Rotavirus A genotype G1P[8]: a novel method to distinguish wild-type strains from the Rotarix vaccine strain

Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix?. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix? vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.     

Reelin and stk25 have opposing roles in neuronal polarization and dendritic Golgi deployment

The Reelin ligand regulates a Dab1-dependent signaling pathway required for brain lamination and normal dendritogenesis, but the specific mechanisms underlying these actions remain unclear. We find that Stk25, a modifier of Reelin-Dab1 signaling, regulates Golgi morphology and neuronal polarization as part of an LKB1-Stk25-Golgi matrix protein 130 (GM130) signaling pathway. Overexpression of Stk25 induces Golgi condensation and multiple axons, both of which are rescued by Reelin treatment. Reelin stimulation of cultured neurons induces the extension of the Golgi into dendrites, which is suppressed by Stk25 overexpression. In vivo, Reelin and Dab1 are required for the normal extension of the Golgi apparatus into the apical dendrites of hippocampal and neocortical pyramidal neurons. This demonstrates that the balance between Reelin-Dab1 signaling and LKB1-Stk25-GM130 regulates Golgi dispersion, axon specification, and dendrite growth and provides insights into the importance of the Golgi apparatus for cell polarization.     

Experimental Adaptation of Burkholderia cenocepacia to Onion Medium Reduces Host Range

It is unclear whether adaptation to a new host typically broadens or compromises host range, yet the answer bears on the fate of emergent pathogens and symbionts. We investigated this dynamic using a soil isolate of Burkholderia cenocepacia, a species that normally inhabits the rhizosphere, is related to the onion pathogen B. cepacia, and can infect the lungs of cystic fibrosis patients. We hypothesized that adaptation of B. cenocepacia to a novel host would compromise fitness and virulence in alternative hosts. We modeled adaptation to a specific host by experimentally evolving 12 populations of B. cenocepacia in liquid medium composed of macerated onion tissue for 1,000 generations. The mean fitness of all populations increased by 78% relative to the ancestor, but significant variation among lines was observed. Populations also varied in several phenotypes related to host association, including motility, biofilm formation, and quorum-sensing function. Together, these results suggest that each population adapted by fixing different sets of adaptive mutations. However, this adaptation was consistently accompanied by a loss of pathogenicity to the nematode Caenorhabditis elegans; by 500 generations most populations became unable to kill nematodes. In conclusion, we observed a narrowing of host range as a consequence of prolonged adaptation to an environment simulating a specific host, and we suggest that emergent pathogens may face similar consequences if they become host-restricted.Some emergent pathogens, such as Pseudomonas and Burkholderia species, persist in a wide range of plant and animal hosts, suggesting that the virulence factors needed to infect plants and animals are similar (5, 40). Yet whether adaptation to a new niche tends to compromise niche breadth or, in this case, host range is an open question. Adaptation to a novel host may restrict host range to various degrees, whether by diminishing host-specific virulence traits without affecting host colonization or by reducing the ability to initiate infection in alternative hosts. However, if factors needed to colonize plant and animal hosts are similar, then why are some bacterial populations restricted to a narrow host range while others are not? One explanation for a limited host range may be the result of genetic trade-offs associated with adaptation to a specific host (7, 18). Another explanation may be that prolonged adaptation to a specific host casts a “selective shadow” over unused functions that are relevant to colonizing other hosts but decay by genetic drift (7, 18). To address these possibilities, we quantified the direct and correlated effects of specific host adaptation by the opportunistic pathogen Burkholderia cenocepacia.Members of the Burkholderia cepacia complex (Bcc), which are ubiquitous in the environment, were once used as biocontrol and bioremedial agents but now are banned from these applications because of the potential of some members to cause plant and human disease (39). The type species B. cepacia is well known as a pathogen of the common yellow onion, Allium cepa, in which it causes a characteristic yellow or brown rot. Another species, B. cenocepacia, can also infect onions as well as a range of plants and animals, including humans (2, 6, 26, 36). Bcc bacteria can cause serious infection in the lungs of cystic fibrosis (CF) patients (6, 26). These infections, called “cepacia syndrome,” are highly contagious among CF patients, and infections produce many negative effects on an already poor quality of life, including longer hospital stays, removal from lung transplant lists, blood poisoning, and eventual death (24). B. cenocepacia, one of the two Bcc species most commonly isolated from lung infections, is especially threatening and is associated with more severe cepacia syndrome (35). However, the mechanisms allowing B. cenocepacia to adapt to colonize both human and plant hosts are unclear. Several putative virulence mechanisms have been identified by random mutagenic screens or by knockouts of candidate genes (2, 12, 20, 25, 29, 35, 43, 46), but these mechanisms generally have not been shown to function in host adaptation. One way to directly study adaptation of bacterial populations to susceptible hosts is by experimental evolution, in which bacterial populations evolve in a controlled laboratory setting that enables study of the adaptive process over time (7).We experimentally evolved populations of B. cenocepacia HI2424 to study the extent to which adaptation to the common yellow onion A. cepa affects host range. B. cenocepacia HI2424 is a soil isolate and is classified as part of the PHDC strain lineage, the strain first characterized as responsible for an outbreak of Bcc infections in large treatment centers located in the mid-Atlantic region of the United States (33). We found that adaptation of B. cenocepacia to the onion model was associated with reduced virulence but did not compromise the capacity to colonize (or be consumed by) the nematode Caenorhabditis elegans, and the coincidence of these events suggests that a genetic trade-off (antagonistic pleiotropy) between fitness in onion medium and nematode virulence exists. We also characterized several phenotypes potentially associated with adaptation to the onion or nematode virulence. Most phenotypes varied significantly among replicate populations, suggesting that adaptation to the onion model may follow several different pathways.     

Brief communication: Ectocranial suture closure in Pongo: Pattern and phylogeny

Ectocranial suture fusion patterns have been shown to contain biological and phylogenetic information. Previously the patterns of Homo, Pan, and Gorilla have been described. These data reflect the phylogenetic relationships among these species. In this study, we applied similar methodology to Pongo to determine the suture synostosis progression of this genus, and to allow comparison to previously reported data on other large‐bodied hominoids. We hypothesized these data would strengthen the argument that suture synostosis patterns reflect the phylogeny of primate taxa. Results indicate that the synostosis of vault sutures in Pongo is similar to that reported for Gorilla (excluding Pan and Homo). However, the lateral‐anterior pattern of fusion, in which there is a strong superior to inferior pattern, for Pongo is unique among these species, reflecting its phylogenetic distinctness among great ape taxa. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Development of homogeneous luminescence assays for histone demethylase catalysis and binding

Covalent modifications to histones play important roles in chromatin dynamics and the regulation of gene expression. The JumonjiC (JmjC)-containing histone demethylases (HDMs) catalyze the demethylation of methylated lysine residues on histone tails. Here we report the development of homogeneous luminescence-based assay methods for measuring the catalytic activity and the binding affinities of peptides to HDMs. The assays use amplified luminescent proximity homogeneous assay (ALPHA) technology, are sensitive and robust, and can be used for small molecule inhibitor screening of HDMs. We have profiled known inhibitors of JMJD2E and demonstrate a correlation between the inhibitor potencies determined by the ALPHA and other types of assays. Although this study focuses on the JMJD2E isoform, the catalytic turnover and binding assays described here can be used in studies on other HDMs. The assays should be useful for the development of small molecule inhibitors selective for HDM isoforms.     

A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.     

Mixed model approaches for the identification of QTLs within a maize hybrid breeding program

Two outlines for mixed model based approaches to quantitative trait locus (QTL) mapping in existing maize hybrid selection programs are presented: a restricted maximum likelihood (REML) and a Bayesian Markov Chain Monte Carlo (MCMC) approach. The methods use the in-silico-mapping procedure developed by Parisseaux and Bernardo (2004) as a starting point. The original single-point approach is extended to a multi-point approach that facilitates interval mapping procedures. For computational and conceptual reasons, we partition the full set of relationships from founders to parents of hybrids into two types of relations by defining so-called intermediate founders. QTL effects are defined in terms of those intermediate founders. Marker based identity by descent relationships between intermediate founders define structuring matrices for the QTL effects that change along the genome. The dimension of the vector of QTL effects is reduced by the fact that there are fewer intermediate founders than parents. Furthermore, additional reduction in the number of QTL effects follows from the identification of founder groups by various algorithms. As a result, we obtain a powerful mixed model based statistical framework to identify QTLs in genetic backgrounds relevant to the elite germplasm of a commercial breeding program. The identification of such QTLs will provide the foundation for effective marker assisted and genome wide selection strategies. Analyses of an example data set show that QTLs are primarily identified in different heterotic groups and point to complementation of additive QTL effects as an important factor in hybrid performance.