Factors affecting phage D29 infection: a tool to investigate different growth states of mycobacteria

Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay

The Future of the Exclusionary Rule: An Alternative Analysis for the Adjudication of Individual Rights

The benefits accrued through the use of computer and technological advances unfortunately sometimes infringe upon personal liberties. This comment examines the range and scope of the Exclusionary Rule in those instances when computer or technological errors supply improper evidence about individuals to police. The author focuses on the Supreme Court decision in United States v. Evans, and then places Evans in its context in criminal procedure jurisprudence

Characterizing the Cool KOIs. IV. Kepler-32 as a Prototype for the Formation of Compact Planetary Systems throughout the Galaxy

The Kepler space telescope has opened new vistas in exoplanet discovery space by revealing populations of Earth-sized planets that provide a new context for understanding planet formation. Approximately 70% of all stars in the Galaxy belong to the diminutive M dwarf class, several thousand of which lie within Kepler's field of view, and a large number of these targets show planet transit signals. The Kepler M dwarf sample has a characteristic mass of 0.5 M_☉ representing a stellar population twice as common as Sun-like stars. Kepler-32 is a typical star in this sample that presents us with a rare opportunity: five planets transit this star, giving us an expansive view of its architecture. All five planets of this compact system orbit their host star within a distance one-third the size of Mercury's orbit, with the innermost planet positioned a mere 4.3 stellar radii from the stellar photosphere. New observations limit possible false positive scenarios, allowing us to validate the entire Kepler-32 system making it the richest known system of transiting planets around an M dwarf. Based on considerations of the stellar dust sublimation radius, a minimum mass protoplanetary nebula, and the near period commensurability of three adjacent planets, we propose that the Kepler-32 planets formed at larger orbital radii and migrated inward to their present locations. The volatile content inferred for the Kepler-32 planets and order of magnitude estimates for the disk migration rates suggest that these planets may have formed beyond the snow line and migrated in the presence of a gaseous disk. If true, then this would place an upper limit on their formation time of ~10 Myr. The Kepler-32 planets are representative of the full ensemble of planet candidates orbiting the Kepler M dwarfs for which we calculate an occurrence rate of 1.0 ± 0.1 planet per star. The formation of the Kepler-32 planets therefore offers a plausible blueprint for the formation of one of the largest known populations of planets in our Galaxy

Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle

Background Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne’s disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne’s disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture. Results Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step. Conclusions This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne’s disease progression by warranting further research on the presence of MAP in blood

An investigation of gram-positive pathogens in powdered foods

The purpose of the project was to obtain and test a range powdered food products that are marketed for consumption by individuals that may be immunocompromised. Hence seventeen infant formula milks (0-6months), twelve over-the-counter, elderly, build-up products and nineteen sports powdered protein shakes were examined. These products were tested for the presence of four different Gram-positive pathogens: Two spore formers; Bacillus cereus and Clostridium perfringens and two non-spore formers; Listeria monocytogenes and Staphylococcus aureus. Products were tested according to the ISO standardised methods of testing for described for each of the organisms and samples plated on different diagnostic agars. The presumptively positive organisms that formed characteristic colonies were then further identified. This identification was confirmed in two ways; biochemically, using a wide range of tests including API, and molecularly using PCR-based assays. The results from the project showed that from the 48 products tested; 16 contained B. cereus, nine S. aureus, three L. monocytogenes and one with C. perfringens. To further investigate whether the non-spore forming organisms that survived in these products were more resilient than expected, a heat inactivation experiment was carried out. A simulated high-temperature-short-time (HTST) pasteurisation was set up and the results gained suggested that the S. aureus isolate was able to survive pasteurisation but not subsequent cold and heat shock. In contrast the L. monocytogenes was sub-lethally damaged by the heat treatment but then recovered during cold storage. Thus postulations about how or where these organisms entered food could be made

Initial phases of massive star formation in high infrared extinction clouds. I. Physical parameters

The earliest phases of massive star formation are found in cold and dense infrared dark clouds (IRDCs). Since the detection method of IRDCs is very sensitive to the local properties of the background emission, we present here an alternative method to search for high column density in the Galactic plane by using infrared extinction maps. We find clouds between 1 and 5 kpc, of which many were missed by previous surveys. By studying the physical conditions of a subsample of these clouds, we aim at a better understanding of the initial conditions of massive star formation. We made extinction maps of the Galactic plane based on the 3.6-4.5 microns color excess between the two shortest wavelength Spitzer IRAC bands, reaching to visual extinctions of ~100 mag and column densities of 9x10^22 cm^-2. From this we compiled a new sample of cold and compact high extinction clouds. We used the MAMBO array at the IRAM 30m telescope to study the morphology, masses and densities of the clouds and the dense clumps within them. The latter were followed up by pointed ammonia observations with the 100m Effelsberg telescope, to determine rotational temperatures and kinematic distances. Extinction maps of the Galactic plane trace large scale structures such as the spiral arms. The 1.2 mm emission maps reveal that the high extinction clouds contain extended cold dust emission, from filamentary structures to still diffuse clouds. Most of the clouds are dark in 24 microns, but several show already signs of star formation via maser emission or bright infrared sources, suggesting that the high extinction clouds contain a variety of evolutionary stages. The observations suggest an evolutionary scheme from dark, cold and diffuse clouds, to clouds with a stronger 1.2 mm peak and to finally clouds with many strong 1.2 mm peaks, which are also warmer, more turbulent and already have some star formation signposts.Comment: 26 pages, 14 figures, 9 tables. Accepted for publication in Astronomy and Astrophysics, small corrections implemente

Planet Populations as a Function of Stellar Properties

Exoplanets around different types of stars provide a window into the diverse environments in which planets form. This chapter describes the observed relations between exoplanet populations and stellar properties and how they connect to planet formation in protoplanetary disks. Giant planets occur more frequently around more metal-rich and more massive stars. These findings support the core accretion theory of planet formation, in which the cores of giant planets form more rapidly in more metal-rich and more massive protoplanetary disks. Smaller planets, those with sizes roughly between Earth and Neptune, exhibit different scaling relations with stellar properties. These planets are found around stars with a wide range of metallicities and occur more frequently around lower mass stars. This indicates that planet formation takes place in a wide range of environments, yet it is not clear why planets form more efficiently around low mass stars. Going forward, exoplanet surveys targeting M dwarfs will characterize the exoplanet population around the lowest mass stars. In combination with ongoing stellar characterization, this will help us understand the formation of planets in a large range of environments.Comment: Accepted for Publication in the Handbook of Exoplanet

An investigation of gram-positive pathogens in powdered foods

The purpose of the project was to obtain and test a range powdered food products that are marketed for consumption by individuals that may be immunocompromised. Hence seventeen infant formula milks (0-6months), twelve over-the-counter, elderly, build-up products and nineteen sports powdered protein shakes were examined. These products were tested for the presence of four different Gram-positive pathogens: Two spore formers; Bacillus cereus and Clostridium perfringens and two non-spore formers; Listeria monocytogenes and Staphylococcus aureus. Products were tested according to the ISO standardised methods of testing for described for each of the organisms and samples plated on different diagnostic agars. The presumptively positive organisms that formed characteristic colonies were then further identified. This identification was confirmed in two ways; biochemically, using a wide range of tests including API, and molecularly using PCR-based assays. The results from the project showed that from the 48 products tested; 16 contained B. cereus, nine S. aureus, three L. monocytogenes and one with C. perfringens. To further investigate whether the non-spore forming organisms that survived in these products were more resilient than expected, a heat inactivation experiment was carried out. A simulated high-temperature-short-time (HTST) pasteurisation was set up and the results gained suggested that the S. aureus isolate was able to survive pasteurisation but not subsequent cold and heat shock. In contrast the L. monocytogenes was sub-lethally damaged by the heat treatment but then recovered during cold storage. Thus postulations about how or where these organisms entered food could be made

Development of rapid phage based detection methods for mycobacteria

MAP is the causative agent of a wasting disease in ruminants and other animals called Johne’s disease. Culture of the organism can take months and in the case of some sheep strains of MAP, culture can take up to a year. It can take several years for an animal infected with MAP to show clinical symptoms of disease. During this subclinical stage of infection, MAP can be shed into the environment contaminating their surroundings and infecting other animals. As well as this Johne’s disease is particularly difficult to diagnose during the subclinical stage of infection. Culture is very difficult and takes too long to be a viable method to diagnose Johne’s disease. Microscopic methods can be used on histological samples to detect MAP, however common acid-fast stains used are not specific for MAP and other mycobacteria and acid-fast organisms can be detected. Molecular methods, such as PCR, exist to rapidly detect the signature DNA sequences of these organisms, however they have the disadvantage of not being able to distinguish between live and dead organisms. Other immunological methods, such as ELISA tests, exist and are routinely used to diagnose Johne’s disease, however their sensitivity is very poor especially during the subclinical stage of disease. The aim of these studies was to develop novel rapid methods of detecting MAP to act as an alternative to methods already available. Sample processing using magnetic separation was carried out to allow good capture of MAP cells and to allow efficient phage infection. Using the phage assay, a specific, sensitive phage based method was developed that could detect approximately 10 cells per ml of blood within 24 h in the laboratory with a sensitive, specific plaque-PCR. This optimised detection method was then used to determine whether MAP cells could be detected in clinical blood samples of cattle suffering from Johne’s disease. The results suggest that animals experimentally and naturally infected with MAP harboured cells in their blood during subclinical and clinical stages of infection. A novel high-throughput method of detecting mycobacteria was also developed. Using phage D29 as a novel mycobacterial DNA extraction tool, viable MAP cells were detected within 8 h and the format of the assay means that it can be adapted to be used in a high-throughput capacity. Factors affecting phage infection and phage-host interactions were investigated to make sure the phage based methods of detection were as efficient as possible. It was found that periods of recovery were often necessary to not only make sure the phage were not inhibited but to also allow the host cells to be metabolically active as it was found that phage D29 can only infect mycobacteria cells that are metabolically active. A fluorescent fusion-peptide capable of specifically labelling MAP cells was also developed to be used as an alternative to acid-fast staining. Peptides that were found to specifically bind to MAP cells were fused with green fluorescent protein and cells mounted on slides were specifically labelled with the fluorescent fusion protein. This resulted in a good alternative to the generic acid-fast staining methods. The blood phage assay has shown that viable MAP cells can be found in the blood of animals suffering from Johne’s disease within 24 h and this can be confirmed using a MAP specific plaque-PCR protocol. A novel faster method to detect MAP was also developed, to cut down the time to detection of viable MAP cells to 8 h, which can be formatted to be used in a high-throughput capacity. The phage assay was used as a tool to determine different metabolic states of mycobacteria, and helped investigate optimal detection conditions when using the phage assay. Finally a novel fluorescent label was developed to detect MAP as an alternative to insensitive acid-fast staining. The development of these novel methods to rapidly, specifically and sensitively detect MAP will push further the understanding of Johne’s disease and help control it