Phenotypic Effects of Predicted SigI on Virulence in Bacillus anthracis

Alternative sigma factors play a key role in the physiology of Bacillus anthracis by regulating the transcription of the appropriate genes required for adaptation and survival. Under specific conditions, alternative sigma factors activate transcription by binding to the promoter of the genes relevant to the condition and initiate synthesis of RNA. Here we report that the transcription of predicted sigI gene in B. anthracis, BAS3231, is induced by elevated temperatures and involved in the regulation of virulence gene expression. We show that BAS3231 is required for cell viability at elevated temperatures. We have also demonstrated that mutation in the BAS3231 gene results in a decrease in virulence gene expression. Our study provides new insight into the role of alternative sigma factors in B. anthracis

The kinetics of ER fusion protein activation in vivo.

Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the hormone-binding domain of the modified oestrogen receptor (ER(TAM)) can be regulated by provision or removal of 4-hydroxytamoxifen (4-OHT). Despite the widespread use of ER(TAM) fusions in vivo, inadequate data are available as to the most efficacious routes for systemic tamoxifen delivery. In this study, we have used two well-characterized ER(TAM) fusion proteins, both reversibly activated by 4-OHT, to compare the effectiveness and kinetics of 4-OHT delivery in mice in vivo by either tamoxifen in food or by intraperitoneal injection. Our data indicate that dietary tamoxifen offers an effective, facile and ethically preferable means for long-term activation of ER(TAM) fusion proteins in vivo.This is the final published version. It's also available from the publishers at: http://www.nature.com/onc/journal/vaop/ncurrent/full/onc201478a.html

Regionally acquired intestinal failure data suggest an underestimate in national service requirements

Objectives, setting and patients: With complete case referral for prolonged parenteral nutrition (PN) beyond term equivalent, serving a stable population of 1.25 million people, we describe the long-term outcome and survival of patients referred to an intestinal failure (IF) nutrition support team over the first 8 years of existence at a regional paediatric centre, and extrapolate to potential numbers of national home parenteral nutrition (HPN) cases and intestinal transplantation data. Design and outcome measures: Retrospective analysis detailing patient demographics, interventions, use of HPN, occurrence of intestinal failure-associated liver disease (IFALD), and outcomes of enteral adaptation, survival, and referral for and receipt of organ transplantation. Results: 23 patients were referred over 8 years, 20 being PN dependent within the neonatal period. Diagnoses included short bowel syndrome (SBS) (18), neuromuscular abnormalities (4) and congenital enterocyte disorder (1). 12 696 days of PN were delivered with 314 confirmed episodes of sepsis at a median of 12 episodes per patient. 144 central venous catheters (CVCs) were required at a median of four per patient. IFALD occurred in 17 (73%) patients, with 10 (44%) referred for transplant assessment. Thirteen (56%) children received HPN. Overall mortality was 44%. A significant predictor for survival in the SBS group was residual bowel >40 cm (82% vs 28%, p = 0.049). Conclusions: Survival for IF at 56% was lower than reported from non-UK supra-regional centres, and nationally collected data, possibly reflecting pre-selected referral populations. Data from regional centres with complete ascertainment may be important both when counselling parents and when planning regional and national HPN and IF specialist services

Heterogeneity of Myc expression in breast cancer exposes pharmacological vulnerabilities revealed through executable mechanistic modeling

Cells with higher levels of Myc proliferate more rapidly and supercompetitively eliminate neighboring cells. Nonetheless, tumor cells in aggressive breast cancers typically exhibit significant and stable heterogeneity in their Myc levels, which correlates with refractoriness to therapy and poor prognosis. This suggests that Myc heterogeneity confers some selective advantage on breast tumor growth and progression. To investigate this, we created a traceable MMTV-Wnt1-driven in vivo chimeric mammary tumor model comprising an admixture of low-Myc- and reversibly switchable high-Myc-expressing clones. We show that such tumors exhibit interclonal mutualism wherein cells with high-Myc expression facilitate tumor growth by promoting protumorigenic stroma yet concomitantly suppress Wnt expression, which renders them dependent for survival on paracrine Wnt provided by low-Myc-expressing clones. To identify any therapeutic vulnerabilities arising from such interdependency, we modeled Myc/Ras/p53/Wnt signaling cross talk as an executable network for low-Myc, for high-Myc clones, and for the 2 together. This executable mechanistic model replicated the observed interdependence of high-Myc and low-Myc clones and predicted a pharmacological vulnerability to coinhibition of COX2 and MEK. This was confirmed experimentally. Our study illustrates the power of executable models in elucidating mechanisms driving tumor heterogeneity and offers an innovative strategy for identifying combination therapies tailored to the oligoclonal landscape of heterogenous tumors

Performance of the 2007 WHO Algorithm to diagnose Smear-negative Pulmonary Tuberculosis in a HIV prevalent setting

The 2007 WHO algorithm for diagnosis of smear-negative pulmonary tuberculosis (PTB) including Mycobacterium tuberculosis (MTB) culture was evaluated in a HIV prevalent area of Kenya

Myc Expression Drives Aberrant Lipid Metabolism in Lung Cancer

MYC-mediated pathogenesis in lung cancer continues to attract interest for new therapeutic strategies. In this study, we describe a transgenic mouse model of KRAS-driven lung adenocarcinoma that affords reversible activation of MYC, used here as a tool for lipidomic profiling of MYC-dependent lung tumors formed in this model. Advanced mass spectrometric imaging and surface analysis techniques were used to characterize the spatial and temporal changes in lipid composition in lung tissue. We found that normal lung tissue was characterized predominantly by saturated phosphatidylcholines and phosphatidylglycerols, which are major lipid components of pulmonary surfactant. In contrast, tumor tissues displayed an increase in phosphatidylinositols and arachidonate-containing phospholipids that can serve as signaling precursors. Deactivating MYC resulted in a rapid and dramatic decrease in arachidonic acid and its eicosanoid metabolites. In tumors with high levels of MYC, we found an increase in cytosolic phospholipase A2 (cPLA2) activity with a preferential release of membrane-bound arachidonic acid, stimulating the lipoxygenase (LOX) and COX pathways also amplified by MYC at the level of gene expression. Deactivating MYC lowered cPLA2 activity along with COX2 and 5-LOX mRNA levels. Notably, inhibiting the COX/5-LOX pathways in vivo reduced tumor burden in a manner associated with reduced cell proliferation. Taken together, our results show how MYC drives the production of specific eicosanoids critical for lung cancer cell survival and proliferation, with possible implications for the use of COX and LOX pathway inhibitors for lung cancer therapy.This research was funded by the Medical Research Council (Lipid Profiling and Signaling, MC UP A90 1006 & Lipid Dynamics and Regulation, MC PC 13030) and Cancer Research UK (program grant A12077)

Steady-state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B

Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone

Theory of disk accretion onto supermassive black holes

Accretion onto supermassive black holes produces both the dramatic phenomena associated with active galactic nuclei and the underwhelming displays seen in the Galactic Center and most other nearby galaxies. I review selected aspects of the current theoretical understanding of black hole accretion, emphasizing the role of magnetohydrodynamic turbulence and gravitational instabilities in driving the actual accretion and the importance of the efficacy of cooling in determining the structure and observational appearance of the accretion flow. Ongoing investigations into the dynamics of the plunging region, the origin of variability in the accretion process, and the evolution of warped, twisted, or eccentric disks are summarized.Comment: Mostly introductory review, to appear in "Supermassive black holes in the distant Universe", ed. A.J. Barger, Kluwer Academic Publishers, in pres

Microevolution of Helicobacter pylori during prolonged infection of single hosts and within families

Our understanding of basic evolutionary processes in bacteria is still very limited. For example, multiple recent dating estimates are based on a universal inter-species molecular clock rate, but that rate was calibrated using estimates of geological dates that are no longer accepted. We therefore estimated the short-term rates of mutation and recombination in Helicobacter pylori by sequencing an average of 39,300 bp in 78 gene fragments from 97 isolates. These isolates included 34 pairs of sequential samples, which were sampled at intervals of 0.25 to 10.2 years. They also included single isolates from 29 individuals (average age: 45 years) from 10 families. The accumulation of sequence diversity increased with time of separation in a clock-like manner in the sequential isolates. We used Approximate Bayesian Computation to estimate the rates of mutation, recombination, mean length of recombination tracts, and average diversity in those tracts. The estimates indicate that the short-term mutation rate is 1.4×10−6 (serial isolates) to 4.5×10−6 (family isolates) per nucleotide per year and that three times as many substitutions are introduced by recombination as by mutation. The long-term mutation rate over millennia is 5–17-fold lower, partly due to the removal of non-synonymous mutations due to purifying selection. Comparisons with the recent literature show that short-term mutation rates vary dramatically in different bacterial species and can span a range of several orders of magnitude