A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.     

Multiblock Copolymers with Highly Sulfonated Blocks Containing Di‐ and Tetrasulfonated Arylene Sulfone Segments for Proton Exchange Membrane Fuel Cell Applications

Multiblock copoly(arylene ether sulfone)s with different block lengths and ionic contents are tailored for durable and proton‐conducting electrolyte membranes. Two series of fully aromatic copolymers are prepared by coupling reactions between non‐sulfonated hydrophobic precursor blocks and highly sulfonated hydrophilic precursor blocks containing either fully disulfonated diarylsulfone or fully tetrasulfonated tetraaryldisulfone segments. The sulfonic acid groups are exclusively introduced in ortho positions to the sulfone bridges to impede desulfonation reactions and give the blocks ion exchange capacities (IECs) of 4.1 and 4.6 meq. g^?1, respectively. Solvent cast block copolymer membranes show well‐connected hydrophilic nanophase domains for proton transport and high decomposition temperatures above 310 °C under air. Despite higher IEC values, membranes containing tetrasulfonated tetraaryldisulfone segments display a markedly lower water uptake than the corresponding ones with disulfonated diarylsulfone segments when immersed in water at 100 °C, presumably because of the much higher chain stiffness and glass transition temperature of the former segments. The former membranes have proton conductivities in level of a perfluorosulfonic acid membrane (NRE212) under fully humidified conditions. A membrane with an IEC of 1.83 meq. g^?1 reaches above 6 mS cm^?1 under 30% relative humidity at 80 °C, to be compared with 10 mS cm^?1 for NRE212 under the same conditions.     

Whole genome duplication of intra- and inter-chromosomes in the tomato genome

Whole genome duplication(WGD)events have been proven to occur in the evolutionary history of most angiosperms.Tomato is considered a model species of the Solanaceae family.In this study,we describe the details of the evolutionary process of the tomato genome by detecting collinearity blocks and dating the WGD events on the tree of life by combining two different methods:synonymous substitution rates(Ks)and phylogenetic trees.In total,593 collinearity blocks were discovered out of 12 pseudo-chromosomes constructed. It was evident that chromosome 2 had experienced an intra-chromosomal duplication event.Major inter-chromosomal duplication occurred among all the pseudo-chromosome.We calculated the Ks value of these collinearity blocks.Two peaks of Ks distribution were found,corresponding to two WGD events occurring approximately 36-82 million years ago(MYA)and 148-205 MYA.Additionally, the results of phylogenetic trees suggested that the more recent WGD event may have occurred after the divergence of the rosidasterid clade,but before the major diversification in Solanaceae.The older WGD event was shown to have occurred before the divergence of the rosid-asterid clade and after the divergence of rice-Arabidopsis(monocot-dicot).     

Antidromic propagation of action potentials in branched axons: implications for the mechanisms of action of deep brain stimulation

Electrical stimulation of the central nervous system creates both orthodromically propagating action potentials, by stimulation of local cells and passing axons, and antidromically propagating action potentials, by stimulation of presynaptic axons and terminals. Our aim was to understand how antidromic action potentials navigate through complex arborizations, such as those of thalamic and basal ganglia afferents-sites of electrical activation during deep brain stimulation. We developed computational models to study the propagation of antidromic action potentials past the bifurcation in branched axons. In both unmyelinated and myelinated branched axons, when the diameters of each axon branch remained under a specific threshold (set by the antidromic geometric ratio), antidromic propagation occurred robustly; action potentials traveled both antidromically into the primary segment as well as "re-orthodromically" into the terminal secondary segment. Propagation occurred across a broad range of stimulation frequencies, axon segment geometries, and concentrations of extracellular potassium, but was strongly dependent on the geometry of the node of Ranvier at the axonal bifurcation. Thus, antidromic activation of axon terminals can, through axon collaterals, lead to widespread activation or inhibition of targets remote from the site of stimulation. These effects should be included when interpreting the results of functional imaging or evoked potential studies on the mechanisms of action of DBS.     

Late Paleozoic faunal, climatic, and geographic changes in the Baoshan block as a Gondwana-derived continental fragment in southwest China

The Carboniferous and Permian of the Baoshan block consist of three major depositional sequences: a Lower Carboniferous carbonate sequence, a Lower Permian siliciclastic sequence, and a Middle Permian carbonate sequence. These three sequences were interrupted by two major regressive events: first, the Namurian Uplift ranging in age from Serpukovian to Gzhelian, and second, the Post-Sakmarian Regression occurring probably at Artinskian time in the Baoshan block, although the precise time interval of the latter event is still unclear. The Baoshan block is characterized by warm-water, highly diverse and abundant faunas during the Early Carboniferous, by cold-water and low diversity faunas during the Early Permian, and by possibly warm-water but low diversity faunas during the Middle Permian. The Sweetognathus bucaramangus conodont fauna constrains the upper boundary of the diamictite-bearing siliciclastic deposits (Dingjiazhai Formation) to the Sakmarian to early Artinskian, as well as the eruption of the rifting basalts (Woniusi Formation) to, at least, the post-early Artinskian. Paleozoogeographically, affiliation of the faunas in the Baoshan block changed from Eurasian in the Early Carboniferous, to Peri-Gondwanan in the Early Permian, and to Marginal Cathaysian/Cimmerian in the Middle Permian. Cimmerian blocks have more or less comparable geohistory to one another in the Carboniferous and Permian. During the Middle Permian, the eastern Cimmerian blocks such as Sibumasu (s.s), Baoshan, and Tengchong are not far from the palaeoequator, but apparently more distant than the western Cimmerian blocks based on the presence or absence of some index taxa such as the fusulinaceans Eopolydiexodina and Neoschwagerina, and the corals Thomasiphyllum and Wentzellophyllum persicum.