ArsP: a methylarsenite efflux permease

Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3‐nitro‐4‐hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study, C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III) > Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus, ArsP is the first identified efflux system specific for trivalent organoarsenicals.     

Microbial ecosystems are dominated by specialist taxa

Abundance and specificity are two key characteristics of species distribution and biodiversity. Theories of species assembly aim to reproduce the empirical joint patterns of specificity and abundance, with the goal to explain patterns of biodiversity across habitats. The specialist‐generalist paradigm predicts that specialists should have a local advantage over generalists and thus be more abundant. We developed a specificity index to analyse abundance–specificity relationships in microbial ecosystems. By analysing microbiota spanning 23 habitats from three very different data sets covering a wide range of sequencing depths and environmental conditions, we find that habitats are consistently dominated by specialist taxa, resulting in a strong, positive correlation between abundance and specificity. This finding is consistent over several levels of taxonomic aggregation and robust to errors in abundance measures. The relationship explains why shallow sequencing captures similar β‐diversity as deep sequencing, and can be sufficient to capture the habitat‐specific functions of microbial communities.     

Modeling of biopterin-dependent pathways of eNOS for nitric oxide and superoxide production

Endothelial dysfunction is associated with increase in oxidative stress and low NO bioavailability. The endothelial NO synthase (eNOS) uncoupling is considered an important factor in endothelial cell oxidative stress. Under increased oxidative stress, the eNOS cofactor tetrahydrobiopterin (BH₄) is oxidized to dihydrobiopterin, which competes with BH₄ for binding to eNOS, resulting in eNOS uncoupling and reduction in NO production. The importance of the ratio of BH₄ to oxidized biopterins versus absolute levels of total biopterin in determining the extent of eNOS uncoupling remains to be determined. We have developed a computational model to simulate the kinetics of the biochemical pathways of eNOS for both NO and O₂^•− production to understand the roles of BH₄ availability and total biopterin (TBP) concentration in eNOS uncoupling. The downstream reactions of NO, O₂^•−, ONOO⁻, O₂, CO₂, and BH₄ were also modeled. The model predicted that a lower [BH₄]/[TBP] ratio decreased NO production but increased O₂^•− production from eNOS. The NO and O₂^•− production rates were independent above 1.5 μM [TBP]. The results indicate that eNOS uncoupling is a result of a decrease in [BH₄]/[TBP] ratio, and a supplementation of BH₄ might be effective only when the [BH₄]/[TBP] ratio increases. The results from this study will help us understand the mechanism of endothelial dysfunction.     

Funding translational work in cell-based therapy

The cell therapy branch of the regenerative medicine field has been innovative in developing new models of delivery and development and identifying alternative sources of funding. We discuss the implications of these changes for pharmaceutical companies and the opportunities they offer to a new entrepreneur.     

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function^1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.Download video file.^{(49M, mov)}     

Combined millimeter wave and cyclophosphamide therapy of an experimental murine melanoma

The objective of the present studies was to investigate whether millimeter wave (MMW) therapy can increase the efficacy of cyclophosphamide (CPA), a commonly used anti-cancer drug. The effect of combined MMW-CPA treatment on melanoma growth was compared to CPA treatment alone in a murine model. MMWs were produced with a Russian made YAV-1 generator. The device produced 42.2 +/- 0.2 GHz modulated wave radiation through a 10 x 20 mm rectangular output horn. The animals, SKH-1 hairless female mice, were irradiated on the nasal area. Peak SAR and incident power density were measured as 730 +/- 100 W/kg and 36.5 +/- 5 mW/cm2, respectively. The maximum skin surface temperature elevation measured at the end of 30 min irradiation was 1.5 degrees C. B16F10 melanoma cells (0.2 x 10(6)) were implanted subcutaneously into the left flank of mice on day 1 of the experiment. On days 4-8, CPA was administered intraperitoneally (30 mg/kg/day). MMW irradiation was applied concurrently with, prior to or following CPA administration. A significant reduction (P < .05) in tumor growth was observed with CPA treatment, but MMW irradiation did not provide additional therapeutic benefit as compared to CPA alone. Similar results were obtained when MMW irradiation was applied both prior to and following CPA treatment.     

Structural and photoluminescence study of Mn2+‐activated CaYAl3O7 blue phosphors

The structural and photoluminescence properties of CaYAl₃O₇ phosphor material doped with varying concentration of Mn²⁺ have been studied. The phosphor material was synthesized by the combustion method at 500 °C and was characterized using X‐ray diffraction, Fourier transform infrared spectroscopy and photoluminescence spectroscopy (PL). X‐ray diffraction showed that the crystallites have average sizes in the range of ~58–70 nm. Corresponding Fourier transform infrared spectroscopy investigations confirm the phase formation and the presence of aluminate group (Al‐O bands) in CaYAl₃O₇:Mn²⁺ phosphor. Under the excitation at 356 nm wavelength, the PL spectra show the occurrence of two emission peaks obtained in the blue region at 389 nm and 412 nm, which is attributed to the 4 T1(G) → 6A1 transition of Mn²⁺ ion. Upon increasing Mn²⁺ concentration, the relative PL intensity shows an initial decrement followed by an increase displaying the effect of concentration quenching. Overall the results suggest the possibility of using this material in white lighting devices and plasma display panels. Copyright © 2013 John Wiley & Sons, Ltd.     

Kinetic and structural properties of disulfide engineered phospholipase A2: insight into the role of disulfide bonding patterns

The family of secreted 14 kDa phospholipase A(2) (PLA2) enzymes have a common motif for the catalytic site but differ in their disulfide architecture. The functional significance of such structural changes has been analyzed by comparing the kinetic and spectroscopic properties of a series of disulfide mutants engineered into the sequence of pig pancreatic IB PLA2 to resemble the mammalian paralogues of the PLA2 family [Janssen et al. (1999) Eur. J. Biochem. 261, 197-207, 1999]. We report a detailed comparison of the functional parameters of pig iso-PLA2, as well as several of the human homologues, with these disulfide engineered mutants of pig IB PLA2. The crystal structure of the ligand free and the active site inhibitor-MJ33 bound forms of PLA2 engineered to have the disulfide bonding pattern of group-X (eng-X) are also reported and compared with the structure of group-IB and human group-X PLA2. The engineered mutants show noticeable functional differences that are rationalized in terms of spectroscopic properties and the differences detected in the crystal structure of eng-X. A major difference between the eng-mutants is in the calcium binding to the enzyme in the aqueous phase, which also influences the binding of the active site directed ligands. We suggest that the disulfide architecture of the PLA2 paralogues has a marginal influence on interface binding. In this comparison, the modest differences observed in the interfacial kinetics are attributed to the changes in the side chain residues. This in turn influences the coupling of the catalytic cycle to the calcium binding and the interfacial binding event.