A compact instrument for gamma-ray burst detection on a CubeSat platform II: Detailed design, assembly and validation.

The Gamma-ray Module, GMOD, is a miniaturised novel gamma-ray detector which will be the primary scientific payload on the Educational Irish Research Satellite (EIRSAT-1) 2U CubeSat mission. GMOD comprises a compact (25 mm × 25 mm × 40 mm) cerium bromide scintillator coupled to a tiled array of 4 × 4 silicon photomultipliers, with front-end readout provided by the IDE3380 SIPHRA. This paper presents the detailed GMOD design and the accommodation of the instrument within the restrictive CubeSat form factor. The electronic and mechanical interfaces are compatible with many off-the-shelf CubeSat systems and structures. The energy response of the GMOD engineering qualification model has been determined using radioactive sources, and an energy resolution of 5.4% at 662 keV has been measured. EIRSAT-1 will perform on-board processing of GMOD data. Trigger results, including light-curves and spectra, will be incorporated into the spacecraft beacon and transmitted continuously. Inexpensive hardware can be used to decode the beacon signal, making the data accessible to a wide community. GMOD will have scientific capability for the detection of gamma-ray bursts, in addition to the educational and technology demonstration goals of the EIRSAT-1 mission. The detailed design and measurements to date demonstrate the capability of GMOD in low Earth orbit, the scalability of the design for larger CubeSats and as an element of future large gamma-ray missions.

Balloon flight test of a CeBr3 detector with silicon photomultiplier readout.

Recent advances in silicon photomultiplier (SiPM) technology and new scintillator materials allow for the creation of compact high-performance gamma-ray detectors which can be deployed on small low-cost satellites. A small number of such satellites can provide full sky coverage and complement, or in some cases replace the existing gamma-ray missions in detection of transient gamma-ray events. The aim of this study is to test gamma-ray detection using a novel commercially available CeBr3 scintillator combined with SiPM readout in a near-space environment and inform further technology development for a future space mission. A prototype gamma-ray detector was built using a CeBr3 scintillator and an array of 16 J-Series SiPMs by ON Semiconductor. SiPM readout was performed using SIPHRA, a radiation-tolerant low-power integrated circuit developed by IDEAS. The detector was flown as a piggyback payload on the Advanced Scintillator Compton Telescope balloon flight from Columbia Scientific Balloon Facility. The payload included the detector, a Raspberry Pi on-board computer, a custom power supply board, temperature and pressure sensors, a Global Navigation Satellite System receiver and a satellite modem. The balloon delivered the detector to 37 km altitude where its detection capabilities and readout were tested in the radiation-intense near-space environment. The detector demonstrated continuous operation during the 8-hour flight and after the landing. It performed spectral measurements in an energy range of 100 keV to 8 MeV and observed the 511 keV gamma-ray line arising from positron annihilation in the atmosphere with full width half maximum of 6.8%. During ascent and descent, the detector count rate peaked at an altitude of 16 km corresponding to the point of maximum radiation intensity in the atmosphere. Despite several engineering issues discovered after the flight test, the results of this study confirm the feasibility of using CeBr3 scintillator, SiPMs, and SIPHRA in future space missions.

A compact instrument for gamma-ray burst detection on a CubeSat platform I: Design drivers and expected performance.

The Educational Irish Research Satellite 1 (EIRSAT-1) is a 2U CubeSat being developed under ESA's Fly Your Satellite! programme. The project has many aspects, which are primarily educational, but also include space qualification of new detector technologies for gamma-ray astronomy and the detection of gamma-ray bursts (GRBs). The Gamma-ray Module (GMOD), the main mission payload, is a small gamma-ray spectrometer comprising a 25 mm × 25 mm × 40 mm cerium bromide scintillator coupled to an array of 16 silicon photomultipliers. The readout is provided by IDE3380 (SIPHRA), a low-power and radiation tolerant readout ASIC. GMOD will detect gamma-rays and measure their energies in a range from tens of keV to a few MeV. Monte Carlo simulations were performed using the Medium Energy Gamma-ray Astronomy Library to evaluate GMOD's capability for the detection of GRBs in low Earth orbit. The simulations used a detailed mass model of the full spacecraft derived from a very high-fidelity 3D CAD model. The sky-average effective area of GMOD on board EIRSAT-1 was found to be 10 cm2 at 120 keV. The instrument is expected to detect between 11 and 14 GRBs, at a significance greater than 10σ (and up to 32 at 5σ), during a nominal one-year mission. The shape of the scintillator in GMOD results in omni-directional sensitivity which allows for a nearly all-sky field of view.

The influence of reduced oxygen availability on pathogenicity and gene expression in Mycobacterium tuberculosis.

We investigated how Mycobacterium tuberculosis responded to a reduced oxygen tension in terms of its pathogenicity and gene expression by growing cells under either aerobic or low-oxygen conditions in chemostat culture. The chemostat enabled us to control and vary the oxygen tension independently of other environmental parameters, so that true cause-and-effect relationships of reduced oxygen availability could be established. Cells grown under low oxygen were more pathogenic for guinea pigs than those grown aerobically. The effect of reduced oxygen on global gene expression was determined using DNA microarray. Spearman rank correlation confirmed that microarray expression profiles were highly reproducible between repeat cultures. Using microarray analysis we have identified genes that respond to a low-oxygen environment without the influence of other parameters such as nutrient depletion. Some of these genes appear to be involved in the biosynthesis of cell wall precursors and their induction may have contributed to increased infectivity in the guinea pig. This study has shown that a combination of chemostat culture and microarray presents a biologically robust and statistically reliable experimental approach for studying the effect of relevant and specific environmental stimuli on mycobacterial virulence and gene expression.

Increased transcription of a potential sigma factor regulatory gene Rv1364c in Mycobacterium bovis BCG while residing in macrophages indicates use of alternative promoters.

Alternative sigma factors are key global regulators that coordinate bacterial responses to environmental changes necessary for adaptation and survival. In turn these sigma factors are controlled by regulators such as anti-sigma and anti-anti-sigma factors. In this report, using a cDNA-total RNA subtractive hybridisation strategy that we have developed previously, we identified increased transcription of a potential sigma factor regulatory gene, Rv1364c, in Mycobacterium bovis BCG upon phagocytosis by macrophages and this was confirmed by Northern blot analysis. Primer extension analysis revealed the use of alternative promotors, P1 and P2, and that the increased expression inside macrophages coincided with promoter switching from P2 to P1. Rv1364c (653 amino acids), originally annotated as RsbU, contains structural domains homologous to the PAS redox sensor, the protein phosphatases anti-anti-sigma factor RsbU/SpoIIE, the protein kinase anti-sigma factor RsbW/SpoIIAB and the anti-anti-sigma factor RsbV/SpoIIAA found in other bacteria. These findings have important implications for understanding coordination of the expression of sigma factors under intra-macrophage conditions. Other potentially differentially expressed genes, including genes for fatty acid metabolism, membrane transportors, heat shock proteins, potential sigma factors and energy metabolic pathways are also listed and their biological significance discussed.

Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment.

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2-deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon gamma- and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and beta-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of sigmaE-dependent, sodium dodecyl sulfate-regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.

Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays.

Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass alpha-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heat-shock response and is related to a major antigen induced in response to anaerobic stress.

The heat shock response of Mycobacterium tuberculosis: linking gene expression, immunology and pathogenesis.

The regulation of heat shock protein (HSP) expression is critically important to pathogens such as Mycobacterium tuberculosis and dysregulation of the heat shock response results in increased immune recognition of the bacterium and reduced survival during chronic infection. In this study we use a whole genome spotted microarray to characterize the heat shock response of M. tuberculosis. We also begin a dissection of this important stress response by generating deletion mutants that lack specific transcriptional regulators and examining their transcriptional profiles under different stresses. Understanding the stimuli and mechanisms that govern heat shock in mycobacteria will allow us to relate observed in vivo expression patterns of HSPs to particular stresses and physiological conditions. The mechanisms controlling HSP expression also make attractive drug targets as part of a strategy designed to enhance immune recognition of the bacterium.

cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG.

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis.

Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae.

Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitro.