Detection and Real-time PCR Assay for the Quantification of Carbapenemase Gene blablaNDM-1 in Hospital Effluent

This study aims to isolate gram-negative bacteria (GNB) harboring the gene NDM-1 from the tertiary care hospital effluents. Also, aims to evaluate the relative copy number of blaNDM-1 carried by the positive isolates. The study isolated 215 GNB from 40 effluent samples. The antibiotic susceptibility tests for carbapenems were performed using disc diffusion assay. The isolates resistant to either meropenem or imipenem were checked for the existence of MBL by phenotypic methods. The isolates carrying NDM-1 gene were genotypically confirmed by Polymerase chain reaction (PCR). The gene copy number of blaNDM- were determined by quantative real-time PCR. A total of 22 isolates showed phenotypic resistance to carbapenems and were characterized by biochemical methods. Among them, 12 harbored NDM-1 gene by PCR; these bacteria were subjected to qPCR for determining the absolute copy numbers of the NDM-1 gene on it. The gene abundance in the strains was in the range of 3.28× 105 to 6.05× 106 copies/ ng of DNA. Hospital effluents are important pool of antibiotic-resistant bacteria harboring the blaNDM-1 and infections caused by these bacteria are difficult to treat. Hence, the present study stresses the need for stringent antibiotic use and efficient wastewater treatment policies in these hospital settings, which is paramount in achieving sustainable health.

Single nucleotide polymorphisms and copy-number variations in the Trypanosoma brucei repeat (TBR) sequence can be used to enhance amplification and genotyping of Trypanozoon strains

The Trypanosoma brucei repeat (TBR) is a tandem repeat sequence present on the Trypanozoon minichromosomes. Here, we report that the TBR sequence is not as homogenous as previously believed. BLAST analysis of the available T. brucei genomes reveals various TBR sequences of 177 bp and 176 bp in length, which can be sorted into two TBR groups based on a few key single nucleotide polymorphisms. Conventional and quantitative PCR with primers matched to consensus sequences that target either TBR group show substantial copy-number variations in the TBR repertoire within a collection of 77 Trypanozoon strains. We developed the qTBR, a novel PCR consisting of three primers and two probes, to simultaneously amplify target sequences from each of the two TBR groups into one single qPCR reaction. This dual probe setup offers increased analytical sensitivity for the molecular detection of all Trypanozoon taxa, in particular for T.b. gambiense and T. evansi, when compared to existing TBR PCRs. By combining the qTBR with 18S rDNA amplification as an internal standard, the relative copy-number of each TBR target sequence can be calculated and plotted, allowing for further classification of strains into TBR genotypes associated with East, West or Central Africa. Thus, the qTBR takes advantage of the single-nucleotide polymorphisms and copy number variations in the TBR sequences to enhance amplification and genotyping of all Trypanozoon strains, making it a promising tool for prevalence studies of African trypanosomiasis in both humans and animals.

Molecular alterations in the culture of lung cancer cells H1299 after exposure to high doses of ionizing radiation

Objective To study the copy number of genes-components of signaling cascades involved in DNA repair, cell cycle regulation and apoptosis under the influence of high doses of ionizing radiation.Material and Мethods The study was carried out on a culture of H1299 non-small cell lung cancer cells. Cell lines were cultured in a Binder incubator (Germany) for 24 h (at 37 °C, 5% CO2 ), and then the groups were divided into therapeutic and control. The first one was irradiated with a NovalisTx, Varian linear accelerator at doses from 18 to 24 Gy, the second was not exposed to radiation. During the study, we monitored cell viability and evaluated apoptotic activity, then each sample was amplified in two iterations. During the study, cell viability was monitored, apoptotic activity was assessed, and then each sample was amplified in two replicates. The relative copy number of genetic loci was determined by Real-Time qPCR (RT-qPCR).Results When comparing the relative copy number in the genetic loci of the H1299 non-small cell lung cancer cell culture after exposure to a high dose of ionizing radiation, a statistically significant decrease in the relative copy number of the CASP3 and RBBP8 genes was found, which may indicate a decrease in the potential of caspase-mediated tumor repopulation and an increase in the radiosensitivity of tumor cells.Conclusion Exposure to high doses of ionizing radiation leads to a detrimental effect on tumor cells and allows to overcome one of the mechanisms of radioresistance – tumor cell repopulation.

Nuclear-cytoplasmic balance: whole genome duplications induce elevated organellar genome copy number

The plant genome is partitioned across three distinct subcellular compartments: the nucleus, mitochondria, and plastids. Successful coordination of gene expression among these organellar genomes and the nuclear genome is critical for plant function and fitness. Whole genome duplication events (WGDs) in the nucleus have played a major role in the diversification of land plants and are expected to perturb the relative copy number (stoichiometry) of nuclear, mitochondrial, and plastid genomes. Thus, elucidating the mechanisms whereby plant cells respond to the cytonuclear stoichiometric imbalance that follow WGDs represents an important yet underexplored question in understanding the evolutionary consequences of genome doubling. We used droplet digital PCR (ddPCR) to investigate the relationship between nuclear and organellar genome copy numbers in allopolyploids and their diploid progenitors in both wheat and Arabidopsis. Polyploids exhibit elevated organellar genome copy numbers per cell, largely preserving the cytonuclear stoichiometry observed in diploids despite the change in nuclear genome copy number. To investigate the timescale over which cytonuclear stoichiometry may respond to WGD, we also estimated organellar genome copy number in Arabidopsis synthetic autopolyploids and in a haploid-induced diploid line. We observed corresponding changes in organellar genome copy number in these laboratory-generated lines, indicating that at least some of the cellular response to cytonuclear stoichiometric imbalance is immediate following WGD. We conclude that increases in organellar genome copy numbers represent a common response to polyploidization, suggesting that maintenance of cytonuclear stoichiometry is an important component in establishing polyploid lineages.

Increased copy number of syncytin-1 in the trophectoderm is associated with implantation of the blastocyst

Background A key step in embryo implantation is the adhesion to and invasion of the endometrium by the blastocyst trophectoderm. The envelope proteins of HERV-W and -FRD (human endogenous retrovirus-W and -FRD), syncytin-1 and syncytin-2, are mainly distributed in the placenta, and play important roles in the development of the placenta. The placenta originates from the trophectoderm of the blastocyst. It is unclear whether the envelope proteins of HERV-W and -FRD have an effect on the development of the trophectoderm and whether they have any association with the implantation of the blastocyst. Methods The whole-genome amplification products of the human blastocyst trophectoderm were used to measure the copy number of syncytin-1 and syncytin-2 using real time qPCR. In addition, clinical data associated with the outcome of pregnancies was collected, and included age, body mass index (BMI), basic follicle stimulating hormone(bFSH), rate of primary infertility and oligo-astheno-teratospermia, the thickness of the endometrium on the day of endometrial transformation, the levels of estrogen and progestin on the transfer day, the days and the morphological scores of the blastocysts. The expression of mRNA and the copy numbers of syncytin-1 and syncytin-2 in H1 stem cells, and in differentiated H1 cells, induced by BMP4, were measured using real time qPCR. Results The relative copy number of syncytin-1 in the pregnant group (median: 424%, quartile: 232%–463%, p < 0.05) was significantly higher than in the non-pregnant group (median: 100%, quartile: 81%–163%). There was a correlation (rs = 0.681, p < 0.001) between the copy number of syncytin-1 and blastocyst implantation after embryo transfer. As the stem cells differentiated, the expression of NANOG mRNA decreased, and the expression of caudal type homeobox 2(CDX2) and β-human chorionic gonadotropin (β-hCG) mRNAs increased. Compared to the undifferentiated cells, the relative expression of the syncytin-1 mRNA was 1.63 (quartile: 0.59–6.37, p > 0.05), 3.36 (quartile: 0.85–14.80, p > 0.05), 10.85 (quartile: 3.39–24.46, p < 0.05) and 67.81 (quartile: 54.07–85.48, p < 0.05) on day 1, 3, 5 and 7, respectively, after the differentiation. The relative expression of syncytin-2 was 5.34 (quartile: 4.50–10.30), 7.90 (quartile: 2.46–14.01), 57.44 (quartile: 38.35–103.87) and 344.76 (quartile: 267.72–440.10) on day 1, 3, 5 and 7, respectively, after the differentiation (p < 0.05). The copy number of syncytin-1 increased significantly during differentiation. Conclusion Preceding the transfer of frozen embryos, the increased copy number of syncytin-1 in the blastocyst trophectoderm was associated with good outcomes of pregnancies.

The prognostic role of aberrant copy number of DDB1, PRPF19, CDKN1B, and с-Myc genes in ovarian cancer patients

Rationale: Ovarian cancer is the leading death cause in gynecological malignancies. More than 70% of the patients are diagnosed with progressing disease extending to outside the true pelvis. The 5-year survival of ovarian cancer patients remains low (about 47%) due to frequent relapses and drug resistance. Identification of markers for early diagnosis and relapse prediction could improve the outcomes of the disease.Aim: To assess relative copy number of cancer-associated genetic loci c-Myc, CDK12, CDKN1B, PRPF19, ERBB2, DDB1, GAB2, COL6A3 in the tumor cells of ovarian cancer, in order to identify potential prognostic oncomarkers in ovarian cancer patients.Materials and methods: The study included 50 women aged 27 to 70 years with ovarian cancer T1-3сN0-1M0-1, Gr. 2 (stages I–IV), who received their elective treatment in the National Medical Research Centre for Oncology in 2015 to 2019. The study was based on samples of genomic DNA from paraffinized blocks of tumor and “healthy” tissues. Relative copy numbers of 8 genetic loci (c-Myc, CDK12, CDKN1B, PRPF19, ERBB2, DDB1, GAB2, COL6A3) was assessed by RT-qPCR technique. Relative copy quantitation of a genetic locus was calculated as 2-ΔCt. The dose of the locus studied was considered equal to diploid set (2n) if RCQtumor/healthy was about 1. If RCQtumor/healthy was>1.5 or<0.5, then the locus dose was considered increased (≥ 3n) or decreased (≤ 1n), respectively.Results: For all genetic loci, an increase of relative copy quantitation in the ovarian tumor cells was observed compared to that in “healthy” tissues. There was a significant (р<0.05) aberrant copy quantitation of 4 genes: c-Myc (р=0.001), DDB1 (р=0.002), PRPF19 (р=0.0001), and CDKN1B (р=0.001). We identified differential thresholds for these genes that made it possible to predict an unfavorable disease course in the patients (р<0.05). The strongest association with the risk of adverse outcomes was found for increased copy number of PRPF19 (odds ratio (OR) 7.3; р=0.0001) and c-Myc (OR 6.8; р=0.001). Conclusion: In this study, we determined the prognostic value of 4 oncogenic drivers, namely, DDB1, PRPF19, CDKN1B, and с-Myc, whose increased copy number was associated with an adverse disease prognosis in ovarian cancer patients.

HER2 Testing in Invasive Breast Cancer: A Comparison Between Immunohistochemistry and Fluorescence In Situ Hybridization Assays

Background: HER2 status testing in breast cancer is crucial for the detection of eligible patients for trastuzumab therapy. In this study, the relative copy number of HER2 gene, in patients with breast cancer, was determined by fluorescence in situ hybridization (FISH) and the results were compared with those of immunohistochemistry (IHC) to obtain the concordance rate between these two methods. Material and Methods: HER2 status of 31 invasive breast cancer samples was compared using IHC and FISH techniques. The ratio of HER2/CEP17 was used to determine the amplification of the HER2 gene. If the ratio of HER2/CEP17 is greater than 2.2, HER2 gene amplification has occurred in the cancer cells. Then, a comparative analysis is performed to estimate the concordance rate between FISH and IHC results. Results: The gene amplification of HER2 was observed in 26% of cases by FISH. The IHC and FISH results showed 100%, 36.36%, and 85.71% concordance rates for cases with IHC scores of 3+, 2+, and 0/+1, respectively. The overall concordance between the two methods was 80%. Based on statistical analysis, HER2 status showed a considerable correlation with tumor grade (P= 0.02). No correlation was observed between HER2 gene status and the size and type of tumor, characteristics of lymph node, and patients’ age. Conclusion: The data suggested that IHC results are reliable for HER2 status testing in cases with IHC scores 0/+1 and 3+. However, in patients with an IHC score of +2, it is necessary to perform a complimentary test to evaluate HER2 status to avoid haphazard treatment with trastuzumab in negative cases and identifying positive cases for suitable treatment.

Analysis of gene copy number in esophageal patient-derived tumor xenografts.

e13648 Background: Patient-derived tumor xenograft (PDX) models are a valuable resource for studying cancer biology and antitumor drug evaluation. The suitability of tumor models in vivo depends on how accurately they mimic a human disease and reproduce the histotype and molecular genetic features of a human tumor. The purpose of the study was to create a PDX model of human cancer and analyze its characteristics. Methods: PDX models of esophageal cancer were obtained by transplanting a tumor fragment from a patient with esophageal squamous cell carcinoma to the BALB/c Nude athymic mice (n = 10 for one PDX generation). Preservation of the tumor histotype was confirmed histologically (hematoxylin and eosin staining). 5 PDX were generated. An analysis of the relative copy number of the YAP1 and KDM6A genes (Real-Time qPCR) in xenograft and donor tumor tissues was performed in each generation. Results: A decrease in the copy numbers of the YAP1 and KDM6A genes by 3.8 and 2.2 times, respectively, was observed in PDX tumor samples (F3 generation) compared to normal donor tissues (p < 0.05). This trend maintained in F4 and F5 generation PDX samples. No changes in the copy numbers of the YAP1 and KDM6A genes were detected in PDX tumor samples in F1 and F2 generations. A cluster analysis (Hierarchical Clustering, Euclidean distance) demonstrated that samples of the first and second generations of esophageal cancer PDX models were closest to the patient tumor tissues in gene copy numbers. Conclusions: Later generations of esophageal cancer PDX models are characterized by changes in the genes copy numbers due to changes in the tumor and clonal selection. Early PDX generations better reproduce genetic, molecular and morphological features of tumors.

Relative gene copy numbers in various patterns of cervical cancer growth.

e18029 Background: Numerous studies on cervical cancer confirm an important role of specific genomic changes in the onset and development of cervical intraepithelial neoplasia and their effect on the progression of cervical cancer. Solid tumors are characterized by genomic changes leading to a change in the DNA sequence copy number. The purpose of the study was to reveal changes in the relative copy number of the ESR1, ESR2, GPER1, STS, SULT1A1, SULT1E1, CYP1A1, CYP1A2 genes responsible for the reception and metabolism of estrogens in cervical tissues in endophytic and exophytic patterns of tumor growth in order to find predictive markers of malignancy. Methods: The study included 40 patients aged 28-65 years with cervical cancer of endophytic (n = 20) and exophytic (n = 20) growth patterns. Eligibility criteria included a morphologically confirmed cervical squamous cell cancer T1b-2aN0M0, stage I-II. The Thermo Scientific GeneJET FFPE DNA Purification Kit was used for the DNA extraction from FFPE blocks of tumor and healthy tissues. DNA concentrations were measured on the Qubit 2.0 fluorimeter (Invitrogen, USA) using the Quant-iT dsDNA High-Sensitivity (HS) Assay Kit (Invitrogen, USA). Results: The relative copy number of the GPER1, SULT1A1, CYP1A1 genes in tumor samples increased (p < 0.05) compared with normal tissues in the total sample of patients diagnosed with cervical cancer. In contrast to the total sample, an increase in the SULT1A1 gene dosage did not reach a statistically significant level in any group (p = 0.242 and p = 0.157); the copy number of the GPER1 locus significantly increased only in the group with the endophytic growth pattern (p = 0.040), as well as the CYP1A2 gene dosage (p = 0.025). Patients of 36-55 years with endophytic tumors showed a statistically significant (p < 0.05) increase in the GPER1 and CYP1A1 gene copy numbers with the rates of 41.7% and 66.7%, respectively, as well as an increased amplification of the CYP1A2 gene in 41.67% of patients. In women of 56-75 years with endophytic tumors, an increase in the copy numbers of the ESR2, GPER1, SULT1A1 genes was observed with a frequency of 50%, 100% and 75%, respectively. Patients aged 20-35 and 36-55 years with exophytic tumors showed a statistically significant (p < 0.05) increase in the CYP1A1 gene copy numbers in 33.33% and 45.45%, respectively. Conclusions: The results suggest the use of the GPER1, SULT1A1 and CYP1A1 gene copy numbers as biomarkers of cervical tumors.

Cloning and functional expression of a food-grade circular bacteriocin, plantacyclin B21AG, in probiotic Lactobacillus plantarum WCFS1

There is an increasing consumer demand for minimally processed, preservative free and microbiologically safe food. These factors, combined with risks of antibiotic resistance, have led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food preservatives and as protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a novel circular bacteriocin produced by Lactobacillus plantarum B21. Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when tested against the WCFS1 (pTRKH2) indicator strain, indicating functional expression of plantacyclin B21AG. Real-time PCR analysis revealed that the relative copy number of pB21AG01 was 7.60 ± 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 ± 0.05 and 25.19 ± 2.68 copies, respectively in WCFS1. This indicates that the bacteriocin gene cluster is located on a highly stable, low copy number plasmid pB21AG01 in L. plantarum B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 may result in similar inhibitory zones observed in both wild type and recombinant hosts despite the low copy number of pCycB21.