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b Multidrug-resistant Mycobacterium tuberculosis strains are widespread and present a challenge to effective treatment of this infection. The need for a low-cost and rapid detection method for clinically relevant mutations in Mycobacterium tuberculosis that confer multidrug resistance is urgent, particularly for developing countries. We report here a novel test that detects the majority of clinically relevant mutations in the beta subunit of the RNA polymerase (rpoB) gene that confer resistance to rifampin (RIF), the treatment of choice for tuberculosis (TB). The test, termed TB ID/R, combines a novel target and temperature-dependent RNase H2-mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a rpoB gene target sequence. Amplified products are detected by probes arrayed on a modified silicon chip that permits visible detection of both RIF-sensitive and RIF-resistant strains of M. tuberculosis. DNA templates of clinically relevant single-nucleotide mutations in the rpoB gene were created to validate the performance of the TB ID/R test. Except for one rare mutation, all mutations were unambiguously detected. Additionally, 11 RIF-sensitive and 25 RIF-resistant clinical isolates were tested by the TB ID/R test, and 35/36 samples were classified correctly (96.2%). This test is being configured in a low-cost test platform to provide rapid diagnosis and drug susceptibility information for TB in the point-of-care setting in the developing world, where the need is acute.

Background Plant chloroplasts and mitochondria utilize nuclear encoded proteins to replicate their DNA. These proteins are purposely built for replication in the organelle environment and are distinct from those involved in replication of the nuclear genome. These organelle-localized proteins have ancestral roots in bacterial and bacteriophage genes, supporting the endosymbiotic theory of their origin. We examined the interactions between three of these proteins from Arabidopsis thaliana : a DNA helicase-primase similar to bacteriophage T7 gp4 protein and animal mitochondrial Twinkle, and two DNA polymerases, Pol1A and Pol1B. We used a three-pronged approach to analyze the interactions, including Yeast-two-hybrid analysis, Direct Coupling Analysis (DCA), and thermophoresis. Results Yeast-two-hybrid analysis reveals residues 120–295 of Twinkle as the minimal region that can still interact with Pol1A or Pol1B. This region is a part of the primase domain of the protein and slightly overlaps the zinc-finger and RNA polymerase subdomains located within. Additionally, we observed that Arabidopsis Twinkle interacts much more strongly with Pol1A versus Pol1B. Thermophoresis also confirms that the primase domain of Twinkle has higher binding affinity than any other region of the protein. Direct-Coupling-Analysis identified specific residues in Twinkle and the DNA polymerases critical to positive interaction between the two proteins. Conclusions The interaction of Twinkle with Pol1A or Pol1B mimics the minimal DNA replisomes of T7 phage and those present in mammalian mitochondria. However, while T7 and mammals absolutely require their homolog of Twinkle DNA helicase-primase, Arabidopsis Twinkle mutants are seemingly unaffected by this loss. This implies that while Arabidopsis mitochondria mimic minimal replisomes from T7 and mammalian mitochondria, there is an extra level of redundancy specific to loss of Twinkle function. Electronic supplementary material The online version of this article (10.1186/s12870-019-1854-3) contains supplementary material, which is available to authorized users.

The plasma protein binding of phenytoin was investigated in 100 epileptic patients, using equilibrium dialysis at 37°C. The unbound fractions of phenytoin in plasma formed a skewed distribution, with a range of 9.7 to 24.7% and a median value of 12.3%. Most (80%) patients appeared to form one group with free phenytoin fractions from 9.7 to 14.5%, while the remainder formed a group with elevated free fractions (> 14.5%). The total and unbound plasma concentrations of phenytoin were strongly correlated (r = 0.95, P < 0.0001). There was a weak correlation between increasing age and the unbound phenytoin fraction (r = 0.28, P < 0.01). The results indicate that measurement of the total phenytoin concentration in plasma should usually provide a reliable index of anticonvulsant effect.

1. Single doses of 1,2,4-trimethylbenzene (124TMB) or 14C-124TMB were administered orally to rats for metabolism and distribution studies. 2. 14C-124TMB was rapidly and widely distributed throughout the body with the highest levels in adipose tissue. No other preferential uptake of 14C-124TMB by any of the organs or tissues examined was evident. 3. Tissue levels declined rapidly within 24 h after dosage, with more than 99% of the administered radioactivity recovered in the urine during this period. 4. A complex mixture of isomeric trimethylphenols, dimethylbenzyl alcohols, dimethylbenzoic acids and dimethylhippuric acids excreted in the urine accounted for more than 81% of the administered dose. The major metabolites were 3,4-dimethylhippuric acid (30.2% dose), 2,4-dimethylbenzyl alcohol (12.7% dose, primarily as sulphate and glucuronide conjugates) and 2,5-dimethylbenzyl alcohol (11.7% dose, primarily as sulphate and glucuronide conjugates).