Development of laminar flow control wing surface porous structure

It was concluded that the chordwise air collection method, which actually combines chordwise and spanwise air collection, is the best of the designs conceived up to this time for full chord laminar flow control (LFC). Its shallower ducting improved structural efficiency of the main wing box resulting in a reduction in wing weight, and it provided continuous support of the chordwise panel joints, better matching of suction and clearing airflow requirements, and simplified duct to suction source minifolding. Laminar flow control on both the upper and lower surfaces was previously reduced to LFC suction on the upper surface only, back to 85 percent chord. The study concludes that, in addition to reduced wing area and other practical advantages, this system would be lighter because of the increase in effective structural wing thickness

Evaluating the Sensitivity of Mycobacterium tuberculosis to Biotin Deprivation Using Regulated Gene Expression

In the search for new drug targets, we evaluated the biotin synthetic pathway of Mycobacterium tuberculosis (Mtb) and constructed an Mtb mutant lacking the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase, BioA. In biotin-free synthetic media, ΔbioA did not produce wild-type levels of biotinylated proteins, and therefore did not grow and lost viability. ΔbioA was also unable to establish infection in mice. Conditionally-regulated knockdown strains of Mtb similarly exhibited impaired bacterial growth and viability in vitro and in mice, irrespective of the timing of transcriptional silencing. Biochemical studies further showed that BioA activity has to be reduced by approximately 99% to prevent growth. These studies thus establish that de novo biotin synthesis is essential for Mtb to establish and maintain a chronic infection in a murine model of TB. Moreover, these studies provide an experimental strategy to systematically rank the in vivo value of potential drug targets in Mtb and other pathogens

A Riboswitch-Based Inducible Gene Expression System for Mycobacteria

Research on the human pathogen Mycobacterium tuberculosis (Mtb) would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb

Simultaneous Analysis of Multiple Mycobacterium tuberculosis Knockdown Mutants In Vitro and In Vivo

Mycobacterium tuberculosis (Mtb) represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags) and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL), the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen was cleared from the lungs of most mice

Metabolic Network for the Biosynthesis of Intra- and Extracellular alpha-Glucans Required for Virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis synthesizes intra- and extracellular alpha-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the alpha-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of alpha-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in alpha-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that alpha-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block alpha-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some alpha-glucan is exported to form the alpha-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate alpha-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADPglucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying alpha-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of alpha-glucan could be constructed, showing a direct link between the GlgE pathway, alpha-glucan biosynthesis and virulence in a mouse infection model

Practical data acquisition strategy for time-lapse experiments using crosshole GPR and full-waveform inversion

Crosshole ground penetrating radar (GPR) methods are increasingly used in time-lapse studies of flow in the uppermost near subsurface with important implications for our understanding of e.g., water infiltration in the unsaturated zone, and fluid flow in the saturated zone. A particular challenge in such time-lapse crosshole studies is the trade-off between collecting sufficient data to be able to resolve how a tracer moves, and, minimizing the data acquisition time such that the data approximates a static state. We test how dense recording geometries are needed for resolving a gas bubble injected in a highly heterogeneous chalk reservoir analogue using a full-waveform inversion (FWI) approach for modelling the crosshole GPR data. We show that even relatively sparse geometries provide sufficient resolution of the permittivity contrast caused by the gas bubble, provided that the detailed background permittivity structure is known from prior (before gas injection) FWI analysis of densely recorded high-resolution data. The conductivity contrast caused by the gas is more challenging to recover and the resolution suffers to a higher degree when reducing the survey geometry or at higher noise levels. As long as the permittivity change during the time-lapse experiment is the main target, a significant reduction in acquisition time is therefore possible as compared to the time needed to record the background permittivity structure. This reduced acquisition time has important practical implications for time-lapse experiments under realistic conditions. Our results are based on synthetic analysis based on a realistic subsurface scenario closely linked to characterization of heterogeneous chalk reservoirs. However, our findings also have important implications for planning of future time-lapse studies in other settings

How an agonist peptide mimics the antibiotic tetracycline to induce Tet repressor

A 16 residue peptide, called Tip, induces the tetracycline repressor TetR as efficiently as the antibiotic tetracycline when fused to the N or C terminus of another protein. This is unusual because the majority of in vitro selected peptides, such as Tip, inhibit protein function, and agonist peptides are only rarely identified. We elucidated the atomic mechanism of TetR induction by Tip from crystal structures of TetR in complex with Tip and of free TetR. Peptide induction ultimately results in the same movements of DNA reading heads, but Tip accomplishes this by very different molecular interactions from tetracycline involving important contacts to the TetR surface. As a direct consequence, an alternate pathway of allostery becomes possible that makes ample use of intersubunit interactions. For the first time it is possible to show in atomic detail how a small molecule controlled bacterial transcription factor such as TetR becomes responsive to protein protein interactions, characteristic of gene transcription regulation in higher organism

Measuring Soil Water Content with Ground Penetrating Radar: A Decade of Progress

Tremendous progress has been made with respect to ground penetrating radar (GPR) equipment, data acquisition, and processing since the establishment of GPR as a tool for soil water content determination in vadose zone hydrology about 25 yr ago. In this update, we aim to provide a critical overview of recent advances in vadose zone applications of GPR with a particular focus on new possibilities for multi-offset and borehole GPR measurements, the development of quantitative off-ground GPR methods, full-waveform inversion of GPR measurements, the potential of time-lapse GPR measurements for process investigations and hydrological parameter estimation, and recent improvements in GPR instrumentation. We hope that this update encourages the soil hydrology, groundwater, and critical zone community to embrace GPR as a viable tool for soil water content determination and the elucidation of subsurface hydrological processes