Identification of novel Xanthomonas euvesicatoria type III effector proteins by a machine‐learning approach

The Gram‐negative bacterium Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Xcv pathogenicity depends on a type III secretion (T3S) system that delivers effector proteins into host cells to suppress plant immunity and promote disease. The pool of known Xcv effectors includes approximately 30 proteins, most identified in the 85‐10 strain by various experimental and computational techniques. To identify additional Xcv 85‐10 effectors, we applied a genome‐wide machine‐learning approach, in which all open reading frames (ORFs) were scored according to their propensity to encode effectors. Scoring was based on a large set of features, including genomic organization, taxonomic dispersion, hypersensitive response and pathogenicity (hrp)‐dependent expression, 5′ regulatory sequences, amino acid composition bias and GC content. Thirty‐six predicted effectors were tested for translocation into plant cells using the hypersensitive response (HR)‐inducing domain of AvrBs2 as a reporter. Seven proteins (XopAU, XopAV, XopAW, XopAP, XopAX, XopAK and XopAD) harboured a functional translocation signal and their translocation relied on the HrpF translocon, indicating that they are bona fide T3S effectors. Remarkably, four belong to novel effector families. Inactivation of the xopAP gene reduced the severity of disease symptoms in infected plants. A decrease in cell death and chlorophyll content was observed in pepper leaves inoculated with the xopAP mutant when compared with the wild‐type strain. However, populations of the xopAP mutant in infected leaves were similar in size to those of wild‐type bacteria, suggesting that the reduction in virulence was not caused by impaired bacterial growth.     

Parametric analysis of the biomechanical response of head subjected to the primary blast loading – a data mining approach

Traumatic brain injury due to primary blast loading has become a signature injury in recent military conflicts and terrorist activities. Extensive experimental and computational investigations have been conducted to study the interrelationships between intracranial pressure response and intrinsic or ‘input’ parameters such as the head geometry and loading conditions. However, these relationships are very complicated and are usually implicit and ‘hidden’ in a large amount of simulation/test data. In this study, a data mining method is proposed to explore such underlying information from the numerical simulation results. The heads of different species are described as a highly simplified two-part (skull and brain) finite element model with varying geometric parameters. The parameters considered include peak incident pressure, skull thickness, brain radius and snout length. Their interrelationship and coupling effect are discovered by developing a decision tree based on the large simulation data-set. The results show that the proposed data-driven method is superior to the conventional linear regression method and is comparable to the nonlinear regression method. Considering its capability of exploring implicit information and the relatively simple relationships between response and input variables, the data mining method is considered to be a good tool for an in-depth understanding of the mechanisms of blast-induced brain injury. As a general method, this approach can also be applied to other nonlinear complex biomechanical systems.     

Enhanced generation of intraluminal vesicles in neuronal late endosomes in the brain of a Down syndrome mouse model with endosomal dysfunction

Down syndrome (DS) is a human genetic disease caused by trisomy of chromosome 21 and characterized by early developmental brain abnormalities. Dysfunctional endosomal pathway in neurons is an early event of DS and Alzheimer's disease. Recently, we have demonstrated that exosome secretion is upregulated in human DS postmortem brains, in the brain of the trisomic mouse model Ts[Rb(12.17¹⁶)]2Cje (Ts2) and by DS fibroblasts as compared with disomic controls. High levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Partially blocking exosome secretion by DS fibroblasts exacerbated a pre‐existing early endosomal pathology. We thus hypothesized that enhanced CD63 expression induces generation of intraluminal vesicles (ILVs) in late endosomes/multivesicular bodies (MVBs), increasing exosome release as an endogenous mechanism to mitigate endosomal abnormalities in DS. Herein, we show a high‐resolution electron microscopy analysis of MVBs in neurons of the frontal cortex of 12‐month‐old Ts2 mice and littermate diploid controls. Our quantitative analysis revealed that Ts2 MVBs are larger, more abundant, and contain a higher number of ILVs per neuron compared to controls. These findings were further corroborated biochemically by Western blot analysis of purified endosomal fractions showing higher levels of ILVs proteins in the same fractions containing endosomal markers in the brain of Ts2 mice compared to controls. These data suggest that upregulation of ILVs production may be a key homeostatic mechanism to alleviate endosomal dysregulation via the endosomal–exosomal pathway.     

Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker.     

Proximity of transmembrane segments M3 and M1 of the alpha subunit of Na+,K+-ATPase revealed by specific oxidative cleavage mediated by a complex of Cu2+ ions and 4,7-diphenyl-1,10-phenanthroline

This paper describes a novel approach to specific oxidative cleavage of Na(+),K(+)-ATPase, mediated by Cu(2+) ions and a hydrophobic phenanthroline, 4,7-diphenyl-1,10-phenanthroline (DPP), in the presence of ascorbate and H(2)O(2). The cleavage produces two major fragments of the alpha subunit, with apparent molecular masses of 96.5 and 76 kDa, and N-termini near the cytoplasmic entrance of transmembrane segments M1 and M3, respectively, The kinetics indicate that both cleavages are mediated by a single Cu(2+)-DPP complex. We infer that M3 and M1 are in proximity near the cytoplasmic surface. The yields of 96.5 and 76 kDa fragments are not significantly affected by ligands that stabilize different E(1) and E(2) conformations. In E(2)(K) and E(2)P conformations, a minor 5.5 kDa fragment with its N-terminus in M10 is also observed. The 96.5 and 76 kDa fragments are indistinguishable from two fragments near M3 and M1 produced by Fe(2+)-catalyzed cleavage described previously [Goldshleger, R., and Karlish, S. J. D. (1999) J. Biol. Chem. 274, 16213-16221], whereas other Fe(2+)-catalyzed cleavage fragments in the cytoplasmic P and A domains are not observed with the Cu(2+)-DPP complex. These findings provide experimental support for the concept of two separate Fe(2+) sites. A homology model, with Na(+),K(+)-ATPase residues within transmembrane segments and connecting loops substituted into the crystal structure of Ca(2+)-ATPase, shows the proximity between the sequences HFIH in M3 and EVWK in M1, near the cytoplasmic surface. Thus, the model strongly supports the conclusions based on cleavages mediated by the Cu(2+)-DPP complex (or Fe(2+) at site 2). As a corollary, the cleavages provide evidence for similar packing of M1 and M3 of Na(+),K(+)-ATPase and Ca(2+)-ATPase.     

Evolution of Microsatellites in the Yeast Saccharomyces cerevisiae: Role of Length and Number of Repeated Units

The observed and expected frequencies of occurrence of microsatellites in the yeast Saccharomyces cerevisiae were investigated. In all cases, the observed frequencies exceeded the expected ones. In contrast to predictions by Messier et al. (1996), there is no critical number of repeats beyond which the observed frequencies of microsatellites significantly exceed the frequencies expected in a random DNA sequence of the same size. Rather, the degree of deviation from expectation was found to be dependent on the length of the microsatellite. That is, a fourfold concatemeric repeat of 3 bp was found to deviate from expectation as much as threefold concatemeric repeat of 4 bp, unlike the deviation of a fourfold concatemeric repeat of 4 bp. These findings suggest that microsatellites evolve through strand-slippage events, rather than recombination events. This, in turn, suggests that the chances of erroneous hybridizations leading to strand-slippage are length dependent. Received: 1 June 1998 / Accepted: 16 September 1998     

Analysis in vitro of the enzyme CRTISO establishes a poly-cis-carotenoid biosynthesis pathway in plants

Most enzymes in the central pathway of carotenoid biosynthesis in plants have been identified and studied at the molecular level. However, the specificity and role of cis-trans-isomerization of carotenoids, which occurs in vivo during carotene biosynthesis, remained unresolved. We have previously cloned from tomato (Solanum lycopersicum) the CrtISO gene, which encodes a carotene cis-trans-isomerase. To study the biochemical properties of the enzyme, we developed an enzymatic in vitro assay in which a purified tomato CRTISO polypeptide overexpressed in Escherichia coli cells is active in the presence of an E. coli lysate that includes membranes. We show that CRTISO is an authentic carotene isomerase. Its catalytic activity of cis-to-trans isomerization requires redox-active components, suggesting that isomerization is achieved by a reversible redox reaction acting at specific double bonds. Our data demonstrate that CRTISO isomerizes adjacent cis-double bonds at C7 and C9 pairwise into the trans-configuration, but is incapable of isomerizing single cis-double bonds at C9 and C9'. We conclude that CRTISO functions in the carotenoid biosynthesis pathway in parallel with zeta-carotene desaturation, by converting 7,9,9'-tri-cis-neurosporene to 9'-cis-neurosporene and 7'9'-di-cis-lycopene into all-trans-lycopene. These results establish that in plants carotene desaturation to lycopene proceeds via cis-carotene intermediates.