Research progress on reversal of drug resistance to Sorafenib in primary liver cancer / 国际外科学杂志

Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence rate and mortality worldwide. However, most patients are not suitable for radical treatment at the time of first diagnosis. As one of the important schemes for the treatment of HCC, one of the most representative drug is Sorafenib, which has certain survival benefits for HCC patients at different stages. However, the drug resistance of HCC to Sorafenib greatly limits its efficacy. So far, people have found that some natural substances, experimental agents and biological macromolecules can reverse the drug resistance of HCC to Sorafenib through tumor cell microenvironment, metabolism and other mechanisms. This article will summarize the above substances and their mechanism in order to provide research ideas for the improvement of Sorafenib′s treatment program.

Differential expression of sigma factor of Mycobacterium tuberculosis under in vivo infection and in vitro culture conditions / 公共卫生与预防医学

Objective To investigate the gene expression of sigma factors in vivo, and to explore the sigma factors that may be closely related to the virulence of pathogenic Mycobacterium tuberculosis Methods Tuberculosis (TB) patients diagnosed in the outpatient department of Tianjin Tuberculosis Control Center from January to December 2018 were selected, and 20 sputum-positive specimens were randomly selected from TB patients confirmed with Xpert-positive for the present study. Two immediate sputum specimens were collected from each case of pulmonary tuberculosis before treatment, one for RNA extraction and one for in vitro culture. In vitro cultured strains in the logarithmic phase of growth were harvested for RNA extraction. The specific primers for 13 sigma factors were designed. The differential expression of the 13 sigma factors between sputum isolates and in vitro cultured strains was analyzed by fluorescence quantitative PCR. Taking ribosomal 16s as the reference gene, the transcription level of sigma factors was analyzed by 2ΔCt. Using the stably expressed sigA as the control reference, the expression differences of other sigma factors were analyzed by one-way ANOVA. Results Within 0 days, stress-associated sigma factors have a different expression profile in clinical isolate strains vs H37Rv or in vitro. All the sigma factors induced up regulation in sputum ,while no difference transcription between clinical isolate strains vs H37Rv（P＞0.05）. When compared to in vitro culture ,only sigM transcript highest in sputum(P＜0.05). Conclusion SigM plays an important role in the initial stages of bacterial infection, but its exact role is unclear.We assumed it could have a role in the interplay between the host immune defenses and the bacterial escape mechanisms.

Significance of cancer testis?associated antigens (SPAG9 and FBXO39) in colon cancer

Background: Cancer testis antigens (CTA) are normally expressed in immune privileged tissues such as the testis. They are considered tumor-associated antigens because they are specifically expressed in different cancers. Their distinct nature rendered them appealing targets for cancer diagnosis, prognosis. and immunotherapy. We aimed to identify the association of two CTA genes with colon cancer (CC) in a cohort of Egyptian patients. Methods: We measured the relative gene expression levels of two CTAs: SPAG9 and FBXO39 in colonic tumor tissue and adjacent normal-appearing mucosa in 50 newly diagnosed colon cancer patients by real-time reverse transcription polymerase chain reaction. Gene expression was also studied in relation to demographic and pathological criteria. Results: SPAG9 and FBXO39 were overexpressed in 22% and 40% of cases, respectively. Overexpression of both genes was evident in 14% of cases. We report the significant expression of FBXO39 (P < 0.01) in tumor tissue compared to normal tissue. SPAG9 was significantly increased in large sized tumors compared to smaller sized tumors. Otherwise, there was no significant association between gene expression and the evaluated clinicopathological features (P > 0.05). Conclusions: SPAG9 and FBXO39 are possible CC diagnostic biomarkers. Further studies are warranted to validate our findings.

Covid 19, the Kerala experience: an observational, single centre retrospective study of outcome in covid positive pregnancies

Background: Covid 19 has spread across the world at an alarming rate. Approximately 4.05 million people have got infected worldwide resulting in around 279,000 deaths. Over 1 million people have recovered worldwide. Aim of this study was to determine whether course and severity of covid 19 is altered in pregnant women and whether covid 19 seemed to worsen the prognosis in pregnant women.Methods: Around 50 covid positive patients were admitted to this study hospital, a tertiary care referral hospital and medical college, between march and May 2020, 11 were pregnant. Authors collected their data retrospectively to understand the course of their disease till the period of recovery.Results: There were 6 patients above 31 weeks of whom one had elective repeat caesarean section, one had full term vaginal delivery, one is under follow up. Three patients had foetal distress necessitating emergency caesarean section. Of the remaining 5 patients with periods of gestation between 9-13 weeks, 1 of 24 weeks, 6 patients above 31 weeks, one had a miscarriage. Rest pregnancies are continuing and under follow up. 6 women had been symptomatic at admission, with mild symptoms of low-grade fever, sore throat and rhinitis. All were treated with hydroxychloroquine (HCQs). Those with respiratory symptoms like cough were also treated with oseltamivir. In view of high prevalence of H1N1 in the region. None of the women developed severe disease. The disease did not appear to worsen prognosis in pregnant women. The rate of recovery in pregnant women was similar to that seen in non-pregnant women and also men under the age of 40 years admitted in this study hospital.Conclusions: Covid 19 did not seem to worsen the prognosis in pregnant individuals when compared to rest of the population. The foetal outcomes also seemed favorable. However larger studies are required before concrete guidelines could be formulated for management of the disease in pregnancy.

Identification of a novel multidrug-resistance mutation in reverse transcription region of hepatitis B virus / 解放军医学杂志

Objective To analyze the clinical occurrence and phenotypic characteristics of rtA181S-related novel multidrug-resistance mutation in reverse transcription region of hepatitis B virus (HBV). Methods The clinical data and sequence information of 16 443 patients with chronic HBV infection who received nucleos(t)ide analogues (NAs)-resistance testing at the original PLA 302 Hospital from 2012 to 2017 were retrospectively analyzed. rtA181S-related mutation patterns were analyzed and cloning-sequencing (≥20 clones/sample) was performed on the mutation samples of rtA181S+T184I+M204I with the highest detection rate. Phenotypic analysis was performed to evaluate the viral replication capacity and drug susceptibility. Results The rtA181S mutation was detected in 0.75% (124/16 443) of the patients. Among them, 21 patients were detected with coexistence of lamivudine (LAM)-resistance mutation and 74 patients were detected with coexistence of entecavir-resistance (ETVr) mutation. The rtA181S+T184I+M204I novel mutation accounted for 77.0% (57/74) of the rtA181S+ETVr mutation. Dynamic clinical data analysis showed that the rtA181S+T184I+M204I mutation emerged after adefovir dipivoxil (ADV), ETV, and LAM/telbivudine (LdT) treatment, accompanied by virological breakthrough or inadequate virological response. Compared to wild-type strain, rtA181S+T184I+M204I mutant had 53.7% decreased replication capacity, over 1000-, 3.9-, and 383.3-fold increased LAM, ADV, and ETV resistance, respectively, and remained sensitive to tenofovir (TDF). Conclusions rtA181S+T184I+M204I mutation is a novel multidrug-resistance mutation, which is related to the ADV, ETV or LAM/LdT sequential or combined treatment. TDF-based rescue therapy should be considered for patients harboring this mutation in clinical practice.

Rapid detection of deformed wing virus in honeybee using ultra-rapid qPCR and a DNA-chip

Phaseolus vulgaris genome possesses CAMTA genes, and phavuCAMTA1 contributes to the drought tolerance

Calmodulin-binding transcription activators (CAMTAs) are a family of transcription factors that play an important role in plants’ response to the various biotic and abiotic stresses. The common bean (Phaseolus vulgaris L.) is one of the most important crops in the world and plays a pivotal role in sustainable agriculture. To date, the composition of CAMTA genes in genomes of Phaseolus species and their role in resistance to drought stress are not known. In this study, five PhavuCAMTA genes were characterized in common bean genome through bioinformatics analysis, the morphological and biochemical response of 23 Ph.vulgaris genotypes to different levels of drought stress were evaluated and the expression patterns of PhCAMTA1 in the leaf tissues of sensitive and tolerant genotypes were analysed. Gene structure, protein domain organization and phylogenetic analyses showed that the CAMTAs of Phaseolus were structurally similar and clustered into three groups as other plant CAMTAs. Genotypes showeda differential response to drought stress. Thus, the plant height, number of nodes and flower, total chlorophyll and total protein content, and activity of antioxidant enzymes (ascorbate peroxidase and catalase) in plants significantly influenced by genotype and drought stress interaction. Moreover, the resistant and susceptible genotypes were identified according to three-dimensional plots and the expression patterns of PhavuCAMTA1 gene were studied using real-time quantitative polymerase chain reaction. The results of the present study serve as the basis for future functional studies on the Phaseolus CAMTA family.

miR-146a is deregulated in gastric cancer

Background: Gastric cancer is one of the most significant reasons for cancer-related death. miR-146a is one of the dysregulated factors associated with gastric tumorigenesis. However, deregulation of this microRNA (miRNA) has become controversial. Moreover, the inflammation-mediating role of this miRNA implies that miR-146a might be dysregulated by gastric cancer-related pathogens, such as Helicobacter pylori. However, the dysregulation of miR-146a in H. pylori-infected gastric tumors has not been widely studied. Objectives: We aimed to analyze the expression level of miR-146a in gastric cancer tissues and then to assess any potential association between miR-146a and H. pylori infection and other clinical characteristics. Materials and Methods: miR-146a expression level was quantitatively studied by reverse transcription quantitative polymerase chain reaction, in 144 fresh tissues including 44 normal and 100 gastric cancer samples. Results: A dramatic overexpression of miR-146a was observed in primary gastric tumors. miR-146a showed lower expression in progressed tumors with greater stages and lymph node metastasis. Conclusion: miR-146a is highly expressed in primary gastric tumor independent of H. pylori infection. It is highly expressed in the lower stages and lymph node-negative tumors. It might suggest the importance of upregulation and downregulation of this miRNA in the initiating/promoting and progressive steps of gastric tumorigenesis, respectively

Quantitative Expression of RNA from Frozen Organs and Formaldehyde-fixed and Paraffin-embedded Tissues / 法医学杂志

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Etiology analysis on a laboratory confirmed human rabies case with atypical symptoms / 中华实验和临床病毒学杂志

Objective@#To investigate and analyze the clinical features, epidemiologic information and pathogenic characteristics of a rabies patient.@*Methods@#Clinical data of the patient(boy) was collected and epidemiological survey was conducted, fluorescence quantitative reverse transcription-polymerase chain reaction (FQRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the samples of saliva, cerebrospinal fluid (CSF), skin tissue with hair follicle at the back of the neck for rabies laboratory diagnosis.@*Results@#Early symptoms of the boy were vomiting, diarrhea, fever and irritability, followed by coma and death. The boy had nasal trauma one month ago and the domestic dog died of illness during the same period. He did not accept the rabies post-exposure prophylaxis (PEP). The result of the saliva sample was positive by FQRT-PCR. The predicted segments of the glycoprotein(G), nucleoprotein (N) genes of rabies virus were amplified from the positive saliva sample of the patient by RT-PCR. Compared with rabies virus strains in Henan province, the nucleotide homology and amino acid homology in G gene segment were 96.5%-98.8% and 96.5%-99.2% respectively.@*Conclusions@#The case was diagnosed in laboratory as rabies case. The pathogenic rabies virus strain was endemic in Henan province. The nasal trauma, the dead domestic dog were probably related to the infection of the boy.

Application of multiple MLL gene rearrangement detection techniques for children with acute mononuclear leukemia / 中华医学遗传学杂志

Objective@#To assess the value of detecting multiple rearrangements of MLL gene in children with acute mononuclear leukemia (AML).@*Methods@#Eighty six children with AML were analyzed by fluorescence in situ hybridization (FISH), chromosomal karyotyping and multiplex reverse transcription-PCR (RT-PCR).@*Results@#Cross signals were detected by FISH in 26 cases, and 30.2% were detected with MLL gene rearrangements. R-band karyotyping analysis revealed 14 translocations with breakages involving 11q23 and 5 other aberrations, which yielded an overall detection rate of 22.1%. Multiple RT-PCR has detected 12 fusion genes produced by the MLL translocation, which yielded a detection rate of 14.0%. A significant difference was found in the detection rate of the three methods (P<0.05).@*Conclusion@#Combined use of FISH, chromosomal karyotyping and multiplex RT-PCR can improve the detection of MLL gene rearrangements and provide important clues for clinical diagnosis, treatment and prognosis of AML.

Characteristics and Outcomes of Patients with Pulmonary Acute Respiratory Distress Syndrome Infected with Influenza versus Other Respiratory Viruses / 결핵

BACKGROUND: Although the frequency of respiratory viral infection in patients with pulmonary acute respiratory distress syndrome (ARDS) is not uncommon, clinical significance of the condition remains to be further elucidated. The purpose of this study was to compare characteristics and outcomes of patients with pulmonary ARDS infected with influenza and other respiratory viruses. METHODS: Clinical data of patients with pulmonary ARDS infected with respiratory viruses January 2014–June 2018 were reviewed. Respiratory viral infection was identified by multiplex reverse transcription–polymerase chain reaction (RT-PCR). RESULTS: Among 126 patients who underwent multiplex RT-PCR, respiratory viral infection was identified in 46% (58/126): 28 patients with influenza and 30 patients with other respiratory viruses. There was no significant difference in baseline and clinical characteristics between patients with influenza and those with other respiratory viruses. The use of extracorporeal membrane oxygenation (ECMO) was more frequent in patients with influenza than in those with other respiratory viruses (32.1% vs 3.3%, p=0.006). Co-bacterial pathogens were more frequently isolated from respiratory samples of patients with pulmonary ARDS infected with influenza virus than those with other respiratory viruses. (53.6% vs 26.7%, p=0.036). There were no significant differences regarding clinical outcomes. In multivariate analysis, acute physiology and chronic health evaluation II was associated with 30-mortality (odds ratio, 1.158; 95% confidence interval, 1.022–1.312; p=0.022). CONCLUSION: Respiratory viral infection was not uncommon in patients with pulmonary ARDS. Influenza virus was most commonly identified and was associated with more co-bacterial infection and ECMO therapy.

cGAS recognizes HTLV-1 RTI and induces STING-dependent innate immune responses / 中华微生物学和免疫学杂志

Objective To investigate whether cyclic GMP-AMP synthase ( cGAS ) , a cytosolic DNA sensor, could recognize the reverse transcription intermediate and induce the subsequent signaling path-way during the infection of human T cell leukemia virus type 1 ( HTLV-1 ) . Methods Biotin-labeled ssDNA90, a reverse transcription intermediate of HTLV-1, was transfected into HeLa cells and the interac-tion between it and cGAS was detected by co-immunoprecipitation experiments. HeLa cells were co-cultured with HTLV-1-positive MT2 cells and the interaction between cGAS and stimulator of interferon genes ( STING) was analyzed by co-immunoprecipitation experiments. The expression of STING in HeLa cells was silenced by siRNA. cGAS was transfected into the HeLa cells 24 h after the silencing and after 24 h, these cells were co-cultured with MT2 cells for another 24 h. Real-time PCR assay was used to measure the ex-pression of IFN-β, RANTES ( regulated upon activation, normal T-cell expressed, and secreted) , TNF-α, HTLV-1 protein Tax, p19 and HBZ. Immunoblot assay was performed to evaluate the phosphorylation of IRF3 and p65 in HeLa cells. Results cGAS interacted with ssDNA90. cGAS interacted with STING in the cytoplasm. In STING-silenced HeLa cells, cGAS transfection had no influence on the expression of IFN-β, RANTES , TNF-α, Tax , p19 or HBZ , nor did it affect the phosphorylation of IRF3 or p65 . Conclusions cGAS interacted with HTLV-1 RTI ssDNA90 and activated STING-dependent innate immune responses.

Obtaining vacuolar protein sorting 34 gene in Sporothrix globosa by RACE method and analysis of its expression during the mycelium-to-yeast transition / 中华微生物学和免疫学杂志

Objective To obtain the full-length sequence of the vacuolar protein sorting 34 coding gene (vps34) of Sporothrix globosa (S. globosa) and to investigate the role of vps34 gene during the phase transition from mycelium to yeast in S. globosa. -ethods The 3′ end and 5′ end of the vps34 gene of S. globosa were amplified by rapid amplification of cDNA ends ( RACE ) . The obtained sequences were spliced and analyzed by bioinformatics software. Quantitative reverse transcription PCR ( qRT-PCR ) was used to analyze the expression of vps34 gene in mycelial and yeast phases. Results The vps34 gene of S. globosa was 3228 bp in length. The coding sequence was 3000 bp and encoded 999 amino acids with a mo-lecular mass of 111. 49×103 and an isoelectric point of 6. 38. It contained three domains including C2 PI3K class Ⅲ, PI3Ka Ⅲ and PI3Kc Ⅲ. The results of qRT-PCR showed that the expression of vps34 gene in yeast-phase S. globosa was higher than that in mycelial phase at 24 h (P<0. 05), and the greatest difference between them was observed at 48 h (P<0. 01). Conclusions Vps34p participates in the process of dimor-phic transformation of S. globosa. The obtainment of the full-length vps34 gene of S. globosa lays the founda-tions for further study on the function of Vps34p.

Epidural Fat-Derived Mesenchymal Stem Cell: First Report of Epidural Fat-Derived Mesenchymal Stem Cell

STUDY DESIGN: Experimental study. PURPOSE: To determine whether epidural fat (EF) tissue contains mesenchymal stem cells (MSC). OVERVIEW OF LITERATURE: Spine surgeons are unaware of the contents of EF tissue and the reason for its presence between the ligamentum flavum and the dura mater; therefore, EF tissues are routinely eliminated during surgical procedures. However, EF removal causes certain postoperative problems, such as post-laminectomy syndrome. We hypothesized that the EF tissue may play a significant supportive role for the neural structures and other nearby conditions. METHODS: EF tissues were obtained from consenting patients (n=3) during posterior decompression surgery of the lumbar spine. The primary cells were isolated and cultured as per previously described methods with some modifications, and the cell morphology and cumulation were examined. Thereafter, reverse transcription–polymerase chain reaction (RT-PCR), a fluorescence-activated cell sorting (FACS) analysis, and differentiation potency for differentiation into osteoblasts, chondroblasts, and adipocytes were investigated to identify whether the cells derived from EF are MSC. RESULTS: The cells from the EF tissue had a fibroblast or neuron-like morphology that persisted until the senescence at p18. MSC-specific genes, such as OCT4, SOX2, KLF4, MYC, and GAPDH were expressed in the RT-PCR study, while MSC-specific surface markers such as CD105, CD90, and CD73 were exhibited in the FACS analysis. The differentiation properties of EF-MSC for differentiation into the three types of cells (osteoblast, chondroblast, and adipocyte) were also confirmed. CONCLUSIONS: Based on the cell culture, FACS analysis, RT-PCR analysis, and differentiation potent outcomes, all the features of the cells corresponded to MSC. This is the first study to identify EF-MSC derived from the EF tissue.

Study on the level of neutrophil autophagy in peripheral blood of patients with tuberculosis infection / 国际检验医学杂志

Objective To investigate the autophagy level of peripheral blood neutrophils in patients with tuberculosis infection.Methods According to the standard for tuberculosis diagnosis, the subjects were divided into normal control group (16 cases) and active tuberculosis group (24 cases), and peripheral blood was collected.The autophagy of neutrophils and mononuclear cells of the two groups were detected by flow cytometry:The total RNA of neutrophils was extracted by Trizol and the relative quantification of Beclin1 expression was calculated by using 2-△△Ct in real-time quantitative PCR.Results There was no significant difference in age between the active tuberculosis and normal control group (P=0.165 5, P＞0.05);the proportion of male in active tuberculosis group (79.2％) was significantly higher than that of normal control group (37.5％), and the difference was statistically significant (P=0.031 5, P＜0.05).The level of neutrophil autophagy in the active tuberculosis group was significantly lower than that of the normal control group (P=0.013 8, P＜0.05), and there was no difference of mononuclear cells between the two groups (P=0.784 2, P＞0.05);active tuberculosis group the mRNA expression of Beclin1△ Ct=-1.254±0.40 was lower than that of the normal control group△Ct=-0.10±0.48, the difference was statistically significant (P＜0.001).Conclusion The incidence of active tuberculosis in male is higher than that of female;the autophagy level of neutrophils in peripheral blood of patients with tuberculosis infection decreased, and the decline of autophagy-related gene Beclin1 mRNA expression may be related to the occurrence and development of tuberculosis.

Canonical Wnt pathway gene expression and their clinical correlation in oral squamous cell carcinoma

Aim: The aim of this study is to explore the prognostic significance and clinicopathological correlations of the Wnt pathway genes in a cohort of surgically treated patients with oral squamous cell carcinoma (OSCC) patients. Settings and Design: A prospective genetic study on patients with OSCC was carried out during the period from July 2014 to January 2016. Informed consent from patients and institutional ethical approval for the study was obtained and the guidelines were strictly followed for collection of samples. Subjects and Methods: Clinical data and mRNA expression analysis of ten genes in the canonical Wnt pathway were evaluated and their relationships with clinical and demographic variables were studied in 58 tissue samples. Wnt-3a, β-catenin, secreted frizzled-related proteins sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wnt inhibitory factor 1, dickkopf-1, c-MYC, and cyclin-D1 from cancer (n = 29) and normal (n = 29) tissue samples were investigated using quantitative reverse transcription-polymerase chain reaction. Statistical Analysis: Descriptive statistics were used to summarize the sample characteristics and clinical variables. If the data were normal, then parametric tests were used; otherwise, nonparametric alternatives were used. All the analyses were carried out using SPSS version 23.0 (IBM SPSS Inc., USA). Results: Expression of sFRP-1, sFRP-2, and sFRP-5 in control samples and expression of c-MYC and cyclin D1 in cancer samples showed statistical significance. Significant expression of Wnt3A was observed among patients who had recurrence and were deceased. Conclusion: Wnt3A, β-catenin, and cyclin D1 are recognized as key components of Wnt/β-catenin signaling. However, in this study, there was no significant expression of all the three genes in OSCC. The proto-oncogene c-MYC showed statistically significant upregulation in cancer tissue samples suggesting that the OSCC among South Indian population is primarily not mediated by the canonical Wnt signaling pathway.

Evaluation of real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus in patients

Background & objectives: West Nile virus (WNV) is a mosquito-borne flavivirus. The disease can be diagnosed by isolation followed by fluorescent antibody tests, enzyme-linked immunosorbent assay and polymerase chain reaction (PCR) assay. These diagnostic methods are laborious and time-consuming. The present study was aimed to evaluate the real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, early and accurate diagnosis of WNV. Methods: A one-step single tube accelerated quantitative RT-LAMP assay was evaluated by targeting the Env gene of WNV. The gene amplification was accomplished by incubating the reaction mixture at 63°C for 60 min in both real time turbidimeter as well as routine laboratory water bath/dry heating bath. To rule out contamination issues, proper negative controls, including no template, no primer; and no enzyme, were always kept alongside each run. The RT-LAMP assay was evaluated on 105 clinical samples from individuals having ocular infection. Results: Of the 105 samples tested, 27 were positive for WNV by RT-LAMP assay. The comparative evaluation with conventional RT-PCR revealed 100 per cent accordance with sensitivity and specificity of 100 and 95 per cent, respectively. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya. Interpretation & conclusions: The RT-LAMP test seemed to be a sensitive and specific method for rapid detection of WNV infection and would be useful for rapid screening of a large number of clinical samples in endemic areas during outbreaks.

Evaluation of 5′-untranslated region amplification and sequencing for enterovirus serotypes identification diagnosis / 中华实验和临床病毒学杂志

Objective@#To evaluate an assay permitting amplification of target 5′-untranslated region (5′-UTR) sequences directly from clinical specimens and distinction among serotypes of enterovirus (EV).@*Methods@#A total of 518 rectal swabs and 148 nasal swabs tested positive by pan-enterovirus real-time PCR were collected. 5′-UTR and the viral protein 1 (VP1) gene fragments were amplified and sequenced separately for serotyping. The inconsistent samples by 5′-UTR and VP1 serotyping were further determined by using the serotype-specific RT-PCR.@*Results@#A total of 553 (83.0%) samples were detected by 5′-UTR serotyping and 318 (47.7%) were detected by VP1 serotyping in all 666 positive specimens, and there was significant difference in the detection rates between two methods in rectal and nasal swabs (P<0.001). For the rectal swabs, the mainly detected serotypes were CoxA6 (217), CoxA16 (88), EVA71 (40), CoxA10 (28) and CoxA4 (27) by 5′-UTR serotyping. Compared with the VP1 serotyping, the sensitivity and specificity of 5′-UTR serotyping were 57.1%-100% and 67.4%-98.1% respectively, with varied consistence with serotypes (kappa value 0.214-0.283). For the nasal swabs, the most frequently detected serotype was EVD68, with the sensitivity of 100%, the specificity of 91.1%, and the poor consistence (kappa value 0.217). CoxA6, CoxA16, EVA71, CoxA10 and EVD68 were further confirmed by serotype-specific RT-PCR. Using VP1 serotyping combined with serotype-specific RT-PCR as a reference method , the effect of performance of 5′-UTR serotyping on diagnosis was increased.@*Conclusions@#The performances of 5′-UTR serotyping in enterovirus vary with serotypes. The application of 5′-UTR serotyping should be considered comprehensively according to the purpose of the study.