Data on Spectrum-Based Fluorescence Resonance Energy Transfer Measurement of \u3cem\u3eE. coli\u3c/em\u3e Multidrug Transporter AcrB

This paper presented the dataset of correction parameters used in the determination of the energy transfer efficiencies from the spectrum-based fluorescence resonance energy transfer (FRET) measurement in a trimeric membrane protein AcrB. The cyan fluorescent protein (CFP) and yellow fluorescent protein (YPet) were used as the donor and acceptor, respectively. Two AcrB fusion proteins were constructed, AcrB-CFP and AcrB-YPet. The proteins were co-expressed in Escherichia coli cells, and energy transfer efficiency were determined in live cells. To obtain reliable energy transfer data, a complete set of correction parameters need to be first determined to accommodate for factors such as background fluorescence and spectra overlap. This paper described the methodology and determination of the correction factors, which are useful data and reference points for researchers working on fluorescence measurement of membrane protein complexes in live bacteria cells. Further interpretation and discussion of these data can be found in “Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter” (Wang et al., in press)

Thin film calixresorcinarene membranes for chemical sensing.

Novel applications for calix[4]recorcinarene (C[4]RA) sensing membranes have been investigated. A comprehensive deposition study was carried out, encompassing casting, spin coating and Langmuir Blodgett (LB) deposition. The spin coating thickness depended on the angular velocity w, following the description of d = c.wx with coefficients of x - 0.44 and - 0.48 for concentrations of 1 mg/ml and 2mg/ml respectively. The analysis of the LB deposited films established a thickness of 0.95 nm for a monolayer. Furthermore the C[4]RA, was successfully employed as a deposition matrix for a non surface active polymer, poly-ortho-methoxy aniline (POMA), which can otherwise not be deposited by LB. The composite film showed good homogeneity and based on thickness and UV measurements a structural model for it was developed, in which two polymer strands aligned themselves per C[4]RA layer, resulting in a monolayer thickness of 2.1 - 2.2 nm.The response of the C[4]RA and the composite membranes to a variety of organic and inorganic gaseous pollutants was investigated by Surface Plasmon Resonance studies, conductivity and capacitance measurements and UV spectroscopic studies. The integration of cast films into the gate of a charge flow transistor, is the first application of pure calixarenes in a conduction based sensor. The turn on response of the transistor is modulated by a variety of organic vapours, at the saturation vapour pressure, showing selectivity between polar and non-polar solvents, i.e. chloroform, methanol, acetone and hexane, with no cross sensitivity to water vapour. The modulation lies within factors of 45 for CHCI3 and 13 for CH3OH. The conductivity increase is partially attributed to micro-condensation of the vapours inside the micro-porous membrane. A successful application of this implementation as an explosion guard sensor for acetone is demonstrated.Gold electrodes with and without C[4]RA LB films have been characterised using impedance spectroscopy and cyclic voltammography. The modification of the gold electrodes by the C[4]RA film changes their constant phase element impedance, givenby 1/(w.Q)" with w being the angular velocity. The observed values were from Q =0.725-10 -5 Farad and n = 0.87 to Q = 0.828-10-6 Farad and n = 0.82. Organic analyteslike, chloroform and acetone in water can be successfully detected with these electrodes. It is shown by cyclic voltammetry, that the permeability of the C[4]RA LB films is modulated by the organic solvents

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe pokes the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization

NMR Spectroscopy Analysis Reveals Differential Metabolic Responses in Arabidopsis Roots and Leaves Treated with a Cytokinesis Inhibitor

In plant cytokinesis, de novo formation of a cell plate evolving into the new cell wall partitions the cytoplasm of the dividing cell. In our earlier chemical genomics studies, we identified and characterized the small molecule endosidin-7, that specifically inhibits callose deposition at the cell plate, arresting late-stage cytokinesis in arabidopsis. Endosidin-7 has emerged as a very valuable tool for dissecting this essential plant process. To gain insights regarding its mode of action and the effects of cytokinesis inhibition on the overall plant response, we investigated the effect of endosidin-7 through a nuclear magnetic resonance spectroscopy (NMR) metabolomics approach. In this case study, metabolomics profiles of arabidopsis leaf and root tissues were analyzed at different growth stages and endosidin-7 exposure levels. The results show leaf and root-specific metabolic profile changes and the effects of endosidin-7 treatment on these metabolomes. Statistical analyses indicated that the effect of endosidin-7 treatment was more significant than the developmental impact. The endosidin-7 induced metabolic profiles suggest compensations for cytokinesis inhibition in central metabolism pathways. This study further shows that long-term treatment of endosidin-7 profoundly changes, likely via alteration of hormonal regulation, the primary metabolism of arabidopsis seedlings. Hormonal pathway-changes are likely reflecting the plant’s responses, compensating for the arrested cell division, which in turn are leading to global metabolite modulation. The presented NMR spectral data are made available through the Metabolomics Workbench, providing a reference resource for the scientific community

Four-dimensional quantitative analysis of cell plate development in Arabidopsis using lattice light sheet microscopy identifies robust transition points between growth phases.

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process

Root vacuolar sequestration and suberization are prominent responses of Pistacia spp. rootstocks during salinity stress

Understanding the mechanisms of stress tolerance in diverse species is needed to enhance crop performance under conditions such as high salinity. Plant roots, in particular in grafted agricultural crops, can function as a boundary against external stresses in order to maintain plant fitness. However, limited information exists for salinity stress responses of woody species and their rootstocks. Pistachio (Pistacia spp.) is a tree nut crop with relatively high salinity tolerance as well as high genetic heterogeneity. In this study, we used a microscopy-based approach to investigate the cellular and structural responses to salinity stress in the roots of two pistachio rootstocks, Pistacia integerrima (PGI) and a hybrid, P. atlantica x P. integerrima (UCB1). We analyzed root sections via fluorescence microscopy across a developmental gradient, defined by xylem development, for sodium localization and for cellular barrier differentiation via suberin deposition. Our cumulative data suggest that the salinity response in pistachio rootstock species is associated with both vacuolar sodium ion (Na+) sequestration in the root cortex and increased suberin deposition at apoplastic barriers. Furthermore, both vacuolar sequestration and suberin deposition correlate with the root developmental gradient. We observed a higher rate of Na+ vacuolar sequestration and reduced salt-induced leaf damage in UCB1 when compared to P. integerrima. In addition, UCB1 displayed higher basal levels of suberization, in both the exodermis and endodermis, compared to P. integerrima. This difference was enhanced after salinity stress. These cellular characteristics are phenotypes that can be taken into account during screening for sodium-mediated salinity tolerance in woody plant species

Investigation of Salt Tolerance Mechanisms across a Root Developmental Gradient in Almond Rootstocks

The intensive use of groundwater in agriculture under the current climate conditions leads to acceleration of soil salinization. Given that almond is a salt-sensitive crop, selection of salt-tolerant rootstocks can help maintain productivity under salinity stress. Selection for tolerant rootstocks at an early growth stage can reduce the investment of time and resources. However, salinity-sensitive markers and salinity tolerance mechanisms of almond species to assist this selection process are largely unknown. We established a microscopy-based approach to investigate mechanisms of stress tolerance in and identified cellular, root anatomical, and molecular traits associated with rootstocks exhibiting salt tolerance. We characterized three almond rootstocks: Empyrean-1 (E1), Controller-5 (C5), and Krymsk-86 (K86). Based on cellular and molecular evidence, our results show that E1 has a higher capacity for salt exclusion by a combination of upregulating ion transporter expression and enhanced deposition of suberin and lignin in the root apoplastic barriers, exodermis, and endodermis, in response to salt stress. Expression analyses revealed differential regulation of cation transporters, stress signaling, and biopolymer synthesis genes in the different rootstocks. This foundational study reveals the mechanisms of salinity tolerance in almond rootstocks from cellular and structural perspectives across a root developmental gradient and provides insights for future screens targeting stress response

Report and preliminary results of SONNE cruise SO175, Miami - Bremerhaven, 12.11 - 30.12.2003 : (GAP, Gibraltar Arc Processes)

Expedition SO175 using FS Sonne aimed for a multidisciplinerary geoscientific approach with an international group of researchers. Methods covered the entire span from geophysical data acquisition (seafloor mapping, echography, seismic reflection), sediment coring at sites of active fluid venting, in situ heat flow measurements across the entire length of the Gibraltar thrust wedge, the deformation front, landslide bodies, and mud volcanoes, and finally the deployment of a long-term pore pressure probe. Video-supported operations helped to identify fluid vent sites, regions with tectonic activity, and other attractive high priority targets. Qualitative and quantitative examinations took place on board and are continued on land with respect to pore pressure variation, geomicrobiology, sediment- and fluid mobilization, geochemical processes, faunal assemblages (e.g. cold water corals), and gas hydrates (flammable methane-ice-crystals). Main focus of the expedition has been a better understanding of interaction between dynamic processes in a seismically active region region with slow plate convergence. In the context of earthquake nucleation and subduction zone processes, the SO175 research programme had a variety of goals, such as: • To test the frictional behaviour of the abyssal plain sediments. • To explore the temperature field of the 1755 thrust earthquake event via heat flow measurements. • To assess the role of fluid venting and gas hydrate processes control slope stability and mud volcanic activity along the Iberian continental margin. • To measure isotope geochemistry of pore waters and carbonates of deep fluids. • To quantify microbial activity in Gibraltar wedge sediments. • To test whether microseismicity in the area corresponds to in situ pore pressure changes. • To find out if enhanced heat flow max be indicative of active subduction. Initial tentative results during the cruise suggest that there is a component of active thrusting at the base of the wedge, as attested by heat flow data. Based on mostly geochemical evidence, mud volcanism was found less active than previously assumed. Highlights from post-cruise research include the successful deployment of the long-term station and high frictional resistance of all incoming sediment on the three abyssal plains