Functional Analysis of the Genes Encoding Diaminopropionate Ammonia Lyase in Escherichia coli and Salmonella enterica Serovar Typhimurium

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on DL-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on DL-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on DL-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on DL-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of DL-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by DL-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by DL-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.

Synthesis and supramolecular assembly of the bifunctional borinic acid 1,2-fcB(OH)](2)

Treatment of the chloro-substituted diboradiferrocene derivative 1 with Me3SiOMe and subsequent hydrolysis resulted in formation of the novel organometallic bis(borinic acid) derivative 3. The assembly of 3 into supramolecular structures via hydrogen bonding and reversible covalent boron-oxygen bond formation was explored. Upon crystallization from acetone or THF one-dimensional chains form in which molecules of 3 alternately serve as hydrogen bond donors and acceptors. The additional OH hydrogens that are not involved in hydrogen bonding within the polymeric chains undergo hydrogen bonding to the solvent molecules. Removal of the solvent was achieved at moderate temperature under high vacuum. While the polymeric chains remain intact, in the absence of the solvent as a hydrogen bond acceptor, short contacts to the Cp rings of neighboring polymer strands lead to a network-like structure. At higher temperatures, further dehydration occurs with formation of B-O-B linkages as confirmed by MALDI-TOF mass spectrometry. Oligomers with up to 15 repeating units (30 ferrocenes) were detected.

Electrocatalytic oxidation of C-3-aliphatic alcohols on electrodeposited Pd-PEDOT nanodendrites in Alkaline medium

Nanodendritic Pd is electrodeposited on poly(3,4-ethylenedioxythiophene) (PEDOT) coated carbon paper electrode. Electrodeposited Pd is non-dendritic in the absence of PEDOT. The electrooxidation of C-3-aliphatic alcohols, namely, propanol (PA), 1,2- propanediol (1, 2-PD), 1, 3-propanediol (1, 3-PD), and glycerol (GL) is studied in 1.0 M NaOH. The catalytic activity of nanodendritic Pd is greater than that of non-dendritic Pd for oxidation of the four alcohols molecules. Among those molecules the oxidation rate increases as: PA< 1, 2-PD < 1, 3-PD < GL. The cyclic voltammetric oxidation current peak appearing in the reverse direction of the sweep is greatly influenced by the nature of alcohol. The reduction of oxide film on Pd surface is attributed to affect the magnitude of backward peak current density. The amperometry and repeated cyclic voltammetry data suggest a high stability of nanodendritic Pd in alkaline medium. Glycerol is expected to be an appropriate alcohol for application as a fuel in alkaline fuel cells at nanodendritic electrodeposited Pd.

Numerical simulations of CO2 migration during charnockite genesis

Charnockite is considered to be generated either through the dehydration of granitic magma by CO2 purging or by solid-state dehydration through CO2 metasomatism during granulite facies metamorphism. To understand the extent of dehydration, CO2 migration is quantitatively modeled in silicate melt and metasomatic fluid as a function of temperature, H2O wt%, pressure, basal CO2 flux and dynamic viscosity. Numerical simulations show that CO2 advection through porous and permeable high-grade metamorphic rocks can generate dehydrated patches close to the CO2 flow path, as illustrated by the occurrences of ``incipient charnockites.'' CO2 reaction-front velocity constrained by field observations is 0.69 km/m.y., a reasonable value, which matches well with other studies. On the other hand, temperature, rate of cooling, and basal CO2 flux are the critical parameters affecting CO2 diffusion through a silicate melt. CO2 diffusion through silicate melt can only occur at temperature greater than 840 degrees C and during slow cooling (<= 3.7 x 10(-5) degrees C/yr), features that are typical of magma emplacement in the lower crust. Stalling of CO2 fluxing at similar to 840 degrees C explains why some deep-level plutons contain both hydrous and anhydrous (charnockitic) mineral assemblages. CO2 diffusion through silicate melt is virtually insensitive to pressure. Addition of CO2 basal flux facilitates episodic dehydrated melt migration by generating fracture pathways.

Identification and characterization of starvation induced msdgc-1 promoter involved in the c-di-GMP turnover

C-di-GMP Bis-(3'-5')-cyclic-dimeric-guanosine monophosphate], a second messenger is involved in intracellular communication in the bacterial species. As a result several multi-cellular behaviors in both Gram-positive and Gram-negative bacteria are directly linked to the intracellular level of c-di-GMP. The cellular concentration of c-di-GMP is maintained by two opposing activities, diguanylate cyclase (DGC) and phosphodiesterase (PDE-A). In Mycobacterium smegmatis, a single bifunctional protein MSDGC-1 is responsible for the cellular concentration of c-di-GMP. A better understanding of the regulation of c-di-GMP at the genetic level is necessary to control the function of above two activities. In this work, we have characterized the promoter element present in msdgc-1 along with the + 1 transcription start site and identified the sigma factors that regulate the transcription of msdgc-1. Interestingly, msdgc-1 utilizes SigA during the initial phase of growth, whereas near the stationary phase SigB containing RNA polymerase takes over the expression of msdgc-1. We report here that the promoter activity of msdgc-1 increases during starvation or depletion of carbon source like glucose or glycerol. When msdgc-1 is deleted, the numbers of viable cells are similar to 10 times higher in the stationary phase in comparison to that of the wild type. We propose here that msdgc-1 is involved in the regulation of cell population density. (C) 2013 Elsevier B.V. All rights reserved.

Modulation of redox regulatory molecules and electron transport chain activity in muscle of air breathing fish Heteropneustes fossilis under air exposure stress

Responses of redox regulatory system to long-term survival (> 18 h) of the catfish Heteropneustes fossilis in air are not yet understood. Lipid and protein oxidation level, oxidant (H2O2) generation, antioxidative status (levels of superoxide dismutase, catalase, glutathione peroxidase and reductase, ascorbic acid and non-protein sulfhydryl) and activities of respiratory complexes (I, II, III and IV) in mitochondria were investigated in muscle of H. fossilis under air exposure condition (0, 3, 6, 12 and 18 h at 25 A degrees C). The increased levels of both H2O2 and tissue oxidation were observed due to the decreased activities of antioxidant enzymes in muscle under water deprivation condition. However, ascorbic acid and non-protein thiol groups were the highest at 18 h air exposure time. A linear increase in complex II activity with air exposure time and an increase up to 12 h followed by a decrease in activity of complex I at 18 h were observed. Negative correlation was observed for complex III and V activity with exposure time. Critical time to modulate the above parameters was found to be 3 h air exposure. Dehydration induced oxidative stress due to modulation of electron transport chain and redox metabolizing enzymes in muscle of H. fossilis was clearly observed. Possible contribution of redox regulatory system in muscle tissue of the fish for long-term survival in air is elucidated. Results of the present study may be useful to understand the redox metabolism in muscle of fishes those are exposed to air in general and air breathing fishes in particular.

Structural Insights into Proton Conduction in Gallic Acid-Isoniazid Cocrystals

Hydrated cocrystal of gallic acid-isoniazid displays a single crystal-to-single crystal transformation upon dehydration, resulting in a difference of three orders of magnitude in proton conduction. The conduction pathway is shown to follow the Grotthus mechanism, supported by theoretical (DFT) calculations.

Multimodal shape oscillations of droplets excited by an air stream

The shape dynamics of droplets exposed to an air jet at intermediate droplet Reynolds numbers is investigated. High speed imaging and hot-wire anemometry are employed to examine the mechanism of droplet oscillation. The theory that the vortex shedding behind the droplet induces oscillation is examined. In these experiments, no particular dominant frequency is found in the wake region of the droplet. Hence the inherent free-stream disturbances prove to be driving the droplet oscillations. The modes of droplet oscillation show a band of dominant frequencies near the corresponding natural frequency, further proving that there is no particular forcing frequency involved. In the frequency spectrum of the lowest mode of oscillation for glycerol at the highest Reynolds number, no response is observed below the threshold frequency corresponding to the viscous dissipation time scale. This selective suppression of lower frequencies in the case of glycerol is corroborated by scaling arguments. The influence of surface tension on the droplet oscillation is studied using ethanol as a test fluid. Since a lower surface tension reduces the natural frequency, ethanol shows lower excited frequencies. The oscillation levels of different fluids are quantified using the droplet aspect ratio and correlated in terms of Weber number and Ohnesorge number. (C) 2014 Elsevier Ltd. All rights reserved.

Oscillation dynamics of sessile droplets subjected to substrate vibration

Sessile droplets on a vibrating substrate are investigated focusing on axisymmetric oscillations with pinned contact line. Proper orthogonal decomposition is employed to identify the different modes of droplet shape oscillation and quantitatively assess the droplet oscillation and spectral response. We offer the first experimental evidence for the analogy of an oscillating sessile droplet with a non-linear spring mass damper system. The qualitative and quantitative agreement of amplitude response and phase response curves and limit cycles of the model dynamical system with that observed experimentally suggest that the bulk oscillations in the fundamental mode of a sessile droplet can be very well modeled by a Duffing oscillator with a hard spring, especially near the resonance. The red shift of the resonance peak with an increase in the glycerol concentration is clearly evidenced by both the experimental and predicted amplitude response curves. The influence of various operational parameters such as excitation frequency and amplitude and fluid properties on the droplet oscillation characteristics is adequately captured by the model. (C) 2014 Elsevier Ltd. All rights reserved.

Fabrication of a novel biomaterial with enhanced mechanical and conducting properties

Conducting polymers have the combined advantages of metal conductivity with ease in processing and biocompatibility; making them extremely versatile for biosensor and tissue engineering applications. However, the inherent brittle property of conducting polymers limits their direct use in such applications which generally warrant soft and flexible material responses. Addition of fillers increases the material compliance, but is achieved at the cost of reduced electrical conductivity. To retain suitable conductivity without compromising the mechanical properties, we fabricate an electroactive blend (dPEDOT) using low grade PEDOT: PSS as the base conducting polymer with polyvinyl alcohol as filler and glycerol as a dopant. Bulk dPEDOT films show a thermally stable response till 110 degrees C with over seven fold increase in room temperature conductivity as compared to 0.002 S cm(-1) for pristine PEDOT: PSS. We characterize the nonlinear stress-strain response of dPEDOT, well described using a Mooney-Rivlin hyperelastic model, and report elastomer-like moduli with ductility similar to fives times its original length. Dynamic mechanical analysis shows constant storage moduli over a large range of frequencies with corresponding linear increase in tan(delta). We relate the enhanced performance of dPEDOT with the underlying structural constituents using FTIR and AFM microscopy. These data demonstrate specific interactions between individual components of dPEDOT, and their effect on surface topography and material properties. Finally, we show biocompatibility of dPEDOT using fibroblasts that have comparable cell morphologies and viability as the control, which make dPEDOT attractive as a biomaterial.

Optically transparent polymer devices for in situ assessment of cell electroporation

In order to study cell electroporation in situ, polymer devices have been fabricated from poly-dimethyl siloxane with transparent indium tin oxide parallel plate electrodes in horizontal geometry. This geometry with cells located on a single focal plane at the interface of the bottom electrode allows a longer observation time in both transmitted bright-field and reflected fluorescence microscopy modes. Using propidium iodide (PI) as a marker dye, the number of electroporated cells in a typical culture volume of 10-100 mu l was quantified in situ as a function of applied voltage from 10 to 90 V in a series of 2-ms pulses across 0.5-mm electrode spacing. The electric field at the interface and device current was calculated using a model that takes into account bulk screening of the transient pulse. The voltage dependence of the number of electroporated cells could be explained using a stochastic model for the electroporation kinetics, and the free energy for pore formation was found to be kT at room temperature. With this device, the optimum electroporation conditions can be quickly determined by monitoring the uptake of PI marker dye in situ under the application of millisecond voltage pulses. The electroporation efficiency was also quantified using an ex situ fluorescence-assisted cell sorter, and the morphology of cultured cells was evaluated after the pulsing experiment. Importantly, the efficacy of the developed device was tested independently using two cell lines (C2C12 mouse myoblast cells and yeast cells) as well as in three different electroporation buffers (phosphate buffer saline, electroporation buffer and 10 % glycerol).

Cryogenian magmatism and crustal reworking in the Southern Granulite Terrane, India

Understanding Neoproterozoic crustal evolution is fundamental to reconstructing the Gondwana supercontinent, which was assembled at this time. Here we report evidence of Cryogenian crustal reworking in the Madurai Block of the Southern Granulite Terrane of India. The study focuses on a garnet-bearing granite-charnockite suite, where the granite shows in situ dehydration into patches and veins of incipient charnockite along the contact with charnockite. The granite also carries dismembered layers of Mg-Al-rich granulite. Micro-textural evidence for dehydration of granite in the presence of CO2-rich fluids includes the formation of orthopyroxene by the breakdown of biotite, neoblastic zircon growth in the dehydration zone, at around 870 degrees C and 8kbar. The zircon U-Pb ages suggest formation of the granite, charnockite, and incipient charnockite at 836 +/- 73, 831 +/- 31, and 772 +/- 49Ma, respectively. Negative zircon epsilon Hf (t) (-5 to -20) values suggest that these rocks were derived from a reworked Palaeoproterozoic crustal source. Zircon grains in the Mg-Al-rich granulite record a spectrum of ages from ca. 2300 to ca. 500Ma, suggesting multiple provenances ranging from Palaeoproterozoic to mid-Neoproterozoic, with neoblastic zircon growth during high-temperature metamorphism in the Cambrian. We propose that the garnet-bearing granite and charnockite reflect the crustal reworking of aluminous crustal material indicated by the presence of biotite+quartz+aluminosilicate inclusions in the garnet within the granite. This crustal source can be the Mg-Al-rich layers carried by the granite itself, which later experienced high-temperature regional metamorphism at ca. 550Ma. Our model also envisages that the CO2 which dehydrated the garnet-bearing granite generating incipient charnockite was sourced from the proximal massive charnockite through advection. These Cryogenian crustal reworking events are related to prolonged tectonic activities prior to the final assembly of the Gondwana supercontinent.

Linking carbon metabolism to carotenoid production in mycobacteria using Raman spectroscopy

Bacteria can utilize multiple sources of carbon for growth, and for pathogenic bacteria like Mycobacterium tuberculosis, this ability is crucial for survival within the host. In addition, phenotypic changes are seen in mycobacteria grown under different carbon sources. In this study, we use Raman spectroscopy to analyze the biochemical components present in M. smegmatis cells when grown in three differently metabolized carbon sources. Our results show that carotenoid biosynthesis is enhanced when M. smegmatis is grown in glucose compared to glycerol and acetate. We demonstrate that this difference is most likely due to transcriptional upregulation of the carotenoid biosynthesis operon (crt) mediated by higher levels of the stress-responsive sigma factor SigF. Moreover, we find that increased SigF and carotenoid levels correlate with greater resistance of glucose-grown cells to oxidative stress. Thus, we demonstrate the use of Raman spectroscopy in unraveling unknown aspects of mycobacterial physiology and describe a novel effect of carbon source variation on mycobacteria.

Cybernetic Modeling Revisited: A Method for Inferring the Cybernetic Variables u(i) from Experimental Data

The cybernetic modeling framework for the growth of microorganisms provides for an elegant methodology to account for the unknown regulatory phenomena through the use of cybernetic variables for enzyme induction and activity. In this paper, we revisit the assumption of limited resources for enzyme induction (Sigma u(i) = 1) used in the cybernetic modeling framework by presenting a methodology for inferring the individual cybernetic variables u(i) from experimental data. We use this methodology to infer u(i) during the simultaneous consumption of glycerol and lactose by Escherichia coli and then model the fitness trade-offs involved in the recently discovered predictive regulation strategy of microorganisms.

Gold catalyzed intramolecular hydroalkoxylation assisted ring opening of furans to the corresponding saturated gamma-keto esters

A facile ring opening of furans in furyl propargyl alcohols to the corresponding saturated gamma-keto esters is observed in the gold(III) chloride catalyzed reaction with MeOH. It is found that the ring opening of furan is driven by the intramolecular hydroalkoxylation. Mitigating the intramolecular hydroalkoxylation led to the expected conjugated enyne resulting from the dehydration. (C) 2015 Elsevier Ltd. All rights reserved.