Allicin causes fragmentation of the peptidoglycan coat in Staphylococcus aureus by effecting synthesis and aiding hydrolysis: a determination by MALDI-TOF mass spectrometry on whole cells.

PURPOSE: To determine the effect of allicin on Staphylococcus aureus cell wall peptidoglycans by the application of MALDI-TOF mass spectrometry on whole cells and to relate this to current knowledge of wall-processing enzymes. METHODOLOGY: Two different S. aureus strains were grown for 48 h after which period each culture was split into two, one part was then treated with sub-inhibitory levels of allicin while the other part left untreated as a control. After a further 24 h whole cells were recovered and analysed by MALDI-TOF mass spectrometry. RESULTS: Changes in the mass spectra between the treated and untreated cells revealed fragmented peptidoglycans identified by mass calculation only in the treated cells. These peptidoglycan fragments where identified as the products of specific peptidoglycan hydrolases. CONCLUSIONS: Allicin is known to target cysteine thiol groups. These are absent in peptidoglycan hydrolases and we might have expected identical results in both of the treated and untreated cells. Peptidoglycan synthesis enzymes such as the Fem family of enzymes do contain cysteines. Fem enzymes A, B and X all have a conserved conformation of 99 % for over 100 S. aureus strains and are therefore potential targets for allicin. Examination of FemA structure showed that cysteine102 is accessible from the surface. We propose that allicin has an inhibitory mechanism alongside others of targeting FemA and possibly other Fem enzymes by curtailing glycine bridging and leading to fragmentation. This study provided an insight into yet another antimicrobial mechanism of allicin.

Transcriptomic analysis of Rhizobium leguminosarum bacteroids in determinate and indeterminate nodules.

Two common classes of nitrogen-fixing legume root nodules are those that have determinate or indeterminate meristems, as in Phaseolus bean and pea, respectively. In indeterminate nodules, rhizobia terminally differentiate into bacteroids with endoreduplicated genomes, whereas bacteroids from determinate nodules are less differentiated and can regrow. We used RNA sequencing to compare bacteroid gene expression in determinate and indeterminate nodules using two Rhizobium leguminosarum strains whose genomes differ due to replacement of the symbiosis (Sym) plasmid pRP2 (strain Rlp4292) with pRL1 (strain RlvA34), thereby switching symbiosis hosts from Phaseolus bean (determinate nodules) to pea (indeterminate nodules). Both bacteroid types have gene expression patterns typical of a stringent response, a stressful environment and catabolism of dicarboxylates, formate, amino acids and quaternary amines. Gene expression patterns were indicative that bean bacteroids were more limited for phosphate, sulphate and iron than pea bacteroids. Bean bacteroids had higher levels of expression of genes whose products are predicted to be associated with metabolite detoxification or export. Pea bacteroids had increased expression of genes associated with DNA replication, membrane synthesis and the TCA (tricarboxylic acid) cycle. Analysis of bacteroid-specific transporter genes was indicative of distinct differences in sugars and other compounds in the two nodule environments. Cell division genes were down-regulated in pea but not bean bacteroids, while DNA synthesis was increased in pea bacteroids. This is consistent with endoreduplication of pea bacteroids and their failure to regrow once nodules senesce.

Anomalous material-dependent transport of focused, laser-driven proton beams.

Intense lasers can accelerate protons in sufficient numbers and energy that the resulting beam can heat materials to exotic warm (10 s of eV temperature) states. Here we show with experimental data that a laser-driven proton beam focused onto a target heated it in a localized spot with size strongly dependent upon material and as small as 35 µm radius. Simulations indicate that cold stopping power values cannot model the intense proton beam transport in solid targets well enough to match the large differences observed. In the experiment a 74 J, 670 fs laser drove a focusing proton beam that transported through different thicknesses of solid Mylar, Al, Cu or Au, eventually heating a rear, thin, Au witness layer. The XUV emission seen from the rear of the Au indicated a clear dependence of proton beam transport upon atomic number, Z, of the transport layer: a larger and brighter emission spot was measured after proton transport through the lower Z foils even with equal mass density for supposed equivalent proton stopping range. Beam transport dynamics pertaining to the observed heated spot were investigated numerically with a particle-in-cell (PIC) code. In simulations protons moving through an Al transport layer result in higher Au temperature responsible for higher Au radiant emittance compared to a Cu transport case. The inferred finding that proton stopping varies with temperature in different materials, considerably changing the beam heating profile, can guide applications seeking to controllably heat targets with intense proton beams.

Ultra-narrow linewidth quantum dot coherent comb lasers with self-injection feedback locking.

We have used an external cavity self-injection feedback locking (SIFL) system to simultaneously reduce the optical linewidth of over 39 individual wavelength channels of an InAs/InP quantum dot (QD) coherent comb laser (CCL). Linewidth reduction from a few MHz to less than 200 kHz is observed. Measured phase noise spectra clearly indicate a significant decrease in phase noise in the frequency range above 2 kHz. The RF beating signal between two adjacent channels also shows a substantial reduction in 3-dB linewidth from 10 kHz to 300 Hz with the SIFL system, and a corresponding drop in baseline level (-27 dB to -50 dB).

High performance InAs/InP quantum dot 34.462-GHz C-band coherent comb laser module.

We have developed an InAs/InP quantum dot (QD) C-band coherent comb laser (CCL) module with actively stabilized absolute wavelength and power, and channel spacing of 34.462 GHz with ± 100 ppm accuracy. The total output power is up to 46 mW. The integrated average relative intensity noise (RIN) values of the lasing spectrum and a filtered single channel at 1540.19 nm were -165.6 dB/Hz and -130.3 dB/Hz respectively in the frequency range from 10 MHz to 10 GHz. The optical linewidth of the 45 filtered individual channels between 1531.77 nm to 1543.77 nm ranged from 850 kHz to 2.16 MHz. We have also analyzed the noise behaviors of each individual channel.

Experiment and simulation of novel liquid crystal plasma mirrors for high contrast, intense laser pulses.

We describe the first demonstration of plasma mirrors made using freely suspended, ultra-thin films formed dynamically and in-situ. We also present novel particle-in-cell simulations that for the first time incorporate multiphoton ionization and dielectric models that are necessary for describing plasma mirrors. Dielectric plasma mirrors are a crucial component for high intensity laser applications such as ion acceleration and solid target high harmonic generation because they greatly improve pulse contrast. We use the liquid crystal 8CB and introduce an innovative dynamic film formation device that can tune the film thickness so that it acts as its own antireflection coating. Films can be formed at a prolonged, high repetition rate without the need for subsequent realignment. High intensity reflectance above 75% and low-field reflectance below 0.2% are demonstrated, as well as initial ion acceleration experimental results that demonstrate increased ion energy and yield on shots cleaned with these plasma mirrors.

Experimental capabilities of 0.4 PW, 1 shot/min Scarlet laser facility for high energy density science.

We report on the recently completed 400 TW upgrade to the Scarlet laser at The Ohio State University. Scarlet is a Ti:sapphire-based ultrashort pulse system that delivers >10 J in 30 fs pulses to a 2 µm full width at half-maximum focal spot, resulting in intensities exceeding 5×1021 W/cm2. The laser fires at a repetition rate of once per minute and is equipped with a suite of on-demand and on-shot diagnostics detailed here, allowing for rapid collection of experimental statistics. As part of the upgrade, the entire laser system has been redesigned to facilitate consistent, characterized high intensity data collection at high repetition rates. The design and functionality of the laser and target chambers are described along with initial data from commissioning experimental shots.

Microengineering Laser Plasma Interactions at Relativistic Intensities.

We report on the first successful proof-of-principle experiment to manipulate laser-matter interactions on microscales using highly ordered Si microwire arrays. The interaction of a high-contrast short-pulse laser with a flat target via periodic Si microwires yields a substantial enhancement in both the total and cutoff energies of the produced electron beam. The self-generated electric and magnetic fields behave as an electromagnetic lens that confines and guides electrons between the microwires as they acquire relativistic energies via direct laser acceleration.

Medical service redesign shares the load saving 6000 bed days and improving morale.

BACKGROUND AND AIMS: In 2010, demand on the Auckland City Hospital general medical service exceeded capacity. A review by the Royal Australasian College of Physicians was critical of training offered to registered medical officers, and low morale was a problem across the service. Management offered support for an improved model that would solve these problems. METHODS: A project to redesign the general medical service was undertaken. Baseline analysis found uneven workload and insufficient capacity at peak times for patient presentations. Workshops involving the entire service led to a new model that splits workload and teams into patients likely to have a short stay from those requiring longer, ward-based care. Admissions are now distributed over 12 teams on weekdays and 4 on the weekends. There was an increase of approximately 2.5 in consultant full time equivalents but no change in registrar or house officer staffing. RESULTS: Since the introduction of the new model, the average length of stay has fallen from 3.7 to 3.2 days (14%) and the median length of stay by 28%, resulting in a saving of 6000 bed days per year. Readmission, inpatient and 30-day mortality rates are unchanged. These results have been sustained over 18 months with signs of continuing improvement. CONCLUSION: This project owes its success to the following factors - management support; iterative engagement of a range of staff; provision of timely data analysis; increases in senior medical officer staffing and reorganisation leading to more predictable and fair work practices. One challenge is discontinuity, whether between doctors and patients or within the medical team.

Interplay between crystal phase purity and radial growth in InP nanowires.

The interplay between crystal phase purity and radial growth in InP nanowires is investigated. By modifying the growth rate and V/III ratio, regions of high or low stacking fault density can be controllably introduced into wurtzite nanowires. It is found that regions with high stacking fault density encourage radial growth. Through careful choice of growth conditions pure wurtzite InP nanowires are then grown which exhibit narrow 4.2 K photoluminescence linewidths of 3.7 meV at 1.490 meV, and no evidence of emission related to stacking faults or zincblende insertions.

Enhanced photonic crystal cavity-waveguide coupling using local slow-light engineering.

This Letter introduces an enhanced cavity-waveguide coupling architecture based upon slow-light engineering in a two-port photonic crystal system. After analyzing the system transmittance using coupled-mode theory, the system is probed experimentally and shown to have increased transmittance due to the enhanced cavity-waveguide coupling. Such a coupling architecture may facilitate next-generation planar lightwave circuitry such as onchip quantum information processing or high precision light-matter sensing applications.

The PTS(Ntr) system globally regulates ATP-dependent transporters in Rhizobium leguminosarum.

Mutation of ptsP encoding EI(Ntr) of the PTS(Ntr) system in Rhizobium leguminosarum strain Rlv3841 caused a pleiotropic phenotype as observed with many bacteria. The mutant formed dry colonies and grew poorly on organic nitrogen or dicarboxylates. Most strikingly the ptsP mutant had low activity of a broad range of ATP-dependent ABC transporters. This lack of activation, which occurred post-translationally, may explain many of the pleiotropic effects. In contrast proton-coupled transport systems were not inhibited in a ptsP mutant. Regulation by PtsP also involves two copies of ptsN that code for EIIA(Ntr) , resulting in a phosphorylation cascade. As in Escherichia coli, the Rlv3841 PTS(Ntr) system also regulates K(+) homeostasis by transcriptional activation of the high-affinity ATP-dependent K(+) transporter KdpABC. This involves direct interaction of a two-component sensor regulator pair KdpDE with unphosphorylated EIIA(Ntr) . Critically, ptsP mutants, which cannot phosphorylate PtsN1 or PtsN2, had a fully activated KdpABC transporter. This is the opposite pattern from that observed with ABC transporters which apparently require phosphorylation of PtsN. These results suggest that ATP-dependent transport might be regulated via PTS(Ntr) responding to the cellular energy charge. ABC transport may be inactivated at low energy charge, conserving ATP for essential processes including K(+) homeostasis.

Mutation of GOGAT prevents pea bacteroid formation and N2 fixation by globally downregulating transport of organic nitrogen sources.

Mutation of gltB (encoding glutamate oxoglutarate amidotransferase or GOGAT) in RU2307 increased the intracellular Gln:Glu ratio and inhibited amino acid transport via Aap and Bra. The mechanism probably involves global post-translational inhibition independent of Ntr. Transport was separately restored by increased gene expression of Aap or heterologous transporters. Likewise, second site suppressor mutations in the RNA chaperone Hfq elevated transport by Aap and Bra by increasing mRNA levels. Microarrays showed Hfq regulates 34 ABC transporter genes, including aap, bra and opp. The genes coding for integral membrane proteins and ABC subunits aapQMP braDEFGC were more strongly elevated in the hfq mutants than solute-binding proteins (aapJ braC). aapQMP and braDEFG are immediately downstream of stem-loops, indicating Hfq attenuates downstream translation and stability of mRNA, explaining differential expression of ABC genes. RU2307 nodulated peas and bacteria grew down infection threads, but bacteroid development was arrested and N(2) was not fixed. This probably results from an inability to synthesize or transport amino acids. However, GOGAT and GOGAT/AldA double mutants carrying suppressor mutations that increased amino acid uptake fixed N(2) on pea plants. Thus de novo ammonium assimilation into amino acids is unnecessary in bacteroids demonstrating sufficient amino acids are supplied by plants.

Selective epitaxy of semiconductor nanopyramids for nanophotonics.

We present a detailed study of the parameters which affect the geometrical perfection of nanopyramids used for the site-selected nucleation of quantum dots. Through an understanding of crystal facet formation, we demonstrate that undesirable high index planes can be suppressed using carefully optimized lithography together with properly orientated source fluxes in the growth reactor. High quality InP nanopyramids are reported with individual InAs/InP quantum dots positioned with high precision. This represents an important milestone for the fabrication of complex quantum dot based nanophotonic devices.

An L-band monolithic InAs/InP quantum dot mode-locked laser with femtosecond pulses.

We have developed an InAs/InP quantum dot (QD) gain material using a double cap growth procedure and GaP sublayer to tune QDs into the L-band. By using it, a passive L-band mode-locked laser with pulse duration of 445 fs at the repetition rate of 46 GHz was demonstrated. The 3-dB linewidth of the RF spectrum is less than 100 KHz. The lasing threshold injection current is 24 mA with an external differential quantum efficiency of 22% and an average output power of 27 mW. The relationship between pulse duration and 3-dB spectral bandwidth as a function of injection current was investigated.

Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids.

One of the largest contributions to biologically available nitrogen comes from the reduction of N(2) to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids, and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N(2) fixation by Rhizobium leguminosarum bv viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N(2) fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N(2) fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis.

Transcriptomic analysis of Rhizobium leguminosarum biovar viciae in symbiosis with host plants Pisum sativum and Vicia cracca.

Rhizobium leguminosarum bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (Pisum sativum) and vetch (Vicia cracca), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by R. leguminosarum bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is gamma-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins. fix genes were induced early but continued to increase in mature bacteroids, while nif genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn5 mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.

General physicians: born or made? The use of a tracking database to answer medical workforce questions.

BACKGROUND: The aim of the study was to use a tracking database to investigate the perceived influence of various factors on career choices of New Zealand medical graduates and to examine specifically whether experiences at medical school may have an effect on a decision to become a general physician. METHODS: Questionnaires were distributed to medical students in the current University of Auckland programme at entry and exit points. The surveys have been completed by two entry cohorts and an exit one since 2006. RESULTS: The response rates were 70 and 88% in the entry and exit groups, respectively. More than 75% of exiting students reported an interest in pursuing a career in general internal medicine. In 42%, this is a 'strong interest' in general medicine compared with 23% in the entry cohort (P < 0.0001). There is correlation between a positive experience in a clinical rotation and the reported level of interest in that specialty with those indicating a good experience likely to specify career intentions in that area. Having a positive experience in a clinical rotation, positive role models and flexibility in training are the most influential factors affecting career decisions in Auckland medical students. Only 11% of study respondents reported that student loan burden has a significant influence on career decisions. CONCLUSION: Quality experiences on attachments seem essential for undergraduates to promote interest in general medicine. There is potential for curriculum design and clinical experiences to be formulated to promote the 'making' of these doctors. Tracking databases will assist in answering some of these questions.

Characterization of a {gamma}-aminobutyric acid transport system of Rhizobium leguminosarum bv. viciae 3841.

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.