Application of singular value decomposition to the analysis of time-resolved macromolecular x-ray data

Singular value decomposition (SVD) is a technique commonly used in the analysis of spectroscopic data that both acts as a noise filter and reduces the dimensionality of subsequent least-squares fits. To establish the applicability of SVD to crystallographic data, we applied SVD to calculated difference Fourier maps simulating those to be obtained in a time-resolved crystallographic study of photoactive yellow protein. The atomic structures of one dark state and three intermediates were used in qualitatively different kinetic mechanisms to generate time-dependent difference maps at specific time points. Random noise of varying levels in the difference structure factor amplitudes, different extents of reaction initiation, and different numbers of time points were all employed to simulate a range of realistic experimental conditions. Our results show that SVD allows for an unbiased differentiation between signal and noise; a small subset of singular values and vectors represents the signal well, reducing the random noise in the data. Due to this, phase information of the difference structure factors can be obtained. After identifying and fitting a kinetic mechanism, the time-independent structures of the intermediates could be recovered. This demonstrates that SVD will be a powerful tool in the analysis of experimental time-resolved crystallographic data.     

Molecular characterization of bla(IMP-5), a new integron-borne metallo-beta-lactamase gene from an Acinetobacter baumannii nosocomial isolate in Portugal

Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0-17.9 per microg circular DNA using 10(7) protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.     

Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.     

Ultrastructural aspects of the myxosporean Henneguya astyanax n. sp. (Myxozoa: Myxobolidae), a parasite of the Amazonian teleost Astyanax keithi (Characidae)

This study reports light and electron microscopical aspects of a myxosporean found in the gills of the freshwater teleost Astyanax keithi Géry, Planquete & Le Bail, 1996 (family Characidae), collected from the estuarine region of the Amazon River, near Belém, Brazil. The prevalence of infection was 23%. In interlamellar spaces of the gills, ellipsoidal whitish cyst-like plasmodia structures were present, which contained spores. The spores had a spermatozoa-like appearance (47.8 +/- 0.71 microm in total length) with a fusiform body (15.2 +/- 0.77 pm in length, 5.7 +/- 0.71 microm in width and 4.2 +/- 0.31 microm in thickness), and each of the 2 valves presented a tapering tail (32.6 +/- 1.11 microm in length). The valves surrounded a binucleate sporoplasm cell and 2 polar capsules (5.0 +/- 0.13 microm in length, 1.5 +/- 0.07 microm in width) that contained 8 to 9 coils of the polar filament. In the sporoplasm, several unique sporoplasmosomes were visible. A synoptic table of spore measurements of known Brazilian Henneguya species is presented. The spores differed from those of previously described species. Based on spore morphology, it is concluded that this species belongs to the family Myxobolidae, genus Henneguya, and that it constitutes a new species: H. astyanax n. sp.     

Subtyping for TNFa microsatellite sequence variation

The microsatellite locus TNFa is frequently used as an additional genetic marker in studies of the major histocompatibility complex (MHC). Novel sequence variations at the TNFa locus have been described, and which may have implications for genetic analyses. In this study, we set up a nested polymerase chain reaction-sequence-specific primer (PCR-SSP) approach to type for these TNFa sequence variations. First, sequencing analysis of workshop B lymphoblastoid cell lines (n=13) showed the presence of three sequence variations upstream of the dinucleotide repeat at TNFa. Using nested PCR-SSP, we were able to detect these variations in a larger B lymphoblastoid cell line panel (n=34). Furthermore, we were able to show that TNFa alleles a7 and a10 are present in two distinct conformations leading to "splitting" of TNFa alleles exhibiting identical fragment lengths. To establish the frequency of the TNFa alleles and their variants, we performed microsatellite typing of a large panel of random individuals from the Dutch population (n=272). Subsequent nested PCR-SSP typing showed the presence of three previously described sequence variations in the Dutch population. Furthermore, the presence of a fourth subtype was established. The described variations of allele TNFa7 and TNFa10 are present in the random population with significant frequencies. Haplotyping analysis between HLA-DR, TNFa, and HLA-B showed that allele TNFa7.2 is present in an extended DR7-TNFa7.2-B13 haplotype. In this way, we were able to show that the additional sequence variations behave like distinct TNFa alleles.     

Diversity and physiological characterization of d-xylose-fermenting yeasts isolated from the brazilian amazonian forest

Background

This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates.

Methodology/Principal Findings

A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L·h to 0.75 g/L·h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L·h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers.

Conclusions/Significance

This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.     

Resistance of Oryza sativa and Oryza glaberrima Genotypes to RBe24 Isolate of Rice Yellow Mottle Virus in Benin and Effects of Silicon on Host Response

Rice yellow mottle virus (RYMV) is the most harmful virus that affects irrigated and lowland rice in Africa. The RBe24 isolate of the virus is the most pathogenic strain in Benin. A total of 79 genotypes including susceptible IR64 (Oryza sativa) and the resistant TOG5681 (O. glaberrima) as checks were screened for their reactions to RBe24 isolate of RYMV and the effects of silicon on the response of host plants to the virus investigated. The experiment was a three-factor factorial consisting of genotypes, inoculation level (inoculated vs. non-inoculated), and silicon dose (0, 5, and 10 g/plant) applied as CaSiO₃ with two replications and carried out twice in the screen house. Significant differences were observed among the rice genotypes. Fifteen highly resistant and eight resistant genotypes were identified, and these were mainly O. glaberrima. Silicon application did not affect disease incidence and severity at 21 and 42 days after inoculation (DAI); it, however, significantly increased plant height of inoculated (3.6% for 5 g CaSiO₃/plant and 6.3% for 10 g CaSiO₃/plant) and non-inoculated (1.9% for 5 g CaSiO₃/plant and 4.9% for 10 g CaSiO₃/plant) plants at 42 DAI, with a reduction in the number of tillers (12.3% for both 5 and 10 g CaSiO₃/plant) and leaves (26.8% for 5 g CaSiO₃/plant and 28% for 10 g CaSiO₃/plant) under both inoculation treatments. Our results confirm O. glaberrima germplasm as an important source of resistance to RYMV, and critical in developing a comprehensive strategy for the control of RYMV in West Africa.     

Requirements for signal peptide peptidase-catalyzed intramembrane proteolysis

The presenilin-type aspartic protease signal peptide peptidase (SPP) can cleave signal peptides within their transmembrane region. SPP is essential for generation of signal peptide-derived HLA-E epitopes in humans and is exploited by Hepatitis C virus for processing of the viral polyprotein. Here we analyzed requirements of substrates for intramembrane cleavage by SPP. Comparing signal peptides that are substrates with those that are not revealed that helix-breaking residues within the transmembrane region are required for cleavage, and flanking regions can affect processing. Furthermore, signal peptides have to be liberated from the precursor protein by cleavage with signal peptidase in order to become substrates for SPP. We propose that signal peptides require flexibility in the lipid bilayer to exhibit an accessible peptide bond for intramembrane proteolysis.