Real-time PCR Method Research Articles

Suitability of different cytological preparations for molecular analysis of advanced NSCLC.

Around 85% of non-small cell lung cancers (NSCLCs) are diagnosed at an advanced stage (IIIB to IV), where therapeutic options depend on molecular analysis. However, diagnostic material for molecular testing is often represented by cytological samples which are generally scarce and span a wide range of preparation types. Thus, the primary objective is to efficiently manage materials for molecular profiling. This study aims to evaluate the suitability of different cytological samples to assess morphological and molecular characteristics of advanced NSCLC. Sixty-seven cytological samples obtained from patients with advanced NSCLC were utilized. The series encompassed different procedure types (fine-needle aspiration cytology, transbronchial needle aspiration, effusions) processed by cell blocks in 54% (n=36), direct smears in 33% (n=22), and Liquid Based Cytology (LBC) in 13% (n=9). Cytological diagnoses were routinely performed and molecular analysis were conducted using NGS and RT-PCR methods. Adequate quantity and quality of nucleic acids were obtained from all the samples, allowing molecular profiling. Combined Next Generation Sequencing (NGS) and Real Time-Polymerase Chain Reaction (RT-PCR) analysis showed wild-type profiles in 62.7% (n=42) and mutated profiles in 37.3% (n=25) of the samples. Kirsten Rat Sarcoma Virus (KRAS) mutations were identified in 19.5% (n=13) of samples, Epidermal Growth Factor Receptor (EGFR) mutations in 10.4% (n=7) and v-raf murine sarcoma viral oncogene homolog B (BRAF) mutations in 2.9% (n=2). Identified chromosomal alterations were v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2) duplication in 2,9% (n=2). The cytological sample types examined in this study proved to be suitable for molecular testing, in addition to conventional morphologic diagnosis, showing versatility and adaptability to different clinical contexts. Molecular testing on cytological samples is accurate and fast, representing a valid tool for molecular profiling of advanced NSCLC.

Prognostic impact of clinical and genetic factors on delayed elimination of methotrexate in children with acute lymphoblastic leukemia

Background. Methotrexate (MTX) in high doses (1000–5000 mg/m2), occupies one of the leading places in modern programs of therapy of acute lymphoblastic leukemia (ALL) in children. Achievement of high long-term survival rates in children with ALL has become possible thanks to this drug. However, no less important problem is the toxicity of the treatment and prediction of its efficacy and safety, in this regard, the role of pharmacogenetic studies in the identification of polymorphisms in candidate genes affecting the pharmacokinetics of MTX is increasing.Objective. To determine predictors of delayed elimination of methotrexate using pharmacokinetic biomarkers in children with acute lymphoblastic leukemia.Materials and methods. We prospectively analyzed the database of pediatric patients with ALL within the framework of an observational (cohort) singlecenter study. The study included 124 children diagnosed with ALL who received therapy according to the ALL-IC BFM 2009 protocol including high-dosed MTX. Real-time PCR method was used to study polymorphisms of ABCB1 and SLCO1B1 genes. The study material was peripheral blood. Statistical analysis ofpharmacogenetic biomarkers influence on toxicity and efficacy of therapy was performed using SPSS Statistics 26.0 program (USA). To form mathematical prognostic models, we used the method of logistic function construction using binary logistic regression with step-by-step selection of factors and, if necessary, additional construction of ROC-curves with subsequent ROC-analysis. Differences were considered significant at p < 0.05; at p ≥ 0.05, differences were considered unlikely and statistically insignificant.Results. Based on the results of the conducted complex analysis of efficacy and safety of high-dosed MTX therapy, a reliable (p < 0.001) prognostic model with high sensitivity, specificity and efficacy (>70 %, respectively) was developed, demonstrating the interrelationships of clinical and genetic factors influencing the delay of MTX elimination in children with ALL, which confirms the necessity of implementing pharmacogenetic testing in real clinical practice.Conclusion. Determination of polymorphisms of genes providing transport and metabolism of cytostatics should be used in practical work of oncohematological clinics for individualization of therapy and ensuring its safety.

Certified reference materials for GMO analysis-more than 25 years of GMO CRM production at EC JRC.

Certified reference materials (CRMs) play a crucial role in ensuring the quality of analytical measurements. Particularly, the EU labelling legislation on genetically modified organisms (GMOs) in food and feed products explicitly requires CRMs for its implementation. The CRMs are used to calibrate and control the quantitative real-time polymerase chain reaction (qPCR) method and support official control laboratories, such as National Reference Laboratories (NRLs), in maintaining their ISO/IEC 17025 accreditation. The Joint Research Centre of the European Commission (EC JRC) is specialized in the production of reference materials and has been producing GMO CRMs since 1998. Together with a validated event-specific qPCR method, these GMO CRMs form the EU reference system for the quantification of EU-authorized GMO events in food and feed products and ensure a common GMO labelling threshold. This article gives a retrospective view on the more than 25 years of GMO CRM production at JRC. It describes requirements for GMO CRMs in view of an EU market authorization under (EC) No. 1829/2003. The evolution and major improvements of the production of GMO CRMs at JRC are summarized as well as the current understanding of theEU's GMO reference system for GMO quantification and its impact on commutability. It provides insights into GMO CRM sales and their worldwide distribution. This information may be useful for policymakers and researchers in understanding the current EU GMO measurement landscape and to anticipate possible future demands related to GMO events based on new genomic techniques (NGTs).

Glut-1 exerts anti-tumor activity in glioma by regulating AKT / mTOR signaling pathway

Objective Glioma is a common tumor in neurosurgery. Glut-1 is the main carrier of glucose uptake by cells and can provide energy through the glycolytic pathway. However, the role and related mechanisms of Glut-1 in glioma have not yet been elucidated. Methods Real time PCR and Western blot were done to assess Glut-1 level in glioma tumor tissues and adjacent tissues. Glioma U87 cells were separated into control group; Glut-1 negative control (NC group); and Glut-1 siRNA group followed by analysis of Glut-1 expression, cell proliferation by MTT assay, cell invasion, cell apoptosis and cycle by flow cytometry and AKT / mTOR signaling proteins level by Western blot. Results Glut-1 expression was significantly increased in the tissues of glioma patients (P < 0.05) compared to adjacent tissues and its level was related to tumor size, pathological grade and survival. Down-regulating the expression of Glut-1 can significantly inhibit tumor cell proliferation and invasion, increase apoptosis and induce G0 phase of cell cycle arrest, and inhibit the expression of AKT / mTOR signaling proteins phosphorylation (P < 0.05). Conclusions Glut-1 level in glioma tissues is significantly increased, which is related to the pathological features. Down-regulating Glut-1 can inhibit glioma by regulating the AKT / mTOR signaling pathway.

A novel ready-to-use loop-mediated isothermal amplification (LAMP) method for detection of Burkholderia mallei and B. pseudomallei

BackgroundGlanders and melioidosis are contagious zoonotic diseases caused by Burkholderia mallei and B. pseudomallei, respectively. Bacterial isolation and polymerase chain reaction (PCR) have been used to detect these bacteria in animals suspected of infection; however, both methods require skilled experimental techniques and expensive equipment. These obstacles make it difficult to diagnose B. mallei and B. pseudomallei infections in areas where reagents and equipment are difficult to procure. To solve this problem, we developed an easy and ready-to-use dried-format diagnostic tool based on loop-mediated isothermal amplification (LAMP) method.ResultsThe primer set targeting the internal transcribed spacer (ITS) region detected 10 genomic copies of B. mallei DNA and B. pseudomallei DNA using the conventional liquid LAMP method. This primer set did not detect any other Burkholderia species. Using this novel primer set, a dried-format in-house LAMP method with high sensitivity and specificity was developed. This method was used to test for the presence of B. mallei DNA in swabs collected from the nasal cavity and ulcerated skin of 19 B. mallei-infected horses and five uninfected horses and was compared with the real-time PCR method. These two tests showed 87.5% agreement for the positive samples and 100% agreement for the negative samples. This method detected all tested B. pseudomallei clinical isolates.ConclusionsWe established the first dry LAMP method for the detection of B. mallei and B. pseudomallei. This study provided a simple, rapid, cost-effective, and sensitive diagnostic tool for glanders and melioidosis.

INVESTIGATION OF DNA GYRASE RESISTANCE GENES IN ACINETOBACTER BAUMANNII ISOLATES

Objective: It is known that bacteria resistant to antimicrobial agents can spread to the community in the hospital environment. Acinetobacter baumannii is a difficult-to-treat pathogen due to its antibiotic resistance genes. It is resistant to many antibiotics, including fluoroquinolones. By identifying mutations in DNA gyrase and topoisomerase IV genes, quinolone resistance of A. baumanni is found. Our study, aimed to determine mutations in DNA gyrase resistance genes that cause quinolone resistance (gyrA, gyrB, parC) in A. baumannii isolates using the real-time PCR method. Material and Method: 73 A. baumannii resistant clinical isolates were used in the study. A. baumannii was amplified using primers for gyrA, parC, gyrB genes. Using HRM analysis, gyrA, gyrB, parC gene Real-Time PCR amplicons were grouped according to their DNA sequences. A. baumannii isolates were grouped as P1, P2, P3, GA1, GA2, GA3, GB1 and GB2 according to different HRM profiles, DNA sequence analysis of 2 different Real-Time PCR products from each group was performed. Result and Discussion: Mutations were detected in GA1, GA2, GA3 genotypes. No mutations were observed in the P1, P2, P3 and, GB1, GB2 genotypes.

The VDR rs1544410 and rs11568820 Variants and the Risk of Osteoporosis in the Polish Population.

Vitamin D affects bone metabolism and calcium-phosphate metabolism. Its deficiency leads to bone mineralization disorders and is the cause of abnormal skeletal development from fetal life to the period of completed skeletal growth. In later periods of life, vitamin D deficiency leads to bone metabolism disorders, i.e., osteoporosis. Disturbance of the balance between osteoblasts responsible for bone formation and osteoclasts associated with bone resorption results in reduced bone mass and bone weakening, and consequently leads to susceptibility to fractures. Analysis of genetic variants of the vitamin D receptor (VDR) concerns their relationship with metabolic bone diseases, and the results of previous studies assessing the relationship of polymorphisms with bone mineral density, fracture risk, or osteoporosis are not clear. Therefore, the aim of our study was to determine the effect of rs1544410 and rs11568820 polymorphisms of the VDR gene on the risk of developing osteoporosis in the Polish population. The study included 197 women with osteoporosis, 98 women with osteopenia, and 147 healthy controls. The real-time PCR method was used to determine the rs1544410 and rs11568820 polymorphisms of the VDR1 gene. Analysis of the results in the group with osteopenia showed that for the rs1544410 polymorphism, heterozygous GA genotypes occurred in 37.8% of the study group and 47.6% of the controls (OR = 0.60; 95%CI: 0.34-1.05), and homozygous AA in 15.3% of the study group and 17.0% of the controls (OR = 0.68; 95%CI: 0.32-1.44) (p = 0.185, AIC = 332.4; AIC-Akaike information criterion). The best model for this variant turned out to be the dominant model OR = 0.62; 95%CI: 0.37-1.04; p = 0.071, AIC = 330.5. In the case of the rs11568820 polymorphism of the VDR gene, the GG genotype was more common in women with osteopenia compared to controls (75.5% vs. 70.1%). Genotypes containing at least one mutant A allele were present in 24.5% of women with osteopenia and 29.9% of controls (OR = 0.76; 95%CI: 0.43-1.36; p = 0.349; AIC = 332.9). Analyzing the rs1544410 polymorphism in women with osteoporosis, the GA genotype was present in 42.1% of the study group and 47.6% of patients with normal bone density (OR = 0.74; 95%CI: 0.46-1.19), and the AA genotype in 15.7% of the study group and 17.0% of controls (OR = 0.78; 95%CI: 0.41-1.46) (p = 0.441). In the case of the rs11568820 polymorphism, the GA genotype occurred in 22.3% of the study subjects and 27.2% of the control patients (OR = 0.76; 95%CI: 0.46-1.25), and the AA genotype in 2.0% of the study subjects and 2.7% of the controls (OR = 0.69; 95%CI: 0.17-2.83) (p = 0.511). For both variants, the model with the lowest AIC value was the dominant model, in which for the rs1544410 variant OR = 0.75; 95%CI: 0.48-1.17; p = 0.203; AIC = 472.0 was obtained, while for rs11568820-OR = 0.75; 95%CI: 0.47-1.22; p = 0.250; AIC = 472.3. The obtained results indicate that the rs1544410 and rs11568820 polymorphisms of the VDR gene do not affect the development of osteoporosis in the Polish population.

Hypermethylation at 45S rDNA promoter in cancers.

The ribosomal genes (rDNA genes) encode 47S rRNA which accounts for up to 80% of all cellular RNA. At any given time, no more than 50% of rDNA genes are actively transcribed, and the other half is silent by forming heterochromatin structures through DNA methylation. In cancer cells, upregulation of ribosome biogenesis has been recognized as a hallmark feature, thus, the reduced methylation of rDNA promoter has been thought to support conformational changes of chromatin accessibility and the subsequent increase in rDNA transcription. However, an increase in the heterochromatin state through rDNA hypermethylation can be a protective mechanism teetering on the brink of a threshold where cancer cells rarely successfully proliferate. Hence, clarifying hypo- or hypermethylation of rDNA will unravel its additional cellular functions, including organization of genome architecture and regulation of gene expression, in response to growth signaling, cellular stressors, and carcinogenesis. Using the bisulfite-based quantitative real-time methylation-specific PCR (qMSP) method after ensuring unbiased amplification and complete bisulfite conversion of the minuscule DNA amount of 1 ng, we established that the rDNA promoter was significantly hypermethylated in 107 breast, 65 lung, and 135 colon tumour tissue samples (46.81%, 51.02% and 96.60%, respectively) as compared with their corresponding adjacent normal samples (26.84%, 38.26% and 77.52%, respectively; p < 0.0001). An excessive DNA input of 1 μg resulted in double-stranded rDNA remaining unconverted even after bisulfite conversion, hence the dramatic drop in the single-stranded DNA that strictly required for bisulfite conversion, and leading to an underestimation of rDNA promoter methylation, in other words, a faulty hypomethylation status of the rDNA promoter. Our results are in line with the hypothesis that an increase in rDNA methylation is a natural pathway protecting rDNA repeats that are extremely sensitive to DNA damage in cancer cells.

GeneXpert MTB/RIF Ultra in Pediatric Tuberculosis: How Disease Characteristics Modify Test Performances.

The diagnosis of pediatric tuberculosis (TB) is challenging, due to the lower sensitivity of microbiological tests, such as culture and microscopy, compared to their performance in adult cases. Guidelines have introduced molecular tests, including GeneXpert MTB/ RIF and GeneXpert MTB/RIF Ultra. These tests use a real-time polymerase chain reaction method and provide information on M. tuberculosis detection and drug-resistance-associated mutations in less than 2 hours. This retrospective single-center study aimed to evaluate the accuracy of GeneXpert and GeneXpert Ultra for the diagnosis of pediatric TB. This retrospective study was conducted on a total of 95 children diagnosed with probable or confirmed TB disease (74 diagnosed with pulmonary TB, 21 with extrapulmonary TB), who referred to the infectious disease unit at Meyer Children's Hospital in Florence, Italy, and tested with GeneXpert MTB/RIF or GeneXpert Ultra, from 2013 to 2023. GeneXpert and GeneXpert Ultra demonstrated a detection rate of 0.357 (95% CI 0.180 to 0.535) and 0.537 (95% CI 0.417-0.657), respectively. No child was tested with both tests. Patients' characteristics, including age and sex, did not significantly influence the test's performances. Notably, GeneXpert Ultra had a significantly higher detection rate in children with extrapulmonary TB (0.813, 95% CI 0.621 to 1.004) compared to that in children with pulmonary TB (p = 0.020). Gastric aspirate was the most tested specimen. Specimens that did not require invasive procedures for collection (including stool) yielded poor results. GeneXpert and GeneXpert Ultra permitted rapid evaluation of genotypic drug-sensitivity testing (DST), even though limited to rifampicin resistance, making necessary confirmation through phenotypic DST (performed on culture). The introduction of GeneXpert and GeneXpert Ultra improved TB diagnosis in children, by providing microbiological information in a short time, complementing results from culture, which remains the reference test in pediatric TB diagnosis.

Evaluation of the toxicity of perfluorooctanoic acid toward human colorectal cancer cells using multi-dimensional approaches

While human exposure to perfluorooctanoic acid (PFOA) can lead to ulcerative colitis, the molecular mechanisms responsible for PFOA-induced intestinal toxicity are unclear. Herein, we examined the toxicity of PFOA toward human colorectal cancer cells (HCT116) from three dimensions: the cytotoxic phenotype, cell respiration, and transcription levels of metabolism-related genes. Formazan was used to assess how PFOA exposure affects HCT116-cell relative viability, after which the mitochondrial respiratory activities of these cells were determined by analyzing extracellular flux. The quantitative real-time polymerase chain reaction (qPCR) method was used to detect metabolism-related gene expression levels. The cytotoxicity assay revealed that the HCT116 showed significantly inhibited relative activities compared to those of the control when exposed to 300 μmol/L PFOA for 48 h (p<0.01), with most cells retained at the G0/G1 stage. In contrast, the mitochondrial respiratory activities of the HCT116 were promoted by concentrations of PFOA as low as 50 μmol/L. Two genes related to cellular metabolism (dipeptidase 1 (DPEP1) and sphingosine kinase 1 (SPHK1)) were found to be related to the PFOA-promoted formation of ulcerative colitis using our self-developed Metabolic Gene and Pathway Query software and Comparative Toxicogenomics Database (CTD). The qPCR studies revealed that DPEP1 and SPHK1 expression levels were enhanced by 8-10 times in HCT116 exposed to 300 μmol/L PFOA relative to the control, whereas this trend was not observed for HCT116 exposed to 50 μmol/L PFOA. Collectively, these results suggest that the respiratory activity of cellular mitochondria may serve as an index for determining the interference effects associated with PFOA and that metabolic pathways mediated by DPEP1 and SPHK1 may be involved in the development of PFOA-induced ulcerative colitis. Future studies should investigate the relationships between changes in metabolism-related genes (DPEP1 and SPHK1) and the mitochondrial respiratory activities of intestinal cells, and verify the roles played by the DPEP1 and SPHK1 genes in PFOA-induced intestinal inflammation using in-vivo models.

Development of a droplet digital PCR method for the detection of Ureaplasma urealyticum.

Human infection with Ureaplasma urealyticum(UU) is mainly manifested as non-gonococcal urethritis, where it can lead to cervicitis, premature rupture of membranes and abortion in women, as well as infertility in males, which becomes a major problem in clinical diagnosis and treatment. At present, real-time fluorescence quantitative PCR and culture are the two main methods for detecting UU. The real-time fluorescence quantitative PCR method is cumbersome and cannot accomplish absolute quantification on nucleic acids, while the cultivation method has limitations such as low sensitivity and being time-consuming. The aim of this study is to establish a more rapid and accurate droplet digital PCR(ddPCR) method for the detection of UU. Primers were designed for the ParC gene of UU. Nucleic acids from a standard strain of UU were extracted. Specificity, sensitivity, and repeatability detection was performed using ddPCR, and the detection performance of ddPCR was evaluated. The detection process could be completed in 92min. It has a high sensitivity of up to 3.8pg/μL. With a high specificity, no positive microdrop were detected in eight negative control pathogens in this experiment. In addition, ddPCR detection of UU has good repeatability, and the calculated CV is 2.1%. Our data indicated that ddPCR detection technology has the characteristics of absolute quantification, high stability, high specificity and high sensitivity of UU. It can promote the accurate detection of UU, providing a more scientific basis for clinical diagnosis and treatment.

Development and Clinical Detection of Rapid Molecular Diagnostic System for Pathogenic Dermatophytes of Tinea Capitis of Multiple Centres in China.

Tinea capitis remains a common fungal infection in children worldwide. Species identification is critical for determining the source of infection and reducing transmission. In conventional methods, macro- and microscopic analysis is time-consuming and results in slow fungal growth or low specificity. We propose a rapid real-time diagnostic PCR method that allows species-specific identification of dermatophytes, including the Microsporum canis complex, Trichophyton mentagrophytes complex, Trichophyton rubrum complex and Trichophyton tonsurans, in patients with tinea capitis. Hair and scrapings samples were collected from 231 patients with tinea capitis who were positive for fungal elements via direct microscopy with potassium hydroxide. Each sample was subjected to a two-step real-time PCR (RT-PCR) assay, which was designed on the basis of differences in the DNA fragments of the internal transcribed spacer (ITS) and β-tubulin covering the Microsporum canis complex, T. mentagrophyte complex, T. rubrum complex, T. tonsurans, T. verrucosum, T. schoenleinii and N. gypseum. In total, 186/231 samples (80.52%) were positive for fungal culture. The two-step RT-PCR was positive in 215/231 samples (93.07%), among which 179 were culture positive. The combined efficacy was 96.81%, which was significantly different when the RT-PCR assays were performed in parallel with fungal culture. A total of 126 samples (54.55%) were identified as Microsporum canis by fungal culture, among which the positive rate of M. canis complex RT-PCR was 97.62% (123/126). A total of 45 samples were negative for fungal culture, of which 80.0% (36/45) were positive by RT-PCR, and the percentage of M. canis complex-positive samples was 53.33% (24/45). The RT-PCR assays were negative for 16/231 samples, among which 7 were culture positive, including M. canis (n = 3), T. violaceum (n = 3) and N. gypseum (n = 1). We developed a new diagnostic assay system using a rapid real-time TaqMan PCR assay with specific primers that can be applied in routine laboratory practice for hair and skin samples of tinea capitis to detect dermatophytes and increase diagnostic efficiency.

Description of Mitochondrial Gene Mutations in Pakistani Patients with Coronary Artery Disease: An Investigation of Genetic Susceptibility

Background: Mutations in mitochondrial DNA (mtDNA) were the important causes of cardiovascular diseases. However, little was known regarding the role of mitochondrial DNA (mt-DNA) mutations in coronary artery disease (CAD). To investigated the association between mitochondrial genes mutations and CAD in Pakistani population. Methods: Approximately, 11 related CAD patients and 29 control subjects were recruited in this study; we performed PCRRFLP to amplify mitochondrial genes and subsequently sequenced the PCR products. In addition, the pathogenicity scoring system was used to evaluate the deleterious roles of these genes mutations. We also used real-time PCR method to determine the mtDNA content in CAD patients carrying these mutations. Results: Three mutations were identified by PCR-Sanger sequencing; these mutations included mt-ND4 12133C&gt;T, mt-ND5 12372G&gt;A and mt-CYB 15884G&gt;C. These mutations were localized at the highly conserved nucleotides, may cause the failure in mt-DNA metabolism. Furthermore, CAD group showed a clear reduction in mitochondrial copy number by comparing with the controls. Conclusions: Mutations in mt-DNA genes were the important causes of CAD, our findings provided novel insight into the pathophysiology of CAD that were manifested by mitochondrial dysfunction.

Quantitative Real-Time PCR for Circular RNA Detection and Analysis.

In eukaryotes, nearly 2% of the genome represented by the coding proteins. However, emerging evidence suggest more than 75% of the human genome referred to as noncoding part also plays a crucial role in governing major regulatory pathways. Noncoding RNAs can be categorized into several groups, such as microRNAs (miRNAs), small nuclear RNA (snRNAs), small nucleolar RNA (snoRNAs), transfer RNA (tRNA), and circular RNA (circRNAs), which contribute to this regulatory landscape. Circular RNAs (circRNAs) are identified as a new class of regulatory noncoding RNAs with gene regulatory roles by acting as miRNA or RNA binding protein sponges or interacting with proteins. Researchers employ quantitative real-time PCR methods to examine circular RNA expression utilizing divergent primers for identification and quantification.

Evaluating the Association Between Methylenetetrahydrofolate Reductase (Rs1801131 and Rs1801133) Gene Polymorphisms and Severity of Coronary Lesions in Patients With STEMI and NSTEMI: A Retrospective Cross-Sectional Study.

Mounting evidence have implicated that rs1801131 and rs1801133, located in the Methylenetetrahydrofolate reductase (MTHFR) gene, may emerge as novel biomarkers for coronary artery disease (CAD). The Synergy between Percutaneous Coronary Intervention with Taxus and Cardiac Surgery (SYNTAX) score is also an appropriate predictor for revascularization strategy in patients with complex CAD. The aim of this study is to investigate the correlation between rs1801131 and rs1801133 with the severity of coronary lesions in patients with ST‑Elevation Myocardial Infarction (STEMI) and Non‑ST‑Elevation Myocardial Infarction (NSTEMI) based on the SYNTAX score. This retrospective cross-sectional study included 96 patients diagnosed with STEMI and NSTEMI from Razi University Hospital between April and September 2019. Ninety-six patients were diagnosed with STEMI (N = 43) and NSTEMI (N = 53) were recruited from South Khorasan, Iran. The angiographical characteristics of CAD were defined by the SYNTAX score. Genomic DNA was isolated from peripheral blood and genotyped for rs1801131 and rs1801133 using the TaqMan real-time PCR method. The results of the one-way analysis of variance indicated that there is no association between rs1801131 and rs1801133 with the severity of coronary lesions in patients with STEMI (p = 0.44) and NSTEMI (p = 0.91). However, the two-way analysis of variance comparison and post-hoc test demonstrated that rs1801133 in the presence of rs1801131 is correlated with the SYNTAX score in NSTEMI (p = 0.03) and total patients (p = 0.03). In conclusion, our study reveals a significant association between the MTHFR polymorphism rs1801133 and CAD severity, particularly in NSTEMI patients. While rs1801131 showed no correlation, rs1801133 may serve as a valuable genetic biomarker for assessing CAD severity. Further research with larger populations is needed to confirm these findings.

A Rapid On-Site Detection Method for Plasmodium falciparum Carried by Mosquitoes Using Disc Microfluidic Isothermal Amplification.

Rapidly identifying Anopheles-carrying malaria parasites is crucial for imported malaria prevention. However, suitable methods still lack quick detection in limited-resource situations. In this study, disc microfluidic isothermal amplification integrating loop-mediated isothermal amplification (LAMP) and microfluidic chip technology were applied to develop rapid and precise detection with low resource requirements. Primer set EMP1G2, which is specific to Plasmodium falciparum (P. falciparum) erythrocyte membrane protein 1, and primer set 18sG2, which is specific to ribosomal 18s subunit RNA, were screened for optimal LAMP-specific primer sets. The minimum detection limits were 125 copies/µL for the EMP1G2 and 6,562 copies/µL for the 18sG2. Subsequently, optimal primer sets were evaluated for specificity with nucleic acid from other mosquito-borne pathogens and arthropod vectors. No nonspecific amplification was observed in optimal amplification-specific primer sets with the DNA of Anopheles mosquitoes and morphologically similar arthropods or with the copy DNA of Zika virus, yellow fever virus, or dengue virus 1. The detection method was evaluated in a simulated scenario and demonstrated a robust capacity for rapid on-site detection. Additionally, polymerase chain reaction (PCR) and quantitative real-time PCR methods were compared using this method. In this study, a rapid detection method based on disc microfluidic isothermal amplification was developed that could be used to detect P. falciparum carried by mosquitoes in a field setting under limited resource conditions.

The effect of biosynthesized zinc oxide nanoparticles on gene expression and apoptosis in triple-negative breast cancer cells.

Some forms of breast cancer such as triple-negative phenotype, are serious challenge because of high metastatic cases, high mortality and resistance to conventional therapy motivated the search for alternative treatment approaches. Nanomaterials are promising candidates and suitable alternatives for improving tumor and cancer cell treatments. Biosynthesis of ZnO NPs by help of Berberis integerrima fruit extract, has been done. Analysis of Zinc Oxide NPs using DLS, FTIR, SEM, and EDS techniques have been performed. Moreover, biological activities of ZnO NPs evaluated through MTT method, Flow cytometry, and real time PCR methods. Biocatalytic and apoptotic activity of ZnO NPs on healthy HFF (human fibroblast cell line), MDA-MB 231, and MDA-MB 468 (triple negative breast cancer cell lines, (TNBC)) evaluated. Furthermore, Bax, Bcl-2 and caspase-3 apoptotic genes expression changes in cancer cells assessed in compare to GAPDH as a house keeping gene. Physico-chemical investigation demonstrated ZnO NPs were confirmed by Berberis integerrima fruit extract for the first time. The MTT assay and Flow cytometry results indicated biocompatibility of the ZnO NPs in normal cell line and high anticancer potential against TNBC MDA-MB-231 and MDA-MB-468 cell lines. The IC50 of ZnO NPs were 104.4 and 44.86, 20.96 after 24 hours for HFF, MDA-MB-231 and MDA-MB-468 cells, respectively. The current research showed a fast, cost effective and ecofriendly method for ZnO NPs nanoparticle synthesis. Furthermore, In vitro data analysis demonstrated biocompatibility and highly anticancer effects of biosynthesized ZnO NPs against TNBC cancerous cells.

Molecular genetic characteristic of pulmonary tuberculosis associated with ABCB1 gene expression of multidrugresistance protein P-gp

Background: Tuberculous inflammation is mediated by a complex molecular signaling pathway, the analysis of which makes it possible to identify promising biomarkers and targets for the development of new diagnostic, prognostic and pharmacological approaches in order to improve the effectiveness of anti-tuberculosis chemotherapy. Determining the relationship between key inflammatory cytokines, the multidrug-resistant protein P-gp and the activity of specific inflammation in the surgical material of patients with pulmonary tuberculosis may prove to be a novel tool in the development of pathogenetic therapy and personalized medicine. Aims: to characterize molecular and genetic profiles of tuberculomas and identify genes that correlate with the expression of the ABCB1 gene of the P-gp protein in the surgical material of patients with pulmonary tuberculosis. Research objectives: 1) to obtain molecular and genetic characteristics of tuberculosis by real-time PCR and compare it with the activity of tuberculous inflammation; 2) to carry out a correlation analysis between the expression of the ABCB1 gene and key cytokines of the tuberculosis process: IL-6, IL-10, IFN-γ, TGF-β, TNF-α, IL-1β. Materials and methods: A prospective cohort study was conducted on the basis of the FSBI CTRI. The object of the study was the surgical material of 35 patients diagnosed with multiple pulmonary tuberculomas. Histological examination methods were used for the morphological assessment of the surgical material. A real-time quantitative PCR method was used to analyze gene expression. Statistical processing was performed using the GraphPad Prism Version 7.04 software package (GraphPad Software, USA). The data is presented as a median with an interquartile range. The nonparametric Mann-Whitney U-test was used to compare the two groups. All p-values were two-sided and p 0.05 was considered statistically significant. The correlation between the variables was estimated using the Spearman correlation coefficient. The correlation analysis was carried out in the Microsoft Office Excel 2010 software. Results: The study revealed that the highest level of expression of ABCB1 gene of the P-gp protein is observed in tuberculomas with high activity of tuberculous inflammation, and its expression is correlated with the expression of the IL6 gene (p0.001) and the expression of the IL10 gene (p0.01). Tuberculomas of this group are also characterized by higher expression of the TGFB1, TNF and IL1B genes, compared with the group of moderate activity of specific inflammation. Conclusions: The data obtained indicate that in addition to pro-/anti-inflammatory cytokines, the P-gp protein plays an important role in the pathogenesis of tuberculous inflammation, especially with its high activity. Further clarification of the P-gp role in tuberculous inflammation may be an important step for the development of new approaches to treat tuberculosis using methods of HDT and personalized medicine.

Investigation of the Relationship Between ACTN3 rs1815739 Polymorphism and Openbite Cases: A Prospective Study.

The aim of this study was to examine whether ACTN3 rs1815739 polymorphism, which causes the deficiency of the alpha-actinin-3 muscle protein, is related to the formation of open bite malocclusion. Fifty-eight participants (18.5 ± 3.6 years old) with anterior open bite (n = 29) and normal overbite (n = 29) who presented to Marmara University, Department of Orthodontics for treatment were included in the study. Initial cephalometric radiographs were used for the diagnosis of malocclusion. The case group was divided into three subgroups according to degree of open bite. For DNA isolation, oral epithelial cells were collected with buccal swabs (Van Allen Way, Carlsbad, USA), and the real-time PCR method was used for the genotyping of all polymorphisms. The results were statistically analysed, and the threshold for statistical significance was set at p < 0.05. The frequencies of RR, RX and XX genotypes of ACTN3 rs1815739 polymorphism were found as 6 (20.7%), 14 (48.3%) and 9 (31.0%) in the control group and 8 (8%), 9 (31.0%) and 12 (41.4%) in the case group, respectively. There was no statistically significant difference between the groups in terms of the presence of the examined polymorphism (p > 0.05). However, the intra-group evaluation of case group revealed a significant difference in the prevalence of XX genotype (83.3%) for the subgroup with an open bite of -5 mm or above. Although no significant difference was observed between the case and control groups, a possible association was identified between ACTN3 rs1815739 polymorphism and an increased severity of open bite malocclusion.

Potential Effect of Defatted Mealworm Hydrolysate on Muscle Protein Synthesis in C2C12 Cells and Rats

(1) Background: the objective of this study was to examine the impact of defatted mealworm hydrolysate (DMH), formulated through protein hydrolysis, on muscle protein synthesis in C2C12 cells and rats; (2) Methods: C2C12 cells were treated with dexamethasone and DMH, and cell viability was quantified using the MTT assay. Twenty-four Sprague–Dawley rats were divided into three groups (control, DEX, and DEX + DMH) and treated for 8 weeks. The DEX and DEX + DMH groups were administered intraperitoneal injections of DEX at a concentration of 2.25 mg/kg over a 3-d period. The control and DEX groups were fed a control diet, whereas the DMH group had part of the protein composition of the control diet replaced with 3.5% of DMH. The impact of DMH on muscle protein synthesis was evaluated through the measurement of grip strength, gastrocnemius and tibialis anterior muscle weights, and the investigation of muscle protein synthesis and degradation factor mRNA expression utilising the real-time PCR method; (3) Results: in vitro experiments demonstrated that treatment with DMH at concentrations greater than 5 mg/mL markedly alleviated DEX-induced injury in C2C12 cells. In vivo experiments demonstrated that the mRNA expression levels of myogenin and myoblast determination proteins, which promote muscle protein synthesis, were significantly increased. Furthermore, rats fed DMH exhibited significantly enhanced grip strength and tibialis anterior weight; (4) Conclusions: these findings indicate that DMH may serve as a functional material capable of promoting muscle protein synthesis and that the utilization of proteolytic enzymes is advantageous for the effective utilization of mealworms.