Diversity and evolution of the small multidrug resistance protein family

<p>Abstract</p> <p>Background</p> <p>Members of the small multidrug resistance (SMR) protein family are integral membrane proteins characterized by four α-helical transmembrane strands that confer resistance to a broad range of antiseptics and lipophilic quaternary ammonium compounds (QAC) in bacteria. Due to their short length and broad substrate profile, SMR proteins are suggested to be the progenitors for larger α-helical transporters such as the major facilitator superfamily (MFS) and drug/metabolite transporter (DMT) superfamily. To explore their evolutionary association with larger multidrug transporters, an extensive bioinformatics analysis of SMR sequences (> 300 Bacteria taxa) was performed to expand upon previous evolutionary studies of the SMR protein family and its origins.</p> <p>Results</p> <p>A thorough annotation of unidentified/putative SMR sequences was performed placing sequences into each of the three SMR protein subclass designations, namely small multidrug proteins (SMP), suppressor of <it>groEL </it>mutations (SUG), and paired small multidrug resistance (PSMR) using protein alignments and phylogenetic analysis. Examination of SMR subclass distribution within Bacteria and Archaea taxa identified specific Bacterial classes that uniquely encode for particular SMR subclass members. The extent of selective pressure acting upon each SMR subclass was determined by calculating the rate of synonymous to non-synonymous nucleotide substitutions using Syn-SCAN analysis. SUG and SMP subclasses are maintained under moderate selection pressure in comparison to integron and plasmid encoded SMR homologues. Conversely, PSMR sequences are maintained under lower levels of selection pressure, where one of the two PSMR pairs diverges in sequence more rapidly than the other. SMR genomic loci surveys identified potential SMR efflux substrates based on its gene association to putative operons that encode for genes regulating amino acid biogenesis and QAC-like metabolites. SMR subclass protein transmembrane domain alignments to Bacterial/Archaeal transporters (BAT), DMT, and MFS sequences supports SMR participation in multidrug transport evolution by identifying common TM domains.</p> <p>Conclusion</p> <p>Based on this study, PSMR sequences originated recently within both SUG and SMP clades through gene duplication events and it appears that SMR members may be evolving towards specific metabolite transport.</p

High-throughput metal susceptibility testing of microbial biofilms

BACKGROUND: Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated. RESULTS: This high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO(3)(2-)) than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic cell E. coli JM109 to metals was time-dependent. CONCLUSION: This method results in accurate, easily reproducible comparisons between the susceptibility of planktonic cells and biofilms to metals. Further, it was possible to make direct comparisons of the ability of different microbial strains to withstand metal toxicity. The data presented here also indicate that exposure time is an important variable in metal susceptibility testing of bacteria

Nonparametric estimation of correlation functions in longitudinal and spatial data, with application to colon carcinogenesis experiments

In longitudinal and spatial studies, observations often demonstrate strong correlations that are stationary in time or distance lags, and the times or locations of these data being sampled may not be homogeneous. We propose a nonparametric estimator of the correlation function in such data, using kernel methods. We develop a pointwise asymptotic normal distribution for the proposed estimator, when the number of subjects is fixed and the number of vectors or functions within each subject goes to infinity. Based on the asymptotic theory, we propose a weighted block bootstrapping method for making inferences about the correlation function, where the weights account for the inhomogeneity of the distribution of the times or locations. The method is applied to a data set from a colon carcinogenesis study, in which colonic crypts were sampled from a piece of colon segment from each of the 12 rats in the experiment and the expression level of p27, an important cell cycle protein, was then measured for each cell within the sampled crypts. A simulation study is also provided to illustrate the numerical performance of the proposed method.Comment: Published in at http://dx.doi.org/10.1214/009053607000000082 the Annals of Statistics (http://www.imstat.org/aos/) by the Institute of Mathematical Statistics (http://www.imstat.org

Hormonal contraceptive methods and risk of HIV acquisition in women : a systematic review of epidemiological evidence

Peer reviewedPublisher PD

Super stellar clusters with a bimodal hydrodynamic solution: an Approximate Analytic Approach

We look for a simple analytic model to distinguish between stellar clusters undergoing a bimodal hydrodynamic solution from those able to drive only a stationary wind. Clusters in the bimodal regime undergo strong radiative cooling within their densest inner regions, which results in the accumulation of the matter injected by supernovae and stellar winds and eventually in the formation of further stellar generations, while their outer regions sustain a stationary wind. The analytic formulae are derived from the basic hydrodynamic equations. Our main assumption, that the density at the star cluster surface scales almost linearly with that at the stagnation radius, is based on results from semi-analytic and full numerical calculations. The analytic formulation allows for the determination of the threshold mechanical luminosity that separates clusters evolving in either of the two solutions. It is possible to fix the stagnation radius by simple analytic expressions and thus to determine the fractions of the deposited matter that clusters evolving in the bimodal regime blow out as a wind or recycle into further stellar generations.Comment: 5 pages, 4 figures, accepted by A&

Shock Speed, Cosmic Ray Pressure, and Gas Temperature in the Cygnus Loop

Upper limits on the shock speeds in supernova remnants can be combined with post-shock temperatures to obtain upper limits on the ratio of cosmic ray to gas pressure (P_CR / P_G) behind the shocks. We constrain shock speeds from proper motions and distance estimates, and we derive temperatures from X-ray spectra. The shock waves are observed as faint H-alpha filaments stretching around the Cygnus Loop supernova remnant in two epochs of the Palomar Observatory Sky Survey (POSS) separated by 39.1 years. We measured proper motions of 18 non-radiative filaments and derived shock velocity limits based on a limit to the Cygnus Loop distance of 576 +/- 61 pc given by Blair et al. for a background star. The PSPC instrument on-board ROSAT observed the X-ray emission of the post-shock gas along the perimeter of the Cygnus Loop, and we measure post-shock electron temperature from spectral fits. Proper motions range from 2.7 arcseconds to 5.4 arcseconds over the POSS epochs and post-shock temperatures range from kT ~ 100-200 eV. Our analysis suggests a cosmic ray to post-shock gas pressure consistent with zero, and in some positions P_CR is formally smaller than zero. We conclude that the distance to the Cygnus Loop is close to the upper limit given by the distance to the background star and that either the electron temperatures are lower than those measured from ROSAT PSPC X-ray spectral fits or an additional heat input for the electrons, possibly due to thermal conduction, is required.Comment: Submitted to ApJ, 7 color figure

Cigarette smoke exposure mediated generation of Platelet-activating factor agonists induces systemic immunosuppression

poster abstractThe ubiquitous environmental pollutant cigarette smoke (CS) is known to exert immodulatory effects. CS also acts as a potent pro-oxidative stressor. Several studies including ours have characterized the importance of various pro-oxidative stressors including UVB to inhibit host immunity and an importance of the platelet-activating factor (1-alkyl-2-acetyl-glycerophosphocholine; PAF), a potent lipid mediator in this process. PAF is produced enzymatically in a tightly-controlled process. In addition, oxidative stressors can act directly on glycerophosphocholines (GPC) to produce oxidized GPC which are potent PAF-R agonists. The present studies employed model systems consisting of PAF-receptor (PAF-R)-expressing (KBP) and–deficient (KBM) cells and mice (wild type [WT] and Pafr-/-) to determine whether CS exposure could generate PAF-R agonists in blood and whether it could suppress contact hypersensitivity reactions in a PAF-R-dependent manner. We show that lipid extracts derived from the blood of CS-treated WT mice resulted in immediate intracellular calcium (Ca2+2+mice. This inhibitory effect of CS in WT mice were similar to those induced by a PAF-R agonist, CPAF or histamine. Furthermore, this inhibition of CHS by CS in WT mice was blocked by antioxidants vitamin C and N-acetyl cysteine. These findings indicate that CS exposure induces systemic immunosuppression in a PAF-R-dependent manner. These studies provide the first evidence that the pro-oxidative stressor CS can modulate cutaneous immunity via the generation of PAF agonists through lipid oxidation.) mobilization response only in KBP cells. However, no Camobilization response was detected with lipid extracts from non-smoked (sham) mice both in KBP and KBM cells. In addition, lipid extracts only from CS-treated mice induced an increase in IL-8 secretion in KBP cells indicating that CS generates systemic PAF-R agonists. CS exposure also inhibited contact hypersensitivity to the allergen dinitrofluorobenzene (DNFB) selectively in WT but not inPafr-/