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.
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 Educational Irish Research Satellite (EIRSAT-1) is a 2U CubeSat developed at University College Dublin. The project aims to build, test, launch, and operate Ireland’s first satellite and to ...perform in-orbit demonstrations of three novel payloads developed in-house. To reduce risk within the mission, the project employs a prototype model philosophy in which two models of the spacecraft exist: an engineering qualification model (EQM) and a flight model (FM). This paper presents the verification approach of the functional tests implemented for the EIRSAT-1 project. The activities of the FlatSat and system level full functional tests of the EQM are presented and the results obtained during the test campaigns are discussed. Four test anomalies were encountered during the full functional test campaign resulting in two minor redesigns, and subsequent reassembly, of the CubeSat. The functional test campaigns highlighted the importance of FlatSat level testing of CubeSats to ensure compatibility of all subsystems prior to assembly and of thorough documentation to diagnose any unexpected behaviour of the hardware efficiently. The functional verification of the EQM proved that the system conformed to its design, verifying 57 mission requirements, and is a crucial step towards the development of the EIRSAT-1 FM.
The compact, standardised form factor of CubeSats allows for the use of commercial off-the-shelf components, reducing traditional barriers to entry, such as cost and development time. More than 1500 ...of these small spacecraft have been launched in the past 20 years, with improving capabilities that enable a wide range of mission profiles. The Educational Irish Research Satellite, EIRSAT-1, is a CubeSat being developed by a student-led team with goals that span education, technology demonstration and science. A comprehensive mission test plan, in which in-flight conditions are simulated, has been developed for EIRSAT-1 and implemented using an engineering qualification model of the spacecraft. In addition to verifying 41 mission requirements, the successful execution of the mission test plan established that the full satellite system can perform the intended mission. Mission testing also proved to be an invaluable tool to prepare for launch and operations, providing the team with a more complete understanding of the satellite’s expected on-orbit behaviour. This work presents a detailed description of the mission test planning process and implementation, as well as key results and lessons learned. In doing so, this work aims to improve the on-orbit reliability of CubeSats by disseminating resources and good practice around mission testing.
CubeSats facilitate rapid development and deployment of missions for educational, technology demonstration, and scientific purposes. However, they are subject to a high failure rate, with a leading ...cause being the lack of system-level verification. The Educational Irish Research Satellite (EIRSAT-1) is a CubeSat mission under development in the European Space Agency’s (ESA) Fly Your Satellite! Programme. EIRSAT-1 is a 2U CubeSat with three novel payloads and a bespoke antenna deployment module, which all contribute to the complexity of the project. To increase the likelihood of mission success, a prototype model philosophy is being employed, where both an engineering qualification model (EQM) and a flight model of EIRSAT-1 are being built. Following the assembly of the EQM, the spacecraft underwent a successful full functional test and month-long mission test. An environmental test campaign in ESA Education Office’s CubeSat Support Facility was then conducted with the EQM where both vibration and thermal verification test campaigns were performed. The focus of this paper is the thermal testing and verification of the EIRSAT-1 EQM. Over three weeks, the EQM was subjected to one non-operational cycle, three and a half operational cycles, and a thermal balance test in a thermal vacuum chamber. After dwelling at each temperature extreme, functional tests were performed to investigate the performance of the spacecraft in this space representative environment. The approach to planning and executing the thermal testing is described in detail including the documentation required, set up of the test equipment, and determination of the test levels. Overall, the campaign demonstrated that the mission can successfully operate in a space environment similar to that expected in orbit, despite encountering a number of issues. These issues included a payload displaying anomalous behaviour at cold temperatures and needing to redefine test levels due to an insufficient understanding of the internal dissipation in the spacecraft. A total of two major and three minor non-conformances were raised. Crucially, these issues could not have been found without thermal testing, despite the comprehensive ambient tests performed. The main results and lessons learned during this thermal test campaign are presented with the aim of guiding future missions on optimal approaches in organising and executing the thermal testing of their CubeSats.
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.
Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly ...consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.
Campylobacter jejuni is the leading cause of bacterial food-borne diarrhoeal disease throughout the world, and yet is still a poorly understood pathogen. Whole genome microarray comparisons of 11 C. ...jejuni strains of diverse origin identified genes in up to 30 NCTC 11168 loci ranging from 0.7 to 18.7 kb that are either absent or highly divergent in these isolates. Many of these regions are associated with the biosynthesis of surface structures including flagella, lipo-oligosaccharide, and the newly identified capsule. Other strain-variable genes of known function include those responsible for iron acquisition, DNA restriction/modification, and sialylation. In fact, at least 21% of genes in the sequenced strain appear dispensable as they are absent or highly divergent in one or more of the isolates tested, thus defining 1300 C. jejuni core genes. Such core genes contribute mainly to metabolic, biosynthetic, cellular, and regulatory processes, but many virulence determinants are also conserved. Comparison of the capsule biosynthesis locus revealed conservation of all the genes in this region in strains with the same Penner serotype as strain NCTC 11168. By contrast, between 5 and 17 NCTC 11168 genes in this region are either absent or highly divergent in strains of a different serotype from the sequenced strain, providing further evidence that the capsule accounts for Penner serotype specificity. These studies reveal extensive genetic diversity among C. jejuni strains and pave the way toward identifying correlates of pathogenicity and developing improved epidemiological tools for this problematic pathogen.
Radiation damage of J-series silicon photomultipliers (SiPMs) has been studied in the context of using these photodetectors in future space-borne scintillation detectors. Several SiPM samples were ...exposed to 101.4 MeV protons, with 1 MeV neutron equivalent fluence ranging from 1.27×108 neq/cm2 to 1.23×1010 neq/cm2. After the irradiation, the SiPMs experienced a large increase in the dark current and noise, which may pose problems for long-running space missions in terms of power consumption, thermal control and detection of low-energy events. Measurements performed with a CeBr3 scintillator crystal showed that after exposure to 1.23×1010 neq/cm2 and following room-temperature annealing, the dark noise of a single 6 mm square SiPM at room temperature increased from 0.1 keV to 2 keV. Because of the large SiPM noise, the gamma-ray detection threshold increased to approximately 20 keV for a CeBr3 detector using a 4-SiPM array and 40 keV for a detector using a 16-SiPM array. Only a small effect of the proton irradiation on the average detector signal was observed, suggesting no or little change to the SiPM gain and photon detection efficiency.
GRB 221009A: The BOAT Burns, Eric; Svinkin, Dmitry; Fenimore, Edward ...
Astrophysical journal. Letters,
03/2023, Volume:
946, Issue:
1
Journal Article
Peer reviewed
Open access
Abstract GRB 221009A has been referred to as the brightest of all time (BOAT). We investigate the veracity of this statement by comparing it with a half century of prompt gamma-ray burst ...observations. This burst is the brightest ever detected by the measures of peak flux and fluence. Unexpectedly, GRB 221009A has the highest isotropic-equivalent total energy ever identified, while the peak luminosity is at the ∼99th percentile of the known distribution. We explore how such a burst can be powered and discuss potential implications for ultralong and high-redshift gamma-ray bursts. By geometric extrapolation of the total fluence and peak flux distributions, GRB 221009A appears to be a once-in-10,000-year event. Thus, it is almost certainly not the BOAT over all of cosmic history; it may be the brightest gamma-ray burst since human civilization began.
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