Small-angle neutron and X-ray scattering analysis of the supramolecular organization of rhodopsin in photoreceptor membrane

The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8 nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering.     

A varying‐coefficient generalized odds rate model with time‐varying exposure: An application to fitness and cardiovascular disease mortality

Varying‐coefficient models have become a common tool to determine whether and how the association between an exposure and an outcome changes over a continuous measure. These models are complicated when the exposure itself is time‐varying and subjected to measurement error. For example, it is well known that longitudinal physical fitness has an impact on cardiovascular disease (CVD) mortality. It is not known, however, how the effect of longitudinal physical fitness on CVD mortality varies with age. In this paper, we propose a varying‐coefficient generalized odds rate model that allows flexible estimation of age‐modified effects of longitudinal physical fitness on CVD mortality. In our model, the longitudinal physical fitness is measured with error and modeled using a mixed‐effects model, and its associated age‐varying coefficient function is represented by cubic B‐splines. An expectation‐maximization algorithm is developed to estimate the parameters in the joint models of longitudinal physical fitness and CVD mortality. A modified pseudoadaptive Gaussian‐Hermite quadrature method is adopted to compute the integrals with respect to random effects involved in the E‐step. The performance of the proposed method is evaluated through extensive simulation studies and is further illustrated with an application to cohort data from the Aerobic Center Longitudinal Study.     

Application of green analytical chemistry to a green chemistry process: Magnetic resonance and Raman spectroscopic process monitoring of continuous ethanolic fermentation

Compact ¹H NMR and Raman spectrometers were used for real-time process monitoring of alcoholic fermentation in a continuous flow reactor. Yeast cells catalyzing the sucrose conversion were immobilized in alginate beads floating in the reactor. The spectrometers proved to be robust and could be easily attached to the reaction apparatus. As environmentally friendly analysis methods, ¹H NMR and Raman spectroscopy were selected to match the resource- and energy-saving process. Analyses took only a few seconds to minutes compared to chromatographic procedures and were, therefore, suitable for real-time control realized as a feedback loop. Both compact spectrometers were successfully implemented online. Raman spectroscopy allowed for faster spectral acquisition and higher quantitative precision, NMR yielded more resolved signals thus higher specificity. By using the software Matlab for automated data loading and processing, relevant parameters such as the ethanol, glycerol, and sugar content could be easily obtained. The subsequent multivariate data analysis using partial linear least-squares regression type 2 enabled the quantitative monitoring of all reactants within a single model in real time.     

Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli

The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but also other Gram-negative bacteria, has captured the interest of the biotechnology industry. In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single-span membrane-embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.     

Double-exponential kinetics of binding and redistribution of the fluorescent dyes in cell membranes witness for the existence of lipid microdomains

New technique of detecting lateral heterogeneity of the plasma membrane of living cells by means of membrane-binding fluorescent dyes is proposed. The kinetics of dye incorporation into the membrane or its lateral diffusion inside the membrane is measured and decomposed into exponential components by means of the Maximum Entropy Method. Two distinct exponential components are obtained consistently in all cases for several fluorescent dyes, two different cell lines and in different types of experiments including spectroscopy, flow cytometry and fluorescence recovery after photobleaching. These components are attributed to the liquid-ordered and disordered phases in the plasma membrane of studied cells in their dynamic equilibrium.     

Fire‐sensitive species dominate seed rain after fire suppression: Implications for plant community diversity and woody encroachment in the Cerrado

Woody encroachment is becoming common in tropical savannas. We studied natural seed rain and performed seed addition experiments in a Brazilian savanna that had not been burned for several decades. We found greater abundance of fire‐sensitive species in the seed rain, likely contributing to woody encroachment. Flexible fire management policies that allow for natural and prescribed fires may be required to maintain savanna diversity.     

Effects of predator exposure on baseline and stress-induced glucocorticoid hormone concentrations in pumpkinseed Lepomis gibbosus

We compared baseline and maximal cortisol concentrations between predator exposure and prey blood samples in pumpkinseed Lepomis gibbosus, captured using a standardised fishing event underneath osprey Pandion haliaetus nests and away from osprey nests. We did not detect differences in cortisol or glucose between sites. These findings suggest that predictable sources of predation risk may not confer stress-related costs in teleosts.     

LAP2 Proteins Chaperone GLI1 Movement between the Lamina and Chromatin to Regulate Transcription

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Genetic polymorphism in Chondrilla (Asteraceae) in southern European Russia and the nature of Chondrilla juncea L

We studied genetic diversity in 54 populations of nine sexual and apomictic species of the genus Chondrilla (C. acantholepis, C. ambigua, C. brevirostris, C. canescens, C. graminea, C. juncea, C. laticoronata, C. latifolia and C. pauciflora) in SE European Russia and neighboring territories of NW Kazakhstan. We analysed the trnT–trnF region of plastid DNA and the internal transcribed spacer of ribosomal DNA (ITS1–5.8S–ITS2) using statistical parsimony, maximum likelihood and neighbor net methods. Two major evolutionary lineages, roughly corresponding to the two subgenera traditionally recognized in the region, were revealed. Within the first evolutionary lineage (subgenus Brachyrhynchus), the sexual diploid C. ambigua and its putatively hybrid apomictic derivatives C. brevirostris, C. laticoronata and C. pauciflora could be recognized. Their identity was also confirmed by analyses of ISSR markers. The second evolutionary lineage (subgenus Chondrilla) comprises C. juncea, C. acantholepis, C. canescens, C. graminea and C. latifolia in European Russia, but analyses of morphological variability and the genealogy of plastid and nuclear markers favor their treatment as the single facultatively apomictic species C. juncea. The results demonstrate that an apomictic mode of reproduction does not necessarily result in the formation of genetically separated microspecies.