Hydrolysis of diadenosine polyphosphates. Exploration of an additional role of Mycobacterium smegmatis MutT1.

Diadenosine polyphosphates (ApnA, n=2-6), particularly Ap4A, are involved in several important physiological processes. The substantial sequence identity of the Nudix hydrolase domain (domain 1) of Mycobacterium smegmatis MutT1 (MsMutT1) with a known Ap4A hydrolase suggested that MsMutT1 could also hydrolyse diadenosine polyphosphates. Biochemical experiments yielded results in conformity with this suggestion, with Ap4A as the best among the substrates. ATP is a product in all experiments; small amounts of ADP were also observed in the experiments involving Ap4A and Ap6A. Hydrolysis was inhibited by fluoride ions in all cases. The mechanism of action and its inhibition in relation to ApnA were explored through the X-ray analysis of the crystals of the MsMutT1 complexes with Ap5A; Ap5A and MnCl2; Ap4A; ATP; and ATP.NaF.MgCl2. The aggregation pattern of molecules in the first four crystals is similar to that found in a majority of MsMutT1-NTP crystals. Substrate molecules occupy the primary binding site and ATP occupies a site at an intermolecular interface, in the first two. ATP occupies both the sites in the third and fourth crystal. The protein-ligand interactions observed in these crystal structures lead to an explanation of the molecular mechanism of hydrolysis of ApnA by MsMutT1. The fifth crystal exhibits a new packing arrangement. The structure of the complex provides an explanation for the fluoride inhibition of the activity of the enzyme. It would thus appear that MutT1 has a major role involving the hydrolysis of diadenosine polyphosphates, which could be elucidated at the molecular level.

Deep learning for single-molecule science.

Exploring and making predictions based on single-molecule data can be challenging, not only due to the sheer size of the datasets, but also because a priori knowledge about the signal characteristics is typically limited and poor signal-to-noise ratio. For example, hypothesis-driven data exploration, informed by an expectation of the signal characteristics, can lead to interpretation bias or loss of information. Equally, even when the different data categories are known, e.g., the four bases in DNA sequencing, it is often difficult to know how to make best use of the available information content. The latest developments in machine learning (ML), so-called deep learning (DL) offer interesting, new avenues to address such challenges. In some applications, such as speech and image recognition, DL has been able to outperform conventional ML strategies and even human performance. However, to date DL has not been applied much in single-molecule science, presumably in part because relatively little is known about the 'internal workings' of such DL tools within single-molecule science as a field. In this Tutorial, we make an attempt to illustrate in a step-by-step guide how one of those, a convolutional neural network (CNN), may be used for base calling in DNA sequencing applications. We compare it with a SVM as a more conventional ML method, and discuss some of the strengths and weaknesses of the approach. In particular, a 'deep' neural network has many features of a 'black box', which has important implications on how we look at and interpret data.

Structural plasticity in Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) and its functional implications.

17 independent crystal structures of family I uracil-DNA glycosylase from Mycobacterium tuberculosis (MtUng) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 5' position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.

An open randomized controlled trial to compare the efficacy of two fixed dose combinations of artemesinin based combinations for uncomplicated falciparum malaria in Bangladesh.

National Malaria Control Program (NMCP) of Bangladesh has introduced Artemisinin Based Combination (ACT), Coartem(R) (Artemether-Lumefantrine (AL), fixed dose combination, in the confirmed cases of uncomplicated P. falciparum malaria since 2004. Despite the reduction of mortality due to malaria, the development and spread of anti-malarial drug resistance wordwide posing a threat to the health services and will make it difficult to control malaria in Bangladesh in future. We need to have an alternative to Coartem which could be Artesunate-amodiaquine (AA) in a fixed dose combination (FDC), a cheaper altenative not yet evidenced to be effective and safe to our population. In this study we compared the efficacy and safety of Artemether + Lumefantrene (FDC, Coartem) with Artesunate +Amodiaquine tablets (100/270 mg FDC) for the treatment of uncomplicated P. falciparum malaria in three high risk multi-drug resistant malaria prevalent areas of Bangladesh. It was an open label randomized controlled trial conducted between December 2008 and November 2009 in 4 upazillas in patients over the age 12 to 60 years diagnosed as a case of uncomplicated P. falciparum malaria. The outcome of the cases were measured as clinical response, parasitological response, defervescence time and parasite clearance time. Drug safety was assessed by comparing the adverse events. A total of 252 cases were randomized to receive Artesunate + Amodiaquine (AA group, 147 cases) and Artemether + Lumefantrene (AL group, 106 cases), one lost to follow up at day 28 in AA group. The distribution of the cases was comparable by age, sex and study sites. Treatment success' response was observed 100% in the AL group and AA group had 99%, two failures with AA were late treatment failures and the difference was not statistically significant (p > .1). The parasitological sensitive (S) response was observed in 97% of cases in AL group and 95% in the AA group, and was not a statistically significant difference. There was no significant difference in deffervescence time and parasite clearance time between two groups of cases. No serious adverse events were observed. The frequencies of minor adverse events were insignificantly different between the two treatment groups. The two ACT regimen, AA and AL had no significant difference in efficacy and safety for treatment of Uncomplicated Malaria in Bangladesh. However, there were few more failures with AA regimen compared to AL regimen, which was not statistically significant. Both these regimens can be used alternatively by the NMCP of Bangladesh as first-line treatment option.

Crystallization and preliminary X-ray studies of MutT1 (MSMEG_2390) from Mycobacterium smegmatis.

MutT1 (MSMEG_2390) from Mycobacterium smegmatis has been crystallized and the crystals have been characterized using X-ray diffraction. The crystals belonged to space group P2(1)2(1)2(1). The Matthews coefficient suggested the possibility of one protein molecule in the asymmetric unit of the orthorhombic unit cell. Solution of the structure using the known three-dimensional structure of a bacterial MutT1 is anticipated.

Re-emergence Nipah - a review.

There was an outbreak of new emergence viral encephalitis caused by Nipah virus among humans in some areas of Bangladesh during 2001 - till to date. The disease affected mainly the young, had increased suspicion to spread from bat to man through eating of the same fruits. The risk of human-to-human transmission is thought to be low though many of the affected individuals belonged to the same family. The disease presented mainly as acute encephalitis with usually a short incubation period of less than two weeks, with the main symptoms of fever, headache, and giddiness followed by coma. Distinctive clinical signs include areflexia, hypotonia, hypertension, tachycardia and segmental myoclonus. Serology was helpful in confirming the diagnosis. Magnetic resonance imaging (MRI) showed distinctive changes of multiple, discrete or confluent small high signal lesions, best seen with fluid-attenuated inversion recovery (FLAIR) sequences. Mortality was as high as 32-92% and death was probably due to severe brainstem involvement. Relapse encephalitis was seen in those who recovered from acute encephalitis, and late-onset encephalitis was seen in those with initial non-encephalitic or asymptomatic diseases. Both these manifested as focal encephalitis arising from recurrent infection.

Plasticity, ligand conformation and enzyme action of Mycobacterium smegmatis MutT1.

Mycobacterium smegmatis MutT1 (MsMutT1) is a sanitation enzyme made up of an N-terminal Nudix hydrolase domain and a C-terminal domain resembling a histidine phosphatase. It has been established that the action of MutT1 on 8-oxo-dGTP, 8-oxo-GTP and diadenosine polyphosphates is modulated by intermolecular interactions. In order to further explore this and to elucidate the structural basis of its differential action on 8-oxo-NTPs and unsubstituted NTPs, the crystal structures of complexes of MsMutT1 with 8-oxo-dGTP, GMPPNP and GMPPCP have been determined. Replacement soaking was used in order to ensure that the complexes were isomorphous to one another. Analysis of the structural data led to the elucidation of a relationship between the arrangements of molecules observed in the crystals, molecular plasticity and the action of the enzyme on nucleotides. The dominant mode of arrangement involving a head-to-tail sequence predominantly leads to the generation of NDPs. The other mode of packing arrangement appears to preferentially generate NMPs. This work also provides interesting insights into the dependence of enzyme action on the conformation of the ligand. The possibility of modulating the enzyme action through differences in intermolecular interactions and ligand conformations makes MsMutT1 a versatile enzyme.

Central nervous system tuberculosis and adjuvant corticosteroid therapy.

Tuberculous involvement of central nervous system is one of the important health issues causing high mortality and morbidity. Uncertainty and doubt dominate all aspects of CNS tuberculosis. Diagnosis is mainly based on clinical features, cerebrospinal fluid changes, and imaging characteristics. Few studies have shown that corticosteroids improve the clinical outcome, although the precise mechanism of action remains tentative. All the cases were selected on strong clinical suspicion of CNS tuberculosis. They were graded according to tuberculous meningitis (TM) severity grades. In this connection, we studied 13 patients in one medicine unit over 12 month's period to see the effect of corticosteroid as part of the outcome. Nine patients (69.23%) were in grade II, three (23.08%) patients were in grade III, and one (7.69%) was in grade I. Seven patients (53.85%) had tuberculous meningitis and six (46.15%) had tuberculoma (CT or MRI). Out of 13 cases 3 patients (23%) died in the hospital and 10 patients (77%) improved, of whom 2 patients (20%) recovered completely and 8 patients (80%) had residual neurological deficit. Our study suggests that the early detection of CNS tuberculosis is the most important prognostic factor. Timely started anti-Koch's treatment with adjuvant corticosteroid therapy has a direct bearing on patient outcome.

Fully automatic cervical vertebrae segmentation framework for X-ray images.

The cervical spine is a highly flexible anatomy and therefore vulnerable to injuries. Unfortunately, a large number of injuries in lateral cervical X-ray images remain undiagnosed due to human errors. Computer-aided injury detection has the potential to reduce the risk of misdiagnosis. Towards building an automatic injury detection system, in this paper, we propose a deep learning-based fully automatic framework for segmentation of cervical vertebrae in X-ray images. The framework first localizes the spinal region in the image using a deep fully convolutional neural network. Then vertebra centers are localized using a novel deep probabilistic spatial regression network. Finally, a novel shape-aware deep segmentation network is used to segment the vertebrae in the image. The framework can take an X-ray image and produce a vertebrae segmentation result without any manual intervention. Each block of the fully automatic framework has been trained on a set of 124 X-ray images and tested on another 172 images, all collected from real-life hospital emergency rooms. A Dice similarity coefficient of 0.84 and a shape error of 1.69 mm have been achieved.

Biochemical and structural studies of Mycobacterium smegmatis MutT1, a sanitization enzyme with unusual modes of association.

Mycobacterium smegmatis MutT1, which is made up of a Nudix domain (domain 1) and a histidine phosphatase domain (domain 2), efficiently hydrolyses 8-oxo-GTP and 8-oxo-dGTP to the corresponding nucleoside diphosphates and phosphate in the presence of magnesium ions. Domain 1 alone hydrolyses nucleoside triphosphates less efficiently. Under high concentrations and over long periods, the full-length enzyme as well as domain 1 catalyses the hydrolysis of the nucleoside triphosphates to the respective nucleoside monophosphates and pyrophosphate. The role of domain 2 appears to be limited to speeding up the reaction. Crystal structures of the apoenzyme and those of ligand-bound enzyme prepared in the presence of 8-oxo-GTP or 8-oxo-dGTP and different concentrations of magnesium were determined. In all of the structures except one, the molecules arrange themselves in a head-to-tail fashion in which domain 1 is brought into contact with domain 2 (trans domain 2) of a neighbouring molecule. The binding site for NTP (site A) is almost exclusively made up of residues from domain 1, while those for NDP (site B) and NMP (site C) are at the interface between domain 1 and trans domain 2 in an unusual instance of intermolecular interactions leading to binding sites. Protein-ligand interactions at site A lead to a proposal for the mechanism of hydrolysis of NTP to NDP and phosphate. A small modification in site A in the crystal which does not exhibit the head-to-tail arrangement appears to facilitate the production of NMP and pyrophosphate from NTP. The two arrangements could be in dynamic equilibrium in the cellular milieu.

Stroke in patients having inadequate or irregular antihypertensive therapy.

One hundred consecutive hypertensive patients with stroke admitted to the medicine units of Dhaka Medical College Hospital were studied. The main objective was to study the extent of drug compliance and control of blood pressure in hypertensive patients who had suffered a stroke. Detailed history and clinical examinations were performed in all patients and outcome was recorded. Of the 100 patients studied there were 73(73%) males, 27 (27%) patients had no formal schooling and 53 (53%) had some education. 48 (48%) patients were from middle class and 36 (36%) were poor, 66 (66%) patients were aware that they were hypertensive though only 8 (12.9%) were taking anti-hypertensive drugs regularly. 62% of the patients had suffered from stroke within 5 years of detection of hypertension, and 15 (15%) patients died in the hospital.

Crystallization and preliminary X-ray characterization of MutT2, MSMEG_5148 from Mycobacterium smegmatis.

Crystallization of MutT2, MSMEG_5148 from Mycobacterium smegmatis, has been carried out and the crystals have been characterized using X-ray diffraction. Matthews coefficient calculation suggests the possibility of one protein molecule in the asymmetric unit of the orthorhombic unit cell, space group P2(1)2(1)2 or P2(1)22. Solution of the structure of the protein by molecular replacement using the known three-dimensional structure of a bacterial Nudix hydrolase is envisaged.

Phase II study of magnesium sulfate in acute organophosphate pesticide poisoning.

BACKGROUND: Acute organophosphorus (OP) poisoning is relatively common and a major cause of death from poisoning in developing countries. Magnesium has been shown to be of benefit in animal models. METHODS: We conducted a phase II study of bolus doses of (MgSO4) in 50 patients with acute organophosphate poisoning. Patients eligible for inclusion had ingested OP and had cholinergic symptoms consistent with moderate or severe poisoning. All patients received standard care of atropinization titrated to control muscarinic symptoms and pralidoxime. The trial was run in 4 sequential groups of patients. Participants in each group received a different total dose of MgSO4 (20%) administered as intermittent bolus doses infused over 10-15 min or placebo. There was one control patient for every 4 patients who received MgSO4. Group A (16 patients) received a total of 4 gm MgSO4 as a single bolus, group B (8 patients) received 8 gm (in two 4 gm doses q4H), group C (8 patients) received 12 gm (in three 4 gm doses q4H) group D (8 patients) received 16 gm (in four 4 gm doses q4H) and control (10 patients) received placebo). Patients were closely monitored for any adverse reaction like significant clinical neuromuscular disturbance and respiratory depression. RESULTS: No adverse reactions to magnesium were observed. The 24 hour urinary magnesium concentration were statistically different between 16 gm (234.74 ± 74.18 mg/dl) and control (118.06 ± 30.76 mg/dl) (p = 0.019), while it was much lower than the 80% of the intravenous magnesium load. Six patients died in control group compared to 3 in 4 gm, 2 in 8 gm and 1 in 12 gm group. There was no mortality in 16 gm group. CONCLUSION: Magnesium was well tolerated in this study. Larger studies are required to examine for efficacy.

Structural diversity based on variability in quaternary association. A case study involving eubacterial and related SSBs.

Eubacterial and related single-stranded DNA-binding proteins (SSBs) exhibit considerable variability in their quaternary association in spite of their having the same tertiary fold. The variability involves differences in the orientation of dimers in the tetrameric molecule (or of two-domain subunits in the dimeric molecule) and that of monomers in each dimer. The presence of an additional strand in mycobacterial and related SSBs, which clamps the dimers together, is a major determinant of the mode of quaternary association in them. The variability in quaternary structure has implications to the stability of the protein and possibly to its mode of DNA binding.