Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis </it>DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product. It shares a common transesterification domain with other type IA DNA topoisomerases. There is, however, no homology between the C-terminal DNA binding domains of <it>Escherichia coli </it>and <it>M. tuberculosis </it>DNA topoisomerase I proteins.</p> <p>Results</p> <p>A new protocol for expression and purification of recombinant <it>M. tuberculosis </it>DNA topoisomerase I (MtTOP) has been developed to produce enzyme of much higher specific activity than previously characterized recombinant enzyme. MtTOP was found to be less efficient than <it>E. coli </it>DNA topoisomerase I (EcTOP) in removal of remaining negative supercoils from partially relaxed DNA. DNA cleavage by MtTOP was characterized for the first time. Comparison of DNA cleavage site selectivity with EcTOP showed differences in cleavage site preferences, but the preferred sites of both enzymes have a C nucleotide in the -4 position.</p> <p>Conclusion</p> <p>Recombinant <it>M. tuberculosis </it>DNA topoisomerase I can be expressed as a soluble protein and purified in high yield from <it>E. coli </it>host with a new protocol. Analysis of DNA cleavage with <it>M. tuberculosis </it>DNA substrate showed that the preferred DNA cleavage sites have a C nucleotide in the -4 position.</p

Transformational leadership and organizational culture: the situational strength perspective

This paper provides a new leadership alignment model incorporating various concepts focusing on leadership styles, organizational leadership, and situational strength. These concepts are brought together to introduce a leadership model, which looks not only at individual leadership and the effect of that on the organization but also at an organization as a system and how leadership behaviour and culture in individual departments or sections in an organization can be explained. Based on the work of Podsakoff et al., a 23-item measure of transformational leadership questionnaire was employed to evaluate transformational leaders. 339 followers from five manufacturing companies were asked to complete the questionnaire about their leaders and it was analysed to identify transformational leaders. The 76 manufacturing leaders then completed the organizational culture assessment instrument and a situational strength questionnaire, which was used to study the hypothesi

The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli: application for identification of potential FRET-dye labeling sites

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis </it>topoisomerase I (MtTOP1) and <it>Escherichia coli </it>topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex.</p> <p>Results</p> <p>We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive <it>topA </it>function of <it>E. coli </it>strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in <it>E. coli</it>. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS) assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate.</p> <p>Conclusions</p> <p>The DNA relaxation and cleavage complex accumulation of <it>M. tuberculosis </it>topoisomerase I can be measured with genetic assays in <it>E. coli</it>, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.</p

Inhaled Nitric Oxide in preterm infants: a systematic review and individual patient data meta-analysis

BACKGROUND: Preterm infants requiring assisted ventilation are at significant risk of both pulmonary and cerebral injury. Inhaled Nitric Oxide, an effective therapy for pulmonary hypertension and hypoxic respiratory failure in the full term infant, has also been studied in preterm infants. The most recent Cochrane review of preterm infants includes 11 studies and 3,370 participants. The results show a statistically significant reduction in the combined outcome of death or chronic lung disease (CLD) in two studies with routine use of iNO in intubated preterm infants. However, uncertainty remains as a larger study (Kinsella 2006) showed no significant benefit for iNO for this combined outcome. Also, trials that included very ill infants do not demonstrate significant benefit. One trial of iNO treatment at a later postnatal age reported a decrease in the incidence of CLD. The aim of this individual patient meta-analysis is to confirm or refute these potentially conflicting results and to determine the extent to which patient or treatment characteristics may explain the results and/or may predict benefit from inhaled Nitric Oxide in preterm infants. METHODS/DESIGN: The Meta-Analysis of Preterm Patients on inhaled Nitric Oxide (MAPPiNO) Collaboration will perform an individual patient data meta-analysis to answer these important clinical questions. Studies will be included if preterm infants receiving assisted ventilation are randomized to receive inhaled Nitric Oxide or to a control group. The individual patient data provided by the Collaborators will be analyzed on an intention-to-treat basis where possible. Binary outcomes will be analyzed using log-binomial regression models and continuous outcomes will be analyzed using linear fixed effects models. Adjustments for trial differences will be made by including the trial variable in the model specification. DISCUSSION: Thirteen (13) trials, with a total of 3567 infants are eligible for inclusion in the MAPPiNO systematic review. To date 11 trials (n = 3298, 92% of available patients) have agreed to participate. Funding was successfully granted from Ikaria Inc as an unrestricted grant. A collaborative group was formed in 2006 with data collection commencing in 2007. It is anticipated that data analysis will commence in late 2009 with results being publicly available in 2010

Developing Countries in the WTO Services Negotiations

The aim of this paper is to analyse developing countries' participation so far in the current round of services negotiations under the Doha Development Agenda. The paper analyses developing countries' negotiating positions, as evidenced by their multilateral negotiating proposals; their initial offers; and, to the extent allowed by the incomplete and sketchy information available, their participation in bilateral market access negotiations. A number of basic themes are raised: the essential role of services for economic development; the high costs imposed by trade protection; the benefits of liberalization; the need to make use of the WTO forum to enhance credibility and sustain domestic regulatory reform programmes; the challenges of regulatory reform and the importance of appropriate sequencing; and the benefits arising from seeking further market access overseas in those areas where developing countries have a comparative advantage

The Beaker phenomenon and the genomic transformation of northwest Europe

From around 2750 to 2500 bc, Bell Beaker pottery became widespread across western and central Europe, before it disappeared between 2200 and 1800 bc. The forces that propelled its expansion are a matter of long-standing debate, and there is support for both cultural diffusion and migration having a role in this process. Here we present genome-wide data from 400 Neolithic, Copper Age and Bronze Age Europeans, including 226 individuals associated with Beaker-complex artefacts. We detected limited genetic affinity between Beaker-complex-associated individuals from Iberia and central Europe, and thus exclude migration as an important mechanism of spread between these two regions. However, migration had a key role in the further dissemination of the Beaker complex. We document this phenomenon most clearly in Britain, where the spread of the Beaker complex introduced high levels of steppe-related ancestry and was associated with the replacement of approximately 90% of Britain’s gene pool within a few hundred years, continuing the east-to-west expansion that had brought steppe-related ancestry into central and northern Europe over the previous centuries

COVID-19 trajectories among 57 million adults in England: a cohort study using electronic health records

BACKGROUND: Updatable estimates of COVID-19 onset, progression, and trajectories underpin pandemic mitigation efforts. To identify and characterise disease trajectories, we aimed to define and validate ten COVID-19 phenotypes from nationwide linked electronic health records (EHR) using an extensible framework. METHODS: In this cohort study, we used eight linked National Health Service (NHS) datasets for people in England alive on Jan 23, 2020. Data on COVID-19 testing, vaccination, primary and secondary care records, and death registrations were collected until Nov 30, 2021. We defined ten COVID-19 phenotypes reflecting clinically relevant stages of disease severity and encompassing five categories: positive SARS-CoV-2 test, primary care diagnosis, hospital admission, ventilation modality (four phenotypes), and death (three phenotypes). We constructed patient trajectories illustrating transition frequency and duration between phenotypes. Analyses were stratified by pandemic waves and vaccination status. FINDINGS: Among 57 032 174 individuals included in the cohort, 13 990 423 COVID-19 events were identified in 7 244 925 individuals, equating to an infection rate of 12·7% during the study period. Of 7 244 925 individuals, 460 737 (6·4%) were admitted to hospital and 158 020 (2·2%) died. Of 460 737 individuals who were admitted to hospital, 48 847 (10·6%) were admitted to the intensive care unit (ICU), 69 090 (15·0%) received non-invasive ventilation, and 25 928 (5·6%) received invasive ventilation. Among 384 135 patients who were admitted to hospital but did not require ventilation, mortality was higher in wave 1 (23 485 [30·4%] of 77 202 patients) than wave 2 (44 220 [23·1%] of 191 528 patients), but remained unchanged for patients admitted to the ICU. Mortality was highest among patients who received ventilatory support outside of the ICU in wave 1 (2569 [50·7%] of 5063 patients). 15 486 (9·8%) of 158 020 COVID-19-related deaths occurred within 28 days of the first COVID-19 event without a COVID-19 diagnoses on the death certificate. 10 884 (6·9%) of 158 020 deaths were identified exclusively from mortality data with no previous COVID-19 phenotype recorded. We observed longer patient trajectories in wave 2 than wave 1. INTERPRETATION: Our analyses illustrate the wide spectrum of disease trajectories as shown by differences in incidence, survival, and clinical pathways. We have provided a modular analytical framework that can be used to monitor the impact of the pandemic and generate evidence of clinical and policy relevance using multiple EHR sources. FUNDING: British Heart Foundation Data Science Centre, led by Health Data Research UK

Deciphering the Chemical Basis of Nestmate Recognition

Social insects maintain colony cohesion by recognizing and, if necessary, discriminating against conspecifics that are not part of the colony. This recognition ability is encoded by a complex mixture of cuticular hydrocarbons (CHCs), although it is largely unclear how social insects interpret such a multifaceted signal. CHC profiles often contain several series of homologous hydrocarbons, possessing the same methyl branch position but differing in chain length (e.g., 15-methyl-pentatriacontane, 15-methyl-heptatriacontane, 15-methyl-nonatriacontane). Recent studies have revealed that within species these homologs can occur in correlated concentrations. In such cases, single compounds may convey the same information as the homologs. In this study, we used behavioral bioassays to explore how social insects perceive and interpret different hydrocarbons. We tested the aggressive response of Argentine ants, Linepithema humile, toward nest-mate CHC profiles that were augmented with one of eight synthetic hydrocarbons that differed in branch position, chain length, or both. We found that Argentine ants showed similar levels of aggression toward nest-mate CHC profiles augmented with compounds that had the same branch position but differed in chain length. Conversely, Argentine ants displayed different levels of aggression toward nest-mate CHC profiles augmented with compounds that had different branch positions but the same chain length. While this was true in almost all cases, one CHC we tested elicited a greater aggressive response than its homologs. Interestingly, this was the only compound that did not occur naturally in correlated concentrations with its homologs in CHC profiles. Combined, these data suggest that CHCs of a homologous series elicit the same aggressive response because they convey the same information, rather than Argentine ants being unable to discriminate between different homologs. This study contributes to our understanding of the chemical basis of nestmate recognition by showing that, similar to spoken language, the chemical language of social insects contains “synonyms,” chemicals that differ in structure, but not meaning