Multiple wavemode scanning for near and far-side defect characterisation

The combination of ultrasonic inspections using different wavemodes can give more information than is available with single mode inspection. In this work, the response of shear and Rayleigh waves to surface-breaking defects propagating on the near-side and far-side of a sample is investigated. The directivity of shear waves generated by a racetrack coil electromagnetic acoustic transducer (EMAT) is identified and used to set an ideal separation for a pair of transmit-receive EMATs. Defects are indicated by a reduction in the transmitted Rayleigh wave amplitude, and by blocking of the shear wave. Used together, these can identify features in the bulk wave behaviour which are due to near-face surface-breaking defects, and give a full picture of both surfaces. By using a combination of the two wavemodes, the angle of propagation and length of any near-side defects can additionally be identified. A scanning method for samples is proposed

Phased electromagnetic acoustic transducer array for Rayleigh wave surface defect detection

A phased electromagnetic acoustic transducer (EMAT) array system has been developed for detection and characterisation of surface breaking defects. An array of four linear coils which are individually controlled are used to generate a Rayleigh wave. The high current electronics combined with the coil designs enables the array to generate either narrowband or broadband signals, and controlling the phase delay between the channels makes it possible to change the ultrasound wavelength without requiring the physical separation of the coils to be changed. Experimental results show that the four-coil phased array is able to generate a wavelength range from 3.0 mm to 11.7 mm. Surface breaking defects were characterised using a transmit-receive set-up with a broadband EMAT detector being used to detect the Rayleigh wave. Machined surface slots with different depths were used for technique validation. The results show that the array is sensitive to surface defects and that a wide depth sensitivity range for defect sizing can be easily achieved by applying phasing to tune the wavelength of operation. A large increase in detection flexibility is immediately shown

The interaction of polymer dispersed liquid crystal sensors with ultrasound

Polymer dispersed liquid crystals (PDLCs) have been shown to be sensitive to ultrasound through the acousto-optic effect. The acousto-optic response of PDLCs was studied over a broad frequency range (0.3–10 MHz). We demonstrate that the displacements required to produce acousto-optic clearing of PDLC films can be as low as a few nanometers, which is at least 103 times smaller than the PDLC droplet size, is 105 times smaller than the PDLC layer thickness, and of the order of the molecular size of the liquid crystal constituents. This suggests that the acousto-optic effect in PDLCs is due to the microscopic effects of the LC reorientation under torques or flows rather than the LC reorientation through macroscopic droplet deformation. The displacement required for clearing is related to the frequency of operation via an exponential decay. We attribute the observed frequency response to a freezing out of the rotational motion around the short axis of the liquid crystal. The reported frequency dependence and displacements required indicate that the effects and materials described here could be used for ultrasound visualization in a non-destructive testing context

Non-linear enhancement of laser generated ultrasonic Rayleigh waves by cracks

Laser generated ultrasound has been widely used for detecting cracks, surface and sub-surface defects in many different materials. It provides a non-contact wideband excitation source which can be focused into different geometries. Previous workers have reported enhancement of the laser generated Rayleigh wave when a crack is illuminated by pulsed laser beam irradiation. We demonstrate that the enhancement observed is due to a combination of source truncation, the free boundary condition at the edge of the crack and interference effects. Generating a Rayleigh wave over a crack can lead to enhancement of the amplitude of the Rayleigh wave signal, a shift in the dominant frequency of the wideband Rayleigh wave and strong enhancement of the high frequency components of the Rayleigh wave

SpoT governs Legionella pneumophila differentiation in host macrophages

During its life cycle, Legionella pneumophila alternates between a replicative and a transmissive state. To determine their contributions to L. pneumophila differentiation, the two ppGpp synthetases, RelA and SpoT, were disrupted. Synthesis of ppGpp was required for transmission, as relA spoT mutants were killed during entry to and exit from macrophages. RelA, which senses amino acid starvation induced by serine hydroxamate, is dispensable in macrophages, as relA mutants spread efficiently. SpoT monitors fatty acid biosynthesis (FAB), since following cerulenin treatment, wild-type and relA strains expressed the flaA transmissive gene, but relA spoT mutants did not. As in Escherichia coli , the SpoT response to FAB perturbation likely required an interaction with acyl-carrier protein (ACP), as judged by the failure of the spoT-A413E allele to rescue transmissive trait expression of relA spoT bacteria. Furthermore, SpoT was essential for transmission between macrophages, since secondary infections by relA spoT mutants were restored by induction of spoT , but not relA . To resume replication, ppGpp must be degraded, as mutants lacking spoT hydrolase activity failed to convert from the transmissive to the replicative phase in either bacteriological medium or macrophages. Thus, L. pneumophila requires SpoT to monitor FAB and to alternate between replication and transmission in macrophages.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73309/1/j.1365-2958.2008.06555.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/73309/2/MMI_6555_sm_Figure_S1.pd

Metabolic Cues and Regulatory Proteins that Govern Legionella Pneumophila Differentiation and Virulence.

In aquatic environments, the gram-negative bacterium Legionella pneumophila likely resides within biofilm communities. However, when protozoa ingest L. pneumophila, the microbes can establish an intracellular niche protected from digestion. Moreover, if humans inhale bacteria-laden aerosols, L. pneumophila can survive and replicate within alveolar macrophages to cause the pneumonia, Legionnaires’ disease. To persist within these diverse niches, L. pneumophila alternates between at least two phenotypic phases: a non-infectious, replicative form required for intracellular growth and an infectious, transmissive form that enhances survival in the extracellular milieu. By exploiting our knowledge of the L. pneumophila biphasic life cycle, we have uncovered novel metabolites and regulatory proteins that govern differentiation. Previous studies established that the LetA/LetS two-component system regulates host transmission through the regulator CsrA, while the stringent response coordinates transcription by producing the alarmone ppGpp. Through genetic, transcription and phenotypic analyses we determined that the architecture of the LetA/LetS two-component system enables L. pneumophila to customize its genotypic and phenotypic profiles. Moreover, our data suggest that the model of the flexible LetA/LetS regulon may be applicable to many two-component systems, and may equip microbes with a mechanism to fine-tune their traits. Since L. pneumophila resides within many different environments, we predicted that various metabolites cue its developmental switch. Using phenotypic microarrays we deduced that perturbations in fatty acid biosynthesis activate the stringent response via an interaction between SpoT and acyl carrier protein, which leads to L. pneumophila differentiation. We propose that by coupling phase differentiation to its metabolic state, L. pneumophila can adapt to environmental fluctuations or stresses encountered in its host, thereby enhancing its overall fitness. Data suggest that nicotinic acid can activate microbial two-component systems, and consequently, modulate the genes and phenotypes governed by these regulatory proteins. By comparing the transcriptional profiles of replicative and transmissive phase L. pneumophila to genes regulated by nicotinic acid, we identified a cohort of genes that are unique for this phenotypic modulator. Taken together, our data suggest that L. pneumophila acclimates to environmental stresses by monitoring metabolic fluctuations and employing a regulatory cascade that enables the bacteria to coordinate an appropriate response.Ph.D.Cellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/61593/1/rlschwar_1.pd

Survey of financial burden of families in the U.S. with children using home mechanical ventilation.

Aim: To describe and quantify the out-of-pocket expenses, employment loss, and other financial impact related to caring for a child using home mechanical ventilation (HMV). METHOD: We conducted a cross-sectional survey of U.S. families with children who used HMV. Eligible participants were invited to complete a questionnaire addressing household and child characteristics, out-of-pocket expenses, employment loss/reduction, and financial stress. Participants were recruited with the help of three national patient registries. RESULTS: Two hundred twenty-six participants from 32 states (152 with children who used invasive ventilation and 74 with children who used noninvasive ventilation) completed the questionnaire. Participants' median reported yearly household income was $90 000 (IQR 70 000-150 000). The median amount paid in out-of-pocket expenses in the previous 3 months to care for their child using HMV totaled$3899 (IQR $2900-4550). Reported levels of financial stress decreased as income increased; 37-60% of participants, depending on income quintile, reported moderate financial stress with "some" of that stress due to their out-of-pocket expenses. A substantial majority reported one or more household members stopped or reduced work and took unpaid weeks off of work to care for their child. CONCLUSION: The financial impact of caring for a child using HMV is considerable for some families. Providers need to understand these financial burdens and should inform families of them to help families anticipate and plan for them

A novel FRET-based screen in high-throughput format to identify inhibitors of malarial and human glucose transporters

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z′ factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum. Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC(50)) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening

A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites

Isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway generates commercially important products and is a target for antimicrobial drug development. MEP pathway regulation is poorly understood in microorganisms. We employ a forward genetics approach to understand MEP pathway regulation in the malaria parasite, Plasmodium falciparum. The antimalarial fosmidomycin inhibits the MEP pathway enzyme deoxyxylulose 5-phosphate reductoisomerase (DXR). Fosmidomycin-resistant P. falciparum are enriched for changes in the PF3D7_1033400 locus (hereafter referred to as PfHAD1), encoding a homologue of haloacid dehalogenase (HAD)-like sugar phosphatases. We describe the structural basis for loss-of-function PfHAD1 alleles and find that PfHAD1 dephosphorylates a variety of sugar phosphates, including glycolytic intermediates. Loss of PfHAD1 is required for fosmidomycin resistance. Parasites lacking PfHAD1 have increased MEP pathway metabolites, particularly the DXR substrate, deoxyxylulose 5-phosphate. PfHAD1 therefore controls substrate availability to the MEP pathway. Because PfHAD1 has homologs in plants and bacteria, other HAD proteins may be MEP pathway regulators

MEPicides: Potent antimalarial prodrugs targeting isoprenoid biosynthesis

AbstractThe emergence of Plasmodium falciparum resistant to frontline therapeutics has prompted efforts to identify and validate agents with novel mechanisms of action. MEPicides represent a new class of antimalarials that inhibit enzymes of the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, including the clinically validated target, deoxyxylulose phosphate reductoisomerase (Dxr). Here we describe RCB-185, a lipophilic prodrug with nanomolar activity against asexual parasites. Growth of P. falciparum treated with RCB-185 was rescued by isoprenoid precursor supplementation, and treatment substantially reduced metabolite levels downstream of the Dxr enzyme. In addition, parasites that produced higher levels of the Dxr substrate were resistant to RCB-185. Notably, environmental isolates resistant to current therapies remained sensitive to RCB-185, the compound effectively treated sexually-committed parasites, and was both safe and efficacious in malaria-infected mice. Collectively, our data demonstrate that RCB-185 potently and selectively inhibits Dxr in P. falciparum, and represents a promising lead compound for further drug development.</jats:p