Real-time Polymerase Chain Reaction Technology Research Articles

3' Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System.

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3′ Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.

Effects of red ginseng extract on gut microbial distribution

BackgroundRed ginseng extract boosts immunity against inflammation and cancer in the human body. However, studies on the effects of red ginseng extract on the gut microbiome remain unexplored. MethodsIn 2019, the positive effects and changes in the gut microbiome after administering 1 pack (3 g) of red ginseng extract per day to 53 adults aged 40 to 75 for 24 weeks were investigated. The gut microbial environment changes were qualitatively and quantitatively analyzed using next-generation sequencing and real-time polymerase chain reaction technology. ResultsOn comparing and analyzing alpha diversity and beta diversity, the microbial pattern showed significant differences (OTUs p = 0.003, chao1 p < 0.001, Bray-Curtis p = 0.001) before and after ingestion of red ginseng extract, indicating that gut microbial richness increased after ingestion. Moreover, after comparing and analyzing the gut microbiome's differences after red ginseng extract intake, significant differences were noted between three strains at the phylum level and among 57 strains at the genus level. ConclusionThis study proposes the potential use of red ginseng extract as a prebiotic after confirming its positive effects, including increasing gut microbiome richness, reducing harm to the gut microbiome, and increasing the number of some strains in the gut microbiome.

Epigenetic Modification of MicroRNA-219-1 and Its Association with Glioblastoma Multiforme.

MicroRNA-219-1 (miR-219-1) acts as a tumor suppressor in a variety of cancers but, the regulatory epigenetic mechanism involved in its gene expression level has not been studied. Using real-time polymerase chain reaction (real-time PCR) and bisulfite genomic sequencing technology, promoter methylation level of miR-219-1 and gene expression levels of miR-219-5p and miR-219-1-3p were determined respectively, in glioblastoma multiforme (GBM) (n = 31), their adjacent normal tissues (n = 31), and GBM U87 cell line. Following treatment of GBM U87 cells with 5-aza-2'-deoxycitidine (5-aza-dC), miR-219-1 promoter methylation, their target mRNA, and protein levels were determined by genomic bisulfite modification, real-time-PCR, and ELISA techniques, respectively. Our results showed that gene expression levels of miR-219-5p and miR-219-1-3p were significantly lower in GBM patients relative to their adjacent normal tissues (p < 0.01). MiR-219-1 promoter had a high level of methylation in GBM tissues (p < 0.01) and a negative correlation was observed between the miRNAs gene expression and methylation levels in GBM tissues (p < 0.01). Treatment of GBM U87 cells by 5-aza-dC decreased the level of miR-219-1 methylation, amount of target mRNA, and levels of cyclin A2 and mucin 4 (MUC4) proteins, and increased the expression levels of miR-219-5p and miR-219-1-3p (p < 0.01). Using external miR-219-5p and miR-219-1-3p, the expression of cyclin A2 and MUC4 were suppressed and proliferative activity of the U87MG cell line was reduced (p < 0.01). These findings suggested that DNA methylation has a crucial role in the regulation of miR-219-1 gene expression and that hypermethylated miR-219-1 may be involved in GBM pathogenesis.

Organic Manure Management Increases Soil Microbial Community Structure and Diversity in the Double-cropping Rice Paddy Field of Southern China

ABSTRACT The soil physicochemical properties and soil microbial community were affected by different fertilizer management. Fertilizer regime was closely related to soil texture and nutrient status in the double-cropping rice (Oryza sativa L.) paddy field of southern China. However, there was limited information about influence of long-term fertilizer management on soil microbial community in the double-cropping rice paddy field. Therefore, the effects of 39-years long-term fertilizer regime on soil bacterial and fungal diversity in the double-cropping rice paddy field of southern China were studied by using Illumina sequencing and quantitative real-time polymerase chain reaction (q-PCR) technology in the present paper. The experiment including four fertilizer treatments: chemical fertilizer alone (MF), rice straw and chemical fertilizer (RF), 30% organic manure and 70% chemical fertilizer (OM), and without fertilizer input as control (CK). The results showed that diversity indices of soil microbial community with RF and OM treatments were higher than that of CK treatment. Application of organic manure and rice straw management increase soil bacterial abundance of the phylum Proteobacteria, Actinobacteria, and Gammaproteobacteria, and soil fungi abundance of the phylum Basidiomycota, Zygomycota, and Tremellales were also increased. Compared with CK treatment, the value of Richness, Shannon and McIntosh indices, and taxonomic diversity were increased with RF and OM treatments. This finding demonstrated that RF and OM treatments modify soil bacterial and fungal diversity. Therefore, the combined application of organic manure or rice straw with chemical fertilizer management significantly increases the abundance of profitable functional bacteria and fungi species in the double-cropping rice paddy field of southern China.

Has_circ_0071106 can be used as a diagnostic marker for type 2 diabetes.

We explored hsa_circ_0071106 as a diagnostic marker for type 2 diabetes (T2DM) in south China, and predicted the functional mechanism of the target circRNA. A total of 107 T2DM patients and 107 healthy reference persons were included as the research objects. In the first stage, the circRNA microarray was used to detect the peripheral blood of 4 T2DM and 4 control groups to screen the differential expression profile of circRNA. In the second stage, four circRNAs were screened from the differential expression profiles of circRNA, and real-time polymerase chain reaction (Q-PCR) technology was used to verify the blood samples of 103 T2DM and 103 controls. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in bioinformatics were used to predict the functional mechanism of target circRNA. Lastly, we found that hsa_circ_0071106 increase the risk of T2DM (OR=2.819 (95% CI: 1.415~5.616)). The area under the ROCcurve hsa_circ_0071106 was 0.690, the sensitivity was 62.1%, and the specificity was 69.9%. The function prediction results showed that hsa_circ_0071106 was involved in biological processes such as protein binding, gene transcription, and may be involved in the pathway of hsa-miR-29a-5p regulating diabetes, hsa_circ_0071106 may be used as a diagnostic marker for T2DM.

Various administration forms of decellularized amniotic membrane extract towards improving corneal repair.

Amniotic membrane (AM) transplantation is often used as a treatment for corneal repair, but AM is prone to dissolving and shedding after surgery; multiple transplants will cause pain and financial burden. In this work, human amniotic membrane was firstly decellularized to obtain an AM extracellular matrix (dAM). This dAM was homogenized and extracted to obtain the dAM extract (simplified as dAME). Different forms of administration for corneal injury were performed as liquid drops (diluted dAME), in situ gels (using temperature-dependent Poloxamer 407 as the matrix), and tablets (poly(vinyl alcohol) as the matrix). The cytocompatibility of dAME was evaluated using corneal epithelial cells, corneal stromal cells and fibroblasts as cell models. The results showed that dAME is biocompatible to all these cells. Cells exhibited normal morphology and growth state at a dAME concentration of up to 160 μg mL-1. In vivo, dAME exhibited increased wound healing efficiency in severe corneal injury, being characterized with a shorter healing time for epithelium and a faster recovery for stromal opacity and thickness, compared with those of the control eyes. Different forms of administration have different effects on corneal repair; among them, in situ gels achieved the best therapeutic efficiency. Their biological mechanism was detected via quantitative real-time polymerase chain reaction (qRT-PCR) technology. It was confirmed that dAME plays important roles in promoting the mRNA expression of leucine-rich and immunoglobulin-like domains 1 (LRIG1) and in inhibiting the mRNA of transforming growth factor-β1 (TGF-β1).

MiR-34c regulates the proliferation and apoptosis of lung cancer cells

Purpose: As miR-34c acts as a tumor suppressant for multiple cancers, the purpose of this study was to investigate that role that miR-34c plays in the proliferation and apoptosis of lung cancer.&#x0D; &#x0D; Methods: The expression of miR-34c in 600 patients with lung cancer was quantitatively analyzed with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) technology and correlated to&#x0D; clinical pathological parameters. The CCK-8 analysis and flow cytometry were carried out to detect cell proliferation and apoptosis in miR-34c-mimic transfected cell lines. Moreover, the regulation of miR-34c to interleukin-6 (IL-6) in cell lines was detected by western blot, qRT-PCR and dual-luciferase reporter assay.&#x0D; &#x0D; Results: The expression of miR-34c was downregulated in lung cancer compared with adjacent normal tissues. The expression level of miR-34c was linked to stromal invasion. Furthermore, overexpressing miR-34c played an active role in effectively inhibiting cell proliferation and inducing apoptosis. In addition, a significant inverse relationship was exhibited between the expression of miR-34c and IL-6 in tumor tissues.&#x0D; &#x0D; Conclusion: At the molecular level, IL-6 can be used as a direct target of miR-34c in the treatment of lung cancer cells and miR-34c can be used as an effective biomarker and therapeutic target for lung cancer.

Expression of MiR-608 in Nonsmall Cell Lung Cancer and Molecular Mechanism of Apoptosis and Migration of A549 Cells.

Objective This Work is aimed at exploring the effect of microRNA (MiR)-608 on the function of nonsmall cell lung cancer (NSCLC) A549 cells and related mechanisms. Methods Blood samples of 106 NSCLC patients (experimental group) as well as 124 normal people (control group) were selected for relevant investigation. Polymerase chain reaction (PCR) as well as DNA sequencing was used to determine the genotyping of the MiR-608 rs4919510 polymorphism. MiR-608 expression in cells was detected by real-time PCR technology. Western blotting was used to detect changes in protein levels. NSCLC tissues as well as adjacent tissues were explored in 33 patients undergoing surgery. Results MiR-608 rs4919510 does not influence the incidence of NSCLC patients. In addition, MiR-608 expression was downregulated in the tumor tissue of NSCLC patients, while the transcription factor activating enhancer-binding protein 4 (TFAP4) expression was upregulated. MiR-608 promotes DOX- (Doxorubicin-) induced apoptosis by negatively regulating TFAP4 expression in NSCLC tissue. TFAP4 can significantly inhibit the migration of A549 cells. Conclusion The findings in this investigation can contribute to the effective treatment of NSCLC patients. Also, the investigation can provide some theoretical support for the application of new targets for NSCLC treatment.

SERS Platform Based on Bimetallic Au-Ag Nanowires-Decorated Filter Paper for Rapid Detection of miR-196ain Lung Cancer Patients Serum

Detecting microRNA (miRNA) biomarkers expression is of great significance for the diagnosis and treatment of lung cancer. Surface-enhanced Raman scattering (SERS) has achieved microRNA sensing for the diagnosis of primary liver cancers. In this work, we developed a SERS technology for the rapid detection of lung cancers-related miRNA (miR-196a) using bimetallic Au-Ag nanowire (AgNW@AuNPs) substrates coupled with the target hairpin DNA. The finite-difference time-domain simulation proved that a large number of “hot spots” were generated between the AgNW and AuNPs, which resulted in a huge enhancement of the signal of Raman reporters. Filter paper treated by hexadecenyl succinic anhydride hydrophobic and modified with AgNWs@AuNPs was used as capturing substrate. The detection limits of miR-196a in PBS and serum were as low as 96.58 aM and 130 aM, respectively. Studies on nonspecific sequence and single-base mismatch of miRNA demonstrated that SERS-based platform was highly selective, excellent uniform, and reproducible. Finally, the platform was used to show that the miR-196a expression in the serum of lung cancer patients was much higher than that in healthy people. The detection results indicated that the SERS platform had potential applications in cancer diagnosis and might be a viable alternative to the conventional miRNA detection method, the real-time polymerase chain reaction (RT-PCR) technology.

Conventional PCR assisted single-component assembly of spherical nucleic acids for simple colorimetric detection of SARS-CoV-2

Continuous identification of suspected infectious cases is crucial to control the recent pandemic caused by the novel human coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Real-time polymerase chain reaction (real-time PCR) technology cannot be implemented easily and in large scale in some communities due to lack of resources and infrastructures. Here, we report a simple colorimetric strategy derived from linker-based single-component assembly of gold nanoparticle-core spherical nucleic acids (AuNP-core SNAs) for visual detection of PCR products of SARS-CoV-2 ribonucleic acid (RNA) template. A palindromic linker is designed based on SARS-CoV-2 specific E gene to program the identical colloidal SNAs into large assemblies along with a distinct red-to-purple color change. The linker acts as a probe of SARS-CoV-2 RNA in conventional PCR reaction. In the presence of the correct template the palindromic linker, which is complementary to a short region within the target amplicon, is cleaved by 5′-exonuclease activity of deoxyribonucleic acid (DNA) polymerase. Cleavage of the palindromic linker during the amplification process inhibits the single-component assembly formation of SNAs. So, positive and negative viral samples produce simply red and purple colors in the post PCR colorimetric test, respectively. Evaluation of the samples obtained from cases with laboratory-confirmed SARS-CoV-2 infection revealed that our assay can rival with real-time PCR method in sensitivity.

Effects of short-term manure nitrogen input on soil microbial community structure and diversity in a double-cropping paddy field of southern China

The soil physicochemical properties and soil microbial communities were affected by different fertilizer management. Fertilizer regime were closely relative to the soil texture and nutrient status in a double-cropping paddy field of southern China. However, there was limited information about the influence of different manure nitrogen (N) input on soil microbial communities in a double-cropping rice (Oryza sativa L.) field. Therefore, the short-term different manure N input rate management on soil bacterial and fungal diversity in a double-cropping paddy field of southern China were studied by using Illumina sequencing and quantitative real-time polymerase chain reaction technology in the present paper. The filed experiment were including 100% N of chemical fertilizer (M0), 30% N of organic manure and 70% N of chemical fertilizer (M30), 50% N of organic manure and 50% N of chemical fertilizer (M50), 100% N of organic manure (M100), and without N fertilizer input as control (CK). The results showed that diversity indices of soil microbial communities with application of organic manure and chemical N fertilizer treatments were higher than that of CK treatment. Application of organic manure and chemical N fertilizer management increase soil bacterial abundance of the phylum Actinobacteria, Proteobacteria and Gammaproteobacteria, and soil fungi abundance of the phylum Basidiomycota and Zygomycota were also increased. Compared with CK treatment, the value of Richness, Shannon and McIntosh indices, and taxonomic diversity were increased with M30, M50 and M100 treatments. This finding demonstrated that M30, M50 and M100 treatments modify soil bacterial and fungal diversity. Therefore, the combined application of organic manure and chemical fertilizer N management could significantly increase the abundance of profitable functional bacteria and fungi species in a double-cropping rice field of southern China.

An improved quantitative real-time polymerase chain reaction technology for Helicobacter pylori detection in stomach tissue and its application value in clinical precision testing

BackgroundHelicobacter pylori (H. pylori) infection is a serious human health threat. The empiric H. pylori treatment paradigm guided by traditional testing technologies has led to antibiotic resistance. Here, we improved the qPCR method to provide technical support for precision H. pylori diagnosis and treatment.MethodsTwo pairs of primers and probes targeting the glmM gene were designed to detect H. pylori, and a multiplex qPCR method was established for virulence factor detection. Then, a rapid urease test (RUT), culturing and qPCR were performed on 141 specimens collected from Xinqiao Hospital of China in 2017 to evaluate the qPCR detection capability. Finally, the H. pylori infectious amount and virulence genes were detected by qPCR.Results1. The improved qPCR method which used two pairs of primers had a higher detection rate (100%) and better accuracy (p = 0.000), compared with the qPCR using a pair of primers. It also had better consistency with the bacterial culture than with RUT (Kappa =0.440, p < 0.001). 2. The H. pylori infectious amount was significantly positively associated with gastritis in corpus (p = 0.003) and gastric erosion (p = 0.043). The H. pylori infectious amount in gastric precancerous patients was significantly lower than that in H. pylori-positive patients (p < 0.05), and the infectious H. pylori-vacA s1+ amount was significantly greater than that of H. pylori-vacA s1- (p < 0.05). 3. The vacA s1 frequency was significantly higher than that of vacA m1/cagA+/babA2+ in chronic superficial gastritis (p = 0.000), peptic ulcer (p = 0.037) and gastric erosion (p = 0.009). The H. pylori-vacA+/cagA+/babA2+ frequency showed a significant positive correlation (p < 0.05).ConclusionsThe H. pylori infectious amount and presence of H. pylori virulence factors showed complex correlations with gastric disease occurrence and development. The improved qPCR with good detection performance can be used for quantitative H. pylori detection and testing for the virulence genes vacA s1, vacA m1, cagA and babA2 simultaneously. These findings will provide valuable information for disease diagnosis and treatment.

CircABCB10 promotes the proliferation and migration of lung cancer cells through down-regulating microRNA-217 expression.

We aimed at studying the role and molecular mechanism of circular RNA circABCB10 in the progression of lung cancer (LCa). We collected LCa tissues using quantitative real-time polymerase chain reaction (qRT-PCR) technology to determine circABCB10 expression and performed survival analysis based on the clinical data of LCa patients. At the same time, the specific effects of circABCB10 on the biological function of LCa cell lines were determined by certain cell function experiments, including cell counting kit-8 (CCK-8) test, plate cloning experiment, transwell and cell wound healing assays. The downstream key gene microRNA-217 of circABCB10 was predicted through bioinformatics analysis and the potential regulation between them was confirmed by luciferase assay. microRNA-217 was knocked down in LCa cell lines to verify its important role in the progression of LCa. CircABCB10 showed abnormally high expression in LCa tissues and cell lines and was related to the poor prognosis of patients. In vitro cell experiments demonstrated that knocking down circABCB10 remarkably suppressed the proliferation and migration ability of LCa cells. In addition, circABCB10 can specifically bind to microRNA-217 and negatively regulate its expression of microRNA-217 in LCa cells. Finally, cell functional experiments showed that microRNA-217 is a key downstream gene that mediates the regulation of circABCB10 on LCa cell function. CircABCB10, abnormally highly expressed in LCa tissues, is able to induce the malignant progression of this cancer.

Detection of Coronavirus Disease (COVID-19) using Radiological Examinations

An outbreak of Coronavirus disease 2019 (COVID-19) occurred in China. The causative agent of COVID-19 is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The disease rapidly spreads from China to the world by trading and traveling. Until April 24, 2020, approximately 2,544,792 cases were confirmed with 175,694 deaths throughout the world. The highest number of cases were identified from the United States of America (USA) whereas the mortality rate is high in Portugal. The diagnosis of COVID-19 is based on Computed Tomography Scanning (CT Scan) and Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Assessing the extension of pathology, the exact location of the area involved, and assessment of the disease severity makes CT scan superior to other modalities. This review shows that real-time polymerase chain reaction and imaging technology both play an important role in the diagnosis of COVID-19. However, imaging modalities have more importance in diagnosis and screening than qRT-PCR. The qRT-PCR was positive in 81.3% whereas CT scan abnormality was observed in 89.8%. Bilateral lobe (51.4%) abnormality was found more than a single lobe (21.5%) in COVID-19 infected patients. The CT scan reports show a high-level abnormality in right lower lung lobe than others in COVID-19 infected patients. The CT scan evaluates different manifestations such as the presence of ground-glass opacities, consolidations, crazy paying linear, cavitation, discrete nodules, pleural effusion, and lymphadenopathy. It is concluded that imaging technology especially CT scan and X-rays play an important role in the screening and diagnosis of COVID-19 infected patients in limited access to qRT-PCR regions. The common radiological manifestation was also determined, which will be helpful for the radiologist to diagnosed COVID-19 infected patients in the early stages. Follow up studies required regarding the radiological examinations.

Clinicopathological and Prognostic Significance of EML4-ALK Rearrangement in Patients with Surgically Resected Lung Adenocarcinoma: A Propensity Score Matching Study.

ObjectiveThe echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene is a key oncogenic driver in non-small cell lung cancer (NSCLC). This study analyzed the clinicopathological characteristics and prognostic significance of EML4-ALK fusion gene in patients with surgically resected adenocarcinoma.MethodsThe clinicopathological characteristics of 1056 consecutive patients with surgically resected stage I–IIIA adenocarcinoma were collected from February 2014 to October 2014, and EML4-ALK rearrangement was detected using real-time polymerase chain reaction (RT-PCR) technology. To compare the imaging and pathological features, a propensity score matching (PSM) method was performed. The follow-up information was collected to evaluate the long-term outcomes of patients with EML4-ALK rearrangement.ResultsThe prevalence of EML4-ALK rearrangement was 6.6% in 1056 consecutive patients. A total of 70 EML4-ALK-positive and 210 EML4-ALK-negative patients were identified after PSM. Imaging and pathological analyses showed that EML4-ALK rearrangement was significantly associated with less ground-glass opacity (GGO) (adjusted OR=1.38, 95% CI=1.03–1.85, Ptrend=0.029) and higher prevalence of non-invasive mucinous adenocarcinoma mucin-laden adenocarcinomas (non-IMA MLA, adjusted OR=6.79, 95% CI=2.69–17.17, P<0.001). EML4-ALK rearrangement was found to be an unfavorable prognostic factor for disease-free survival (DFS) in female patients (HR=2.26, 95% CI=1.13–4.53, P=0.021).ConclusionOur results suggest that adenocarcinomas harboring EML4-ALK fusion gene exhibit specific radiological and pathological characteristics compared with EML4-ALK-negative adenocarcinomas. In female patients, EML4-ALK rearrangement was associated with shorter DFS.

Assessment of respiratory tract viruses in febrile neutropenic etiology in children and comparison with healthy children with upper/lower respiratory tract infection.

OBJECTIVE:This study aims to compare the frequency of respiratory viruses using real-time and multiplex polymerase chain reaction technology and nasopharyngeal swabs taken during exacerbation of patients aged 0–18 years followed for febrile neutropenia (FN) with non-FN children.METHODS:This prospective study included a total of 40 patients with FN and malignancies followed at Eskisehir Osmangazi University, Department of Pediatric Hematology and Oncology. The control group (n=76) consisted of age-matched patients with upper respiratory tract infections (URTIs) or lower respiratory tract infections (LRTIs) who were admitted to the emergency service due to fever.RESULTS:Viral agents were detected in 16 of 53 FN attacks (30.1%). The most commonly isolated viruses were coronavirus (23.7%, n=9), influenza B (18.4%, n=7), and adenovirus (18.4%, n=7). Of 76 children diagnosed with URTI with fever (52.6%) had viral agents, and only 28 of them had a single agent. The most commonly isolated virus was adenovirus (28.6%, n=14). Viral factors were found in 32 of 42 patients (76.1%) patients diagnosed with LRTI, while respiratory syncytial virus was the most common virus in 27 patients (21.7%, n=5).CONCLUSION:Our study results show that viral agents play an important role in the etiology of FN. This is the first study to show that viral agents play an important role in the etiology of this disease and viral factors in non-neutropenic febrile children at the same time period by detecting respiratory viruses in 30% of FN cases. More similar studies provide antiviral therapy in selected patients, as well as these studies lead to reduce the use of antimicrobial agents or allow more selective use of antibiotics and/or the earlier discontinuation of these antibiotics in febrile neutropenic children who have been shown to have viral cause of respiratory tract infection based on clinical and microbiological/molecular diagnostic criteria.

Comparison of CapitalBio™ Mycobacterium nucleic acid detection test and Xpert MTB/RIF assay for rapid diagnosis of extrapulmonary tuberculosis

The CapitalBio™ Mycobacterium nucleic acid detection test is a real-time fluorescent polymerase chain reaction and TaqMan probe technology test for rapid diagnosis of extrapulmonary tuberculosis (EPTB). This test had moderate sensitivity and high specificity for EPTB, like that of Xpert MTB/RIF, especially for lymph node, chest wall, and purulent fluid samples.

High expression of ALDH1A3 might independently influence poor progression-free and overall survival in patients with glioma via maintaining glucose uptake and lactate production.

Recent studies have found that the acetaldehyde dehydrogenase 1A3 (ALDH1A3) gene is a marker of glioma stem cells. A total of 115 brain glioma specimens were collected and classified into grade I-IV, while non-tumor brain tissue specimens, taken from 12 patients of vascular malformation surgery, were used as control. ALDH1A3 gene promoter methylation in glioma tissues was detected by pyrosequencing, while immunohistochemistry and western blot were used to detect ALDH1A3 protein expressions in different grades of glioma tissues and normal brain tissues. The expression of ALDH1A3 in the glioma cell line U87 was detected by quantitative real-time polymerase chain reaction and RNA-Seq technology was applied to investigate differentially expressed genes before and after silencing the ALDH1A3 gene. Among the 115 glioma tissue specimens, 50 (43.48%) showed low and 65 (56.52%) high expression of ALDH1A3, but no expression was detected in the control. Univariate and multivariate COX regression analyses showed that the patient's tumor pathological grade, the methylation status of ALDH1A3 promoter, and the expression of ALDH1A3 protein were risk factors for progression-free survival (PFS) and overall survival (OS) (all P < 0.05) and the OS of mice with silenced ALDH1A3 in a glioma nude mouse model was prolonged. U87 experiments revealed that ALDH1A3 expression had significant effects on apoptosis, proliferation, cell cycle, mitochondrial membrane potential, glucose consumption, lactate production, invasion ability, and expression of the pyruvate kinase M2 (PKM2) and hexokinase 2 (HK2) in glioma cells. ALDH1A3 protein expression is a marker for poor PFS and OS in glioma patients.

Association of Autoimmune Regulator Gene Rs2075876 Variant, but Not Gene Expression with Alopecia Areata in Males: A Case–control Study

ABSTRACTAlopecia areata (AA) is a non-scarring hair loss of autoimmune etiology. The autoimmune regulator (AIRE) gene is believed to be an important driver in AA pathogenesis. Genetic variants can alter mRNA expression levels which may provoke an autoimmune response. A total of 337 males (97 AA patients and 240 controls) were enrolled in the current case–control study. On screening of the most frequent variants in the gene, rs2075876 (A/G) polymorphism in intron 5 was selected and genotyped using Real-Time PCR (polymerase chain reaction) technology. Additionally, circulatory AIRE expression levels were quantified by quantitative reverse-transcription PCR (qRT-PCR). Allelic discrimination analysis revealed GG genotype to be more frequent in patients (90.7% in AA compared to 32.5% in controls, p < .001). G variant conferred increased risk to alopecia under homozygote comparison (GG versus AA: OR = 16.1, 95%CI = 5.57–46.3), dominant model (GG+AG versus AA: OR = 7.24, 95%CI = 2.5–20.5), recessive model (GG versus AG+AA: OR = 20.3, 95%CI = 9.7–42.4), and allelic model (G versus A: OR = 11.6, 95%CI = 6.47–21.1). The expression levels of AIRE gene did not differ significantly between patients and controls and were not related to rs2075876 variant. In conclusion, the intronic variant (rs2075876) is suggested to be a potent susceptibility variant for AA development in the studied population.Abbreviations: AA: Alopecia areata; AIRE: Autoimmune Regulator; APECED: Autoimmune, Polyendocrinopathy Candidiasis Ectodermal Dystrophy; DLQI: Dermatology life quality index questionnaire; MIQE: Minimum information for publication of quantitative real-time PCR experiments; mTEC: Medullary thymic epithelial cells; PHD: Plant homeodomain; qRT-PCR: Quantitative reversetranscription-polymerase chain reaction; RA: Rheumatoid arthritis

Hippocampal neuron loss and astrogliosis in medial temporal lobe epileptic patients with mental disorders.

Hippocampal neuron loss and reactive astrogliosis are pathological features of medial temporal lobe epilepsy. Here, the expression of hippocampal astrogliosis-associated genes are studied in subjects with medial temporal lobe epilepsy and mental disorders (such as depression, anxiety and psychiatric comorbidities). The relationship between functional changes in hippocampus astrocytes and concurrent mental disorders are discussed. Nissl staining identified medial temporal lobe epilepsy-induced neuronal loss in the CA1 region of hippocampus. Quantitative real-time polymerase chain reaction and immunofluorescence technology were used to detect hippocampus glial fibrillary acidic protein, metallothionein, and aquaporin-4. The hippocampus area of subjects with medial temporal lobe epilepsy (with or without mental disorders) were smaller than the control group. Hippocampal neuronal loss and astrogliosis were more obvious in groups of medial temporal lobe epileptic patients with mental disorders. Relative protein levels of glial fibrillary acidic protein, metallothionein-I/II, and aquaporin-4 were significantly higher in subjects with medial temporal lobe epilepsy than seen in controls. Medial temporal lobe epileptic patients with mental disorder or depression had elevated metallothionein-I/II protein level when compared to controls and medial temporal lobe epileptic patients without mental disorder. Protein levels of glial fibrillary acidic protein and aquaporin-4 in medial temporal lobe epileptic patients with mental disorders were significantly lower than that in medial temporal lobe epileptic patients with no mental disorder. It is concluded that functional changes in hippocampus astrocytes are associated with mental disorders in medial temporal lobe epileptic patients and the astrogliosis-related genes of glial fibrillary acidic protein, metallothionein-I/II and aquaporin-4, are involved in this process.