Accessing Bioactive Natural Products from Cultured and Uncultured Microorganisms.

Microbial natural products are an important source of novel medicines in an ongoing war against drug-resistant infections. Their unique chemical structures are capable of affecting new molecular targets that can help slow the ability of bacteria, viruses, fungi, and cancers to develop resistance mechanisms. To maximize the discovery of new medicines, we need to simultaneously explore the natural products of both cultured and currently uncultured microbes. This dissertation explores methodologies to facilitate access to bioactive medicines from both of these microbial sources. To access the natural products of cultured microorganisms, we screened a library of extracts collected from microbial isolates for inhibitors of enzymatic targets involved in siderophore biosynthesis. Bioactivity-guided fractionation of the best hit led to the isolation of a novel class of antibiotics, the baulamycins. Analysis of the bioactivity of these natural products against the original enzymatic targets and several pathogenic microorganisms helped to elucidate their potency, broad-spectrum activity, and mode of inhibition. These new antibiotics from a cultured microbe serve as an important drug lead in the war against antibiotic resistance. To access the natural products of uncultured microorganisms, we selected the clinically approved chemotherapeutic ET-743 as a model system. Researchers have long suspected that the medicine is produced by an uncultured microbial symbiont of a mangrove tunicate. We first sought to understand the biology of the uncultured microbe. We used metagenomic techniques to uncover its complete genome. Detailed analysis of the genome, its primary, and secondary metabolism provided a thorough look at its endosymbiotic lifestyle and the biosynthesis of ET-743. In the last part of my dissertation, we utilized the newfound knowledge of the symbiont’s biology to take the first steps toward more efficient access of the drug. We biochemically analyzed a key enzyme involved in the production of the core of the anti-cancer medicine. This analysis supports our predicted role for the enzyme in ET-743 biosynthesis. It also supports the feasibility of reconstituting ET-743 and more potent or selective analogues in vitro. These collective methodologies can be applied to other systems, expanding our ability to harness the bioactive natural products of cultured and uncultured microorganisms.PHDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111340/1/schofiem_1.pd

Identification and analysis of the bacterial endosymbiont specialized for production of the chemotherapeutic natural product ET‐743

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/115956/1/emi12908.pd

Phenotype ontologies for mouse and man: bridging the semantic gap

A major challenge of the post-genomic era is coding phenotype data from humans and model organisms such as the mouse, to permit the meaningful translation of phenotype descriptions between species. This ability is essential if we are to facilitate phenotype-driven gene function discovery and empower comparative pathobiology. Here, we review the current state of the art for phenotype and disease description in mice and humans, and discuss ways in which the semantic gap between coding systems might be bridged to facilitate the discovery and exploitation of new mouse models of human diseases

The spectrum of BPS branes on a noncompact Calabi-Yau

We begin the study of the spectrum of BPS branes and its variation on lines of marginal stability on O_P^2(-3), a Calabi-Yau ALE space asymptotic to C^3/Z_3. We show how to get the complete spectrum near the large volume limit and near the orbifold point, and find a striking similarity between the descriptions of holomorphic bundles and BPS branes in these two limits. We use these results to develop a general picture of the spectrum. We also suggest a generalization of some of the ideas to the quintic Calabi-Yau.Comment: harvmac, 45 pp. (v2: added references

Surface tension of the isotropic-nematic interface

We present the first calculations of the pressure tensor profile in the vicinity of the planar interface between isotropic liquid and nematic liquid crystal, using Onsager's density functional theory and computer simulation. When the liquid crystal director is aligned parallel to the interface, the situation of lowest free energy, there is a large tension on the nematic side of the interface and a small compressive region on the isotropic side. By contrast, for perpendicular alignment, the tension is on the isotropic side. There is excellent agreement between theory and simulation both in the forms of the pressure tensor profiles, and the values of the surface tension.Comment: Minor changes; to appear in Phys. Rev.

Strategies for Understanding and Reducing the Plasmodium vivax and Plasmodium ovale Hypnozoite Reservoir in Papua New Guinean Children: A Randomised Placebo-Controlled Trial and Mathematical Model

The undetectable hypnozoite reservoir for relapsing Plasmodium vivax and P. ovale malarias presents a major challenge for malaria control and elimination in endemic countries. This study aims to directly determine the contribution of relapses to the burden of P. vivax and P. ovale infection, illness, and transmission in Papua New Guinean children.; From 17 August 2009 to 20 May 2010, 524 children aged 5-10 y from East Sepik Province in Papua New Guinea (PNG) participated in a randomised double-blind placebo-controlled trial of blood- plus liver-stage drugs (chloroquine [CQ], 3 d; artemether-lumefantrine [AL], 3 d; and primaquine [PQ], 20 d, 10 mg/kg total dose) (261 children) or blood-stage drugs only (CQ, 3 d; AL, 3 d; and placebo [PL], 20 d) (263 children). Participants, study staff, and investigators were blinded to the treatment allocation. Twenty children were excluded during the treatment phase (PQ arm: 14, PL arm: 6), and 504 were followed actively for 9 mo. During the follow-up time, 18 children (PQ arm: 7, PL arm: 11) were lost to follow-up. Main primary and secondary outcome measures were time to first P. vivax infection (by qPCR), time to first clinical episode, force of infection, gametocyte positivity, and time to first P. ovale infection (by PCR). A basic stochastic transmission model was developed to estimate the potential effect of mass drug administration (MDA) for the prevention of recurrent P. vivax infections. Targeting hypnozoites through PQ treatment reduced the risk of having at least one qPCR-detectable P. vivax or P. ovale infection during 8 mo of follow-up (P. vivax: PQ arm 0.63/y versus PL arm 2.62/y, HR = 0.18 [95% CI 0.14, 0.25], p < 0.001; P. ovale: 0.06 versus 0.14, HR = 0.31 [95% CI 0.13, 0.77], p = 0.011) and the risk of having at least one clinical P. vivax episode (HR = 0.25 [95% CI 0.11, 0.61], p = 0.002). PQ also reduced the molecular force of P. vivax blood-stage infection in the first 3 mo of follow-up (PQ arm 1.90/y versus PL arm 7.75/y, incidence rate ratio [IRR] = 0.21 [95% CI 0.15, 0.28], p < 0.001). Children who received PQ were less likely to carry P. vivax gametocytes (IRR = 0.27 [95% CI 0.19, 0.38], p < 0.001). PQ had a comparable effect irrespective of the presence of P. vivax blood-stage infection at the time of treatment (p = 0.14). Modelling revealed that mass screening and treatment with highly sensitive quantitative real-time PCR, or MDA with blood-stage treatment alone, would have only a transient effect on P. vivax transmission levels, while MDA that includes liver-stage treatment is predicted to be a highly effective strategy for P. vivax elimination. The inclusion of a directly observed 20-d treatment regime maximises the efficiency of hypnozoite clearance but limits the generalisability of results to real-world MDA programmes.; These results suggest that relapses cause approximately four of every five P. vivax infections and at least three of every five P. ovale infections in PNG children and are important in sustaining transmission. MDA campaigns combining blood- and liver-stage treatment are predicted to be a highly efficacious intervention for reducing P. vivax and P. ovale transmission.; ClinicalTrials.gov NCT02143934

Jet vetoing and Herwig++

We investigate the simulation of events with gaps between jets with a veto on additional radiation in the gap in Herwig++. We discover that the currently-used random treatment of radiation in the parton shower is generating some unphysical behaviour for wide-angle gluon emission in QCD 2 to 2 scatterings. We explore this behaviour quantitatively by making the same assumptions as the parton shower in the analytical calculation. We then modify the parton shower algorithm in order to correct the simulation of QCD radiation.Comment: 18 pages, 11 figure

Molecular Dynamics Study of the Nematic-Isotropic Interface

We present large-scale molecular dynamics simulations of a nematic-isotropic interface in a system of repulsive ellipsoidal molecules, focusing in particular on the capillary wave fluctuations of the interfacial position. The interface anchors the nematic phase in a planar way, i.e., the director aligns parallel to the interface. Capillary waves in the direction parallel and perpendicular to the director are considered separately. We find that the spectrum is anisotropic, the amplitudes of capillary waves being larger in the direction perpendicular to the director. In the long wavelength limit, however, the spectrum becomes isotropic and compares well with the predictions of a simple capillary wave theory.Comment: to appear in Phys. Rev.

Comparison of verona integron-borne metallo-beta-lactamase (VIM) variants reveals differences in stability and inhibition profiles

DUZGUN, AZER OZAD/0000-0002-6301-611X; Abboud, Martine I./0000-0003-2141-5988; Brem, Jurgen/0000-0002-0137-3226; McDonough, Michael A/0000-0003-4664-6942; Rydzik, Anna/0000-0003-3158-0493; DUZGUN, AZER OZAD/0000-0002-6301-611X; McDonough, Michael/0000-0003-4664-6942; Schofield, Christopher/0000-0002-0290-6565; SANDALLI, Cemal/0000-0002-1298-3687WOS: 000376490800025PubMed: 26666919Metallo-beta-lactamases (MBLs) are of increasing clinical significance; the development of clinically useful MBL inhibitors is challenged by the rapid evolution of variant MBLs. the Verona integron-borne metallo-beta-lactamase (VIM) enzymes are among the most widely distributed MBLs, with > 40 VIM variants having been reported. We report on the crystallographic analysis of VIM-5 and comparison of biochemical and biophysical properties of VIM-1, VIM-2, VIM-4, VIM-5, and VIM-38. Recombinant VIM variants were produced and purified, and their secondary structure and thermal stabilities were investigated by circular dichroism analyses. Steady-state kinetic analyses with a representative panel of beta-lactam substrates were carried out to compare the catalytic efficiencies of the VIM variants. Furthermore, a set of metalloenzyme inhibitors were screened to compare their effects on the different VIM variants. the results reveal only small variations in the kinetic parameters of the VIM variants but substantial differences in their thermal stabilities and inhibition profiles. Overall, these results support the proposal that protein stability may be a factor in MBL evolution and highlight the importance of screening MBL variants during inhibitor development programs.Rhodes Trust; Scientific and Technology Council of Turkey; Recep Tayyip Erdogan Universitesi Research FundRecep Tayyip Erdogan University [BAP-2013.102.03.13]; Medical Research CouncilMedical Research Council UK (MRC) [MR/L007665/1]; Medical Research Council/Canadian Grant [G1100135]; Biochemical Society Krebs Memorial Award; Medical Research CouncilMedical Research Council UK (MRC) [G1100135, MR/N002679/1] Funding Source: researchfishThe Rhodes Trust provided funding to Anne Makena. Scientific and Technology Council of Turkey provided funding to Cemal Sandalli. Recep Tayyip Erdogan Universitesi Research Fund provided funding to Aysegul Saral, Aysegul C. Cicek, and Cemal Sandalli under grant number BAP-2013.102.03.13. Medical Research Council provided funding to Jurgen Brem, Michael A. McDonough, Anna M. Rydzik, and Christopher J. Schofield under grant number MR/L007665/1. Medical Research Council/Canadian Grant provided funding to Jurgen Brem, Michael A. McDonough, Anna M. Rydzik, and Christopher J. Schofield under grant number G1100135. Biochemical Society Krebs Memorial Award provided funding to Martine I. Abboud

Inter-decadal variability of phytoplankton biomass along the coastal West Antarctic Peninsula

The West Antarctic Peninsula (WAP) is a climatically sensitive region where periods of strong warming have caused significant changes in the marine ecosystem and food-web processes. Tight coupling between phytoplankton and higher trophic levels implies that the coastal WAP is a bottom-up controlled system, where changes in phytoplankton dynamics may largely impact other food-web components. Here, we analysed the inter-decadal time series of year-round chlorophyll-a (Chl) collected from three stations along the coastal WAP: Carlini Station at Potter Cove (PC) on King George Island, Palmer Station on Anvers Island and Rothera Station on Adelaide Island. There were trends towards increased phytoplankton biomass at Carlini Station (PC) and Palmer Station, while phytoplankton biomass declined significantly at Rothera Station over the studied period. The impacts of two relevant climate modes to the WAP, the El Niño-Southern Oscillation and the Southern Annular Mode, on winter and spring phytoplankton biomass appear to be different among the three sampling stations, suggesting an important role of local-scale forcing than large-scale forcing on phytoplankton dynamics at each station. The inter-annual variability of seasonal bloom progression derived from considering all three stations together captured ecologically meaningful, seasonally co-occurring bloom patterns which were primarily constrained by water-column stability strength. Our findings highlight a coupled link between phytoplankton and physical and climate dynamics along the coastal WAP, which may improve our understanding of overall WAP food-web responses to climate change and variability