Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.     

A susceptibility gene for alveolar lung tumors in the mouse maps between Hsp70.3 and G7 within the H2 complex

Lung tumor susceptibility (LTS) in the mouse is influenced by multiple loci within the H2 complex. We compared the LTS of two H2 congenic strains with intra H2 recombinations, B10.A(1R) and B10.A(2R), whose genetic difference has been reduced to a region of approximately 50 kilobases within the C4-H2D interval, between Hsp70.3 and G7. After transplacental induction with N-ethyl-N-nitrosourea the load of alveolar lung tumors in strain B10.A(2R) is significantly higher than in strain B10.A(1R) (P <0.001). For papillary tumors no significant differences were observed. We conclude that the alveolar lung tumor load is influenced by an LTS gene located within the Hsp70.3-G7 interval.     

Simple and efficient CRISPR/Cas9‐mediated targeted mutagenesis in Xenopus tropicalis

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers. genesis 51:835–843. © 2013 Wiley Periodicals, Inc.     

Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage

Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time‐intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185‐bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per‐amplicon‐variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi‐quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus‐specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species.     

Age-Dependent Changes in Geometry,Tissue Composition and Mechanical Properties of Fetal to Adult Cryopreserved Human Heart Valves

There is limited information about age-specific structural and functional properties of human heart valves, while this information is key to the development and evaluation of living valve replacements for pediatric and adolescent patients. Here, we present an extended data set of structure-function properties of cryopreserved human pulmonary and aortic heart valves, providing age-specific information for living valve replacements. Tissue composition, morphology, mechanical properties, and maturation of leaflets from 16 pairs of structurally unaffected aortic and pulmonary valves of human donors (fetal-53 years) were analyzed. Interestingly, no major differences were observed between the aortic and pulmonary valves. Valve annulus and leaflet dimensions increase throughout life. The typical three-layered leaflet structure is present before birth, but becomes more distinct with age. After birth, cell numbers decrease rapidly, while remaining cells obtain a quiescent phenotype and reside in the ventricularis and spongiosa. With age and maturation–but more pronounced in aortic valves–the matrix shows an increasing amount of collagen and collagen cross-links and a reduction in glycosaminoglycans. These matrix changes correlate with increasing leaflet stiffness with age. Our data provide a new and comprehensive overview of the changes of structure-function properties of fetal to adult human semilunar heart valves that can be used to evaluate and optimize future therapies, such as tissue engineering of heart valves. Changing hemodynamic conditions with age can explain initial changes in matrix composition and consequent mechanical properties, but cannot explain the ongoing changes in valve dimensions and matrix composition at older age.     

Adult neurogenesis and pattern separation in rodents: A critical evaluation of data,tasks and interpretation

The ability to discriminate and store similar inputs as distinct representations in memory is thought to rely on a process called pattern separation in the dentate gyrus of the hippocampus. Recent computational and empirical findings support a role for adult-born granule neurons in spatial pattern separation. We reviewed rodent studies that have manipulated both hippocampal adult neurogenesis and assessed pattern separation. The majority of studies report a supporting role of adult born neurons in pattern separation as measured at the behavioral level. However, closer evaluation of the published findings reveals variation in both pattern separation tasks and in the interpretation of behavioral performance that, taken together, suggests that the role of hippocampal adult neurogenesis in pattern separation may be less established than is currently assumed. Assessment of pattern separation at the network level through the use of immediate early gene expression, optogenetic, pharmacogenetic and/or in vivo electrophysiology studies could be instrumental in further confirming a role of adult born neurons in pattern separation further. Finally, hippocampal adult neurogenesis and pattern separation are not an exclusive pair, as evidence for hippocampal adult neurogenesis contributing to the temporal separation of events in memory, forgetting and cognitive flexibility has also been found. We conclude that whereas current empirical evidence for the involvement of hippocampal adult neurogenesis in pattern separation seems supportive, there is a need for careful interpretation of behavioral findings and an integration of the various proposed functions of adult born neurons.     

Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization

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Internalization-defective mutants of somatostatin receptor subtype 2 exert normal signaling functions in hematopoietic cells

The regulatory peptide somatostatin (SST) acts via a family of G-protein-coupled receptors comprising five subtypes (SSTR1-5). G-protein-coupled receptors activate multiple signaling mechanisms, which variably depend on internalization and intracellular routing of activated receptors. We have recently demonstrated that hematopoietic precursors express SSTR2 and that SST is a chemoattractant for these cells. Herein, we characterize critical regions in SSTR2 involved in endocytosis and describe how ligand-induced internalization impacts on two major signaling functions of SSTR2 in hematopoietic cells, the activation of the Erk pathway and the induction of promigratory responses.     

Salicylic acid-independent plant defence pathways

Salicylic acid is an important signalling molecule involved in both locally and systemically induced disease resistance responses. Recent advances in our understanding of plant defence signalling have revealed that plants employ a network of signal transduction pathways, some of which are independent of salicylic acid. Evidence is emerging that jasmonic acid and ethylene play key roles in these salicylic acid-independent pathways. Cross-talk between the salicylic acid-dependent and the salicylic acid-independent pathways provides great regulatory potential for activating multiple resistance mechanisms in varying combinations.     

Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.