scholarly journals Screening and Absolute Quantification of a β-lactamase Resistance Gene NDM-1 in Lake Sediment

2021 ◽  
Author(s):  
Rajeev Ranjan ◽  
Shashidhar Thatikonda
Keyword(s):  
Lake Sediment ◽  
Copy Number ◽  
Quantitative Polymerase Chain Reaction ◽  
Horizontal Transmission ◽  
Gene Copy Number ◽  
Absolute Quantification ◽  
Gene Copy ◽  
Qpcr Analysis ◽  
Wide Range ◽  
Template Dna

Abstract The extensive usage of antibiotics in humans and veterinary medicine and their discharge into the aquatic environment hasten the growth, selection, and horizontal transmission of ARGs in a given bacterial community. New Delhi Metallo-β-lactamase-1(NDM-1) is an enzyme that hydrolyzes a wide range of β-lactams antibiotics, including carbapenems. The rapid distribution of NDM-1 harboring bacteria accounts for a significant public health menace worldwide. The presence of the NDM-1 inhibited the potential of β–lactam antibiotics for treating infections caused by bacterial strains carrying such resistances, leaving minimal treatment options available. NDM-1 harboring bacteria have been detected in clinical specimens and environmental compartments where bacterial infections are ubiquitous. In this study, identification and absolute quantification of NDM-1 in sixteen lake sediment samples collected in and around Hyderabad, India, was carried out using a real-time quantitative polymerase chain reaction (qPCR) and results were expressed in gene copy number/ng (nanogram) of template DNA. 13 samples (out of 16) shown a positive signal for NDM-1 during qPCR analysis. Durgamcheru lake, Kandi lake, and Singur dam showed a negative signal for the NDM-1 during qPCR analysis among the tested samples. The remaining sampling locations tested positive with the highest gene copy number/ng of template DNA observed in the Amberpet STP (71.8). Hierarchical clustering analysis was performed to categorize the sampling location into different clusters based on pollution sources and observed results expressed in the form of a dendrogram.

Seasonal comparison of potential denitrification rates and nitrogen functional genes with sediment depths in the wetland, Korea

2020 ◽  
Author(s):  
Ji Yeon Han ◽  
Dong-Hun Kim ◽  
Seolran Oh ◽  
Hee Sun Moon
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Denitrification Rate ◽  
Seasonal Effects ◽  
Gene Copy ◽  
Related Gene ◽  
Nosz Gene ◽  
Potential Denitrification ◽  
Qpcr Analysis ◽  
Wide Range

<p>Wetlands provide not only habitats for a wide range of organisms but also ecological functions of degrading and removing pollutants from water body through a variety of physical, chemical and biological processes. Seasonal variation including recent increases in the frequency of floods and droughts have affected the hydrological environment of wetlands. Furthermore, these effects may result in changes in redox conditions in the nitrogen biogeochemical process in wetland sediments. Therefore, in this study, the potential denitrification rate and denitrification-related gene quantitative analysis was performed to investigate seasonal nitrogen dynamics of wetland sediments associated with surface and groundwater interactions in Baekseok reservoir wetlands (Gunsan-si, Jeollabuk Province, Korea). Sediment from two different sites (i.e., PA and PB) in wetland were collected with different depths in June and December 2019 to investigate seasonal effects on denitrification with sediment depths. Potential denitrification rate experiments were performed using the acetylene inhibition technique, and denitrification-related gene quantification was performed by qPCR analysis. As a result of potential denitrification rate, PA sites ranged from 2.67–3.27 ng N<sub>2</sub>O/g/hr and 3.13–15.13 ng N<sub>2</sub>O/g/hr in June and December, respectively. PB sites ranged from 2.43-6.30 ng N<sub>2</sub>O/g/hr and 5.47-6.30 ng N<sub>2</sub>O/g/hr. Overall, higher levels were observed at 0–10 cm, with higher denitrification rates in December than in June. The qPCR analysis showed that the narG, nirS and nosZ gene copy number ranges for the PA site in June showed 1.82 x 10<sup>6</sup> – 6.15 x 10<sup>7</sup> copies/g, and in December 7.71 x 10<sup>5</sup> – 5.97 x 10<sup>8</sup> copies/g. The narG, nirS and nosZ gene copy number ranges for the PB site in June showed 3.53 x 10<sup>5</sup> – 3.86 x 10<sup>8</sup> copies/g, and in December, 1.24 x 10<sup>6</sup> – 3.47 x 10<sup>8</sup> copies/g. Overall, both sites had higher copy numbers in December than in June, corresponding to an increase in potential denitrification rate in December.</p>

scholarly journals Brachyury gene copy number gain and activation of the PI3K/Akt pathway: association with upregulation of oncogenic Brachyury expression in skull base chordoma

2018 ◽  
Vol 128 (5) ◽  
pp. 1428-1437 ◽  
Author(s):  
Ryohei Otani ◽  
Akitake Mukasa ◽  
Masahiro Shin ◽  
Mayu Omata ◽  
Shunsaku Takayanagi ◽  
...  
Keyword(s):  
Cell Line ◽  
Copy Number ◽  
Growth Regulation ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Copy Number ◽  
Copy Number Gain ◽  
High Expression ◽  
Gene Copy ◽  
Akt Pathway ◽  
Expression Levels

OBJECTIVEChordoma is a slow-growing but clinically malignant tumor, and the prognosis remains poor in many cases. There is a strong impetus to develop more effective targeted molecular therapies. On this basis, the authors investigated the potential of Brachyury, a transcription factor involved in notochord development, as a candidate molecular target for the treatment of chordoma.METHODSBrachyury gene copy number and expression levels were evaluated by quantitative polymerase chain reaction in 27 chordoma samples, and the transcriptomes of Brachyury high-expression tumors (n = 4) and Brachyury low-expression tumors (n = 4) were analyzed. A chordoma cell line (U-CH2) was used to investigate the signaling pathways that regulate Brachyury expression.RESULTSAll chordoma specimens expressed Brachyury, and expression levels varied widely. Patients with higher Brachyury expression had significantly shorter progression-free survival (5 months, n = 11) than those with lower expression (13 months, n = 16) (p = 0.03). Somatic copy number gain was confirmed in 12 of 27 (44%) cases, and copy number was positively correlated with Brachyury expression (R = 0.61, p < 0.001). Expression of PI3K/Akt pathway genes was upregulated in Brachyury high-expression tumors, and suppression of PI3K signaling led to reduced Brachyury expression and inhibition of cell growth in the U-CH2 chordoma cell line.CONCLUSIONSActivation of the PI3K/Akt pathway and Brachyury copy number gain are strongly associated with Brachyury overexpression, which appears to be a key event in chordoma growth regulation. These findings suggest that targeting Brachyury and PI3K/Akt signaling may be an effective new approach for treating chordoma.

scholarly journals ABERRANT EXPRESSION OF SELENIUM-CONTAINING GLUTATHIONE PEROXIDASES IN CLEAR CELL RENAL CELL CARCINOMAS

2015 ◽  
Vol 37 (2) ◽  
pp. 105-110 ◽  
Author(s):  
E Rudenko ◽  
O Kondratov ◽  
G Gerashchenko ◽  
Y Lapska ◽  
S Kravchenko ◽  
...  
Keyword(s):  
Renal Cell ◽  
Copy Number ◽  
Clear Cell ◽  
Cancer Genomics ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Copy Number ◽  
Diagnostic Markers ◽  
Gene Copy ◽  
Renal Cell Carcinomas ◽  
Aberrant Expression

Aim: To find putative diagnostic markers for clear cell renal cell carcinomas (ccRCC). Material and methods: Quantitative polymerase chain reaction (Q-PCR), bisulfite treatment, methylation-specific PCR, analysis on cBioPortal for Cancer Genomics. Results: We have found that expression of GPX 1, GPX3, and GPX4 genes was decreased in ccRCC. We have shown that the number of alanine (GCG) repeats at the amino terminus of the GPX1 protein is variable. It was reported earlier that an allele that possess 5 alanine repeats is associated with the increased cancer risk. According to the obtained data, the allele with the 5 alanine repeats was also present in a group of healthy donors. Moreover, the frequency of alleles with repeats was similar among ccRCC patients and healthy individuals. We found that decreased expression of GPXs genes was not associated with promoter methylation. To provide other explanation, an analysis on the gene copy number was performed. We have found the heterozygous deletions for GPX1 gene, amplification for GPX3 gene, and no change in gene copy number for GPX4. Conclusions: Our data support the hypothesis that GPX1, GPX3, and GPX4 genes may play a role in ccRCC cancerogenesis and therefore they might be considered as putative diagnostic markers for ccRCC.

scholarly journals Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes

2000 ◽  
Vol 346 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Michael G. PATON ◽  
S. H. P. Parakrama KARUNARATNE ◽  
Elsa GIAKOUMAKI ◽  
Neil ROBERTS ◽  
Janet HEMINGWAY
Keyword(s):  
Translational Control ◽  
Copy Number ◽  
Gene Copy Number ◽  
Absolute Quantification ◽  
Gene Copy ◽  
Common Mechanism ◽  
Control Mechanisms ◽  
Dot Blot ◽  
Culex Mosquitoes ◽  
Pcr Product

The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estα21 and estβ21 genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estα21 and estβ21 are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estβ21 gene from this amplicon has greater transcription than estα21 in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.

scholarly journals Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Sumin Zhao ◽  
Yaoshen Wang ◽  
Xiuqing Xin ◽  
Zhonghai Fang ◽  
Linlin Fan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Copy Number ◽  
Reliable Method ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Copy Number ◽  
Carrier Screening ◽  
Gene Copy ◽  
Next Generation ◽  
Expanded Carrier Screening ◽  
Generation Sequencing

AbstractSpinal muscular atrophy (SMA) is one of the most common and severe genetic diseases. SMA carrier screening is an effective way to identify couples at risk of having affected children. Next-generation sequencing (NGS)-based expanded carrier screening could detect SMN1 gene copy number without extra experiment and with high cost performance. However, its performance has not been fully evaluated. Here we conducted a systematic comparative study to evaluate the performance of three common methods. 478 samples were analyzed with multiplex ligation probe amplification (MLPA), real-time quantitative polymerase chain reaction (qPCR) and NGS, simultaneously. Taking MLPA-based results as the reference, for 0 copy, 1 copy and ≥ 2 copy SMN1 analysis with NGS, the sensitivity, specificity and precision were all 100%. Using qPCR method, the sensitivity was 100%, 97.52% and 94.30%, respectively; 98.63%, 95.48% and 100% for specificity; and 72.72%, 88.72% and 100% for precision. NGS repeatability was higher than that of qPCR. Moreover, among three methods, NGS had the lowest retest rate. Thus, NGS is a relatively more reliable method for SMN1 gene copy number detection. In expanded carrier screening, compared with the combination of multiple methods, NGS method could reduce the test cost and simplify the screening process.

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