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

Synthesis and structure of sterically overloaded tetra-coordinated yttrium and lanthanum disiloxides

The NSF is thanked for purchase of a JEOL ECS-400 NMR Spectrometer (CRIF-MU CHE-1048553).The synthesis, structures and reactivity of the spirocyclic yttrium and lanthanum disiloxides {[(CH2R2SiO)2]2M}H [M = Ln, Y; R = SiMe(SiMe3)2] 3 and 4 are reported. Compounds 3 and 4 were prepared from reactions of two equivalents of [CH2(R)2SiOH]2 [R = Si(SiMe3)2Me] ( 1 ) with one equivalent of M[N(SiMe3)2]2 (M = Y, La), respectively.PostprintPeer reviewe

Statistical Basis for Predicting Technological Progress

Forecasting technological progress is of great interest to engineers, policy makers, and private investors. Several models have been proposed for predicting technological improvement, but how well do these models perform? An early hypothesis made by Theodore Wright in 1936 is that cost decreases as a power law of cumulative production. An alternative hypothesis is Moore's law, which can be generalized to say that technologies improve exponentially with time. Other alternatives were proposed by Goddard, Sinclair et al., and Nordhaus. These hypotheses have not previously been rigorously tested. Using a new database on the cost and production of 62 different technologies, which is the most expansive of its kind, we test the ability of six different postulated laws to predict future costs. Our approach involves hindcasting and developing a statistical model to rank the performance of the postulated laws. Wright's law produces the best forecasts, but Moore's law is not far behind. We discover a previously unobserved regularity that production tends to increase exponentially. A combination of an exponential decrease in cost and an exponential increase in production would make Moore's law and Wright's law indistinguishable, as originally pointed out by Sahal. We show for the first time that these regularities are observed in data to such a degree that the performance of these two laws is nearly tied. Our results show that technological progress is forecastable, with the square root of the logarithmic error growing linearly with the forecasting horizon at a typical rate of 2.5% per year. These results have implications for theories of technological change, and assessments of candidate technologies and policies for climate change mitigation

Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance.

BACKGROUND: Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. METHODS: To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. RESULTS: The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. CONCLUSIONS: Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance mutations to improve the design of tuberculosis control measures, such as diagnostics, and inform patient management

Case Report: Insulin hypersensitivity in youth with type 1 diabetes

OBJECTIVE: Immediate type I, type III, and delayed type IV hypersensitivity reactions to insulin are rare, but potentially serious complications of exogenous insulin administration required for the treatment of type 1 diabetes (T1D). METHODS: We present four cases of insulin hypersensitivity reactions occurring in youth with T1D and a literature review of this topic. RESULTS: Insulin hypersensitivity reactions included types I, III, and IV with presentations ranging from localized urticaria, erythematous nodules, and eczematous plaques to anaphylaxis with respiratory distress. Reactions occurred in youth with newly diagnosed T1D and in those with long-standing T1D who were using both injection and insulin pump therapy. Multidisciplinary care involving pediatric endocrinology and allergy/immunology utilizing trials of many adjunct therapies yielded minimal improvement. Despite the use of various treatments, including antihistamines, topical therapies, immunosuppressant medications, desensitization trials, and intravenous immune globulin, cutaneous reactions, elevated hemoglobin A1c levels, and negative effects on quality of life remain persistent challenges. One patient became one of the youngest pancreas transplant recipients in the world at age 12 years due to uncontrollable symptoms and intolerable adverse effects of attempted therapies. CONCLUSION: Although rare, insulin hypersensitivity reactions negatively affect glycemic control and quality of life. These cases demonstrate the varying severity and presentation of insulin hypersensitivity reactions along with the limited success of various treatment approaches. Given the life-sustaining nature of insulin therapy, further studies are needed to better understand the underlying pathophysiology of insulin hypersensitivity and to develop targeted treatment approaches

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

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 conducted over the past 25-30 years, and to perform cutting-edge research where knowledge gaps still exist. This knowledge needs to be integrated into affordable and practical tools for farmers and food processors along the chain in order 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-actor partnership (academia, farmers, technology small and medium sized enterprises, food industry and policy stakeholders) to develop novel interventions aimed at achieving a significant reduction in crop losses due to mycotoxin contamination. Besides a field-to-fork approach, MyToolBox also considers safe use options of contaminated 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 the end users along the food and feed chain in a web-based MyToolBox platform (e-toolbox). The project focuses on 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 interventions including investigation of genetic resistance to fungal infection, cultural control (e.g. minimum tillage or crop debris 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-harvest measures includes real-time monitoring during storage, innovative sorting of crops using vision-technology, novel milling technology and studying the effects of baking on mycotoxins at an industrial scale

Rapid Diagnostic Algorithms as a Screening Tool for Tuberculosis: An Assessor Blinded Cross-Sectional Study

Background: A major obstacle to effectively treat and control tuberculosis is the absence of an accurate, rapid, and low-cost diagnostic tool. A new approach for the screening of patients for tuberculosis is the use of rapid diagnostic classification algorithms. Methods: We tested a previously published diagnostic algorithm based on four biomarkers as a screening tool for tuberculosis in a Central European patient population using an assessor-blinded cross-sectional study design. In addition, we developed an improved diagnostic classification algorithm based on a study population at a tertiary hospital in Vienna, Austria, by supervised computational statistics. Results: The diagnostic accuracy of the previously published diagnostic algorithm for our patient population consisting of 206 patients was 54% (CI: 47%–61%). An improved model was constructed using inflammation parameters and clinical information. A diagnostic accuracy of 86% (CI: 80%–90%) was demonstrated by 10-fold cross validation. An alternative model relying solely on clinical parameters exhibited a diagnostic accuracy of 85% (CI: 79%–89%). Conclusion: Here we show that a rapid diagnostic algorithm based on clinical parameters is only slightly improved by inclusion of inflammation markers in our cohort. Our results also emphasize the need for validation of new diagnostic algorithms in different settings and patient populations

Investigating methods of sharing data between police, health, education, and social services: Semi-structured interviews with police service areas in Wales

The Crime and Disorder Act (1998) requires the police, local authorities, NHS, and other organisations to share intelligence and collectively work to reduce violent crime. This paper aimed to explore opinions on linking police data with other agency data. Interviews were undertaken with individuals from police forces in Wales, UK. Barriers to sharing data with other organisations involve differences in the systems used to store police data and uncertainties around what is allowed to be shared. Overcoming barriers would allow data linkage across organisations leading to deeper insights into the causes of violence, and therefore intelligence that supports crime prevention