Annotation and Transcription Start Site Analysis of contig70 in Drosophila biarmipes

The following is my annotation and transcription start site analysis report for contig70 in D. biarmipes to be submitted to the Genomics Education Partnership (GEP) at Washington University in St. Louis. The report was completed in accordance with the guidelines and template set forth by the GEP. There are eight complete genes found in contig70, for which I found the protein coding exon boundaries as well as the transcription start sites for all isoforms. All conclusions and reasoning for them are outlined in the report. The GEP does genomics research, specifically comparing the genomes of all species of Drosophila. Students completing projects for the GEP analyze a portion of a genome of one of the species. The GEP collects all of this information and data and uses it for evolutionary analysis. Comparing the genomes of distinct species illuminates important regions of DNA, which are well conserved between species. Students in CMB 440: Research Applications of Drosophila Genomics are expected to complete GEP reports for their individual projects. I took CMB 440 in Winter 2014, so this is the second project and report I have done. I decided to complete another report for my Honors Senior Project to assist Dr. Martin Burg in advancing the scope of the course. Before this semester, students were not expected to complete transcription start site (TSS) analysis for their projects. To ensure a smoother integration of TSS analysis, Dr. Burg asked me to become well versed in the process so I could teach it to students. I studied GEP materials and instructions over the course of the semester to compile a comprehensive instructional presentation on TSS analysis to give to the class. Along with the presentation, I made myself available once a week in class for students to ask me questions concerning TSS analysis in their individual projects

Mycobacteria, metals, and the macrophage

Mycobacterium tuberculosis is a facultative intracellular pathogen that thrives inside host macrophages. A key trait of M. tuberculosis is to exploit and manipulate metal cation trafficking inside infected macrophages to ensure survival and replication inside the phagosome. Here, we describe the recent fascinating discoveries that the mammalian immune system responds to infections with M. tuberculosis by overloading the phagosome with copper and zinc, two metals which are essential nutrients in small quantities but are toxic in excess. M. tuberculosis has developed multi-faceted resistance mechanisms to protect itself from metal toxicity including control of uptake, sequestration inside the cell, oxidation, and efflux. The host response to infections combines this metal poisoning strategy with nutritional immunity mechanisms that deprive M. tuberculosis from metals such as iron and manganese to prevent bacterial replication. Both immune mechanisms rely on the translocation of metal transporter proteins to the phagosomal membrane during the maturation process of the phagosome. This review summarizes these recent findings and discusses how metal-targeted approaches might complement existing TB chemotherapeutic regimens with novel anti-infective therapies.Microbiology and Molecular Genetic

Late quaternary snowlines and cirque moraines within the Waimakariri watershed

A study of the glacial geology and present and past snowlines of tributaries to the Waimakariri River, South Island, New Zealand, has yielded a glacial chronology from late Pleistocene times up to the present day. Three advances of the ultimate (Otiran) Pleistocene glaciation were recognized and named the Kowai, Porter and Tims stream advances. Ice limits were established for an early Holocene (McGrath) advance and for three following Neoglacial ice resurgences, named Arthur's Pass, O'Malley and Barker advances. Moraine-headwall and area-altitude methods were used to compute past snowline evaluations from which snowline surfaces have been constructed. Compared with the present snowline, depressions of up to 970m for the Pleistocene, 480m for the McGrath advance and 400m, 300m and 200m for the Neoglacial events are indicated. Isopleth maps of the snowline surfaces show a steep upward gradient towards the east, with strong topographically-induced irregularities superimposed upon this pattern. Weathering rind studies on surface boulders of the Holocene deposits showed a systematic increase in thickness with increase in age. Radiocarbon dates with rind thicknesses from landslide deposits provided the basis for a rind thickness growth curve. This curve was used to age and correlate the glacial events. Parallelism of the different snowline surfaces suggests that climatic patterns remained similar throughout the Holocene. Early Holocene deposits suggest a cool wet period between the Tims Stream and McGrath events, while a number of rock glacier deposits indicate a cool drier climate for the beginning of the Neoglacial advances

Sur7 Promotes Plasma Membrane Organization and Is Needed for Resistance to Stressful Conditions and to the Invasive Growth and Virulence of Candida albicans

The human fungal pathogen Candida albicans causes lethal systemic infections because of its ability to grow and disseminate in a host. The C. albicans plasma membrane is essential for virulence by acting as a protective barrier and through its key roles in interfacing with the environment, secretion of virulence factors, morphogenesis, and cell wall synthesis. Difficulties in studying hydrophobic membranes have limited the understanding of how plasma membrane organization contributes to its function and to the actions of antifungal drugs. Therefore, the role of the recently discovered plasma membrane subdomains termed the membrane compartment containing Can1 (MCC) was analyzed by assessing the virulence of a sur7Δ mutant. Sur7 is an integral membrane protein component of the MCC that is needed for proper localization of actin, morphogenesis, cell wall synthesis, and responding to cell wall stress. MCC domains are stable 300-nm-sized punctate patches that associate with a complex of cytoplasmic proteins known as an eisosome. Analysis of virulence-related properties of a sur7Δ mutant revealed defects in intraphagosomal growth in macrophages that correlate with increased sensitivity to oxidation and copper. The sur7Δ mutant was also strongly defective in pathogenesis in a mouse model of systemic candidiasis. The mutant cells showed a decreased ability to initiate an infection and greatly diminished invasive growth into kidney tissues. These studies on Sur7 demonstrate that the plasma membrane MCC domains are critical for virulence and represent an important new target for the development of novel therapeutic strategies

Evidence for Oxidative Stress and Defective Antioxidant Response in Guinea Pigs with Tuberculosis

The development of granulomatous inflammation with caseous necrosis is an important but poorly understood manifestation of tuberculosis in humans and some animal models. In this study we measured the byproducts of oxidative stress in granulomatous lesions as well as the systemic antioxidant capacity of BCG vaccinated and non-vaccinated guinea pigs experimentally infected with Mycobacterium tuberculosis. In non-vaccinated guinea pigs, oxidative stress was evident within 2 weeks of infection as measured by a decrease in the serum total antioxidant capacity and blood glutathione levels accompanied by an increase in malondialdehyde, a byproduct of lipid peroxidation, within lesions. Despite a decrease in total and reduced blood glutathione concentrations, there was an increase in lesion glutathione by immunohistochemistry in response to localized oxidative stress. In addition there was an increase in the expression of the host transcription factor nuclear erythroid 2 p45-related factor 2 (Nrf2), which regulates several protein and non-proteins antioxidants, including glutathione. Despite the increase in cytoplasmic expression of Nrf2, immunohistochemical staining revealed a defect in Nrf2 nuclear translocation within granulomatous lesions as well as a decrease in the expression of the Nrf2-regulated antioxidant protein NQO1. Treating M. tuberculosis–infected guinea pigs with the antioxidant drug N-acetyl cysteine (NAC) partially restored blood glutathione concentrations and the serum total antioxidant capacity. Treatment with NAC also decreased spleen bacterial counts, as well as decreased the lung and spleen lesion burden and the severity of lesion necrosis. These data suggest that the progressive oxidative stress during experimental tuberculosis in guinea pigs is due in part to a defect in host antioxidant defenses, which, we show here, can be partially restored with antioxidant treatment. These data suggest that the therapeutic strategies that reduce oxidant-mediated tissue damage may be beneficial as an adjunct therapy in the treatment and prevention of tuberculosis in humans

Uptake and Accumulation of Oxidized Low-Density Lipoprotein during Mycobacterium tuberculosis Infection in Guinea Pigs

The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically naïve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically naïve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence

Influence of organic molecules on the aggregation of TiO2 nanoparticles in acidic conditions

Engineered nanoparticles released into the environment may interact with natural organic matter (NOM). Surface complexation affects the surface potential, which in turn may lead to aggregation of the particles. Aggregation of synthetic TiO2 (anatase) nanoparticles in aqueous suspension was investigated at pH 2.8 as a function of time in the presence of various organic molecules and Suwannee River fulvic acid (SRFA), using dynamic light scattering (DLS) and high-resolution transmission electron microscopy (TEM). Results showed that the average hydrodynamic diameter and ?-potential were dependent on both concentration and molecular structure of the organic molecule. Results were also compared with those of quantitative batch adsorption experiments. Further, a time study of the aggregation of TiO2 nanoparticles in the presence of 2,3-dihydroxybenzoic acid (2,3-DHBA) and SRFA, respectively, was performed in order to observe changes in ?-potential and particle size over a time period of 9 months. In the 2,3-DHBA-TiO2 system, ?-potentials decreased with time resulting in charge neutralization and/or inversion depending on ligand concentration. Aggregate sizes increased initially to the micrometer size range, followed by disaggregation after several months. No or very little interaction between SRFA and TiO2 occurred at the lowest concentrations tested. However, at the higher concentrations of SRFA, there was an increase in both aggregate size and the amount of SRFA adsorbed to the TiO2 surface. This was in correlation with the ?-potential that decreased with increased SRFA concentration, leading to destabilization of the system. These results stress the importance of performing studies over both short and long time periods to better understand and predict the long-term effects of nanoparticles in the environment

Interplays between copper and Mycobacterium tuberculosis GroEL1

The recalcitrance of pathogenic Mycobacterium tuberculosis, the agent of tuberculosis, to eradication is due to various factors allowing bacteria to escape from stress situations. The mycobacterial chaperone GroEL1, overproduced after macrophage entry and under oxidative stress, could be one of these key players. We previously reported that GroEL1 is necessary for the biosynthesis of phthiocerol dimycocerosate, a virulence-associated lipid and for reducing antibiotic susceptibility. In the present study, we showed that GroEL1, bearing a unique C-terminal histidine-rich region, is required for copper tolerance during Mycobacterium bovis BCG biofilm growth. Mass spectrometry analysis demonstrated that GroEL1 displays high affinity for copper ions, especially at its C-terminal histidine-rich region. Furthermore, the binding of copper protects GroEL1 from destabilization and increases GroEL1 ATPase activity. Altogether, these findings suggest that GroEL1 could counteract copper toxicity, notably in the macrophage phagosome, and further emphasizes that M. tuberculosis GroEL1 could be an interesting antitubercular target

Die physiologische Funktion kanalbildender Proteine der äußeren Membran in Mykobakterien

Mycobacterium tuberculosis is the leading cause of deaths resulting from a single infectious disease with 1.7 million victims annually. The exceptionally low permeability of the outer membrane contributes to the intrinsic resistance of mycobacteria to many antibiotics. Despite the well-documented importance of outer membrane proteins for nutrient uptake, secretion, and host-pathogen interactions in Gram-negative bacteria, only the porin MspA of M. smegmatis and the channel-forming protein OmpA of M. tuberculosis have been characterized as mycobacterial integral outer membrane proteins. By contrast, E. coli uses more than 60 proteins to functionalize its outer membrane, none of which has significant sequence similarity to any M. tuberculosis protein. Rv1698 of M. tuberculosis was discovered by us as an outer membrane channel protein with unknown function. Intracellular copper in an M. tuberculosis mutant lacking Rv1698 was 100-fold increased. An M. smegmatis mutant lacking the close homolog Msm_3747 accumulated 11-fold more copper than the wild-type, while uptake of glucose remained unchanged. These results demonstrated that Rv1698-like channel proteins are required for copper efflux across the mycobacterial outer membrane and that secretion of Cu+ is a mechanism by which M. tuberculosis maintains copper homeostasis to prevent copper toxicity. Rv1698 is the first identified mycobacterial channel protein that is involved in efflux across the outer membrane. In addition, Rv1698 lacks a predicted copper binding motive and there is no energy source in the outer membrane that would support efflux trough the Rv1698 channels against the concentration gradient. Thus, Rv1698 is likely recruited by Cu+ specific inner membrane translocases that determine substrate specificity and provide energy for the transport. These findings indicate that mycobacteria possess multicomponent efflux systems that are functionally similar to those of Gram-negative bacteria. We also found that M. tuberculosis did not grow at Cu2+ concentrations above 25 µM. The amount of copper in phagosomes of macrophages stimulated with interferon-gamma increases to similar concentrations after infection with M. tuberculosis. Thus, macrophages appear to utilize copper to control intracellular growth of M. tuberculosis. Uptake pathways for the essential micronutrient copper are unknown in mycobacteria. However, an M. smegmatis porin mutant did not grow with trace amounts of copper (<1 µM), but was more resistant than wild-type, demonstrating that channel proteins are required for copper uptake across the outer membrane. These outer membrane channels are essential components of a considerably revised model of copper homeostasis in M. tuberculosis. The implications of these findings for our understanding of transport mechanisms and, in particular efflux systems, in mycobacteria are profound.Mykobakterien sind von großer Bedeutung, da Tuberkulose die weltweit verbreitetste Infektionskrankheit mit 1.7 Millionen Todesopfern darstellt. Die außergewöhnlich niedrige Durchlässigkeit der mykobakteriellen äußeren Membran gilt als Hauptursache für deren Widerstandsfähigkeit gegenüber vielen Antibiotika. Äußere Membranproteine in Gram-negativen Bakterien sind wichtig für die Nahrungsaufnahme, Sekretion und deren Infektionsvermögen. Escherichia coli hat mehr als 60 verschiedene äußere Membranproteine. Keines hat Ähnlichkeit mit mykobakteriellen Proteinen. Bisher sind das Porin MspA von Mycobacterium smegmatis und das kanalbildende Protein OmpA von Mycobacterium tuberculosis die einzigen bekannten mykobakteriellen Membranproteine. Rv1698 von M. tuberculosis wurde von uns als Kanalprotein der äußeren Membran mit unbekannter Funktion entdeckt. Zellen der entsprechenden Mutante reicherten 100-mal mehr Kupfer an als der Wildtyp. Eine M. smegmatis Mutante der das Homolog Msm_3747 fehlt, zeigte einen 11-mal höheren Kupfergehalt. Das Aufnahmevermögen für Glukose blieb jedoch unverändert. Diese Ergebnisse zeigen, dass diese Kanalproteine eine wichtige Rolle für die Ausscheidung von Kupferionen über die mykobakterielle äußere Membran spielen. Damit wurde die Ausscheidung von Kupfer als ein wichtiger Mechanismus identifiziert, mit dem Mykobakterien der Ansammlung von giftigen Kupferionen in der Zelle entgegenwirken. Rv1698 ist somit das erste mykobakterielle äußere Membranprotein mit nachgewiesener Ausscheidungs-funktion. Rv1698 hat kein Kupferbindemotiv. Ebenso fehlen der äußeren Membran die Energiequellen für den Transport entgegen dem Konzentrationsgefälle. Darum ist es wahrscheinlich, dass Rv1698 mit Kupfer-spezifischen Transportproteinen der inneren Membran interagiert. Diese sind in der Regel substratspezifisch und stellen die Energie für den Transport bereit. Darum ist zu vermuten, dass die mykobakteriellen Transportsysteme prinzipiell denen in Gram-negativen Bakterien ähneln. Kupfer über einer Konzentration von 25 µM hemmt das Wachstum von M. tuberculosis auf künstlichem Nährmedium. Ähnliche Konzentrationen wurden in Phagosomen von Interferon-gamma stimulierten Makrophagen gemessen. Dies lässt vermuten, dass Makrophagen das Wachstum von M. tuberculosis mit Hilfe von Kupfer eindämmen. Aufnahmemechanismen für Kupferionen sind unbekannt. Wir haben gezeigt, dass Porinmutanten von M. smegmatis im Gegensatz zum Wildtyp auf nahezu kupferfreiem Medium kaum wachsen und eine Toleranz bei erhöhtem Kupfergehalt zeigen. Kupferaufnahme erfolgt somit über Porine. Kanäle in der mykobakteriellen äußeren Membran sind daher unverzichtbar für die Aufnahme und Ausscheidung überschüssiger Kupferionen. Diese Ergebnisse erweitern dramatisch unser Verständnis über mykobakterielle Transportprozesse