370 research outputs found
Single strand conformation polymorphism profiles with biotinylated PCR products to detect mutations in rpoB gene of Mycobacterium tuberculosis
A fragment of the rpoB gene, including the region
shown to be involved in rifampicin resistance, was
amplified from 15 rifampicin-resistant and 6 rifampicin-
sensitive clinical isolates of Mycobacterium
tuberculosis by the polymerase chain reaction (PCR).
One of the primers, employed in PCR, was biotinylated.
The biotinylated strand of the PCR product
was separated from the unbiotinylated strand using
streptavidin magnetic beads. Both the strands were
subjected to single strand conformation polymorphism
in polyacrylamide gel electrophoresis. The DNA bands
were silver stained to study their migration pattern.
A shift in the migration of either of the strands of
the test strain compared to the strands from a control
rifampicin-sensitive strain was considered as indicative
of resistance. This strategy was found to ease the
visualization of shift in the migration of the strands
in 17 of 21 samples and thereby detection of mutations
Use of streptavidin magnetic beads in single strand conformation polymorphism profiles to detect mutations in rpoB gene of M.tuberculosis
Single strand conformation polymorphism (SSCP) is one of the promising
techniques to identify mutations in short pieces of DNA (Orita et al. 1989). In this
technique, DNA of interest is often amplified by the polymerase chain reaction (PCR)
and then denatured by heat or alkali treatment before electrophoresis on a non
denaturing polyacrylamide gel. Differences in mobility of either of the single strands
compared to the control DNA indicate mutations which affect the secondary structure
and alter the mobility of the DNA. We applied PCR-SSCP for the detection of
mutations in the rifampicin resistance determining region (RRDR) of the rpoB gene of
M. tuberculosis (Telenti et al. 1993a; 1993b). A nested PCR was used to amplify the
RRDR. In the first PCR, 293-bp product was amplified and in the second PCR a 103-
bp of the first PCR product was amplified. However, in our experience using
denaturation by alkali or heating, the denatured PCR product most often reannealed to
form a large proportion of double stranded DNA during the electrophoresis
(Selvakumar et al. 1997a). After visualisation by staining with ethidium bromide or
silver staining, most of the DNA was in the double stranded form, with very little or
no single stranded DNA. The single strands that could be observed often ran close
together, making analysis of any difference in mobility difficult. Therefore an attempt
was made to generate biotinylated PCR product using a biotinylated forward primer
and later the biotinylated strand was separated using sterptavidin magnetic beads. The
separated strands eliminated the problem of strand reannealing during SSCP and were
silver stained to detect the shift in the mobility. Since the nested PCR requires more
time and is more expensive. a biotinylated PCR product was generated in a single PCR
using a biotinylated forward primer and an unbiotinylated reverse primer. This
simplified protocol was applied to clinical isolates in an attempt to detect rifampicin
resistance
Safe food and feed through an integrated toolbox for mycotoxin management: the MyToolBox approach
There is a pressing need to mobilise the wealth of knowledge from the international mycotoxin research conductedover the past 25-30 years, and to perform cutting-edge research where knowledge gaps still exist. This knowledgeneeds to be integrated into affordable and practical tools for farmers and food processors along the chain inorder to reduce the risk of mycotoxin contamination of crops, feed and food. This is the mission of MyToolBox – a four-year project which has received funding from the European Commission. It mobilises a multi-actorpartnership (academia, farmers, technology small and medium sized enterprises, food industry and policystakeholders) to develop novel interventions aimed at achieving a significant reduction in crop losses due tomycotoxin contamination. Besides a field-to-fork approach, MyToolBox also considers safe use options ofcontaminated batches, such as the efficient production of biofuels. Compared to previous efforts of mycotoxin reduction strategies, the distinguishing feature of MyToolBox is to provide the recommended measures to theend users along the food and feed chain in a web-based MyToolBox platform (e-toolbox). The project focuseson small grain cereals, maize, peanuts and dried figs, applicable to agricultural conditions in the EU and China. Crop losses using existing practices are being compared with crop losses after novel pre-harvest interventionsincluding investigation of genetic resistance to fungal infection, cultural control (e.g. minimum tillage or cropdebris treatment), the use of novel biopesticides suitable for organic farming, competitive biocontrol treatment and development of novel modelling approaches to predict mycotoxin contamination. Research into post-harvestmeasures includes real-time monitoring during storage, innovative sorting of crops using vision-technology, novelmilling technology and studying the effects of baking on mycotoxins at an industrial scale
N-acetylcysteine (NAC), an anti-oxidant, does not improve bone mechanical properties in a rat model of progressive chronic kidney disease-mineral bone disorder
Individuals with chronic kidney disease have elevated levels of oxidative stress and are at a significantly higher risk of skeletal fracture. Advanced glycation end products (AGEs), which accumulate in bone and compromise mechanical properties, are known to be driven in part by oxidative stress. The goal of this study was to study effects of N-acetylcysteine (NAC) on reducing oxidative stress and improving various bone parameters, most specifically mechanical properties, in an animal model of progressive CKD. Male Cy/+ (CKD) rats and unaffected littermates were untreated (controls) or treated with NAC (80 mg/kg, IP) from 30 to 35 weeks of age. Endpoint measures included serum biochemistries, assessments of systemic oxidative stress, bone morphology, and mechanical properties, and AGE levels in the bone. CKD rats had the expected phenotype that included low kidney function, elevated parathyroid hormone, higher cortical porosity, and compromised mechanical properties. NAC treatment had mixed effects on oxidative stress markers, significantly reducing TBARS (a measure of lipid peroxidation) while not affecting 8-OHdG (a marker of DNA oxidation) levels. AGE levels in the bone were elevated in CKD animals and were reduced with NAC although this did not translate to a benefit in bone mechanical properties. In conclusion, NAC failed to significantly improve bone architecture/geometry/mechanical properties in our rat model of progressive CKD
Differential control of proto-oncogene c-myc and c-fos expression in lymphocytes and fibroblasts
Machine learning predicts accurately mycobacterium tuberculosis drug resistance from whole genome sequencing data
Background: Tuberculosis disease, caused by Mycobacterium tuberculosis, is a major
public health problem. The emergence of M. tuberculosis strains resistant to existing
treatments threatens to derail control efforts. Resistance is mainly conferred by mutations
in genes coding for drug targets or converting enzymes, but our knowledge of these
mutations is incomplete. Whole genome sequencing (WGS) is an increasingly common
approach to rapidly characterize isolates and identify mutations predicting antimicrobial
resistance and thereby providing a diagnostic tool to assist clinical decision making.
Methods: We applied machine learning approaches to 16,688 M. tuberculosis isolates
that have undergone WGS and laboratory drug-susceptibility testing (DST) across 14
antituberculosis drugs, with 22.5% of samples being multidrug resistant and 2.1% being
extensively drug resistant. We used non-parametric classification-tree and gradientboosted-tree models to predict drug resistance and uncover any associated novel putative
mutations. We fitted separate models for each drug, with and without “co-occurrent
resistance” markers known to be causing resistance to drugs other than the one of interest.
Predictive performance was measured using sensitivity, specificity, and the area under the
receiver operating characteristic curve, assuming DST results as the gold standard.
Results: The predictive performance was highest for resistance to first-line drugs,
amikacin, kanamycin, ciprofloxacin, moxifloxacin, and multidrug-resistant tuberculosis
(area under the receiver operating characteristic curve above 96%), and lowest for thirdline drugs such as D-cycloserine and Para-aminosalisylic acid (area under the curve below
85%). The inclusion of co-occurrent resistance markers led to improved performance
for some drugs and superior results when compared to similar models in other largescale studies, which had smaller sample sizes. Overall, the gradient-boosted-tree models
performed better than the classification-tree models. The mutation-rank analysis detected
no new single nucleotide polymorphisms linked to drug resistance. Discordance between
DST and genotypically inferred resistance may be explained by DST errors, novel rare
mutations, hetero-resistance, and nongenomic drivers such as efflux-pump upregulation.
Conclusion: Our work demonstrates the utility of machine learning as a flexible approach
to drug resistance prediction that is able to accommodate a much larger number of
predictors and to summarize their predictive ability, thus assisting clinical decision
making and single nucleotide polymorphism detection in an era of increasing WGS data
generation
Bacteriophage- based tests for the detection of Mycobacterium tuberculosis in clinical specimens: a systematic review and meta- analysis
BACKGROUND: Sputum microscopy, the most important conventional test for tuberculosis, is specific in settings with high burden of tuberculosis and low prevalence of non tuberculous mycobacteria. However, the test lacks sensitivity. Although bacteriophage-based tests for tuberculosis have shown promising results, their overall accuracy has not been systematically evaluated. METHODS: We did a systematic review and meta-analysis of published studies to evaluate the accuracy of phage-based tests for the direct detection of M. tuberculosis in clinical specimens. To identify studies, we searched Medline, EMBASE, Web of science and BIOSIS, and contacted authors, experts and test manufacturers. Thirteen studies, all based on phage amplification method, met our inclusion criteria. Overall accuracy was evaluated using forest plots, summary receiver operating (SROC) curves, and subgroup analyses. RESULTS: The data suggest that phage-based assays have high specificity (range 0.83 to 1.00), but modest and variable sensitivity (range 0.21 to 0.88). The sensitivity ranged between 0.29 and 0.87 among smear-positive, and 0.13 to 0.78 among smear-negative specimens. The specificity ranged between 0.60 and 0.88 among smear-positive and 0.89 to 0.99 among smear-negative specimens. SROC analyses suggest that overall accuracy of phage-based assays is slightly higher than smear microscopy in direct head-to-head comparisons. CONCLUSION: Phage-based assays have high specificity but lower and variable sensitivity. Their performance characteristics are similar to sputum microscopy. Phage assays cannot replace conventional diagnostic tests such as microscopy and culture at this time. Further research is required to identify methods that can enhance the sensitivity of phage-based assays without compromising the high specificity
A novel pathway producing dimethylsulphide in bacteria is widespread in soil environments
The volatile compound dimethylsulphide (DMS) is important in climate regulation, the sulphur cycle and signalling to higher organisms. Microbial catabolism of the marine osmolyte dimethylsulphoniopropionate (DMSP) is thought to be the major biological process generating DMS. Here we report the discovery and characterisation of the first gene for DMSP-independent DMS production in any bacterium. This gene, mddA, encodes a methyltransferase that methylates methanethiol (MeSH) and generates DMS. MddA functions in many taxonomically diverse bacteria including sediment-dwelling pseudomonads, nitrogen-fixing bradyrhizobia and cyanobacteria, and mycobacteria, including the pathogen Mycobacterium tuberculosis. The mddA gene is present in metagenomes from varied environments, being particularly abundant in soil environments, where it is predicted to occur in up to 76% of bacteria. This novel pathway may significantly contribute to global DMS emissions, especially in terrestrial environments, and could represent a shift from the notion that DMSP is the only significant precursor of DMS
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