Measurement of proton-driven antiport in Escherichia coli

Secondary active transport of substrates across the inner membrane is vital to the bacterial cell. Of the secondary active transporter families, the ubiquitous major facilitator superfamily (MFS) is the largest and most functionally diverse (Reddy et al., 2012). Recently, it was reported that the MFS multidrug efflux protein MdtM from Escherichia coli (E. coli) functions physiologically in protection of bacterial cells against bile salts (Paul et al., 2014). The MdtM transporter imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H+) to the efflux of bile salts from the cell interior via an antiport reaction. This protocol describes, using fluorometry, how to detect the bile salt/H+ antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆pH. This method exploits the changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the pH-sensitive dye acridine orange in response to changes in [H+] in the vesicular lumen. Due to low levels of endogenous transporter expression that would normally make the contribution of individual transporters such as MdtM to proton-driven antiport difficult to detect, the method typically necessitates that the transporter of interest be overexpressed from a multicopy plasmid. Although the first section of the protocol described here is very specific to the overexpression of MdtM from the pBAD/Myc-His A expression vector, the protocol describing the subsequent measurement of bile salt efflux by MdtM can be readily adapted for measurement of antiport of other substrates by any other antiporter that exchanges protons for countersubstrate.

Beam profiles of proton and carbon ions in the relativistic transparency regime

Ion acceleration from relativistic laser solid interactions has been of particular interest over the last decade. While beam profiles have been studied for target normal sheath acceleration (TNSA), such profiles have yet to be described for other mechanisms. Here, experimental data is presented, investigating ion beam profiles from acceleration governed by relativistic transparent laser plasma interaction. The beam shape of carbon C6+ ions and protons has been measured simultaneously with a wide angle spectrometer. It was found that ion beams deviate from the typical Gaussian-like shape found with TNSA and that the profile is governed by electron dynamics in the volumetric laser-plasma interaction with a relativistically transparent plasma; due to the ponderomotive force electrons are depleted from the center of the laser axis and form lobes affecting the ion beam structure. The results are in good agreement with high resolution three-dimensional-VPIC simulations and can be used as a new tool to experimentally distinguish between different acceleration mechanisms.

Transport of laser accelerated proton beams and isochoric heating of matter

The acceleration of intense proton and ion beams by ultra-intense lasers has matured to a point where applications in basic research and technology are being developed. Crucial for harvesting the unmatched beam parameters driven by the relativistic electron sheath is the precise control of the beam. We report on recent experiments using the PHELIX laser at GSI, the VULCAN laser at RAL and the TRIDENT laser at LANL to control and use laser accelerated proton beams for applications in high energy density research. We demonstrate efficient collimation of the proton beam using high field pulsed solenoid magnets, a prerequisite to capture and transport the beam for applications. Furthermore we report on two campaigns to use intense, short proton bunches to isochorically heat solid targets up to the warm dense matter state. The temporal profile of the proton beam allows for rapid heating of the target, much faster than the hydrodynamic response time thereby creating a strongly coupled plasma at solid density. The target parameters are then probed by X-ray Thomson scattering (XRTS) to reveal the density and temperature of the heated volume. This combination of two powerful techniques developed during the past few years allows for the generation and investigation of macroscopic samples of matter in states present in giant planets or the interior of the earth.

Determination of 3,4-methylenedioxyamphetamine analogues (''ecstasy'') by proton nuclear magnetic resonance spectrometry

A procedure for the determination of three commonly encountered ecstasy type drugs has been demonstrated using proton nuclear magnetic resonance spectrometry (H-1-NMR).

Visible-Wavelength Survey of Jupiter-Family Cometary Nuclei as Part of SEPPCoN

We present observations of a statistically-significant number of Jupiter-family cometary nuclei as part of SEPPCoN (Survey of the Ensemble Physical Properties of Cometary Nuclei). We present preliminary results on distributions of albedos and shapes.

Visible-wavelength Observations of Jupiter-family Comet Nuclei as Part of Seppcon.

We present the latest analysis and results from SEPPCoN (Survey of Ensemble Physical Properties of Cometary Nuclei). This on-going survey involves studying 100 JFCs - about 25% of the known population - at both mid-infrared and visible wave-lengths to constrain the distributions of sizes, shapes, spins, and albedos of this population. Having earlier reported results from measuring thermal emissions of our sample nuclei [1,2,3,4], we report here progress on the visible-wavelength observations that we have obtained at many ground-based facilities in Chile, Spain, and the United States. To date we have attempted observations of 91% of our sample of 100 JFCs, and at least 64 of those were successfully detected. In most cases the comets were at heliocentric distances between 3.0 and 6.5 AU so as to decrease the odds of a comet having a coma. Of the 64 detected comets, 48 were apparently bare, having no extended emission. Our datasets are further augmented by archival data and photometry from the NEAT program [5]. An important goal of SEPPCoN is to accumulate a large comprehensive set of high quality physical data on cometary nuclei in order to make accurate statistical comparisons with other minor-body populations such as Trojans, Centaurs, and Kuiper-belt objects. Information on the size, shape, spin-rate, albedo and color distributions is critical for understanding their origins and evolutionary processes affecting them.

Solar phase functions of cometary nuclei

Relatively few measurements of the solar phase function of cometary nuclei exist, despite the importance of this parameter in determining accurate sizes and its use in modeling surface properties. We make use of robotic telescopes and servicemode observing to monitor cometary nuclei over months at a time, combining intensive observations at a single epoch with regular short light-curve segments to efficiently account for brightness changes due to both nucleus rotation and changing solar phase angle. We present our latest results on comets 8P/Tuttle, 14P/Wolf, 67P/Churyumov- Gerasimenko and 110P/Hartley 3.

A Hundred Comets: The Visual-Wavelength Observations of the Survey of Ensemble Physical Properties of Cometary Nuclei (SEPPCoN)

We present new results from SEPPCoN, a Survey of Ensemble Physical Properties of Cometary Nuclei. This project is currently surveying 100 Jupiter-family comets (JFCs) to measure the mid-infrared thermal emission and visible reflected sunlight of the nuclei. The scientific goal is to determine the distributions of radius, geometric albedo, thermal inertia, axial ratio, and color among the JFC nuclei. In the past we have presented results from the completed mid-IR observations of our sample [1]; here we present preliminary results from ongoing, broadband visible-wavelength observations of nuclei obtained from a variety of ground-based facilities (Mauna Kea, Cerro Pachon, La Silla, La Palma, Apache Point, Table Mtn., and Palomar Mtn.), including contributions from the Near Earth Asteroid Telescope project (NEAT) archive. The nuclei were observed at high heliocentric distance (usually over 4 AU) and so many comets show either no or little contamination from dust coma. While several nuclei have been observed as snapshots, we have multiepoch photometry for many of our targets. With our datasets we are building a large database of photometry, and such a database is essential to the derivation of albedo and shape of a large number of nuclei, and to the understanding of biases in the survey. Support for this work was provided by NSF and the NASA Planetary Astronomy program. Reference: [1] Fernandez, Y.R., et al. 2007, BAAS 39, 827.

Proton irradiation of DNA nucleosides in the gas phase

The four DNA nucleosides guanosine, adenosine, cytidine and thymidine have been produced in the gas phase by a laser thermal desorption source, and irradiated by a beam of protons with 5 keV kinetic energy. The molecular ions as well as energetic neutrals formed have been analyzed by mass spectrometry in order to shed light on the ionization and fragmentation processes triggered by proton collision. A range of 8-20 eV has been estimated for the binding energy of the electron captured by the proton. Glycosidic bond cleavage between the base and sugar has been observed with a high probability for all nucleosides, resulting in predominantly intact base ions for guanosine, adenosine, and cytidine but not for thymidine where intact sugar ions are dominant. This behavior is influenced by the ionization energies of the nucleobases (G < A < C < T), which seems to determine the localization of the charge following the initial ionization. This charge transfer process can also be inferred from the production of protonated base ions, which have a similar dependence on the base ionization potential. Other dissociation pathways have also been identified, including further fragmentation of the base and sugar moieties for thymidine and guanosine, respectively, and partial breakup of the sugar ring without glycosidic bond cleavage mainly for adenosine and cytidine. These results show that charge localization following ionization by proton irradiation is important in determining dissociation pathways of isolated nucleosides, which could in turn influence direct radiation damage in DNA.

Three motAB stator gene products in Bdellovibrio bacteriovorus contribute to motility of a single flagellum during predatory and prey-independent growth

The predatory bacterium Bdellovibrio bacteriovorus uses flagellar motility to locate regions rich in Gram-negative prey bacteria, colliding and attaching to prey and then ceasing flagellar motility. Prey are then invaded to form a "bdelloplast" in a type IV pilus-dependent process, and prey contents are digested, allowing Bdellovibrio growth and septation. After septation, Bdellovibrio flagellar motility resumes inside the prey bdelloplast prior to its lysis and escape of Bdellovibrio progeny. Bdellovibrio can also grow slowly outside prey as long flagellate host-independent (HI) cells, cultured on peptone-rich media. The B. bacteriovorus HD100 genome encodes three pairs of MotAB flagellar motor proteins, each of which could potentially form an inner membrane ion channel, interact with the FliG flagellar rotor ring, and produce flagellar rotation. In 2004, Flannagan and coworkers (R. S. Flannagan, M. A. Valvano, and S. F. Koval, Microbiology 150:649-656, 2004) used antisense RNA and green fluorescent protein (GFP) expression to downregulate a single Bdellovibrio motA gene and reported slowed release from the bdelloplast and altered motility of the progeny. Here we inactivated each pair of motAB genes and found that each pair contributes to motility, both predatorily, inside the bdelloplast and during HI growth; however, each pair was dispensable, and deletion of no pair abolished motility totally. Driving-ion studies with phenamil, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and different pH and sodium conditions indicated that all Mot pairs are proton driven, although the sequence similarities of each Mot pair suggests that some may originate from halophilic species. Thus, Bdellovibrio is a "dedicated motorist," retaining and expressing three pairs of mot genes.

Massive stars exploding in a He-rich circumstellar medium - VI. Observations of two distant Type Ibn supernova candidates discovered by La Silla-QUEST

We present optical observations of the peculiar stripped-envelope supernovae (SNe) LSQ12btw and LSQ13ccw discovered by the La Silla-QUEST survey. LSQ12btw reaches an absolute peak magnitude of M-g = -19.3 +/- 0.2, and shows an asymmetric light curve. Stringent pre-discovery limits constrain its rise time to maximum light to less than 4 d, with a slower post-peak luminosity decline, similar to that experienced by the prototypical SN Ibn 2006jc. LSQ13ccw is somewhat different: while it also exhibits a very fast rise to maximum, it reaches a fainter absolute peak magnitude (M-g =-18.4 +/- 0.2), and experiences an extremely rapid post-peak decline similar to that observed in the peculiar SN Ib 2002bj. A stringent pre-discovery limit and an early marginal detection of LSQ13ccw allow us to determine the explosion time with an uncertainty of +/- 1 d. The spectra of LSQ12btw show the typical narrow He I emission lines characterizing Type Ibn SNe, suggesting that the SN ejecta are interacting with He-rich circumstellar material. The He I lines in the spectra of LSQ13ccw exhibit weak narrow emissions superposed on broad components. An unresolved H alpha line is also detected, suggesting a tentative Type Ibn/IIn classification. As for other SNe Ibn, we argue that LSQ12btw and LSQ13ccw likely result from the explosions of Wolf-Rayet stars that experienced instability phases prior to core collapse. We inspect the host galaxies of SNe Ibn, and we show that all of them but one are hosted in spiral galaxies, likely in environments spanning a wide metallicity range.

Massive stars exploding in a He-rich circumstellar medium - V. Observations of the slow-evolving SN Ibn OGLE-2012-SN-006

We present optical observations of the peculiar Type Ibn supernova (SN Ibn) OGLE-2012-SN-006, discovered and monitored by the Optical Gravitational Lensing Experiment-IV survey, and spectroscopically followed by Public ESO Spectroscopic Survey of Transient Objects (PESSTO) at late phases. Stringent pre-discovery limits constrain the explosion epoch with fair precision to JD = 245 6203.8 +/- 4.0. The rise time to the I-band light-curve maximum is about two weeks. The object reaches the peak absolute magnitude M-I = -19.65 +/- 0.19 on JD = 245 6218.1 +/- 1.8. After maximum, the light curve declines for about 25 d with a rate of 4 mag (100 d)(-1). The symmetric I-band peak resembles that of canonical Type Ib/c supernovae (SNe), whereas SNe Ibn usually exhibit asymmetric and narrower early-time light curves. Since 25 d past maximum, the light curve flattens with a decline rate slower than that of the Co-56-Fe-56 decay, although at very late phases it steepens to approach that rate. However, other observables suggest that the match with the Co-56 decay rate is a mere coincidence, and the radioactive decay is not the main mechanism powering the light curve of OGLE-2012-SN-006. An early-time spectrum is dominated by a blue continuum, with only a marginal evidence for the presence of He I lines marking this SN type. This spectrum shows broad absorptions bluewards than 5000 angstrom, likely O II lines, which are similar to spectral features observed in superluminous SNe at early epochs. The object has been spectroscopically monitored by PESSTO from 90 to 180 d after peak, and these spectra show the typical features observed in a number of SN 2006jc-like events, including a blue spectral energy distribution and prominent and narrow (v(FWHM) approximate to 1900 km s(-1)) He I emission lines. This suggests that the ejecta are interacting with He-rich circumstellar material. The detection of broad (10(4) km s(-1)) O I and Ca II features likely produced in the SN ejecta (including the [OI] lambda lambda 6300,6364 doublet in the latest spectra) lends support to the interpretation of OGLE-2012-SN-006 as a core-collapse event.

Massive stars exploding in a He-rich circumstellar medium - IV. Transitional Type Ibn supernovae

We present ultraviolet, optical and near-infrared data of the Type Ibn supernovae (SNe) 2010al and 2011hw. SN 2010al reaches an absolute magnitude at peak of M-R = -18.86 +/- 0.21. Its early light curve shows similarities with normal SNe Ib, with a rise to maximum slower than most SNe Ibn. The spectra are dominated by a blue continuum at early stages, with narrow P-Cygni He I lines indicating the presence of a slow-moving, He-rich circumstellar medium. At later epochs, the spectra well match those of the prototypical SN Ibn 2006jc, although the broader lines suggest that a significant amount of He was still present in the stellar envelope at the time of the explosion. SN 2011hw is somewhat different. It was discovered after the first maximum, but the light curve shows a double peak. The absolute magnitude at discovery is similar to that of the second peak (M-R = -18.59 +/- 0.25), and slightly fainter than the average of SNe Ibn. Though the spectra of SN 2011hw are similar to those of SN 2006jc, coronal lines and narrow Balmer lines are clearly detected. This indicates substantial interaction of the SN ejecta with He-rich, but not H-free, circumstellar material. The spectra of SN 2011hw suggest that it is a transitional SN Ibn/IIn event similar to SN 2005la. While for SN 2010al the spectrophotometric evolution favours a H-deprived Wolf-Rayet progenitor (of WN-type), we agree with the conclusion of Smith et al. that the precursor of SN 2011hw was likely in transition from a luminous blue variable to an early Wolf-Rayet (Ofpe/WN9) stage.