained by a direct sequencing protocol.RESULTS The LNA-RT-PCR method could identify two rt269L and rt269I polymorphisms of three genotypes,two rt269L types[‘L1’(WT)and‘L2’]and one rt269I type(‘I’)in single(63 samples,72.4%)or mixed forms(24 samples,27.6%)in 87(92.6%sensitivity)of 94 samples from Korean CHB patients.When the results were compared with those obtained by the direct sequencing protocol,the LNA-RT-PCR method showed the same results in all but one of 87 positive detected samples(98.9%specificity).CONCLUSION The newly developed LNA-RT-PCR method could identify two rt269 polymorphisms,rt269L and rt269I,in CHB patients with genotype C2 infections.This method could be effectively used for the understanding of disease progression in genotype C2 endemic areas.

Confusing finding of quantitative fluorescent polymerase chain reaction analysis in invasive prenatal genetic diagnosis:A case report

BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.

Establishment of a rapid detection method of Ureaplasma urealyticum based on recombinant polymerase amplification

Ureaplasma urealyticum(UU),is one of the most vital pathogens causing genitourinary tract infections of the body,and it can result in poor maternal and perinatal outcomes.The aim of this study was to establish a method to detect Ureaplasma urealyticum based on recombinant polymerase amplification(RPA)technique.Specific primers and probes were designed according to the 16sRNA gene sequence of Ureaplasma urealyticum.Six pathogens were detected for real-time fluorescence RPA specificity verification,including Mycoplasma hominis(MH),Chlamydia trachomatis(CT),Neisseria gonorrhoeae(NG),Staphylococcus aureus,Escherichia coli,and Lactobacillus vaginalis.The sensitivity of the method was performed by gradient dilution of the extracted template.A total of 60 clinical samples were detected by the established real-time fluorescence RPA.Detection of Ureaplasma urealyticum can be completed within 20 minutes at 39°C using established RPA method.The minimum detection limit of Ureaplasma urealyticum by real-time fluorescence RPA was 3 pg.The evaluation of 60 clinical samples proved that RPA method was feasible.A high specificity,sensitivity,simplicity and rapidity method for Ureaplasma urealyticum detection was successfully established based on the real-time fluorescence RPA method.

A Molecular Approach to Identification of the Chinese Drug“pu Gong Ying”(Herba Taraxaci)and Six Adulterants byDNA Fingerprinting Using Random Primed PolymeraseChain Resaction(PCR)

DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbitrarily primed polymerase chain reaction(AP-PCR)and random amplified polymorphic DNA(RAPD).Distinctive,reproducible genomic fingerprints from DNA from 7 species belonged to Compositae were generated with two long(20 and 24 mer)and one short(10 mer)randomly chosen primers.The Pu Gong Ying can be differentiated from six species of Tu Gong Ying according to the banding pattems of their amplified DNA on agarose gels.The results showed that AP-PCR and RAPD methods can be used for identifying Chinese drugs.Moreover,the Similarity Indexes of the genomic DNA fingerprints showed that Pu Gong Ying and its adulterants are unrelated.Therefore,AP-PCR and RAPD methods can be used for identifying Chinese drugs.

Development and Clinical Application of a Single-tube Nested PCR Method to Amplify the DNA Polymerase Ⅰ Gene of Treponema Pallidum

Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.

Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing *

AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.

3'-terminus shifted bases degeneracy primers increasing sensitivity of polymerase chain reaction

目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。

Study of differential polymerase chain reaction of C-erbB-2 oncogene amplification in gastric cancer

AIM To study the significance of C-erbB-2 oncogene amplification in gastric cancer.METHODS C-erbB-2 oncogene amplification was examined by using differential polymerase chain reaction (dPCR) in surgical and endoscopic specimens of 83 cases of gastric cancer and 101 metastatic lymph nodes.RESULTS C-erbB-2 amplification was found in 28.9% (24/ 83) surgical specimens and 20.5% (17/ 83) endoscopic ones of gastric cancer patients. The amplification was significant in both types of specimens of advanced cancer cases (P＜0.05) and surgical specimens with lymph node metastasis (P＜0.01). The incidence of C-erbB-2 amplification in lymph nodes with metastasis was higher than in primary sites (surgical specimens, P＜0.05). The patients with amplification tumors had poorer 5-year survival rates than those with unamplification ones in the early cancers and well to moderately differentiated adenocarcinomas (P＜0.05). The same surgical samples were tested again by Southern blot hybridization to ascertain C-erbB-2 amplification, and the positive rate of C-erbB-2 amplification (15.7%) was lower than that of dPCR (28.9%, P＜0.05).CONCLUSION Examining C-erbB-2 amplification by dPCR is a quick, simple, reliable and independent method, and is helpful in predicting prognosis and metastatic potential of gastric cancer.

Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection

AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C.jejuni.The specificity of the primers and probe were tested against a set of Campylobacter spp.and other enteric pathogens.The optimal PCR conditions were determined by testing a series of conditions with standard a C.jejuni template.The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen.Two hundred and forty-two specimens were analyzed for the presence of C.jejuni by direct bacterial culture and real-time PCR.RESULTS:The optimal PCR system was determined using reference DNA templates,1 × uracil-DNA glycosylase,3.5 mmol/L MgCl 2,1.25 U platinum Taq polymerase,0.4 mmol/L PCR nucleotide mix,0.48 μmol/L of each primer,0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL.The PCR reaction was carried as follows:95 ℃ for 4 min,followed by 45 cycles of 10 s at 95 ℃ and 30 s at 59 ℃.The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10 3 CFU/g using DNA from stool specimens.Twenty(8.3%,20/242) C.jejuni strains were isolated from bacterial culture,while 41(16.9%,41/242) samples were found to be positive by realtime PCR.DNA sequencing of the PCR product indicated the presence of C.jejuni in the specimen.One mixed infection of C.jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.CONCLUSION:The sensitivity of detection of C.jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.

Targeted Screening of SiRNA Directed HBV Polymerase Gene for Effective Inhibition of HBV Expression

In order to screen potential mRNA locations of P gene in which targeting siRNAs can effectively inhibit HBV expression, 5 recombinant plasmids containing 4 targeting-specific siRNA fragments and a control were prepared and transfected into 2.2.15 cells respectively. The expression levels of HBx mRNA, HBs mRNA and HBc mRNA were detected by RT-PCR. The concentrations of the hepatitis B virus antigens, including HBsAg and HBeAg harvested from the culture supernatant of transfected 2.2.15 cells, were measured by ELISA. X protein was tested by Western blot. The results showed that four siRNAs against distinct mRNA locations of HBV polymerse gene had different inhibitory effects on their targeted mRNA. The plasmid-derived psiRNA1 and psiRNA2 could effectively inhibit the transcription and translation of HBs gene, whereas the inhibitory efficiency of psiRNA3, psiRNA4 for HBe gene was much higher than that of psiRNA1 and psiR.NA2. In comparison to the rest of psiRNAs in this study, psiRNA4 was the most effective to suppress the transcription and translation of HBx. It is suggested that siRNA can be considered as a powerful therapeutic agent for reducing HBV expression. The siRNAs against HBV polymerase are effective largely depending on the location of targeted sites. To enhance inhibitory efficiency, hunting for high effective target in polymerase gene is necessary and feasible.

Systematic identification of endogenous RNA polymeraseⅢpromoters for efficient RNA guidebased genome editing technologies in maize

Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter(TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%(U6-1) to over 21.0%(U6-6). The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.

Detection of bacterial DNA from cholesterol gallstones by nested primers polymerase chain reaction

AIM To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture. METHODS DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reaction (NP PCR) was used to amplify bacterial gene fragments for identifying the existence of bacteria. The samples of bacterial DNA extracted from potentially causative or unrelated living bacteria were amplified in vitro as the standard markers and comparative 16S ribosomal RNA sequence analysis was made for bacterial identification. RESULTS The gallbladder gallstones of 30 patients were analyzed and bacterial DNA was found in 26 patients. Among them, gallstones with cholesterol content between 30%-69% were seen in 5 (5/5) patients, 70%-90% in 11 (11/14) patients, and more than 90% in 10 (10/11) patients. There was no difference either in cholesterol and water content of gallstones or in harboring bacterial DNA of gallstones. E.coli related DNA fragments appeared in the stones of 8 (26 67%) patients; propionibacteria type DNA in 7 (23 33%); and harbored bacterial gene fragments in 2 patients, similar to Streptococcus pyogenes . A more heterogeneous sequence collection was found in 7 (23 33%) patients, which could belong to multiple bacterial infections. Two (6 67%) patients had bacterial DNA with low molecular weight which might be related to some unidentified bacteria. CONCLUSION Most cholesterol gallstones harbor bacterial DNA. It is important to determine whether these microorganisms are innocent bystanders or active participants in cholesterol gallstone formation.

Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis

Hepatitis C virus(HCV)infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20%of the total population are infected.Initial infection is usually asymptomatic and result in chronic hepatitis that give rise to complications including cirrhosis and hepatocellular carcinoma.The management of HCV infection should not only be focus on therapy,but also to screen carrier individuals in order to prevent transmission.In the present,molecular detection and quantification of HCV genome by real time polymerase chain reaction(PCR)represent the gold standard in HCV diagnosis and plays a crucial role in the management of therapeutic regimens.However,real time PCR is a complicated approach and of limited distribution.On the other hand,isothermal DNA amplification techniques have been developed and offer molecular diagnosis of infectious dieses at point-of-care.In this review we discuss recombinase polymerase amplification technique and illustrate its diagnostic value over both PCR and other isothermal amplification techniques.