Encephalopathy caused by ablation of very long acyl chain ceramide synthesis may be largely due to reduced galactosylceramide levels

Sphingolipids (SLs) act as signaling molecules and as structural components in both neuronal cells and myelin. We now characterize the biochemical, histological, and behavioral abnormalities in the brain of a mouse lacking very long acyl (C22-C24) chain SLs. This mouse, which is defective in the ability to synthesize C22-C24-SLs due to ablation of ceramide synthase 2, has reduced levels of galactosylceramide (GalCer), a major component of myelin, and in particular reduced levels of non-hydroxy-C22-C24-GalCer and 2-hydroxy-C22-C24- GalCer. Noteworthy brain lesions develop with a time course consistent with a vital role for C22-C24-GalCer in myelin stability. Myelin degeneration and detachment was observed as was abnormal motor behavior originating from a subcortical region. Additional abnormalities included bilateral and symmetrical vacuolization and gliosis in specific brain areas, which corresponded to some extent to the pattern of ceramide synthase 2 expression, with astrogliosis considerably more pronounced than microglial activation. Unexpectedly, unidentified storage materials were detected in lysosomes of astrocytes, reminiscent of the accumulation that occurs in lysosomal storage disorders. Together, our data demonstrate a key role in the brain for SLs containing very long acyl chains and in particular GalCer with a reduction in their levels leading to distinctive morphological abnormalities in defined brain regions.     

Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-angstrom resolution

The Cry4Aa delta-endotoxin from Bacillus thuringiensis is toxic to larvae of Culex, Anopheles, and Aedes mosquitoes, which are vectors of important human tropical diseases. With the objective of designing modified toxins with improved potency that could be used as biopesticides, we determined the structure of this toxin in its functional form at a resolution of 2.8 angstroms. Like other Cry delta-endotoxins, the activated Cry4Aa toxin consists of three globular domains, a seven-alpha-helix bundle responsible for pore formation (domain I) and the following two other domains having structural similarities with carbohydrate binding proteins: a beta-prism (domain II) and a plant lectin-like beta-sandwich (domain III). We also studied the effect on toxicity of amino acid substitutions and deletions in three loops located at the surface of the putative receptor binding domain II of Cry4Aa. Our results indicate that one loop is an important determinant of toxicity, presumably through attachment of Cry4Aa to the surface of mosquito cells. The availability of the Cry4Aa structure should guide further investigations aimed at the molecular basis of the target specificity and membrane insertion of Cry endotoxins.     

Specific mutations within the α4–α5 loop of the bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry δ-endotoxins suggests that helices α4 and α5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the α4–α5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-β-d-thiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the α4–α5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.     

Toward an unbiased evolutionary platform for unraveling Xenopus developmental gene networks

The availability of both the Xenopus tropicalis genome and the soon to be released Xenopus laevis genome provides a solid foundation for Xenopus developmental biologists. The Xenopus community has presently amassed expression data for ～2,300 genes in the form of published images collected in the Xenbase, the principal Xenopus research database. A few of these genes have been examined in both X. tropicalis and X. laevis and the cross-species comparison has been proven invaluable for studying gene function. A recently published work has yielded developmental expression profiles for the majority of Xenopus genes across fourteen developmental stages spanning the blastula, gastrula, neurula, and the tail-bud. While this data was originally queried for global evolutionary and developmental principles, here we demonstrate its general use for gene-level analyses. In particular, we present the accessibility of this dataset through Xenbase and describe biases in the characterized genes in terms of sequence and expression conservation across the two species. We further indicate the advantage of examining coexpression for gene function discovery relating to developmental processes conserved across species. We suggest that the integration of additional large-scale datasets--comprising diverse functional data--into Xenbase promises to provide a strong foundation for researchers in elucidating biological processes including the gene regulatory programs encoding development.     

Seeing is believing: new methods for in situ single-cell transcriptomics

New methods employ RNA-seq to study single cells within complex tissues by in situ sequencing or mRNA capture from single photoactivated cells.     

The Central Nervous System as Target of Bacillus anthracis Toxin Independent Virulence in Rabbits and Guinea Pigs

Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC) inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits'' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.     

A cross-sample statistical model for SNP detection in short-read sequencing data

Highly multiplex DNA sequencers have greatly expanded our ability to survey human genomes for previously unknown single nucleotide polymorphisms (SNPs). However, sequencing and mapping errors, though rare, contribute substantially to the number of false discoveries in current SNP callers. We demonstrate that we can significantly reduce the number of false positive SNP calls by pooling information across samples. Although many studies prepare and sequence multiple samples with the same protocol, most existing SNP callers ignore cross-sample information. In contrast, we propose an empirical Bayes method that uses cross-sample information to learn the error properties of the data. This error information lets us call SNPs with a lower false discovery rate than existing methods.