scholarly journals A comparison of cost for mycobacterial load determination in research laboratories

2015 ◽  
Vol 21 (3) ◽  
pp. 55
Author(s):  
A Pooran ◽  
R Meldau ◽  
K Dheda ◽  
R N Van Zyl-Smit
Keyword(s):  
Dynamic Range ◽  
Quantitative Polymerase Chain Reaction ◽  
Quantitative Measure ◽  
Turnaround Time ◽  
Luciferase Reporter ◽  
Reporter Construct ◽  
Luciferase Reporter Construct ◽  
Single Experiment ◽  
Capital Costs ◽  
Solid Media

<p><strong>Background</strong>. Mycobacterial load determination is a critical quantitative measure needed in many clinical and research laboratory studies and selection depends on several factors including sensitivity, dynamic range and turnaround time. However, there are no data about cost, which is an important selection determinant. We therefore performed a cost analysis of five quantitative mycobacterial load assays.</p><p><strong>Methods</strong>. The costs of five mycobacterial quantification techniques were compared in a hypothetical single experiment (control and intervention) performed in triplicate. Assays evaluated were: mycobacterial colony-forming units (MCFU) using 7H10-Middlebrook solid media, automated liquid culture (BACTEC-MGIT-960), [3H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative polymerase chain reaction (PCR) (Xpert-MTB/RIF) using serial dilutions of Mycobacterium tuberculosis. Costs associated with consumables, equipment and personnel were included and expressed in 2015 South African rands and US dollars.</p><p><strong>Results</strong>. The least costly technique was the luminescence reporter construct assay (R85.68/$6.72) whereas the most expensive technique was the Xpert MTB/RIF PCR (R388.02/$30.42). The high cost of the PCR assay was mainly attributable to the costly Xpert MTB/RIF cartridges. Although the MCFU by solid culture had a similar cost compared with uracil incorporation and Xpert MTB/RIF, the purchase price of the equipment required to perform the latter assays was ~2 - 10 times higher.</p><p><strong>Conclusion</strong>. Taking into consideration the turnaround time, capital costs, discriminatory ability, the running costs (excluding staff) of the luminescence reporter assay are the lowest. Where time to result is critical, more expensive techniques such as the Xpert MTB/RIF should be used. In a clinical setting where automated culture and Xpert are routinely performed, quantitative load from time to positivity and cycle thresholds will provide extra data without additional cost.</p>

Mapping and functional analysis of an instability element in phosphoenolpyruvate carboxykinase mRNA

2000 ◽  
Vol 279 (5) ◽  
pp. F866-F873 ◽  
Author(s):  
Omar F. Laterza ◽  
Lynn Taylor ◽  
Shashikala Unnithan ◽  
Ly Nguyen ◽  
Norman P. Curthoys
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Luciferase Activity ◽  
Specific Protein ◽  
Luciferase Reporter ◽  
Reporter Construct ◽  
Luciferase Reporter Construct ◽  
Functional Studies ◽  
Renal Gluconeogenesis ◽  
Shift Analysis ◽  
Nontranslated Region

Phosph oenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of renal gluconeogenesis. The 3′-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3′-nontranslated region of the PEPCK mRNA. Deletion constructs were then used to map the binding interactions to two adjacent RNA segments (PEPCK-6 and PEPCK-7). However, competition experiments established that only the binding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysis indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produce a decrease in luciferase activity equivalent to that observed with the full-length 3′-nontranslated region. Thus the PEPCK-7 segment binds a specific protein that may recruit one or more proteins to form a complex that mediates the rapid decay of the PEPCK mRNA.

scholarly journals OR07-06 The Roles of GNAQ and GNA11 in Calcium-Sensing Receptor (CaSR) Signalling

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Anna K Gluck ◽  
Mark Stevenson ◽  
Sara Falcone ◽  
Asuka Inoue ◽  
Gerda E Breitwieser ◽  
...  
Keyword(s):  
Protein Expression ◽  
G Protein ◽  
Extracellular Calcium ◽  
Luciferase Reporter ◽  
Reporter Construct ◽  
Calcium Sensing Receptor ◽  
Luciferase Reporter Construct ◽  
Response Element ◽  
Calcium Sensing

Abstract The G-protein subunits Gα 11 and Gα q, which share &gt;90% peptide sequence identity and are encoded by the GNA11 and GNAQ genes, respectively, mediate signalling by the calcium-sensing receptor (CaSR), a class C G-protein coupled receptor (GPCR) that regulates extracellular calcium (Ca2+e) homeostasis. Germline Gα 11 inactivating and activating mutations cause familial hypocalciuric hypercalcaemia type-2 (FHH2) and autosomal dominant hypocalcaemia type-2 (ADH2), respectively, but such Gα q mutations have not been reported. We therefore investigated the DiscovEHR cohort database, which has exomes from 51,289 patients with matched phenotyping data, for such GNAQ mutations. The DiscovEHR cohort was examined for rare GNAQ variants, which were transiently expressed in CaSR-expressing HEK293A Gα q/11 knockout cells, and their effects on CaSR-mediated intracellular calcium (Ca2+i) release and MAPK activity, in response to increasing concentrations of extracellular calcium were assessed using a nuclear factor of activated T-cells response element (NFAT-RE) luciferase reporter construct and a serum response element (SRE) luciferase reporter construct, respectively. Responses were compared to those of wild-type (WT), inactivating FHH2-associated GNA11 mutations (Leu135Gln and Phe220Ser), and engineered GNAQ mutations that were equivalent to the FHH2-causing GNA11 mutations. Gα q/11 protein expression was confirmed by Western blot analysis. Six rare missense GNAQ variants (Arg19Trp, Ala110Val, Gln299His, Ala302Ser, Ala331Thr, Val344Ile) were identified in DiscovEHR individuals, all of whom had mean plasma calcium values in the normal range (8.30–10.00 mg/dL). Functional characterisation of all six Gα q variants showed no significant difference to WT Gα q responses, thereby indicating that these variants are unlikely to be disease-causing mutations. In addition, the FHH2-causing GNA11 mutations (Leu135Gln and Phe220Ser) had significantly reduced responses, compared to WT Gα 11; however, this could be compensated by WT Gα q. GNAQ Leu135Gln and Phe220Ser, in contrast to their Gα 11 counterparts, showed no differences in protein expression or signalling responses when compared to WT Gα q. Our study, which provides mechanistic insights into the differences between Gα q and Gα 11, indicates that Gα q, unlike Gα 11, does not play a major role in the pathogenesis of FHH2 or ADH2.

Role of the membrane anchoring and cytoplasmic domains in intracellular transport and localization of viral glycoproteins

1999 ◽  
Vol 77 (3) ◽  
pp. 165-178 ◽  
Author(s):  
Kakoli Ghosh ◽  
Hara P Ghosh
Keyword(s):  
Nitric Oxide ◽  
Intracellular Transport ◽  
Transendothelial Migration ◽  
Interleukin 8 ◽  
Nitric Oxide Donor ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Reporter Construct ◽  
Luciferase Reporter Construct ◽  
Exogenous Nitric Oxide

Nuclear factor-κB (NF-κB) binds to nucleotide sequences between -80 and -70 bp upstream of the transcriptional start site in the interleukin-8 (IL-8) promoter and is crucial for transcription of the IL-8 gene. We showed that exogenous nitric oxide in the form of a nitric oxide donor significantly reduced IL-8 mRNA in cytokine-activated ECV304. Similarly, nitric oxide significantly reduced migration of polymorphonuclear neutrophils through cytokine-activated ECV304 monolayers, an IL-8-dependent process. Using a luciferase reporter construct containing the NF-κB site of the IL-8 gene, we showed that exposing cytokine-activated ECV304 to exogenous nitric oxide resulted in significant reduction of NF-κB binding. Follow-up studies using a luciferase reporter construct possessing a mutated NF-κB site confirmed that the luciferase activity observed in the NF-κB reporter resulted from NF-κB binding. These studies demonstrate that nitric oxide, supplied exogenously into reactions containing activated endothelium, down-regulates pro-inflammatory activity, such as the secretion of chemokines, and functional activity, such as transendothelial migration of neutrophils. Key words: interleukin-8, nuclear factor κ B, transendothelial migration, nitric oxide.

Endotoxin induction of luciferase activity in mice transgenic for a COX-2 promoter-luciferase reporter construct

1999 ◽  
Vol 59 (1-6) ◽  
pp. 5
Author(s):  
Amanda Freeman ◽  
Harvey Herschman ◽  
Olga Voznesensky ◽  
Anita Bhatt ◽  
Steve Clark ◽  
...  
Keyword(s):  
Luciferase Activity ◽  
Luciferase Reporter ◽  
Reporter Construct ◽  
Luciferase Reporter Construct ◽  
Cox 2

AP-1-dependent induction of plasminogen activator inhibitor-1 by nickel does not require reactive oxygen

2001 ◽  
Vol 281 (3) ◽  
pp. L616-L623 ◽  
Author(s):  
Angeline S. Andrew ◽  
Linda R. Klei ◽  
Aaron Barchowsky
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Airway Epithelial Cells ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Reporter Construct ◽  
Luciferase Reporter Construct ◽  
Activator Inhibitor ◽  
Pai 1

Inhalation of nickel dust has been associated with an increased incidence of pulmonary fibrosis. Nickel may promote fibrosis by transcriptionally activating plasminogen activator inhibitor (PAI)-1 and inhibiting fibrinolysis. The current studies examined whether nickel stimulated the PAI-1 promoter though an oxidant-sensitive activator protein (AP)-1 signaling pathway. Addition of nickel to BEAS-2B human airway epithelial cells stimulated intracellular oxidation, induced c-Jun and c-Fos mRNA levels, increased phospho- and total c-Jun protein levels, and elevated PAI-1 mRNA levels over a 24-h time course. Pretreatment of the cells with antioxidants did not affect increased c-Jun protein or PAI-1 mRNA levels. Expression of the dominant negative inhibitor of AP-1, TAM67, prevented nickel-stimulated AP-1 DNA binding, AP-1-luciferase reporter construct activity, and PAI-1 mRNA levels. Overexpression of c-Jun, however, failed to induce the AP-1 luciferase reporter construct or PAI-1 mRNA levels. These data indicated that nickel activated AP-1 through an oxidant-independent pathway and that basal AP-1 is necessary for nickel-induced expression of PAI-1.

scholarly journals Tetracycline-reversible silencing of eukaryotic promoters.

1995 ◽  
Vol 15 (4) ◽  
pp. 1907-1914 ◽  
Author(s):  
U Deuschle ◽  
W K Meyer ◽  
H J Thiesen
Keyword(s):  
Transcriptional Start Site ◽  
Adaptor Protein ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Reporter Construct ◽  
Luciferase Reporter Construct ◽  
Genetic Switch ◽  
Mammalian Genomes ◽  
Eukaryotic Promoters ◽  
Maximal Induction

A tetracycline-controlled transrepressor protein has been engineered to silence transcriptional activities of eukaryotic promoters that are stably integrated into the chromatin of human cells. By fusing the KRAB domain of human Kox1 to the Tet repressor derived from Tn10 of Escherichia coli, a tetracycline-controlled hybrid protein (TetR-KRAB) was generated and constitutively expressed in HeLa cells. The TetR-KRAB protein binds to tet operator (tetO) sequences in the absence but not in the presence of tetracycline. When TetR-KRAB bound to tetO sequences upstream of the immediate-early promoter-enhancer of human cytomegalovirus (CMV), the expression of a CMV-driven luciferase reporter construct (ptetO7-CMV-L) was repressed in transient transfection experiments. This silencing was found to operate on different promoters and from tetO sequences placed more than 3 kb from the transcriptional start site. We constructed a stable, doubly transfected cell line (TIS-10) carrying a chromosomally integrated ptetO7-CMV-L reporter construct and expressing the TetR-KRAB protein. Upon addition of tetracycline, luciferase expression was induced more than 50-fold above the baseline level, with half-maximal induction by 2 days. Furthermore, a protein of around 110 kDa was found to coimmunoprecipitate with the TetR-KRAB fusion protein. This protein might play a role as an adaptor protein mediating the silencing exerted by the TetR-KRAB protein. The TetR-KRAB silencing system should be useful as a genetic switch for regulating the expression of chromosomally integrated heterologous and endogenous genes present in mammalian genomes.

scholarly journals In Vivo Imaging of Transiently Transgenized Mice with a Bovine Interleukin 8 (CXCL8) Promoter/Luciferase Reporter Construct

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e39716 ◽  
Author(s):  
Fabio Franco Stellari ◽  
Valentina Franceschi ◽  
Antonio Capocefalo ◽  
Marcello Ronchei ◽  
Fabrizio Facchinetti ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Interleukin 8 ◽  
Luciferase Reporter ◽  
Reporter Construct ◽  
Luciferase Reporter Construct

Analysis of Regulatory miRNAs of Histone Demethylase JMJD3 In MDS CD34+ Hematoprogenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 609-609
Author(s):  
Yue Wei ◽  
George Calin ◽  
Yu Jia ◽  
Hong Zheng ◽  
Sara McCay ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Histone H3 ◽  
Histone Demethylase ◽  
Hematopoietic Progenitor Cells ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Reporter Construct ◽  
Hematopoietic Progenitor ◽  
Luciferase Reporter Construct ◽  
Cd34 Cells

Abstract Abstract 609 Introduction: Myelodysplastic syndromes (MDS) represent a very complex group of myeloid leukemias with diverse genetic and epigenetic characteristics. We have identified JMJD3, a histone demethylase, aberrantly upregulated in bone marrow CD34+ hematopoietic progenitor cells of MDS (Blood 2009). JMJD3 is a jmjc domain containing histone demethylase that removes methyl groups from Lys-27 of histone H3, and is also known to positively regulate the methylation on Lys-4 of histone H3. This results in activation of upstream regulators of NF-kB signaling, suggesting that JMJD3 plays a crucial role in the pathogenesis of MDS. Therefore, insight on the regulation of JMJD3 expression in MDS is of importance. Aberrant expression of miRNAs, including loss of expression, which leads to up-regulation of the expression of its target genes, has been identified in various types of malignancies including MDS. We hypothesized that alteration of regulatory miRNAs could be involved in the upregulation of JMJD3 in MDS CD34+ cells. Identification of JMJD3 regulatory miRNAs may help further dissect the molecular mechanisms underlying the pathogenesis of MDS. Methods and Results: To identify regulatory miRNAs targeting JMJD3, we performed an analysis using microRNA Genomics Resource (miRGen) and identified 40 candidate miRNAs that potentially interact with the 3′ untranslate region (3′-UTR) of JMJD3 RNA. We then characterized the interactions of these candidate miRNAs to JMJD3 by using a luciferase assay system by cloning the 3′-UTR region of JMJD3 gene into a luciferase reporter construct. By co-transfecting each of the 27 available miRNAs from the 40 candidates together with the luciferase reporter construct into human megakaryocytic blast cell line Meg-O1, we identified 7 mirRNAs (has-mir -29a, -29b, -29c, -99b, -101, -377, and -767-5p) that negatively regulate JMJD3 3′UTR associated luciferase activity. We then examined the expression for these 7 key potential regulatory miRNAs of JMJD3 in a cohort of 36 patient bone marrow CD34+ cells by microRNA Taqman probe based Q-PCR analysis. The median age of these patients was 67 years (32 to 85); 42% had low or INT-1 disease; 21% were diploid and 28% had an alteration of chromosome 5 or 7. Analysis showed that 16 of the 36 MDS CD34+ cell samples examined showed lost expression of mirRNA hsa-mir767-5p, and 7 had down-regulated expression of the same microRNA. For these 23 MDS samples with repressed mir-767-5p expression, 11 of them (~48%) had upregulated expression of JMJD3, as demonstrated by Q-PCR analysis Consistently, in miRNA transfected Meg-O1 cells, Q-PCR analysis indicates that over-expression of mir767-5p is accompanied with decrease of JMJD3 RNA by 68% based on Q-PCR analysis using 3 different probes for JMJD3. Of interest, 4 of the 5 patients with monosomy of chromosome 7 had downregulation of mir767-5p. Conclusion and Future Direction: These results support the hypothesis that mir767-5p is a key regulatory miRNA targeting JMJD3 in hematopoietic progenitor cells. Since one important mechanism for miRNA on target gene expression is through inhibiting the translation from mRNA to protein, we are now developing an antibody against endogenous JMJD3, with which we will be able to further correlate the level of mir767-5p and the expression of JMJD3 protein in both cell lines and primary MDS samples. Furthermore, correlation between alteration of mir767-5p with clinical, cytogenetic and molecular features in MDS patients will be characterized in a larger cohort of patients. These will further determine the role of mir767-5p for the regulation of JMJD3 and its role during the pathogenesis of MDS. By pursuing the studies described above, a potential link between two key epigenetic regulatory components, histone methylation regulator (JMJD3) and microRNAs, involved in pathogenesis of MDS, will be established. Disclosures: No relevant conflicts of interest to declare.

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