RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3′ splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3′ splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF⁶⁵) and small (hU2AF³⁵) subunits. hU2AF⁶⁵ contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF³⁵ has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF⁶⁵. While the requirement for the three RRMs on hU2AF⁶⁵ is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF³⁸) was complexed with the large-subunit (dU2AF⁵⁰) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF⁵⁰. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF⁶⁵. Surprisingly, the RS domain on dU2AF³⁸ was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF⁵⁰ΔRS) severely impaired its binding activity. However, if the dU2AF³⁸ RS domain was supplied in a complex with dU2AF⁵⁰ΔRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.     

THE COMPARATIVE DEMOGRAPHY OF RECIPROCALLY SOWN POPULATIONS OF PHLOX DRUMMONDII HOOK. I. SURVIVORSHIPS,FECUNDITIES, AND FINITE RATES OF INCREASE

Comparative demography of reciprocally sown populations for all stages of the life cycle of the winter annual, Phlox drummondii, was recorded for two seasons in natural sites. The sites investigated covered the natural range of the species and were separated by linear distances ranging from 10 to over 500 kilometers. Interpopulational variation was observed in all stages of the life cycle. Prereproductive survivorship ranged from 0 to 92 percent. Fecundity per plant ranged from 0 to 81 seeds. Finite rates of increase ranged from 0 to 34.1 yr^?1. The experimental populations with low values were growing in sites that received little rainfall or had experienced an insect or fungal infestation. The populations with high growth rates occurred in the northern sites which received greater rainfall. The relative fitness of individuals from the alien populations was compared to the local populations for all stages of the life cycle. The alien relative fitness for survivorship averaged 0.72. The average relative fitness of aliens for fecundity was 0.71 and that for finite rate of increase was 0.57. The life history parameters found in Phlox populations differ from one part of the range to another so that aliens have lower relative fitnesses than individuals indigenous to the site.     

Geographic structure, genetic diversity and source tracking of Spartina alterniflora

Aim To examine the distribution and structure of genetic variation among native Spartina alterniflora and to characterize the evolutionary mechanisms underlying the success of non‐native S. alterniflora. Location Intertidal marshes along the Atlantic, Gulf and Pacific coasts of North America. Methods amova , parsimony analysis, haplotype networks of chloroplast DNA (cpDNA) sequences, neighbour‐joining analysis, Bayesian analysis of population structure, and individual assignment testing were used. Results Low levels of gene flow and geographic patterns of genetic variation were found among native S. alterniflora from the Atlantic and Gulf coasts of North America. The distribution of cpDNA haplotypes indicates that Atlantic coast S. alterniflora are subdivided into ‘northern’ and ‘southern’ groups. Variation observed at microsatellite loci further suggests that mid‐Atlantic S. alterniflora are differentiated from S. alterniflora found in southern Atlantic and New England coastal marshes. Comparisons between native populations on the Atlantic and Gulf coasts and non‐native Pacific coast populations substantiate prior studies demonstrating reciprocal interspecific hybridization in San Francisco Bay. Our results corroborate historical evidence that S. alterniflora was introduced into Willapa Bay from multiple source populations. However, we found that some Willapa Bay S. alterniflora are genetically divergent from putative sources, probably as a result of admixture following secondary contact among previously allopatric native populations. We further recovered evidence in support of models suggesting that S. alterniflora has secondarily spread within Washington State, from Willapa Bay to Grays Harbor. Main conclusions Underlying genetic structure has often been cited as a factor contributing to ecological variation of native S. alterniflora. Patterns of genetic structure within native S. alterniflora may be the result of environmental differences among biogeographical provinces, of migration barriers, or of responses to historical conditions. Interactions among these factors, rather than one single factor, may best explain the distribution of genetic variation among native S. alterniflora. Comprehensive genetic comparisons of native and introduced populations can illustrate how biological invasions may result from dramatically different underlying factors – some of which might otherwise go unrecognized. Demonstrating that invasions can result from several independent or interacting mechanisms is important for improving risk assessment and future forecasting. Further research on S. alterniflora not only may clarify what forces structure native populations, but also may improve the management of non‐native populations by enabling post‐introduction genetic changes and the rapid evolution of life‐history traits to be more successfully exploited.     

Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin

The growing resistance against antibiotics demands the search for alternative treatment strategies. Photodynamic therapy is a promising candidate. The natural intermediate of chlorophyll biosynthesis, protochlorophyllide, was produced, purified and tested as a novel photosensitizer for the inactivation of five model organisms including Staphylococcus aureus, Listeria monocytogenes and Yersinia pseudotuberculosis , all responsible for serious clinical infections. When microorganisms were exposed to white light from a tungsten filament lamp (0.1 mW cm⁻²), Gram-positive S. aureus, L. monocytogenes and Bacillus subtilis were photochemically inactivated at concentrations of 0.5 mg L⁻¹ protochlorophyllide. Transmission electron microscopy revealed a disordered septum formation during cell division and the partial loss of the cytoplasmic cell contents. Gram-negative Y. pseudotuberculosis and Escherichia coli were found to be insensitive to protochlorophyllide treatment due to the permeability barrier of the outer membrane. However, the two bacteria were rendered susceptible to eradication by protochlorophyllide (10 mg L⁻¹) upon addition of polymyxin B nonapeptide at 50 and 20 mg L⁻¹, respectively. The release of DNA and a detrimental rearrangement of the cytoplasm were observed.     

Flexible Ion‐Conducting Composite Membranes for Lithium Batteries

The use of metallic lithium anodes enables higher energy density and higher specific capacity Li‐based batteries. However, it is essential to suppress lithium dendrite growth during electrodeposition. Li‐ion‐conducting ceramics (LICC) can mechanically suppress dendritic growth but are too fragile and also have low Li‐ion conductivity. Here, a simple, versatile, and scalable procedure for fabricating flexible Li‐ion‐conducting composite membranes composed of a single layer of LICC particles firmly embedded in a polymer matrix with their top and bottom surfaces exposed to allow for ionic transport is described. The membranes are thin (<100 μm) and possess high Li‐ion conductance at thicknesses where LICC disks are mechanically unstable. It is demonstrated that these membranes suppress Li dendrite growth even when the shear modulus of the matrix is lower than that of lithium. It is anticipated that these membranes enable the use of metallic lithium anodes in conventional and solid‐state Li‐ion batteries as well as in future Li? S and Li? O₂ batteries.     

Four-Dimensional Characterization of Thrombosis in a Live-Cell,Shear-Flow Assay: Development and Application to Xenotransplantation

Background

Porcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications.

Methodology/Principal Findings

Confluent wild-type (WT, n = 48) and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11) porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm² and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells) and total fluorescence (a metric of clot volume). Aggregation was calculated by the fluorescence/SA ratio (FR). WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2%) and aggregation (FR 120 ± 1 a.u.), indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each); importantly, aggregation was only marginally affected (11% reduction, P < 0.0001).

Conclusions/Significance

This novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to xenogeneic thrombosis, it enables future studies regarding the effect of modifying the porcine genotype on sheer-stress-dependent events that characterize xenograft injury. This in-vitro platform is likely to prove broadly useful to study thrombosis and endothelial interactions under dynamic physiologic conditions.     

LUMOS - A Sensitive and Reliable Optode System for Measuring Dissolved Oxygen in the Nanomolar Range

Most commercially available optical oxygen sensors target the measuring range of 300 to 2 μmol L^-1. However these are not suitable for investigating the nanomolar range which is relevant for many important environmental situations. We therefore developed a miniaturized phase fluorimeter based measurement system called the LUMOS (Luminescence Measuring Oxygen Sensor). It consists of a readout device and specialized “sensing chemistry” that relies on commercially available components. The sensor material is based on palladium(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin embedded in a Hyflon AD 60 polymer matrix and has a K_SV of 6.25 x 10^-3 ppmv^-1. The applicable measurement range is from 1000 nM down to a detection limit of 0.5 nM. A second sensor material based on the platinum(II) analogue of the porphyrin is spectrally compatible with the readout device and has a measurement range of 20 μM down to 10 nM. The LUMOS device is a dedicated system optimized for a high signal to noise ratio, but in principle any phase flourimeter can be adapted to act as a readout device for the highly sensitive and robust sensing chemistry. Vise versa, the LUMOS fluorimeter can be used for read out of less sensitive optical oxygen sensors based on the same or similar indicator dyes, for example for monitoring oxygen at physiological conditions. The presented sensor system exhibits lower noise, higher resolution and higher sensitivity than the electrochemical STOX sensor previously used to measure nanomolar oxygen concentrations. Oxygen contamination in common sample containers has been investigated and microbial or enzymatic oxygen consumption at nanomolar concentrations is presented.