Molecular diagnostics for tuberculosis.

The phenotypic methods of smear microscopy, culture and indirect drug susceptibility testing (DST) remain the 'gold standard' diagnostics for tuberculosis (TB) in 2015. However, this review demonstrates that genotypic methods are in the ascendancy. Current-generation nucleic acid amplification tests (NAATs) are important supplementary tests for the rapid direct detection of (multidrug-resistant) TB in specific clinical settings. Genotypic detection is already the preferred method of detecting rifampicin and pyrazinamide resistance. Next-generation NAATs able to detect about 10 colony forming units/mL of sputum could replace culture as the initial test for detecting TB. Whole genome sequencing could also plausibly replace phenotypic DST but much work is required in method standardisation, database development and elucidation of all resistance gene determinants. The challenge then will be to rollout these increasingly complex and expensive diagnostics in the low-income countries where TB is prevalent.

The presence of ammonium facilitates nitrite reduction under PHB driven simultaneous nitrification and denitrification.

For economic and efficient nitrogen removal from wastewater treatment plants via simultaneous nitrification and denitrification the nitrification process should stop at the level of nitrite such that nitrite rather than nitrate becomes the substrate for denitrification. This study aims to contribute to the understanding of the conditions that are necessary to improve nitrite reduction over nitrite oxidation. Laboratory sequencing batch reactors (SBRs) were operated with synthetic wastewater containing acetate as COD and ammonium as the nitrogen source. Computer controlled operation of the reactors allowed reproducible simultaneous nitrification and denitrification (SND). The oxygen supply was kept precisely at a low level of 0.5 mgL(-1) and bacterial PHB was the only electron donor available for denitrification. During SND little nitrite or nitrate accumulated (< 20% total N), indicating that the reducing processes were almost as fast as the production of nitrite and nitrate from nitrification. Nitrite spiking tests were performed to investigate the fate of nitrite under different oxidation (0.1-1.5 mgL(-1) of dissolved oxygen) and reduction conditions. High levels of reducing power were provided by allowing the cells to build up to 2.5 mM of PHB. Nitrite added was preferentially oxidised to nitrate rather than reduced even when dissolved oxygen was low and reducing power (PHB) was excessively high. However, the presence of ammonium enabled significant reduction of nitrite under low oxygen conditions. This is consistent with previous observations in SBR where aerobic nitrite and nitrate reduction occurred only as long as ammonium was present. As soon as ammonium was depleted, the rate of denitrification decreased significantly. The significance of the observed strongly stimulating effect of ammonium on nitrite reduction under SND conditions is discussed and potential consequences for SBR operation are suggested.

Apoptosis, reproductive failure, and oxidative stress in Chinese hamster ovary cells with compromised genomic integrity.

Chromosomal instability and persistent reproductive cell death show a significant correlation after cells are exposed to ionizing radiation. To examine the possible role of apoptosis in persistent reproductive cell death, we analyzed subsets of chromosomally stable and unstable clones for relationships between chromosome stability, reproductive integrity, and apoptosis. All clones were generated from the GM10115 cell line and derived from single progenitor cells surviving 10 Gy of X-rays, and all measurements were made approximately 60-80 generations after irradiation. The incidence of apoptosis, as measured by both annexin V binding of phosphatidylserine residues and terminal deoxynucleotidyl transferase labeling of DNA strand breaks, was significantly higher in chromosomally unstable clones than it was in chromosomally stable clones (P < 0.05; ANOVA and Student's t test). Furthermore, statistical analyses of the biological end points of persistent reproductive cell death and apoptosis were consistent, showing R2 values of 0.78 and 0.76, respectively. These results suggest that persistent reproductive cell death can, in part, be explained by the predisposition of a fraction of the clonal population to undergo apoptosis or necrosis. Immunological blot analyses of protein levels and DNA bandshift assays confirmed the mutant status of p53 in the host cell line, implying an apoptotic pathway that is independent of p53. Induction of apoptosis by agents such as actinomycin D, etoposide, and staurosporine and induction of necrosis by sodium azide were accompanied by an increase in the level of intracellular peroxy radicals and lipid peroxidation products, two independent end points that are typically associated with oxidative stress. Similar findings were observed in several subclones showing persistent apoptosis. These results suggest that the elevated levels of free radical damage that we detected were derived from the fraction of cells dying by apoptotic or necrotic processes. Possible mechanisms whereby oxidative stress may contribute indirectly to the perpetuation of chromosomal instability are discussed.

Formation of stable cationic lipid/DNA complexes for gene transfer.

Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

Purification and properties of thyroid hormone receptor beta 1 expressed in Escherichia coli as a fusion protein.

Thyroid hormone receptor binds to specific DNA sequences and acts as a hormone-dependent transcriptional regulator. The protein can form homodimers, or heterodimers with the related 9-cis-retinoic acid receptor (RXR) or retinoic acid receptor (RAR) receptor families, leading to complex patterns of regulation. To obtain relatively large quantities of the receptor for biochemical studies, we have inserted the cDNA for human thyroid receptor beta into a variant of the pGEX vector (pGEX-KG) and produced the protein in Escherichia coli as a fusion with glutathione-S-transferase. Conditions for protein production, isolation on glutathione agarose, and thrombin cleavage to generate active receptor were developed. Final yields were approximately 1 mg/liter of culture. Scatchard plots of 125I-triiodothyronine binding data revealed a single class of sites with a Kd of 0.1 nM. An overlay assay was established to measure protein-protein binding and used to show a direct interaction with bacterially expressed RXR receptor. Binding of the purified receptor to DNA response elements measured in a DNA binding assay was increased by RXR to different extents, depending on the DNA sequence. This preparation will be useful in exploring the mechanisms of receptor activity.

T3 receptor suppression of Sp1-dependent transcription from the epidermal growth factor receptor promoter via overlapping DNA-binding sites.

Expression of the human epidermal growth factor receptor (EGFR) gene is inhibited by ligand-activated thyroid hormone receptor (T3R). Binding sites for Sp1 and for the T3R.retinoid X receptor (RXR) complex overlap in a functional core of the EGFR promoter. Sp1 inhibited binding of the T3R complex to this 36-base pair (bp) EGFR element in vitro but did not affect binding of the T3R complex to a positive thyroid hormone response element (TRE). In Drosophila SL2 cells, which lack Sp1 and T3R, function of the EGFR promoter was strongly dependent on Sp1. Sp1-dependent promoter function was inhibited by ligand-activated T3R but not by mutant T3R defective in DNA or T3 binding. RXR increased the extent of inhibition. Sp1 enhanced activity of the 36-bp element placed 5' to a minimal TATA promoter and this enhancement was also repressed by T3R. Mutations in the 36-bp element were unable to separate Sp1 and T3R functions. However, addition of a second half-site 5' to the existing site in an inverted repeat configuration created a positive TRE. In the absence of ligand, T3R inhibited Sp1 stimulation from this altered element; addition of T3 reversed the inhibition. When a dimeric TRE is separated from Sp1-binding sites strong synergism was observed. The nature and location of the TRE thus strongly influence biological responses. A TRE site in the EGFR promoter that overlaps an Sp1-binding site inhibits Sp1 function but is unable to direct positive function.

A nuclear protein is required for thyroid hormone receptor binding to an inhibitory half-site in the epidermal growth factor receptor promoter.

The epidermal growth factor (EGF) receptor (EGFR) promoter is negatively regulated by thyroid hormone and retinoic acid. This regulation can be mapped to a 36-basepair GC-rich region of the promoter (EGFR P/E) that functions autonomously as a promoter and an enhancer when placed in front of the thymidine kinase gene TATA element. Direct high affinity binding of the thyroid hormone receptor (T3R) to this element requires a nuclear protein. Through ion exchange chromatography and gel filtration of HeLa nuclear extract, this activity was identified as a protein of approximately 67 kilodaltons. This protein did not bind to DNA alone, but greatly augmented T3R binding to the EGFR P/E sequence in gel mobility shift and DNA precipitation assays. When combined with the T3R auxillary protein (TRAP), the T3R migrated as a larger complex on the DNA. Chemical cross-linking identified this complex as a heterodimer between T3R and TRAP. T3R-TRAP binds to a 7-basepair site in the EGFR P/E (GGGACTC) that has weak homology to a consensus thyroid response element half-site. Thus, on this element, T3R-TRAP heterodimers contact the DNA primarily on a single site that comprises an inhibitory thyroid response element.