Retroreflection-based sandwich type affinity sensing of isothermal gene amplification product for foodborne pathogen detection

Loop-mediated isothermal amplification (LAMP) is an outstanding method for molecular diagnostics, as the rapid, specific, and sensitive amplification of target genes is possible. However, it is necessary to measure fluorescence...

Gene Amplification as a Mechanism of Yeast Adaptation to Nonsense Mutations in Release Factor Genes

Protein synthesis (translation) is one of the fundamental processes occurring in the cells of living organisms. Translation can be divided into three key steps: initiation, elongation, and termination. In the yeast Saccharomyces cerevisiae, there are two translation termination factors, eRF1 and eRF3. These factors are encoded by the SUP45 and SUP35 genes, which are essential; deletion of any of them leads to the death of yeast cells. However, viable strains with nonsense mutations in both the SUP35 and SUP45 genes were previously obtained in several groups. The survival of such mutants clearly involves feedback control of premature stop codon readthrough; however, the exact molecular basis of such feedback control remain unclear. To investigate the genetic factors supporting the viability of these SUP35 and SUP45 nonsense mutants, we performed whole-genome sequencing of strains carrying mutant sup35-n and sup45-n alleles; while no common SNPs or indels were found in these genomes, we discovered a systematic increase in the copy number of the plasmids carrying mutant sup35-n and sup45-n alleles. We used the qPCR method which confirmed the differences in the relative number of SUP35 and SUP45 gene copies between strains carrying wild-type or mutant alleles of SUP35 and SUP45 genes. Moreover, we compare the number of copies of the SUP35 and SUP45 genes in strains carrying different nonsense mutant variants of these genes as a single chromosomal copy. qPCR results indicate that the number of mutant gene copies is increased compared to the wild-type control. In case of several sup45-n alleles, this was due to a disomy of the entire chromosome II, while for the sup35-218 mutation we observed a local duplication of a segment of chromosome IV containing the SUP35 gene. Taken together, our results indicate that gene amplification is a common mechanism of adaptation to nonsense mutations in release factor genes in yeast.

Differential Regulation of the EGFR/PI3K/AKT/PTEN Pathway between Low- and High-Grade Gliomas

Gliomas represent 70% of all central system nervous tumors and are classified according to the degree of malignancy as low- or high-grade. The permanent activation of the EGFR/PI3K/AKT pathway by various genetic or post-translational alterations of EGFR, PI3KCA, and PTEN has been associated with increased proliferation and resistance to apoptosis. The present study aimed to analyze the molecular/genetic changes in the EGFR/PI3K/AKT/PTEN pathway between low-grade and high-grade gliomas in a sample of Colombian patients. A total of 30 samples were tested for PI3K and PTEN mutations, EGFR, PI3K, and AKT gene amplification, AKT, PI3K, BAX, Bcl2 expression levels, and phosphorylation of AKT and PTEN, EGFR and/or PI3K gene amplification was found in 50% of low-grade and 45% of high-grade ones. AKT amplification was found in 25% of the low-grade and 13.6% of the high-grade. The expression of PI3K, AKT, Bcl2, and BAX was increased particularly to a high degree. AKT phosphorylation was found in 66% of low-grade and 31.8% of high-grade. Increased phosphorylation of PTEN was found in 77% low-grade and 66% high-grade. Our results indicate that alterations in the EGFR/PI3K/AKT/PTEN pathway could be important in the initiation and malignant progression of this type of tumor.

Detection of Nontuberculous Mycobacterial Species from Tissue Samples of Cattle and Buffaloes by PCR and PRA (PCR-RFLP)

Background: Nontuberculous mycobacteria are opportunistic pathogens and some of them may cause disease in humans and animals causing pulmonary infections, mastitis, lesions in respiratory tract and lymph nodes of cattle, due to which they are being recognized worldwide and also interfere with the diagnosis of bovine tuberculosis. Methods: The present study was conducted for detection of nontuberculous mycobacterial species (NTM) in tissue samples (with and without tubercle lesions) in cattle and buffaloes from postmortem hall GADVASU, Ludhiana. Polymerase Chain Reaction and PCR-RFLP which involved hsp65 gene amplification (439 bp) and restriction analysis of amplified product was performed on 30 tissue samples for detection of nontuberculous mycobacterial species. Result: Three out of 30 samples showed hsp65 gene amplification and 2 were identified as M. kansasii using restriction analysis technique and one could not be identified as the RFLP patterns was different from other known PCR-RFLP profiles. NTM such as M. kansasi may cause infection in animals and PRA (PCR-Restriction Fragment Length Polymorphism Analysis) technique was found to be a rapid tool for identification and differentiation of NTM upto species level.

Comparable genetic alteration profiles between gastric cancers with current and past Helicobacter pylori infection

Gastric cancers can develop even after Helicobacter pylori (H. pylori) eradication in 0.2–2.9% cases per year. Since H. pylori is reported to directly activate or inactivate cancer-related pathways, molecular profiles of gastric cancers with current and past H. pylori infection may be different. Here, we aimed to analyze whether profiles of point mutation and gene amplification are different between the two groups. Current or past infection by H. pylori was determined by positive or negative amplification of H. pylori jhpr3 gene by PCR, and past infection was established by the presence of endoscopic atrophy. Among the 90 gastric cancers analyzed, 55 were with current infection, and 35 were with past infection. Target sequencing of 46 cancer-related genes revealed that 47 gastric cancers had 68 point mutations of 15 different genes, such as TP53 (36%), KRAS (4%), and PIK3CA (4%) and that gene amplification was present for ERBB2, KRAS, PIK3CA, and MET among the 26 genes assessed for copy number alterations. Gastric cancers with current and past infection had similar frequencies of TP53 mutations (38% and 31%, respectively; p = 0.652) and oncogene activation (20% and 29%, respectively; p = 0.444). Gastric cancers with current and past infection had comparable profiles of genetic alterations.

Infantile mediastinal neuroblastoma presenting as an oncologic emergency: usefulness of serum-based MYCN gene amplification analysis for risk stratification

We reported two infantile cases of mediastinal neuroblastoma with life-threatening tracheal obstructions presenting as oncologic emergencies that were successfully treated per tentative risk classification using serum-based MYCN gene amplification (MNA) analysis. Tentative risk stratification based on age, tumour location and serum-based MNA status may be useful in patients with neuroblastoma presenting as oncologic emergencies who require urgent therapy stratification but for whom tumor-based molecular diagnoses cannot be established.

Fibroblast growth factor (FGF), FGF receptor (FGFR), and cyclin D1 (CCND1) DNA methylation in head and neck squamous cell carcinomas is associated with transcriptional activity, gene amplification, human papillomavirus (HPV) status, and sensitivity to tyrosine kinase inhibitors

Background Dysregulation of fibroblast growth factor receptor (FGFR) signaling pathway has been observed in head and neck squamous cell carcinoma (HNSCC) and is a promising therapeutic target for selective tyrosine kinase inhibitors (TKIs). Potential predictive biomarkers for response to FGFR-targeted therapies are urgently needed. Understanding the epigenetic regulation of FGF pathway related genes, i.e. FGFRs, FGFs, and CCND1, could enlighten the way towards biomarker-selected FGFR-targeted therapies. Methods We performed DNA methylation analysis of the encoding genes FGFR1, FGFR2, FGFR3, FGFR4, FGF1-14, FGF16-23, and CCND1 at single CpG site resolution (840 CpG sites) employing The Cancer Genome Research Atlas (TCGA) HNSCC cohort comprising N = 530 tumor tissue and N = 50 normal adjacent tissue samples. We correlated DNA methylation to mRNA expression with regard to human papilloma virus (HPV) and gene amplification status. Moreover, we investigated the correlation of methylation with sensitivity to the selective FGFR inhibitors PD 173074 and AZD4547 in N = 40 HPV(−) HNSCC cell lines. Results We found sequence-contextually nuanced CpG methylation patterns in concordance with epigenetically regulated genes. High methylation levels were predominantly found in the promoter flank and gene body region, while low methylation levels were present in the central promoter region for most of the analyzed CpG sites. FGFRs, FGFs, and CCND1 methylation differed significantly between tumor and normal adjacent tissue and was associated with HPV and gene amplification status. CCND1 promoter methylation correlated with CCND1 amplification. For most of the analyzed CpG sites, methylation levels correlated to mRNA expression in tumor tissue. Furthermore, we found significant correlations of DNA methylation of specific CpG sites with response to the FGFR1/3–selective inhibitors PD 173074 and AZD4547, predominantly within the transcription start site of CCND1. Conclusions Our results suggest an epigenetic regulation of CCND1, FGFRs, and FGFs via DNA methylation in HNSCC and warrants further investigation of DNA methylation as a potential predictive biomarker for response to selective FGFR inhibitors in clinical trials.